Antifungal Activities of <em><em>Camellia Sinensis</em></em> Crude Extract on Selected Pathogenic and Mycotoxic Fungi

Research Article

J Bacteriol Mycol. 2015; 2(2): 1015.

Antifungal Activities of Camellia Sinensis Crude Extract on Selected Pathogenic and Mycotoxic Fungi

Cheruiyot SE¹*, Muturi M¹ and Bii C²

¹Department of Medical Laboratory Sciences, School of Medicine, Kenyatta University, Kenya

²Department of Infectious Diseases, Centre for microbiology Research, Kenya Medical Research Institute, Kenya

*Corresponding author: Sigei Erolls Cheruiyot, Department of Medical Laboratory Sciences, School of Medicine, Kenyatta University,

Received: May 13, 2015; Accepted: September 10, 2015; Published: October 01, 2015


The objective of this study was undertaken to evaluate the in vitro antifungal activity, Minimum inhibitory concentration (MICs) and fungicidal/ fungi static property of Camellia Sinensis crude extracts. The aqueous extracts of Camellia Sinensis were investigated against seven fungal species; Green and black tea crude (100mgmL-1) extracts were evaluated for antifungal activities. Quantitative bioassay was done using disc diffusion method and MIC done using broth dilution methods. The fungal isolates used for bioactivity testing were yeasts. Green tea crude extract showed stronger inhibitory effect against the fungal strains tested than black tea crude extract. There was a significant difference in zone of inhibitions (T=4.09, P<0.05). Zone of inhibition exhibited by green tea crude extracts (11.92±0.00mm) were higher than black tea (8.14±0.56mm). The pattern of activity by tea crude extracts against ATCC standard fungal strains and clinical isolates strains were similar. Candidafamata, Candidalusitaniae, Candidatropicalis ATCC 750 and dermatophyte, Trichophytonmentangrophyte were inhibited by green tea crude extract (IZD=15mm). Clinical isolates of Candida albicans (strain 4 and strain 5); Cryptococcus neoformans (strain 3, 5 and 12), showed susceptibility to Camellia Sinensis green crude extracts. The MIC of crude extracts against fungal isolates tested ranged from 50 mg mL-1 to 1.6 mg mL-1. Hot green tea crude extract (mean MIC 12.25mg mL-1) had a higher MIC on clinical fungal isolates than cold green tea crude extract (Mean MIC 12.167 mg mL-1).The studies on Camellia Sinensis have shown remarkable antifungal activity and highlighted its significance as potential health products.

Keywords: Camellia Sinensis; Crude tea extracts; Green tea; Black tea; Fungal species


Human fungal infections pose serious medical issues. Tea is one of the most consumed drinks worldwide where green tea (Camellia Sinensis) comprises of about over 20% of the total tea consumption [1]. Recently, several studies have shown that green tea consumption can protect against diseases that are associated with free radical damage including atherosclerosis, coronary heart disease and cancer [1-3]. The Kenyan tea germ plasm has been observed to be diverse in its polyphenol composition and contents and therefore provides raw material for production of different types of tea products including health drinks [4]. However, the state of research on tea regarding its pharmacological properties in relation to fungi is limited and the majority of work has been conducted on green tea with very little on black and white tea. Beneficial effects of tea have been attributed to the strong antioxidative activity of the tea phenolic compounds known as catechins [5]. In addition, many scientific research reports have presented data on the antimicrobial activity of different types of tea extracts on various pathogenic microorganisms [6,7]. Green tea elicits strong antibacterial activity including potential to inhibit gram positive cocci; gram negative bacilli [8]. Studies have also shown that tea can inhibit and kill a

wide range of pathogenic bacteria at or slightly below typical concentrations found in brewed tea [8]. Various studies have shown significant suppressive effects of green tea polyphenols against many microorganisms [9]. Black tea, a major source of phenolic, [10] including theaflavins and thearubigins, [11] has also been shown to have antimicrobial properties both in vivo and in vitro [12]. This study attempts to facilitate to establish the potentiality of Camellia Sinensis plant product as novel modalities in the line of new drug discoveries.

Material and Methods

Samples of Camellia Sinensis

Processed Commercial Camellia Sinensis (black and Green tea) produced and packed by James Finlay (K) Ltd were purchased off shelf in retail outlet at the factory in Kericho County, Kenya.

Test fungal organisms

The standard test fungi of American Type Culture Collection (ATCC) was sourced from Kenya Medical Research Institute (KEMRI) and included: Cryptococcus neoformans ATCC 66031, Candida albicans ATCC 90028, Candida krusei ATCC 6258, Candida glabrata ATCC 24433, Candida tropicalis ATCC 750, and Candida parapsilosis ATCC22019 as standard organisms. Clinical isolates included: Cryptococcus neoformans, Candida albicans, Candida famata, Candida lusitaniae, Trichophytonmentangrophytes, Microsporum gypseum. Mycotoxigenic fungi included: environmental pathogenic isolates Fusarium moniliforme, Aspergillus flavus, Aspergillus niger and Penicillium chrysogeneum. The selection of test strains was based on their significance as opportunistic pathogens and their resistance to conventional drugs.

Preparation of McFarland standard

McFarland standard is used as a reference to adjust the turbidity of fungal suspension so that fungal organisms will be within a given range. Exactly 0.5 McFarland equivalent turbidity standards was prepared by adding 0.6 ml of 1% barium chloride solution (BaCl2.2H20) to 99.4 ml of 1% sulphuric acid (H2SO4) and mixed thoroughly. A small volume of the turbid solution was transferred to cap tube of the same type that was used to prepare the test and control inocula. It was then stored in the dark at room temperature (25°C). Exactly 0.5 McFarland gives an equivalent approximate density of fungi 1.5 x 108 Colony Forming Units per ml (CFU) mL-1 [13].

Crude extraction of Camellia Sinensis (Teas)

The prepared soluble granules of both black and green tea samples sealed in silver lined sachets stored at room temperature were obtained. Cold aqueous crude extracts were done by soaking weighed amount of dry soluble granules of tea (10 grammes) in 100 mL of sterile distilled water and shaken for half an hour in an electric shaker. The extracts were filtered using Whatman No.1 filter paper to exclude any suspending granules. Crude extract, supernatant, was then transferred to sterile screw cap bottles, labeled and stored under refrigerated condition (40C) until use. For hot aqueous crude extraction, 10 grammes of dry soluble granules of tea samples were extracted with 100 mL of boiling water and filtered using sterile Whatman filter paper No.1 to give a solution that contains 100 mg/ ml [6]. Crude extract, filtrate, was then transferred to sterile screw cap bottles, labeled and stored under refrigerated condition (40C) until use. Only fresh extracts was used in the experiment, as marked chemical changes occurred when tea was allowed to stand [14].

Preparation of tea extracts stock and working solutions

A twofold dilutions were obtained (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml, 1.5625 mg/ml) concentrations. Antifungal activities of the above concentrations were determined.

Preparations of antifungal compounds stock and working solutions

The antifungal compounds were removed from storage (-200C) and allowed to come to room temperature. Each 250 μg of antifungal compound (Fluconazole) was weighed and dissolved in sterile distilled water to make a final 10 mL solution. The stock solutions of azole group of compounds (Fluconazole) used was usually kept at -200C until used. Doubling dilutions of stock solutions were made to obtain working solution.

Antimicrobial assay

The antimicrobial activities of the extracts were evaluated by the disc diffusion method [15]. The use of agar disc diffusion method to screen for antimicrobial activities of the crude tea extracts was done according to the National committee of clinical and laboratory standards [16] now CLSI. Dilutions of several concentrations of the crude tea extracts and azole group of compounds, Fluconazole, were then made in a test tube using sterile distilled water. Positive and negative standard controls were used. Blank sterile paper discs measuring 6mm were impregnated with 20 μL of test concentration of crude tea extract. The discs were air dried and aseptically transferred into respective inoculated plates [17]. The activities of the tea crude extracts were established by the presence zones of inhibition which were measured in mm. Fluconazole discs containing (25 μg) were used as antifungal reference standards. Similarly the sterile distilled water was set as negative controls.

Extracts with activity was serially diluted and re-tested to determine the minimum inhibitory concentrations (MIC). All the assays were carried out in triplicates, average result calculated and recorded against corresponding concentrations as described [18]. Assays were subjected to quality control procedures recommended by clinical laboratory standard institute (CLSI). Fluconazole disc was prepared as described by [19]. Minimum inhibitory concentrations were determined by Broth micro dilution method for the active crude extracts against test fungal organisms. The procedures were done as recommended by the National Committee for Clinical Laboratory Standards (NCCL) now Clinical Laboratory Standard Institute (CLSI) [20]. The MIC was recorded as the lowest extract concentration demonstrating no visible growth as compared to the control broth turbidity [21]. Wells that were not inoculated were set to act as control.


The antifungal activities of green and black tea (Camellia Sinensis) crude extracts having a concentration of 100 mg/ml of extracting solvent (sterile distilled water) on selected yeasts and mould are presented in the Table 1 below. Their inhibitory effects against selected pathogenic and mycotoxic fungi were then compared.