An ethanolic extraction of Galenia africana was prepared. The ethanolic extract indicated antifungal activity by means of broth macro- and microdilutions and disk diffusion method. A minimum inhibitory concentration (MIC) was calculated against fungus, Candida albicans, and was evident by a clear 'zone of inhibition' on six different agars. A Flavonoid structure of Galenia africana was identified, by spectroscopic analysis, as the bioactive component contributing to the anti-candidal activity.
1. STATEMENT OF RESEARCH PROBLEM
Candida albicans is a dimorphic fungus that is found naturally in the human micro biota of the gastrointestinal and respiratory tract, vaginal area, as well as in the mouth. It is a commensal microorganism that, when removed from its natural habitat, has the ability to become pathogenic, expressing its virulence factors producing an opportunistic infection known as candidiosis (Wiley et al, 2011).
The extensive treatment of Candida by means of 'azole (e.g. Fluconazole) and polyene (e.g. Amphotericin B) drugs has led to the emergence of resistant strains as well as a range of other coupled problems due to the severe side effects of these therapeutic drugs (Lee et al, 2011). As a result, we aim to procure a new drug or fungistatic agent that has a low toxic level with a high healing level.
In recent studies, plant extracts have been used as a means of replacement therapy for traditional microbial control strategies, as these plant-compounds are widely and freely available (Lee et al, 2011).
Galenia africana, also known as 'Kraalbos', is a southern African plant that shows high levels of antimycobacterial activity when chewed or used in the form of a paste/liquid made by decoction of the plant material. In the past, a tribe known as the Hottentots used to use the plant for the treatment of toothache, venereal and skin diseases as well as for the relief of inflamed eyes (Pool et al, 2009). Flavonoids found in G. africana, show to have a major contribution to the mode of action against C. albicans by one or more of the following actions; Inhibition of nucleic acid synthesis, cytoplasmic membrane function and inhibition of energy metabolism (Cushnie et al, 2005).
In this paper, we aim to investigate the in vitro effects of Galenia africana ethanolic extracts on the growth of the fungus, Candida albicans, during mode of action studies.
2. LITERATURE REVIEW
2.1. Candida albicans
2.1.1 Epidemiology of Candida albicans
Studies performed have determined that the pattern of transmission of infection by Candida albicans is most commonly vertical (from mother to neonate) (Chapman et al, 2003). Oesophageal and vaginal candidiosis are among the most frequently occurring infections seen in HIV patients and young woman respectively. Oral candidiosis, also known as or thrush, is common among young children, or infants.
2.1.2 Pathogenicity and virulence factors of Candida albicans
C. Albicans is a dimorphic fungus which is capable of a yeast-to-hyphal -phase. It may differ in colony morphology, cell shape cell surface and permeability. These are important properties contributing to candidal pathogenicity (Lewis et al, 2012). Several factors have attributed to the virulence of the Candida species; some include the ability to form hyphae, biofilms, secretion of hydrolases, adherence to host tissue, and their response to environmental changes and morphogenesis. The greatest of all characteristics is their ability to colonise both biological and inanimate objects such as catheters, thus allowing for the formation of a biofilm which can later infect a human host (Lewis et al, 2012).
2.1.3 Morphology of Candida albicans
The opportunistic pathogen can exist in multiple morphological forms depending on its surrounding environment. It most commonly forms buds, but under adjusted conditions is able to form elongated pseudohyphae, long filamentous chains of cells or alternatively large round cell, known as a chlamydospore (Shick et al, 2011).
2.2. Herbal medication and plant derived drugs of Southern Africa
Plants have been used as medical medications for over sixty thousand years and have given rise to some of the most important modern day drugs used today (Ammena Gurib-facim, 2006). South Africa is renowned for their unique and diverse botanical heritage with more than thirty thousand plant species of which three thousand of these are used for therapeutic purposes ( Van Vuuren, 2008). African traditional medication is amongst the oldest medical system, usually referred to as the (cradle of mankind) (Van Wyk et al, 1997). Early literature on KhoiKhoi, Cape Dutch and San (Bushmen) medicinal plants, medical practices and techniques indicate a variety of plants used for healing. Some of these healing processes include massage and aromatherapy. Sheep fat combined with a preparation of plant material was used during massage to relieve pain. Aromatherapy, a key characteristic in the San culture, involves the use of powdered aromatic leaves and sheep fat combined with massage (Van Wyk, 2008). Different forms of preparations have been recorded including masticatories, infusions in water, decoctions (in water or milk), tinctures, poultices and snuffs (Pappe, 1847).
2.2.2 Medicinal uses of plants
Many plants produce substances that are important for health maintenance in humans and animals then consumed. Substances include aromatic compounds of which most are phenolic or oxygen substituted derivatives. Secondary metabolites such as alkaloids, a nitrogenous organic molecule, have pharmacological effects on humans and animals and in many cases, serve as defence mechanisms (wiki, alkaloid). Bee products such as honey and pollen are traditionally used in the treatment of diseases. Traditionally, honey would be used to treat inflammation of the skin or throat. This is due to the large concentration of Flavonoids found in the honey flavoured extract (Candiracci et al, 2012; Al-mamary et al, 2002). Multiple plants derived from South Africa have antimicrobial activity, such as the treatment of mycobacterium related illnesses with Galenia africana (Mativandlela et al, 2009).
2.3. Galenia africana
2.3.1 Ethanolic extraction
The extract obtained from the plant is taken from the leaves (Mabusela et al, 2005) or alternatively the whole plant (Namrita Lall et al, 2014). The leaves of the plant are ground and subsequently mixed with the ethanol. Dried extracts are resuspended in Dimethyl Sulfoxide (DMSO) to obtain a 50% extract. The ethanolic extract can then be used in determining antimicrobial susceptibility by infusion into paper discs (Pool, et al).
2.3.2 Antifungal phyto-constituents and mode of action
Antifungal active extracts were indentified by Mabusela et al against multiple strains of fungi. The structures of the compounds were identified as (1) 5-hydeoxy-7-methoxy-flavanone or pinostobin; (2) dihydroechinoldinin which has been recently isolated from Andrographis echoides; (3) 5,7-dihydroxy-flavonone or pinocembrin; (4) 2',4'-dihydrochalcone and (5) 2',4'-dihydroxydihydrochalcone (Mabusela et al, 2009). A new flavanone has recently been discovered, isolated from a shrub called Eysenhardtia texana. 5,7,4-trihyroxy-8-methy-6-(3-methyl-[2-butenyl])-(25)-flavonone has shown to possess antifungal activity against C.albicans (Cushnie et al, 2005).
2.4. Anti-fungal susceptibility testing
2.4.1 Kirby-Bauer test
The Kirby Bower method also referred to as the disc diffusion susceptibility test (Wiley et al, 2011) involves the inoculation of a petri plate with a standardised amount of the fungus. Small discs containing a pre infused drug are arranged firmly on the inoculated plate. After the discs have been placed, the plates are incubated allowing the pathogen to replicate producing a lawn. The areas around the disc that are clear indicate the sensitivity of the pathogen to the drug. This is known as a (zone of inhibition). The larger the zone of inhibition the greater the degree of sensitivity to the drug tested. If there is no clearing around the disc it is said that the pathogen is resistant to the drug (Lawrence, et al).
2.4.2 Minimum Inhibitory Concentration (MIC)
As suggested by the title, the minimum inhibitory concentration is the smallest amount of the tested drug that will inhibit the reproduction and growth of the pathogen (Wiley et al, 2011). There are multiple methods of determining the minimum inhibitory concentration, including agar and broth dilutions.
2.4.3 MTT and XTT assay
The MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, assay and XTT, sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium, assay are colorimetric assays that are used in the discovery of cell viability. Particular enzymes, namely NAD(P)H-dependent cellular oxidoreductase enzymes, are responsible for the conversion of the yellow MTT dye to an insoluble form of formazan, a purple dye. A solubilizing solution, usually DMSO, is added to the purple solution dissolving it thus resulting in a colour change. The final product can be read using a spectrophotometer, usually between 500-600nmwavelength (Wiki MTT assay, 2014). The XTT assay is a newly adapted assay serving to replace the MTT assay. It has the ability to yield a much higher sensitivity and broader dynamic range resulting in a more soluble formazan dye product. This formazan dye, unlike the MTT assay formazan, is water soluble thus allowing for the elimination of the last solubilization step as in the MTT assay (Wiki MTT assay, 2014). The use of the XTT assay is a method commonly employed to quantify Candidal biofilm growth and response to drug therapy (Nett et al, 2010).
2.5.1 Types of microscopy used
For viewing the morphological characteristics of Candida albicans, scanning electron microscopy (SEM) is used. Other microscopic techniques can be used such as Transmission Electron Microscopes (Shannon,1981). TEM produces a flat (2D) image that have greater resolution and higher magnification than what SEM does. An SEM might be used in preference to a TEM as it produces a three dimensional (3D) image.
3. AIMS OF THE RESEARCH
By doing this study, we aim to answer the following questions;
What is the Minimum Inhibitory Concentration (MIC) of the G. africana extracts required to inhibit the growth of C. albicans?
Which methods can be used to determine the effects of the G. africana extract on the inhibition of C. albicans?
Are the inhibitory effects of the G. africana extract similar to that of the fluconazole antibiotic?
Could one use microscopic studies to evaluate the inhibitory effects of the G. africana extract?
The ethanolic extract of Galenia africana will have a fungistatic and fungicidal on the mode of action of Candida albicans at a known minimum inhibitory concentration.
5. RESEARCH DESIGN AND METHODOLOGY
5.1 Plant extraction
Leaves of the plant are minced (sieve size~ 2-3 mm) after which the leaves are mixed with 94.4% ethanol. The mixture is shaken for brief periods during the overnight extraction. The extracts are then pressed to separate the tincture and the milled leaves. Filtration is carried out to clean the tincture from the plant debris and the extract is stored at 20??C. The extracts are then air dried in a laminar air flow hood and re-suspended in dimethyl sulfoxide (DMSO) to obtain a 50% (w/v) extract.
5.2 Minimum Inhibitory Concentration (MIC)
The MIC`s are determined using the agar well diffusion method as well as the broth serial dilutions as a modified version of Witebsky et al, 1979. Two-fold serial dilutions of the active portion are performed.
5.3 Antifungal susceptibility testing
Antifungal susceptibility testing is performed by disk diffusion susceptibility testing or better known as Kirby-Bauer test. Candida strains are grown on Nutrient agar, Mueller-Hinton agar, Biggy agar, Brilliance candida agar, Corn meal agar, Brilliance candida agar selective supplement, Tryptone Soya agar and Tryptone Soya broth. Disks soaked in the plant extract placed on the inoculated plates and allowed to incubate at 37??C overnight. After incubation the plates are examined for the presence of a zone of inhibition. The zones are measured in millimeters starting from the edge of the well and then the means of each were determined and compared. Determinations all made in triplicate.
Scanning electron microscopy and Transfer Electron Microscopy is then used to view the effect of the extract on the morphology and functionality of the cells.
5.4 MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and XTT [sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium] assay
An MTT as well as XTT assay are performed to assess cell viability. Protocol as per Kuhn et al, 2003.
5.5 Statistical analyisis
Data is expressed as mean ?? S.D. Data was analyzed statistically using dependant and independent t- tests. Values P<0.05 are considered statistically significant. Experiments run in triplicates were analysed by one-way ANOVA, values P<0.001 are considered statistically significant.
5.6 Project milestones
5.7 Dissemination of results
Results of the project will be published as a journal article and disseminated to scientists whom aim to replicate the experiment. Results should also be disseminated to the public as a new form of drug available for the treatment of diseases or infections produced by the fungus Candida albicans.
6. ANTICIPATED IMPACT
A new pharmaceutical discovered, G. africana ethanolic extract, will be an alternative and herbal treatment accessible to patients suffering infections caused by the fungus C. albicans.
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