Antifungal activity of crude tea extracts

1 Department of Biochemistry and Molecular Biology, Egerton University, P. O. Box 536, Egerton, Kenya. 2 Tea Research Foundation of Kenya, P. O. Box 820, Kericho, Kenya. 3 Association for Strengthening Agricultural Research in Eastern and Central Africa, P. O. Box 765 Entebbe, Uganda. 4 Department of Infectious Diseases, Centre for Respiratory Diseases Research, Kenya Medical Research Institute, P. O. Box 54840-00200 Nairobi, Kenya. .


INTRODUCTION
Tea is the single largest agribusiness in Kenya and the crop contributes over 26% of all foreign exchange earnings and over 4% of the gross domestic product (Economic Survey, 2005).In Africa, Kenya specializes in black tea production and processing and making the country the third largest producer of tea in the world after China and India (ITC, 2006).Of the numerous tea products in the market, green tea is mainly consumed in China, Japan and the Middle East, while black tea is mostly consumed in India, Sri-Lanka, European countries and regions of Africa.Currently, tea is the leading foreign exchange earner and export commodity amongst agricultural produces in Kenya.In 2010, the tea industry turnover was Kshs 97 billion in foreign exchange after exporting 398.5 million kilograms of made tea (TBK, 2010).
There is a growing body of knowledge on health benefits of tea.This knowledge has however been largely generated from studies using green tea (Carmen et al., 2006).However, little or no work has been carried out on black tea, yet this is the predominant type of tea produced and consumed in Kenya because of a dire paucity of information on the potential health benefits of black tea.Studies have demonstrated that, processed Kenyan black tea have total polyphenols levels that are as high as those found in green teas produced in Asia (Wachira and Kamunya, 2005) and therefore may be as efficacious as green tea in enhancing human health.*Corresponding author.E-mail: fwachira@yahoo.com.
Therefore, there is a need to promptly initiate research on black tea to establish its beneficial effects on human health.
A few studies have focused on the evaluation of antimicrobial activity of black tea, while several scientific studies have been conducted using green tea (Sharangi, 2009).This study therefore is of the first kind aimed at investigating the antifungal activity of aqueous extracts of black, green and white tea products from Kenyan germplasm and green tea products from Chinese and Japanese germplasm against Candida albicans ATCC 90028 and a clinical isolate of Cryptococcus neoformans.

Fungi
The test fungi of American Type Culture Collection (ATCC) were sourced from the Kenya Medical Research Institute Centre for Respiratory Disease Research (KEMRI-CRDR) and included C. albicans ATCC 90028 and a clinical isolate of C. neoformans.

Biochemical profiling of the tea extracts based on catechins
A modified high performance liquid chromatography method was used to assay for the tea catechins (Zuo et al., 2002).

Estimation of total polyphenols in the tea extracts
Folin-Ciocalteu phenol reagent method was used to determine total polyphenols in the tea extracts according to ISO (BS ISO 14502-1: 2005(E)).

Analysis of total theaflavins content in the tea sample by flavognost method
Black, green, purple and white teas were also assayed for total theaflavins (TF) using the flavognost method of Hilton (1973).

Spectrophotometric determination of total thearubigins in the tea samples
Total thearubigins (TRs) were determined in the tea samples using the method of Roberts and Smith (1961).

Determination of antioxidant activity of tea
The stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was used for determination of free radical scavenging of tea extracts using a modified method of Brand-Williams et al. (1995).The assay is based on the measurement of scavenging ability of antioxidants towards the stable DPPH radical.The percentage inhibition of the DPPH radical was calculated from the absorbance data according to the method of Yen and Duh (1994).

Freeze drying of tea liquors
Tea liquors derived from the processed tea samples were freeze dried according to the method described by Turkmen et al. (2009).Twenty grams (20 g) of the tea sample was mixed with 200 ml of water for 30 min and subjected to continuous hot water extraction at 70°C for 2 h.The resulting water extract was then filtered and subsequently freeze dried using an Edwards Modulyo freeze drier (EF4) to give 2 g of powder, a yield of 20.1%.

Antifungal assays
Fungal isolates of C. albicans ATCC 90028 and a clinical isolate of C. neoformans were used in the study.The agar disc diffusion method was used to screen for antifungal activities of the tea liquors according to standardize method of the National Committee of Clinical and Laboratory Standards (NCCLS, 2011).The discs used absorbed 0.01 ml of the sample hence the concentration of each sample extract was established.

Minimum inhibitory concentrations
Tea liquors that presented inhibitory properties in vitro in the screening activity were evaluated for their minimum inhibitory concentration (MIC) using the disc diffusion test.The MIC was determined as the lowest drug concentration that inhibited growth, as recommended by the National Committee of Clinical and Laboratory Standards (NCCLS, 2011).

Statistical analysis
All determinations were carried out in triplicate and data were subjected to analysis of variance using MSTATC software.The Duncan's multiple range test (DMRT) was used to separate the means.

Antifungal activity
The results of this study showed that different tea extracts had antifungal activity against C. albicans ATCC 90028 and a clinical isolate of C. neoformans at 1 mg/ml after 24 h (Figure 1).There was no significant difference (p≤0.05) in the antifungal activity of Kenyan black tea and purple leaf coloured (aerated) tea extracts with the Chinese and Japanese green tea extracts against C. albicans ATCC 90028.There was also no significant difference (p≤0.05) between black tea processed from terminal tea buds of TRFK 301/5 and AHP S15/10 with green tea extracts from Kenyan, Chinese and Japanese cultivars in the antifungal activity.Unaerated tea from purple leaf coloured and white tea extracts did not differ significantly in the antifungal activity with black tea extracts against C. albicans ATCC 90028.This  corroborated with the results of Sitheeque et al. (2009) who showed antifungal activity of both green and black tea catechins against C. albicans.
The antifungal activity shown by tea extracts is probably due to mainly the catechin EGCG and perhaps EGC in green and white tea and theaflavins and thearubigins in black tea.The results on the antifungal activity also indicated that the green tea products as well as products from the purple leaf coloured (unaerated) tea and white tea (silvery tips) products processed from Kenyan tea cultivars exerted the highest antifungal activities.This may indicate that the presence of the hydroxyl moieties at 3', 4', and 5' on the B ring in the catechin and epicatechin molecules is a major contributing factor that contributed to inhibitory activity of both green, unaerated tea from the purple leaf coloured clone and white tea.The contributions of the other catechins might be limited by the fact that only small amounts are presented.This is in agreement with a study reported by Nance et al. (2006) who concluded that antimicrobial activity of catechins is predominantly as a result of the gallic moiety and hydroxyl group member.
A clinical isolate of C. neoformans was inhibited by all the different types of tea extracts used in this study (Figure 1).White tea extracts from the Kenyan tea cultivars exhibited the highest antifungal activity against the clinical isolate of C. neoformans as compared to black tea, black tea buds, green tea and aerated and unaerated tea extracts from the purple coloured clone.Nystatin (30 µg) was used as a positive standard control while distilled water was used as a negative standard control.

Correlation of biochemical parameters with antifungal activity
The highest antifungal activity also corresponded to the highest total polyphenols content and to antioxidant activity.Several tea polyphenols of black, green and white tea products had significant antifungal activity.These included EC, EGCG, ECG, TFs and TRs.The total polyphenols (r = 0.579*), gallic acid (r = 0.534*), EGCG (r = 0.514*), ECG (r = 0.577*) and total TRs (r = 0.550*) significantly correlated with inhibition zone diameters of C. albicans ATCC 90028 (Table 1).The inhibition zones  for clinical isolate of C. neoformans correlated significantly with total polyphenols (r = 0.765***), antioxidant activity (r = 0.664**), GA (r = 0.533*), EC (r = 0.537*), EGCG (r = 0.718***), ECG (r = 0.620**), total TFs (r = 0.739***) and total TRs (r = 0.769***) as presented in Table 2. Overly, from this study TPP, EGCG, ECG, GA, total TFs and total TR were identified as the most potent antifungal biochemicals in the assayed teas.Results from this study generally revealed that the inhibition zones were significantly and positively correlated to the catechins (EGCG and EGC), total TFs and total TRs.The results obtained in this study are therefore in agreement with those of Gramza and Korczak (2005), who studied the effects of individual catechins separately and found that EGCG and EGC had the highest antioxidant and antimicrobial activity.
Since medicinal plants produce a variety of substances with antimicrobial properties, screening programs are expected to find out new compounds well suited to the development of new antibiotic drugs.Present findings suggest a potential antifungal activity of tea extracts against C. albicans ATCC 90028 and a clinical isolate of C. neoformans.

Figure 1 .
Figure 1.Variation in antifungal activity among different types of tea extracts.

Table 1 .
Correlation coefficients between inhibition zone diameters (IZDs) and various tea biochemical parameters for C. albicans ATCC 90028.

Table 2 .
Correlation coefficients between inhibition zone diameters (IZDs) and various tea biochemical parameters for clinical isolate of C. neoformans.