Immunohistochemical localisation of caffeine in young tea (Camellia sinensis (L.) O. Kuntze) leaves

Abstract

The agricultural product using the top young shoots from the Camellia sinensis bush is known as tea. The leaves have many health benefits and contain high contents of antioxidants. The leaves contain caffeine, which is believed to be a phosphodiesterase inhibitor preventing the disposal of cyclic adenosine monophosphate, and thus when consumed, cellular responses are up regulated, and people experience unwanted effects such as being unable to fall asleep. Caffeine is currently believed to be synthesized within chloroplasts and stored within the vacuoles of parenchyma cells. It is hypothesized that the molecule acts as a chemical defence as well as an aid in the sequestration of catechins and polyphenols within the vacuoles of parenchyma cells. Different methods of decaffeination exist using solvents such as methylene chloride, supercritical CO2, and using hot water. Each method has a specific disadvantage. Currently, the Swiss Water Process is a 100 % chemically free method of decaffeination for coffee beans, and a similar process should be envisaged for the tea decaffeination industry as there are many advantages over the other current methods of decaffeination. The anatomical localisation of caffeine within young C. sinensis leaves was investigated using immunohistochemical methods, and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in Lowicryl K4M resin was deemed the best protocol as it retained most of the caffeine, and allowed for the samples to be sectioned with ease. Immunohistochemical localisation with primary anti-caffeine antibodies, and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localised and accumulated within vascular bundles, mainly the precursor phloem. Immunocytochemical studies using a secondary antibody conjugated to gold were attempted but were inconclusive. With the use of a pressure bomb, xylem sap was analysed by thin-layer chromatography, and the presence of caffeine was determined to be present in a small amount. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defence against various pathogens, and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine’s localisation. Using the knowledge acquired from microscopy analysis, and thin-layer chromatography analysis of xylem sap we investigated three possible methods of decaffeination; vacuum, pressure, and a combination of both. All were based on using the intercellular air within the young leaves to our advantage as this might act as a natural way to ‘squeeze’ caffeine out of its localised areas. Other possible methods of decaffeination were also attempted using hot water, and addition of an external PPO source for black tea production.