Identification of digallated and methylated catechins using UPLC/MS/MS and development of a rapid analysis method for theanine in tea (Camellia sinensis (L.) O. Kuntze) utilizing evaporative light scattering detection

Theanine is a free, non-protein amino acid found in Camellia sinensis (tea) leaves. It is the main amino acid component in tea and contributes 1 to 2% of the dry weight of tea leaves. The content of amino acids is a key green tea quality parameter. High grade teas contain high amounts of theanine. It is responsible for the sweet, brothy umami taste of green tea. To date there is no international standard method for the analysis of theanine in tea samples. Several methods with different advantages and disadvantages have been reported but most of them are unsuitable for routine quality control analysis in commercial tea samples. In this study, a high performance liquid chromatography (HPLC) method with evaporative light scattering detection for the determination of theanine in tea samples has been developed. Chromatographic separation of theanine was achieved by reverse phase HPLC using a phenyl-hexyl column and isocratic water elution within 8 minutes. The method requires no sample derivatization and includes simple sample clean-up. Applying the same chromatographic conditions to a widely available C18 column, separation of theanine was achieved within 10 minutes. The successful application of the C18 column renders this method widely applicable. The method is linear over several orders of magnitude and the LOD are 11.53 and 10.83 μg/ml and the LOQ are 39.44 and 35.10 μg/ml for the phenyl-hexyl and C18 column, respectively. Simple but effective sample preparation and polyvinylpolypyrrolidone pre-treatment, along with the short analysis time facilitates high sample throughput (~40 samples can be prepared in an 8 hour day with overnight analysis by HPLCELSD). The method is selective, precise, accurate and practical for the quantification of theanine in tea extracts and was successfully used to determine theanine content in a variety of tea samples. The sensitivity and simplicity of this method renders it suitable for use in routine theanine analysis in quality control laboratories. Green tea, a simple refreshing beverage, has been believed to have therapeutic uses for centuries. Scientists have recognized that the wide range of physiological functions of green tea is due to the presence of catechins. An increasing body of evidence is showing that methylated forms of catechins have potent inhibitory activities to allergies. In addition to the methylated catechins, another relative novel group of catechins exists, namely digallated catechins. The medicinal value of these compounds is not yet known, but it is reasonable to speculate that the antioxidant capacity of digallated catechins will be superior to that of epigallocatechin gallate due to the 3 additional hydroxyl groups provided by the second gallic acid moiety. In this study reverse phase UPLC/ESI-MS/MS was effectively applied to investigate the presence of digallated and methylated catechins in crude tea extract. Low abundance catechins (-)-epigallocatechin-(O-3”-methyl) gallate ([M + H]+, m/z 473) and (-)-epicatechin-3-(O-3”-methyl) gallate ([M + H]+, m/z 457) were successfully detected at 31.9 and 37.5 minutes, respectively. The 2 digallated catechins investigated could not be detected, possibly due to being present at concentrations below the limit of detection. Compared to conventional HPLC/ESI-MS/MS, increased resolution and sensitivity, 80% less use of solvent and significantly reduced analysis times were observed.Dissertation (MSc)--University of Pretoria, 2009.Biochemistryunrestricte

Detoxification and elimination of nicotine by nectar-feeding birds

Many dilute nectars consumed by bird pollinators contain secondary metabolites, potentially toxic chemicals produced by plants as defences against herbivores. Consequently, nectar-feeding birds are challenged not only by frequent water excess, but also by the toxin content of their diet. High water turnover, however, could be advantageous to nectar consumers by enabling them to excrete secondary metabolites or their transformation products more easily. We investigated how the alkaloid nicotine, naturally present in nectar of Nicotiana species, influences osmoregulation in white-bellied sunbirds Cinnyris talatala and Cape white-eyes Zosterops virens.We also examined the metabolic fate of nicotine in these two species to shed more light on the post-ingestive mechanisms that allow nectar-feeding birds to tolerate nectar nicotine. A high concentration of nicotine (50 μM) decreased cloacal fluid output and increased its osmolality in both species, due to reduced food intake that led to dehydration. White-eyes excreted a higher proportion of the ingested nicotine-containing diet than sunbirds. However, sugar concentration did not affect nicotine detoxification and elimination. Both species metabolised nicotine, excreting very little unchanged nicotine. Cape white-eyes mainly metabolised nicotine through the cotinine metabolic pathway, with norcotinine being the most abundant metabolite in the excreta, while white-bellied sunbirds excreted mainly nornicotine. Both species also utilized phase II conjugation reactions to detoxify nicotine, with Cape white-eyes depending more on the mercapturic acid pathway to detoxify nicotine than white-bellied sunbirds. We found that sunbirds and white-eyes, despite having a similar nicotine tolerance, responded differently and used different nicotine-derived metabolites to excrete nicotine.http://link.springer.com/journal/3602018-05-28hb2017BiochemistryZoology and Entomolog

The metabolic fate of nectar nicotine in worker honey bees

Honey bees (Apis mellifera) are generalist pollinators that forage for nectar and pollen of a very large variety of plant species, exposing them to a diverse range of secondary metabolites produced as chemical defences against herbivory. Honey bees can tolerate high levels of many of these toxic compounds, including the alkaloid nicotine, in their diet without incurring apparent fitness costs. Very little is known about the underlying detoxification processes mediating this tolerance. We examined the metabolic fate of nicotine in newly emerged worker bees using radiolabeled nicotine and LC-MS/MS analysis to determine the kinetic distribution profile of nicotine as well as the absence or presence and identity of any nicotine-derived metabolites. Nicotine metabolism was extensive; virtually no unmetabolised nicotine were recovered from the rectum. The major metabolite found was 4-hydroxy-4-(3-pyridyl) butanoic acid, the end product of 2'C-oxidation of nicotine. It is the first time that 4-hydroxy-4-(3-pyridyl) butanoic acid has been identified in an insect as a catabolite of nicotine. Lower levels of cotinine, cotinine N-oxide, 3'hydroxy-cotinine, nicotine N-oxide and norcotinine were also detected. Our results demonstrated that formation of 4-hydroxy-4-(3-pyridyl) butanoic acid is quantitatively the most significant pathway of nicotine metabolism in honey bees and that the rapid excretion of unmetabolised nicotine does not contribute significantly to nicotine tolerance in honey bees. In nicotine-tolerant insects that do not rely on the rapid excretion of nicotine like the Lepidoptera, it is possible that the 2'C-oxidation of nicotine is the conserved metabolic pathway instead of the generally assumed 5'C-oxidation pathway.The BBSRC, NERC, the Wellcome Trust, Defra, the Scottish Government under the Insect Pollinators Initiative (BB/I000968/1) as well as the National Research Foundation of South Africa and the University of Pretoria.http://www.elsevier.com/locate/jinsphys2018-04-30BiochemistryZoology and Entomolog

Food consumption, lifestyle patterns, and body mass index of a group of white South African students

There is worldwide concern that higher education students are increasingly engaging in unhealthy eating and lifestyle practices. A total of 488 white students participated in a study aimed at investigating the current food consumption and related lifestyle patterns of students at a South African residential university. The respondents’ self‐reported weight and height was used to calculate their body mass index (BMI). Closed and open‐ended questions measured aspects of the respondents’ usual eating patterns and lifestyles. The meal patterns and composition confirmed Western‐orientated food practices, as the majority consumed three meals a day, with in‐between meal snacking, and a different meal pattern over weekends. Respondents’ food intake was further characterized by a low intake of fruit, vegetables, and dairy products and frequent consumption of foods high in fat, sugar, and sodium. Although the majority (66.8%) of the study group were classified as normal weight according to their BMI, when comparing males and females, more males than females were overweight and obese. Only 54% of the males had a normal weight compared to 82% of the females. There was, however, no statistically significant difference (p = 0.149) between how males and females in the different BMI groups felt about their weight. The study also explored the relationships between the respondents’ BMI, gender, food consumption patterns, and type of residence. The features of the food consumption patterns were depicted according to whether breakfast was eaten or not; snacking activity between meals; the consumption of ready‐prepared convenience meals, fast foods; home‐cooked meals; and eating out. There were no statistical significant differences between the BMI categories of males and females regarding their habit to eat breakfast or not; and to snack between meals either during the morning, in the afternoon or after supper. Similarly, no statistical significant differences were noted when relating the BMI categories of the gender groups to the frequency of consumption of ready‐prepared, convenience type meals, fast foods, and home‐cooked meals. However, a statistical significant difference (p‐value 0.006) was found between BMI categories per gender and general frequency of eating out. No statistical differences was noted between BMI categories, gender and place of residence irrespective of the type, whether the student lived with parents, independently in a flat or apartment, or a house with friends or a room, or in a university residence.http://wileyonlinelibrary.com/journal/ijcs2019-09-01hj2018Consumer Scienc

Antibiotic treatment impairs protein digestion in the honeybee, Apis mellifera

Nutritional stress due to habitat transformation and loss is one of several factors contributing to current declines in global bee populations. Bees obtain protein from pollen, which in honeybees is consumed and digested by nurse bees. They then distribute the protein to the rest of the colony in the form of hypopharyngeal gland secretions. Little is known of how efficiently honeybees digest protein. Moreover, antibiotics are used by beekeepers as in-hive treatments for diseases and may interfere with microbial contributions to protein digestion. Caged, newly emerged workers of Apis mellifera scutellata were fed caseinate as protein source, to investigate the effects of protein intake and antibiotic treatment on digestive efficiency. These workers were fed protein:carbohydrate ratios of 1:120, 1:50 and 1:15 or pure sucrose for 9 days. Half the cages received dietary oxytetracycline at a concentration used by beekeepers. Antibiotic exposure did not affect survival or protein consumption. Protein digestive efficiency increased with increasing levels of protein in the diet, although a decrease would have contributed to maintaining nutrient balance. Importantly, we show that antibiotic exposure impaired protein digestive efficiency, especially on low-protein diets. This may be particularly important when colonies are restricted to a single protein deficient source of pollen.The University of Pretoria and by the National Research Foundation of South Africa.http://link.springer.com/journal/135922020-12-02hj2020BiochemistryGeneticsMicrobiology and Plant PathologyZoology and Entomolog

Oxidative damage is influenced by diet but unaffected by selection for early age of oviposition in the Marula fly, ceratitis cosyra (Diptera: Tephritidae)

The expression of life-history traits, such as lifespan or reproductive effort, is tightly correlated with the amount and blend of macronutrients that individuals consume. In a range of herbivorous insects, consuming high protein to carbohydrate ratios (P:C) decreases lifespan but increases female fecundity. In other words, females face a resourcebased trade-off between lifespan and fecundity. Redox metabolism may help mediate this trade-off, if oxidative damage is elevated by reproductive investment and if this damage, in turn, reduces lifespan. Here, we test how diets varying in P:C ratio affect oxidative damage and antioxidant protection in female and male of the marula fly, Ceratitis cosyra (Diptera: Tephritidae). We use replicated lines that have been subjected to experimental evolution and differ in their lifespan and reproductive scheduling. We predicted that high fecundity would be associated with high oxidative damage and reduced antioxidant defences, while longer lived flies would show reduced damage and elevated antioxidant defences. However, higher levels of oxidative damage were observed in longlived control lines than selection lines, but only when fed the diet promoting lifespan. Flies fed diets promoting female fecundity (1:4 and 1:2 P:C) suffered greater oxidative damage to lipids than flies fed the best diet (0:1 P:C) for lifespan. Total antioxidant capacity was not affected by the selection regime or nutrition. Our results reiterate the importance of nutrition in affecting life-history traits, but suggest that in C. cosyra, reactive oxygen species play a minimal role in mediating dietary trade-offs between lifespan and reproduction.A National Research Foundation Competitive Programme for Rated Researchers.https://www.frontiersin.org/journals/physiologydm2022Zoology and Entomolog

Resistance of developing honeybee larvae during chronic exposure to dietary nicotine

The effects of pesticides on honeybee larvae are less understood than for adult bees, even though larvae are chronically exposed to pesticide residues that accumulate in comb and food stores in the hive. We investigated how exposure to a plant alkaloid, nicotine, affects survival, growth and body composition of honeybee larvae. Larvae of Apis mellifera scutellata were reared in vitro and fed throughout development on standard diets with nicotine included at concentrations from 0 to 1000 μg/100 g diet. Overall mortality across all nicotine treatments was low, averaging 9.8 % at the prepupal stage and 18.1 % at the whiteeyed pupal stage, but survival was significantly reduced by nicotine. The mass of prepupae and white-eyed pupae was not affected by nicotine. In terms of body composition, nicotine affected water content but did not influence either protein or lipid stores of white-eyed pupae. We attribute the absence of consistent negative effects of dietary nicotine to detoxification mechanisms in developing honeybees, which enable them to resist both natural and synthetic xenobiotics.The BBSRC, NERC, the Wellcome Trust,Defra, and the Scottish Government under the Insect Pollinators Initiative (BB/I000968/1), National Research Foundation of South Africa and the University of Pretoria.http://www.elsevier.com/locate/jinsphyshb201

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.http://www.nature.com/srephb201

Proteomic and metabolomic analysis reveals rapid and extensive nicotine detoxification ability in honey bee larvae

Despite potential links between pesticides and bee declines, toxicology information on honey bee larvae (Apis mellifera) is scarce and detoxification mechanisms in this development stage are virtually unknown. Larvae are exposed to natural and synthetic toxins present in pollen and nectar through consumption of brood food. Due to the characteristic intensive brood care displayed by honey bees, which includes progressive feeding throughout larval development, it is generally assumed that larvae rely on adults to detoxify for them and exhibit a diminished detoxification ability. We found the opposite. We examined the proteomic and metabolomic responses of in vitro reared larvae fed nicotine (an alkaloid found in nectar and pollen) to understand how larvae cope on a metabolic level with dietary toxins. Larvae were able to effectively detoxify nicotine through an inducible detoxification mechanism. A coordinated stress response complemented the detoxification processes, and we detected significant enrichment of proteins functioning in energy and carbohydrate metabolism, as well as in development pathways, suggesting that nicotine may promote larval growth. Further exploration of the metabolic fate of nicotine using targeted mass spectrometry analysis demonstrated that, as in adult bees, formation of 4-hydroxy-4-(3-pyridyl) butanoic acid, the result of 2’C-oxidation of nicotine, is quantitatively the most significant pathway of nicotine metabolism. We provide conclusive evidence that larvae are capable of effectively catabolising a dietary toxin, suggesting that increased larval sensitivity to specific toxins is not due to diminished detoxification abilities. These findings broaden the current understanding of detoxification biochemistry at different organizational levels in the colony, bringing us closer to understanding the capacity of the colony as a superorganism to tolerate and resist toxic compounds, including pesticides, in the environment.This work was funded jointly by a grant from the Biotechnology and Biological Sciences Research Council (BBSRC), Natural Environment Research Council (NERC), the Wellcome Trust, Defra, and the Scottish Government under the Insect Pollinators Initiative (BB/ I000968/1). We also acknowledge the support of the National Research Foundation of South Africa (73671) and the University of Pretoria.http://www.elsevier.com/locate/ibmb2018-03-31hb2017BiochemistryZoology and Entomolog