Tea is typically made from an infusion of dried leaves or young shoots of the tea bush with hot water . Different methods of leaf processing and alternative brewing methods can affect subsequent antimicrobial activity although these were not investigated these in this study [24, 25].
Levels of copper found in WT following hot water extraction were found to be comparable to those reported for GT which may reflect similar methods of processing and dehydration of these two teas . In BT production leaves are subject to a longer dehydrating process which could account for the higher concentration of copper reported in BT leaf product . Some metal ions are also known to accumulate to varying levels in the tea bush according to cultivar type and growing conditions .
In suspension assays copper (II) sulphate tested at the level found in the WT extract (0.56 μM added to assay, equivalent to level of 0.14 mg L-1 found in WT) was found to have the same effect as a buffer control on the viability of S. aureus NCTC 06571. Copper (II) sulphate tested at higher concentrations produced an increase in bactericidal effect. When WT was added to 4.8 mM copper (II) sulphate the same viability was seen as for 4.8 mM copper (II) sulphate alone (c.a 1 log10 cfu mL-1) suggesting no additional effect on viability from the WT. When GT and BT were combined with 4.8 mM copper (II) sulphate both tea combinations showed a similar viability efficacy to buffer control levels suggesting the GT and BT had negated the bactericidal effect of the copper (II) sulphate. It is possible that GT and BT both reduced the bioavailability of the copper (II) ions by the formation of chemical complexes, which showed no antimicrobial efficacy.
Vitamin C assayed in the absence of teas enhanced the antimicrobial efficacy of copper (II) sulphate against S. aureus NCTC 06571 although vitamin C itself showed no antimicrobial activity when tested alone. Other investigations have shown that although vitamin C has little or no effect against many Gram positive bacteria it does enhance the bactericidal effect of copper (II) sulphate against both Gram positive and negative species [27, 28]. Studies have also shown that vitamin C can further enhance the antimicrobial efficacy of plant products combined with copper (II) sulphate e.g. pomegranate rind extract [28–30].
WTF (< 1000 Da) was prepared to remove large molecular mass compounds suspected of inhibiting low molecular mass components with potential antimicrobial efficacy. WTF combined with 4.8 mM copper (II) sulphate produced a reduction in viability of c.a 3.5 log10 cfu mL-1 whereas whole WT with the same level of copper (II) sulphate produced a lower fall of c.a 1 log10 cfu mL-1, this level in reduction in viability had been previously shown to be due to the effects of the copper (II) sulphate and not the added tea. Since whole WT was found not to affect the antimicrobial efficacy of the copper (II) sulphate it is possible that whole tea contains substances that affect the bioavailability of smaller bactericidal substances.
The antimicrobial activity of the WTF alone and with added agents against S. aureus NCTC 06571 is comparable to the activity found by McCarrell et al. who showed that another plant extract, pomegranate rind extract (PRE) which like tea contains polyphenols, did not reduce S. aureus NCTC 06571 viability when tested alone but was enhanced by added copper (II) sulphate and further by added vitamin C [28, 29]. Comparing the results of this investigation with those of Gould et al. as well as McCarrell et al. it appears that both WTF and PRE in combination with 4.8 mM copper (II) sulphate produce a similar reduction in viability of c.a 3.5 - 4 log10 cfu mL-1 against the Gram positive S. aureus NCTC 06571 [30, 31, 28].
Aspects of the reaction mechanisms of the antimicrobial agents investigated in this study can be deduced. Where samples contained copper (II) sulphate the addition of catalase reversed the bactericidal effects of these samples on S. aureus NCTC 06571 suggesting the main effect of the copper (II) sulphate and other added agents was via the manufacture of hydrogen peroxide. Other investigators have shown that hydrogen peroxide is bactericidal to S. aureus . It is also known that WT contains flavonoids e.g. catechins some of which are known to generate hydrogen peroxide in aqueous solution and more so when copper (II) ions are also present [33, 34]. In this investigation hydrogen peroxide and other reactive oxygen species (ROS) could have been produced by re-oxidation of copper (I) following reduction of the added copper (II) by chemical species present in the mixtures including e.g. water, vitamin C and other compounds within the WTF e.g. flavonoids [33, 34].
To observe a complete rather than partial restoration of buffer viability levels with the addition of catalase to samples containing copper (II) ions was unexpected since other mechanisms besides the manufacture of hydrogen peroxide have been suggested for the antimicrobial action of copper (II) ions on bacterial cells [35, 36]
. In this investigation catalase tested on copper (II) sulphate alone required 300 μg mL-1 of the enzyme to produce buffer levels of viability whereas 600 μg mL-1 was needed to produce the same viability with the WTF/copper (II) combination. This finding suggests that the WTF enhanced the production of hydrogen peroxide by the copper (II) ions since WTF tested in the absence of copper (II) ions had no antimicrobial effect on the viability of S. aureus NCTC 06571. Addition of vitamin C to the same combination did not require further amounts of added catalase for reversal suggesting the vitamin contributed little to the generation of hydrogen peroxide when WTF was also present. When WTF was absent, a combination of copper (II) sulphate and vitamin C required the same level of catalase (600 μg mL-1 f.c.) to produce buffer levels of viability suggesting the vitamin C contributed to the generation of hydrogen peroxide. Possibly the WTF and the vitamin C both interact with the copper (II) ions in a similar way e.g. by reducing them to copper (I) and may both work at the same reaction site. If this were so then it could explain why either agent enhanced the copper (II) ion production of hydrogen peroxide and yet did not show any summative effect on the viability of S. aureus NCTC 06571 when both were present with the copper (II) ions. Such a mechanism is analogous to that of competitive inhibition in enzyme kinetics.
ROS such as superoxide and hydrogen peroxide can cause the collapse of cell viability . S. aureus NCTC 06571 defends itself against the harmful effects of ROS e.g. hydrogen peroxide by synthesising enzymes such as catalase to deactivate ROS usually by catabolic breakdown . The amount of hydrogen peroxide released by the agents in this investigation apparently exceeded the ability of the bacterial cells to adequately defend themselves by the levels of endogenous cellular catalase.
Experiments into the possible effects of pH on the suspension assays were carried out. Following three different pH adjustments (pH values of 3, 5.5, and 9) to control samples of Ringer's solution no reduction in bacterial viability was seen. Samples were tested in the same way and those containing copper (II) sulphate adjusted to pH 9 showed no reduction in viability when compared to controls and to samples adjusted to pH 3 and pH 5.5. In the case of copper (II) sulphate samples adjusted to pH 9 the addition of NaOH was accompanied by the appearance of a thick pale blue precipitate, conceivably copper (II) hydroxide. This precipitation reaction would have reduced the bioavailabilty of the copper (II) ions reducing their mobility as well as preventing them from generating any hydrogen peroxide.
In the pH experiments it was discovered that the addition of WT and WTF to copper (II) sulphate resulted in a fall in pH greater than expected from a dilution effect alone from the weakly acidic tea extracts which suggested that proton release had taken place in each case. The aqueous copper (II) sulphate which contained mainly positively charged free copper (II) ions would have had a high affinity for the negatively-charged oxygen ions within the ionised hydroxyl groups present in some tea components such as polyphenols. Possessing such high affinity copper (II) ions would tend to displace the less positively charged hydrogen ions from the hydroxyl groups in aqueous solution resulting in a greater level of free protons and thus a lower pH as observed.
In an attempt to explain the varying antimicrobial efficacies shown by the WT and WTF when combined with additives UV-vis. was used to investigate the possible formation of chemical complexes which could account for these. Other studies on plant product polyphenols have shown increases in absorbance when transition metal ions form chemical complexes with these agents [39, 40]. It is tempting to speculate that with the teas investigated here the copper (II) ions were possibly absorbed by non-polyphenols which do not absorb in the UV vis. wavelength range investigated and consequently no new UV-vis. absorbance peaks were apparent within this range. WTF showed a lower absorbance and a paler colour than the WT although peak absorbance was in a region typical of polyphenols . The differences seen between the WTF and WT may be attributed to the ultra-filtration process restricting the passage of larger molecules and leaving smaller ones in the filtrate.