Zeno Apostolides

Simultaneous analysis of tea catechins, caffeine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatography (MEKC) method for the simultaneous analysis of five tea catechins, theanine, caffeine, gallic acid and ascorbic acid has been developed. The catechins are (-)-epicatechin, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate. p-Nitrophenol serves as both reference and internal standard. All the components are separated within 13 min with a 57 cm uncoated fused-silica column. On-column detection was carried out at 200 nm. This method has been used to measure these compounds in fresh tea leaves and tea liquor. The limit of detection for all analytes ranged from 1 to 20 microg/ml.

Detoxification mechanisms of honey bees (Apis mellifera) resulting in tolerance of dietary nicotine.

Insecticides are thought to be among the major factors contributing to current declines in bee populations. However, detoxification mechanisms in healthy, unstressed honey bees are poorly characterised. Alkaloids are naturally encountered in pollen and nectar, and we used nicotine as a model compound to identify the mechanisms involved in detoxification processes in honey bees. Nicotine and neonicotinoids have similar modes of action in insects. Our metabolomic and proteomic analyses show active detoxification of nicotine in bees, associated with increased energetic investment and also antioxidant and heat shock responses. The increased energetic investment is significant in view of the interactions of pesticides with diseases such as Nosema spp which cause energetic stress and possible malnutrition. Understanding how healthy honey bees process dietary toxins under unstressed conditions will help clarify how pesticides, alone or in synergy with other stress factors, lead to declines in bee vitality.

Analysis of caffeine and flavan-3-ol composition in the fresh leaf of Camellia sinesis for predicting the quality of the black tea produced in central and Southern Africa

A parameter of fresh tea leaf that correlates with black tea quality is highly desired. Twenty good and 20 poor quality tea clones were selected from the breeding programme at the Tea Research Foundation (Central Africa) (TRF(CA)). The flavan-3-ol profile of fresh tea leaves was analysed by capillary electrophoresis while total theaflavin (TF) content was determined in the black tea manufactured from the same leaves for each clone. The above parameters were correlated with total scores and valuation from two tea tasters with regression analysis. The significance of the differences between the 20 good and 20 poor quality tea clones was determined with the Students t-test. The total TF content of the black tea correlated (r = 0.63, P = 0.0001) well with the value of the tea. Of all the parameters determined in the fresh leaves, the highest correlation was obtained with (−)-epicatechin (EC) (r = 0.65, P = 0.0001). This may facilitate early selection of good quality TRF(CA) clones in the future. © 2000 Society of Chemical Industry

Animal models used for the evaluation of antiretroviral therapies.

Several animal models for the study of HIV/AIDS have been established and characterized and have been widely used to study the pathogenesis of HIV/AIDS as well as vaccine development. The purpose of this study was to review the literature and identify the animal models most frequently used for the evaluation of drugs, drug combinations, plant extracts and drug-plant combinations. Four of these animal models were evaluated namely the SIV model due to its similarities in pathogenesis of disease to humans, the FIV and the LP-BM5 model due to wide availability and the SCID murine model that combines components of both systems. The pathogenesis of disease in each model, application in the evaluation of drugs, drug combinations and plant extracts as well as the inherent advantages and disadvantages of each model are discussed. The LP-BM5 murine AIDS (MAIDS) model with its in vitro equivalent was identified as the animal model, although not identical to HIV/AIDS, most suitable for the rapid and cost effective initial screening of drugs, drug combinations, plant extracts and drug-plant combinations.

Analysis of black tea theaflavins by non-aqueous capillary electrophoresis

In this study a new capillary electrophoresis (CE) method was developed to quantify the four major theaflavins occurring in black tea. Where aqueous based CE methods showed poor selectivity and considerable band broadening, non-aqueous CE achieved baseline separation of the theaflavins within 10 min. The effects of the organic solvent composition and background electrolyte concentration on the separation selectivity and electrophoretic mobilities were investigated. Our optimized separation solution consisted of acetonitrile-methanol-acetic acid (71:25:4, v/v) and 90 mM ammonium acetate. This method was used to analyze three black tea samples.

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

The anatomical localization of caffeine within young Camellia 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 resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine’s localization.

Global Tea Breeding

The tea plant, Camellia sinensis (L.) O. Kuntze, originated in the southwestern part of China, and has been cultivated there for approximately 5,000 years. Now tea plants are cultivated in 52 countries around the world. More than one-half of the population of the world consumes tea. The daily consumption of tea is approximately 3 billion cups all over the world. Tea, coffee and cocoa are the three most popular non-alcoholic beverages in the world. 1.1 Development of the Global Tea Industry The discovery and drinking of tea originated during the “Shen-Nong” era in ancient China, around 5,000 years ago. Originally, tea was used as a medicine for various illnesses and it can be traced back as early as 2737 B.C. in ancient China (Yamanishi, 1995). Tea production has developed rapidly since the Tang Dynasty (618 – 907 A.D.) and has been accepted as a beverage. However, tea has achieved popularity in other parts of the world only since the middle of the 17th century. Commercial cultivation of tea gradually expanded to Indonesia, India and Sri Lanka until the middle of the 19th century (Chen & Yang, 2011). The history of tea cultivation in Africa is relatively short. The first record of cultivation in Africa was in 1850; however, the tea industry developed until the middle of the 20th century. Now, tea plants are distributed worldwide ranging in latitude from 43° N (Georgia) to 27° S (Argentina). It is now grown commercially in tropical, subtropical and temperate climatic regions of Asia, Africa and South America, and 1 Delicious and Healthy Tea: An Overview 2 also in limited areas in North America, Australia and Europe. In 2010, the tea growing area in the world amounted to approximately 3,692 kilohectares and the total output amounted 4,162 kilotonnes (ITC, 2011). The average yield in the world now is around 1,110 kg/ha. The following 8 major tea producing countries, namely China, India, Kenya, Sri Lanka, Vietnam, Turkey, Indonesia and Japan, accounted for 88.9% of the world production (Table 1.1). China is the largest tea producing country once again in the world since 2005. The production in 2010 amounted to 1,475.1 kilotonnes and occupied 35.4% of the total world production. The total world export amounted to 1,728.8 kilotonnes in 2010 and was about 41.5% of total production. Kenya exports the most tea in the world, as 95% of production (ITC, 2011). Virtually all tea produced in Japan and about 74% of that produced in China is green tea. About 60% of consumers prefer black tea and the rest consume green tea and Oolong tea. Green tea is preferred in China, Japan and Middle East countries; Oolong tea is mainly consumed in the eastern part of China and in Japan. In terms of annual tea consumption per capita, Kuwait has the highest value at 2.86 kg (triennial average in the period of 2008 – 2010), followed by Ireland (2.31 kg), Qatar (2.04 kg), Turkey (2.02 kg), Afghanistan (2.01 kg) and United Kingdom (1.97 kg) (ITC, 2011). Table 1.1 Tea production in the major tea producing countries Country 194

Antioxidant supplementation can reduce the survival costs of excess amino acid intake in honeybees.

Over-consuming amino acids is associated with reduced survival in many species, including honeybees. The mechanisms responsible for this are unclear but one possibility is that excessive intake of amino acids increases oxidative damage. If this is the case, antioxidant supplementation may help reduce the survival costs of high amino acid intake. We tested this hypothesis in African honeybees (Apis mellifera scutellata) using the major antioxidant in green tea, epigallocatechin-3-gallate (EGCG). We first determined the dose-range of EGCG that improved survival of caged honeybees fed sucrose solution. We then provided bees with eight diets that differed in their ratio of essential amino acids (EAA) to carbohydrate (C) (0:1, 1:250, 1:100, 1:75, 1:50, 1:25, 1:10, 1:5 EAA:C) and also in their EGCG dose (0.0 or 0.4 mM). We found that bees fed sucrose only solution survived better than bees fed EAA diets. Despite this, bees preferred a diet that contained intermediate ratios of EAA:C (ca. 1:25), which may represent the high demands for nitrogen of developing nurse bees. EGCG supplementation improved honeybee survival but only at an intermediate dose (0.3-0.5 mM) and in bees fed low EAA diets (1:250, 1:100 EAA:C). That EGCG counteracted the lifespan reducing effects of eating low EAA diets suggests that oxidative damage may be involved in the association between EAAs and lifespan in honeybees. However, that EGCG had no effect on survival in bees fed high EAA diets suggests that there are other physiological costs of over-consuming EAAs in honeybees.

Screening of Tea (Camellia sinensis) for Trait-Associated Molecular Markers

This study was done to identify random amplified polymorphic DNA (RAPD) markers that may associate with seven important traits in tea. Sixty RAPD primers were first screened using 18 cultivars under each of the 7 traits, followed by confirmatory screening of 20 promising primers with 32 tea cultivars. Six RAPD primers generated a total of nine specific bands that associated with six desired traits: black tea quality and tolerance to drought, high temperature, low temperature, Phomopsis theae, and high yield. These markers would allow early identification of plant material with the desired traits that can be advanced to the next stage of selection and enhance targeted choice of breeding stocks with the desirable traits. The nine RAPD markers identified in this study could improve precision and efficiency in tea breeding and selection and are an important contribution towards the establishment of marker-assisted selection in tea breeding programmes.

Determination of scoparone in citrus roots by micellar electrokinetic capillary chromatography

A new MEKC method has been developed to determine the amount of the phytoalexin, scoparone, in Citrus roots. The separation and analysis was achieved with a running buffer of 100 mM SDS, 25 mM phosphate and 12% (v/v) methanol pH 6.2. Separation was performed at 12 kV with 25 degrees C and UV detection at 200 nm. A near complete recovery of scoparone was obtained with the extraction procedure. The MEKC method was compared with a fluorescence TLC method. The detection limit for scoparone with the MEKC method (2 mu g/mL) was better than the TLC method (10 mu g/mL).