Caffeine Accumulation Research Articles

Low pharmaceutical pollution of epibenthic organisms from Admiralty Bay, Antarctica (King George Island, South Shetland Islands)

The presence of medicinal products has been reported in the Antarctic marine environment but still little is known about the bioaccumulation of these compounds. This study was set up to detect and quantify pharmaceutical residues in benthic biota from Admiralty Bay, King George Island (South Shetland Islands, Antarctica). Pharmaceuticals and stimulants were analysed using LC-MS/MS in dominant epibenthic macroorganisms that were collected at eight nearshore sublittoral sites (water depth 3–30 m) by a SCUBA diving team during austral summer (February 2023). Out of 22 analytes, only the stimulant caffeine (CAF) and the antiepileptic carbamazepine (CBZ) have been identified in the macrobenthic algae and invertebrates indicating a relatively low contamination level of the bay. The analytes were detected predominantly in the northern part of the bay (Mackellar Inlet and Martel Inlet) which reflects likely their elevated environmental concentrations in this area and suggests that local research stations represent the main source of pharmaceutical contamination. Irrespective of the sampling site, both compounds were found almost exclusively in brown and red macroalgae highlighting their potential for uptake and accumulation of CAF and CBZ. This study provided the first evidence of the presence of medicinal substances in the Antarctic macrobenthic organisms and can serve as a baseline for environmental risk assessment.

The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis.

Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea, cacao, and coffee, but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear. Here, we report a novel transcription factor Camellia sinensis Senescnece 40 (CsS40) as a caffeine biosynthesis regulator, which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase (TCS1) promoter. Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays, a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter. Consistently, immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells. The expression pattern of CsS40 in 'Fuding Dabai' developing leaves was opposite to that of TCS1; and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels, respectively. The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of 'Fuding Dabai'. Furthermore, overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants, meanwhile, increased their susceptibility to aging. CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors. Taken together, we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar 'Fuding Dabai'. The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.

Development of SEDDS formulation containing caffeine for dermal delivery.

The objective of this work was to develop a self-emulsifying drug delivery system (SEDDS) containing caffeine for the treatment of cellulite. SEDDS were prepared using the solution method. 0.5% (w/v) caffeine was added to the previously selected excipients. The system was characterized by droplet size, zeta potential, emulsification time and long-term stability. In vitro release and skin permeation were investigated using Franz-type diffusion cells. The cytotoxicity was evaluated on normal human keratinocytes. Caffeine SEDDS were thermodynamically stable, with a zeta potential less than - 22 mV and droplet size around 30 nm, and were long-term stable. The permeation study showed that the formulation promoted caffeine accumulation in the skin layers, suggesting an increase in local circulation. Cytotoxicity studies on HaCaT cells were not conclusive as the surfactant used indicated false-positive results due to its high molar mass. It was possible to obtain a stable SEDDS that could cause an increase in blood flow in the applied area, resulting in cellulite reduction.

Metabolomic and Transcriptomic Analyses Reveal the Characteristics of Tea Flavonoids and Caffeine Accumulation and Regulation between Chinese Varieties (Camellia sinensis var. sinensis) and Assam Varieties (C. sinensis var. assamica).

Flavonoids and caffeine are the major secondary metabolites with beneficial bioactivity for human health in tea plants, and their biosynthesis pathway and regulatory networks have been well-deciphered. However, the accumulation traits of flavonoids and caffeine in different tea cultivars was insufficient in investigation. In this study, metabolomic and transcriptomic analyses were performed to investigate the differences of flavonoids and caffeine accumulation and regulation between Chinese varieties, including the 'BTSC' group with green leaf, the 'BTZY' group with purple foliage, and the 'MYC' group comprising Assam varieties with green leaf. The results showed that most of the flavonoids were down-regulated in the 'MYC' group; however, the total anthocyanin contents were higher than that of the 'BTSC' group while lower than that of the 'BTZY' group. An ANS (Anthocyanin synthase) was significantly up-regulated and supposed to play a key role for anthocyanin accumulation in the 'BTZY' group. In addition, the results showed that esterified catechins were accumulated in the 'BTSC' and 'BTZY' groups with high abundance. In addition, SCPL1A (Type 1A serine carboxypeptidase-like acyltransferases gene) and UGGT (UDP glucose: galloyl-1-O-β-d-glucosyltransferase gene) potentially contributed to the up-accumulation of catechins esterified by gallic acid. Interestingly, the results found that much lower levels of caffeine accumulation were observed in the 'MYC' group. RT-qPCR analysis suggested that the expression deficiency of TCS1 (Tea caffeine synthase 1) was the key factor resulting in the insufficient accumulation of caffeine in the 'MYC' group. Multiple MYB/MYB-like elements were discovered in the promoter region of TCS1 and most of the MYB genes were found preferentially expressed in 'MYC' groups, indicating some of which potentially served as negative factor(s) for biosynthesis of caffeine in tea plants. The present study uncovers the characteristics of metabolite accumulation and the key regulatory network, which provide a research reference to the selection and breeding of tea varieties.

Characterization of Plant Accumulation of Pharmaceuticals from Soils with Their Concentration in Soil Pore Water.

Predicting plant uptake of pharmaceuticals from soils is very challenging because many pharmaceuticals are ionizable compounds, which experience highly variable sorption/desorption and transformation processes in soils. This study aimed to elucidate how the equilibrium between sorbed and dissolved phases influences radish uptake of 15 pharmaceuticals from three soils with different properties. After 30 days of uptake, the accumulation of acetaminophen, carbamazepine, lamotrigine, carbadox, trimethoprim, and triclosan in radish ranked as Riddles > Capac > Spinks soil. In contrast, radish accumulation of caffeine, lincomycin, monensin, tylosin, sulfadiazine, and sulfamethoxazole exhibited the opposite order of Riddles < Capac < Spinks soil. Oxytetracycline and estrone demonstrated similar accumulation in radish grown in the three soils. Accumulation of pharmaceuticals in radish demonstrated no apparent relation with their concentration in soils. However, we identified strong positive correlation between pharmaceutical accumulation in radish and their corresponding concentration in soil pore water. These results reveal that pharmaceutical in soil pore water is the dominant fraction bioavailable to plant uptake. Relatively constant root concentration factors (RCFs) on the basis of pharmaceutical concentration in soil pore water, compared to the highly variable RCFs derived from soils, suggest that pore water-based RCF is superior for describing pharmaceutical accumulation in plants grown in soils. We recommend that pharmaceuticals in soil pore water should be evaluated and included in modeling their uptake by plants.

Chromosome-level genome of Camellia lanceoleosa provides a valuable resource for understanding genome evolution and self-incompatibility.

The section Oleifera (Theaceae) has attracted attention for the high levels of unsaturated fatty acids found in its seeds. Here, we report the chromosome-scale genome of the sect. Oleifera using diploid wild Camellia lanceoleosa with a final size of 3.00 Gb and an N50 scaffold size of 186.43 Mb. Repetitive sequences accounted for 80.63% and were distributed unevenly across the genome. Camellia lanceoleosa underwent a whole-genome duplication event approximately 65 million years ago (65 Mya), prior to the divergence of C. lanceoleosa and Camellia sinensis (approx. 6-7 Mya). Syntenic comparisons of these two species elucidated the genomic rearrangement, appearing to be driven in part by the activity of transposable elements. The expanded and positively selected genes in C. lanceoleosa were significantly enriched in oil biosynthesis, and the expansion of homomeric acetyl-coenzyme A carboxylase (ACCase) genes and the seed-biased expression of genes encoding heteromeric ACCase, diacylglycerol acyltransferase, glyceraldehyde-3-phosphate dehydrogenase and stearoyl-ACP desaturase could be of primary importance for the high oil and oleic acid content found in C. lanceoleosa. Theanine and catechins were present in the leaves of C. lanceoleosa. However, caffeine can not be dectected in the leaves but was abundant in the seeds and roots. The functional and transcriptional divergence of genes encoding SAM-dependent N-methyltransferases may be associated with caffeine accumulation and distribution. Gene expression profiles, structural composition and chromosomal location suggest that the late-acting self-incompatibility of C. lanceoleosa is likely to have favoured a novel mechanism co-occurring with gametophytic self-incompatibility. This study provides valuable resources for quantitative and qualitative improvements and genome assembly of polyploid plants in sect. Oleifera.

Comprehensive analysis of quality characteristics in main commercial coffee varieties and wild Arabica in Kenya.

Coffee flavor is a complex commercial trait and its generation mechanisms remain largely unclear. Here, we investigated non-volatile and volatile compounds in the AA grade coffee beans of cultivated and wild Coffea arabica accessions in Kenya. An increased accumulation of trigonelline and sucrose along with a decreased accumulation of caffeine and 5-caffeoylquinic acid or chlorogenic acid (CGA) relative to wild Arabica contribute to the improved flavor of commercial varieties. Trigonelline was strongly associated with attributes of coffee brews such as pH, aroma intensity and antioxidant activity, suggesting that it is one of the main flavor precursors. Partial least squares discriminant analysis (PLS-DA) identified 18 volatiles that could potentially define flavor quality of coffee brews, with pyrazines and thiols as the major coffee flavor determinants.

The NAC-like transcription factor CsNAC7 positively regulates the caffeine biosynthesis-related gene yhNMT1 in Camellia sinensis.

Caffeine is an important functional substance and is abundant in tea plant, but little is known about how its biosynthesis is regulated by transcription factors. In this study, the NAC-like transcription factor-encoding gene CsNAC7, which is involved in caffeine synthesis, was isolated from a Yinghong 9 cDNA library using a yeast one-hybrid assay; this gene comprises 1371bp nucleotides and is predicted to encode 456 amino acids. The expression of CsNAC7 at the transcriptional level in tea shoots shared a similar pattern with that of the caffeine synthase gene yhNMT1 in the spring and summer, and its expressed protein was localized in the nucleus. Assays of gene activity showed that CsNAC7 has self-activation activity in yeast, that the active region is at the N-terminus, and that the transient expression of CsNAC7 could significantly promote the expression of yhNMT1 in tobacco leaves. In addition, overexpression or silencing of CsNAC7 significantly increased or decreased the expression of yhNMT1 and the accumulation of caffeine in transgenic tea calli, respectively. Our data suggest that the isolated transcription factor CsNAC7 positively regulates the caffeine synthase gene yhNMT1 and promotes caffeine accumulation in tea plant.

Occurrence and risk assessment of trace organic contaminants and metals in anaerobically co-digested sludge

Anaerobic co-digestion of sludge increases biogas production and maintains anaerobic digestion stability. However, it is unclear whether the addition of co-substrates may increase the concentration of trace organic contaminants (TrOCs) and metals, limiting potential resource recovery opportunities when applied to agricultural land. This study explored the occurrence of 20 TrOCs and 18 metals in wastewater sludge anaerobically co-digested with beverage rejects (cola, beer and juice) and food wastes. TrOCs results showed that cola reject caused an accumulation of caffeine in final digestate. Bisphenol A also significantly increased in food waste co-digestion when compared with the mono-digestion (control). No significant difference in TrOCs was observed in the juice reject co-digestion. Analysis of the metal composition revealed a significant increase in Cr and Al in juice reject co-digested sludge. While restaurant food waste increased concentrations of K and Ca, both of which may be beneficial when applied to land. All metals in this study were below the maximum permissible concentrations specified for agricultural land use in Australia. Environmental risk assessment of sludge when used as soil fertiliser, showed that caffeine, diuron, triclocarban, triclosan, Cu and Zn exhibited high risks, with the largest risk quotient (RQ) posed by caffeine. Estrone and naproxen implied medium risks, and ibuprofen implied a high risk except for the co-digestion using cola reject (RQ = 0.9, medium risk). The results emphasise the importance for wastewater utility operators to understand the impact of co-substrate selection on the quality of sludge to minimise environmental risk from the use of biosolids on agricultural land.

Caffeine as a contaminant of periphyton: ecological changes and impacts on primary producers.

Every day, tons of caffeine is consumed by humans in beverages, medications or supplements, and a significant amount of this stimulant is released in domestic sewage. Once in aquatic environments caffeine interacts directly with the periphytic community, which is responsible for a significant part of primary production in aquatic ecosystems. However, the effects of exposure to caffeine are mostly unknown for both the periphyton and their predators. Aiming to comprehend the interaction between caffeine and the periphytic community, ecotoxicological experiments were performed by exposing a periphytic biofilm cultivated in the laboratory to different concentrations of caffeine, following concentrations found in domestic sewers. The impact of exposure to this contaminant was observed on the structure of the community through taxonomic evaluation, as well a set of physiological variables linked to primary production. After exposure to the highest caffeine concentration (300 µg L-1), the density of the genus Scenedesmus was severely affected, leading to an increase in cyanobacteria and diatoms. Both richness and diversity decreased after exposure, and there was lower photosynthetic activity, with light saturation point changing from 186 µmol m-2 s-1 in the control treatment to 108 µmol m-2 s-1 after exposure. Caffeine accumulation within the biofilm was also observed during the first 24 h, in the concentration of 0.14 µg /cm².

Uptake, subcellular distribution and metabolism of 14C-caffeine in leafy vegetables from water

Irrigation with treated wastewater could lead to the accumulation of caffeine in agricultural fresh. Caffeine is one of the most frequently detected compounds in treated wastewater; however, little is known about its subcellular distribution and metabolism in vegetables. This study reported the uptake, subcellular distribution, and metabolism of 14C-caffeine in Chinese flowering cabbage and water spinach. The results showed that 98% of caffeine lost from solution after 768 h of cultivation. Caffeine was taken up by vegetables and most 14C-activity was accumulated in the bottom leaves. At the subcellular level, 14C-activity was mainly distributed in the organelles in root and stem cells, while in the leafy cells it was dominant in the solution. The metabolism of caffeine was investigated using LC-QTOF-MS. Caffeine underwent demethylation forming xanthine and theobromine, and mineralization to release CO2. Approximately 40.2% of the initially applied caffeine was accumulated in Chinese flowering cabbage as the parent compound (28.3%) and metabolites (11.9%), and 50.9% of the added caffeine was mineralized to CO2 after 768 h of exposure. The knowledge obtained herein is key to evaluating potential risks of caffeine present in treated wastewater, and the quality and safety of agricultural fresh produced by irrigation with treated wastewater.

Effects of temperature on caffeine and carbon nanotubes co-exposure in Ruditapes philippinarum

In the marine environment, organisms are exposed to a high and increasing number of different contaminants that can interact among them. In addition, abiotic factors can change the dynamics between contaminants and organisms, thus increasing or even decreasing the toxic effect of a particular compound. In this study, the effects of caffeine (CAF) and functionalized multi-walled carbon nanotubes (f-MWCNTs) induced in the clam Ruditapes philippinarum were evaluated, acting alone and in combination (MIX), under two temperature levels (18 and 21 °C). To assess the impact of such compounds, their interaction and the possible influence of temperature, biochemical and histopathological markers were investigated. The effects of f-MWCNTs and caffeine appear to be clearly negative at the control temperature, with lower protein content in contaminated clams and a significant decrease in their metabolism when both pollutants were acting in combination. Also, at control temperature, clams exposed to pollutants showed increased antioxidant capacity, especially when caffeine was acting alone, although cellular damages were still observed at CAF and f-MWCNTs treatments. Increased biotransformation capacity at 18 °C and MIX treatment may explain lower caffeine concentration observed. At increased temperature differences among treatments were not so evident as at 18 °C, with a similar biological pattern among contaminated and control clams. Higher caffeine accumulation at MIX treatment under warming conditions may result from clams’ inefficient biotransformation capacity when exposed to increased temperatures.

The analysis of transcription factor CsHB1 effects on caffeine accumulation in tea callus through CRISPR/Cas9 mediated gene editing

Caffeine is an important metabolite in tea. In previous study, transcription factor CsHB1 was cloned from tea plants via yeast one-hybrid using yhNMT1 as bait. In this study, the expression of CsHB1 and yhNMT1 in tea plants was analyzed by qRT-PCR, the effect of CsHB1 on the expression of yhNMT1 was determined by tobacco transient expression system, and the CsHB1 was assembled into pYLCRISPR/Cas9Pubi-H vector followed by transgene to tea callus. Our results showed that the expression of CsHB1 was similar to that of yhNMT1. The overexpression of CsHB1 showed no significant effect on yhNMT1 in tobacco leaves. The constructed pYLCRISPR/Cas9Pubi-H-CsHB1 vector was successfully transformed into tea callus, and CsHB1 gene was mutated directionally. In transgenic callus, the expression of CsHB1 and yhNMT1 decreased by 65 % and 93 %, respectively, and the accumulation of caffeine decreased by 97.5 %, which indicated that yhNMT1 gene was regulated by CsHB1. Our results will help to understand the regulation mechanism of caffeine biosynthesis in tea.

Secondary metabolites of tea leaves and their role in protecting against environmental stressors

The dynamics of a number of secondary metabolites and the activity of guaiacol peroxidase in 3-leaf sprout of tea varieties and forms were studied. In the course of 3-year research we noted the presence of declines and peaks in the activity of the enzyme and the accumulation of flavonoids, carotenoids, caffeine and amino acids associated with the meteorological conditions of each month. It is shown that there is a close inverse relationship between increased enzyme activity, carotenoid content and hydrothermal factors. The most significant correlation was found between the activity of guaiacol peroxidase and carotenoids in a 3-leaf tea flush with precipitation (r = -0.86 and -0.68). At the beginning of vegetation the enzyme activity is low (ranging from 0.363 to 0.607 units/g·s). The decrease in the activity of guaiacol peroxidase in June is due to the biological peculiarities of the tea culture associated with a period of rest, during which the metabolic processes are somewhat slowed down. Increased activity of guaiacol peroxidase is accompanied by rapid synthesis of phenolic compounds, such as flavonoids. We have traced the dynamics of the formation of thearubigins and theaflavins in tea sprout. It is shown that their amount varies depending on the season of collection from 0.075 mg/g (theaflavins) and 1.178 mg/g (thearubigins) to 0.115 mg/g and 1.625 mg/g (theaflavins and thearubigins, respectively) and their dynamics is similar. The content of the routine varies depending on the season of tea leaf collection. The synthesis of caffeine also depends on the growing conditions, the active accumulation of which in experimental plants was observed in July (on average 26.6±1.4 mg/g). The largest amount of amino acids (16 665 mg/g) is synthesized in May, and then their synthesis is halved. Genotypic peculiarities are manifested in the content of secondary metabolites and the activity of guaiacol peroxidase. The revealed patterns are common for all varieties and forms of tea.

Callus culture as a new approach for the production of high added value compounds in Ilex paraguariensis: genotype influence, medium optimization and compounds identification.

Ilex paraguariensis (yerba mate) is a native species from South America and is a rich source of bioactive compounds. There is a lack of research efforts on the phytochemical investigation of callus culture from this species. In the present study, an effort was made to optimize callus culture conditions and to identify secondary compounds. Calli were induced from 10 genotypes using leaf explants and the best genotype was selected to evaluate the effects of cytokinin types and concentrations on callus induction and biomass accumulation. The best genotype and cytokinin treatment were used to conduct one last experiment with sucrose concentrations in culture media and its effects on calli biomass, antioxidant activity and secondary compounds accumulation. Callus initiation was genotype dependent, and the 6-156-6 line had the best response. Zeatin supplemented medium showed higher callus induction rate (82%) and higher biomass accumulation after 120 days (328.2 mg). Higher biomass and secondary compounds accumulation were observed for calli on 3% sucrose medium. Antioxidant activity was not affected by sucrose concentrations. Yerba mate callus culture allowed the accumulation of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, theobromine and caffeine.

De novo transcriptome and phytochemical analyses reveal differentially expressed genes and characteristic secondary metabolites in the original oolong tea (Camellia sinensis) cultivar \u2018Tieguanyin\u2019 compared with cultivar \u2018Benshan\u2019

BackgroundThe two original plants of the oolong tea cultivar (‘Tieguanyin’) are “Wei shuo” ‘Tieguanyin’—TGY (Wei) and “Wang shuo” ‘Tieguanyin’—TGY (Wang). Another cultivar, ‘Benshan’ (BS), is similar to TGY in its aroma, taste, and genetic make-up, but it lacks the “Yin Rhyme” flavor. We aimed to identify differences in biochemical characteristics and gene expression among these tea plants.ResultsThe results of spectrophotometric, high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) analyses revealed that TGY (Wei) and TGY (Wang) had deeper purple-colored leaves and higher contents of anthocyanin, catechins, caffeine, and limonene compared with BS. Analyses of transcriptome data revealed 12,420 differentially expressed genes (DEGs) among the cultivars. According to a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the flavonoid, caffeine, and limonene metabolic pathways were highly enriched. The transcript levels of the genes involved in these three metabolic pathways were not significantly different between TGY (Wei) and TGY (Wang), except for two unigenes encoding IMPDH and SAMS, which are involved in caffeine metabolism. The comparison of TGY vs. BS revealed eight up-regulated genes (PAL, C4H, CHS, F3’H, F3H, DFR, ANS, and ANR) and two down-regulated genes (FLS and CCR) in flavonoid metabolism, four up-regulated genes (AMPD, IMPDH, SAMS, and 5′-Nase) and one down-regulated XDH gene in caffeine metabolism; and two down-regulated genes (ALDH and HIBADH) in limonene degradation. In addition, the expression levels of the transcription factor (TF) PAP1 were significantly higher in TGY than in BS. Therefore, high accumulation of flavonoids, caffeine, and limonene metabolites and the expression patterns of their related genes in TGY might be beneficial for the formation of the “Yin Rhyme” flavor.ConclusionsTranscriptomic, HPLC, and GC-MS analyses of TGY (Wei), TGY (Wang), and BS indicated that the expression levels of genes related to secondary metabolism and high contents of catechins, anthocyanin, caffeine, and limonene may contribute to the formation of the “Yin Rhyme” flavor in TGY. These findings provide new insights into the relationship between the accumulation of secondary metabolites and sensory quality, and the molecular mechanisms underlying the formation of the unique flavor “Yin Rhyme” in TGY.

Quantitative Trait Loci Mapping for Theobromine and Caffeine Contents in Tea Plant ( Camellia sinensis).

Understanding the genetic basis of theobromine and caffeine accumulation in the tea plant is important due to their contribution to tea flavor. Quantitative trait loci (QTL) analyses were carried out to identify genetic variants associated with theobromine and caffeine contents and ratio using a pseudo-testcross population derived from an intervarietal cross between two varieties of Camellia sinensis. A total of 10 QTL controlling caffeine content (CAF), theobromine content (TBR), sum of caffeine and theobromine (SCT), and caffeine-to-theobromine ratio (CTR) were identified over four measurement years. The major QTL controlling CAF, qCAF1, was mapped onto LG01 and validated across years, explaining an average of 20.1% of the phenotypic variance. The other QTL were detected in 1 or 2 years, and of them there were four, two, and three for TBR, SCT, and CTR, respectively. The present results provide valuable information for further fine mapping and cloning functional genes and for genetic improvement in tea plant.

Transcriptomic Analysis Reveals the Molecular Mechanisms of Drought-Stress-Induced Decreases in Camellia sinensis Leaf Quality.

The tea plant [Camellia sinensis (L.) O. Kuntze] is an important commercial crop rich in bioactive ingredients, especially catechins, caffeine, theanine and other free amino acids, which the quality of tea leaves depends on. Drought is the most important environmental stress affecting the yield and quality of this plant. In this study, the effects of drought stress on the phenotype, physiological characteristics and major bioactive ingredients accumulation of C. sinensis leaves were examined, and the results indicated that drought stress resulted in dehydration and wilt of the leaves, and significant decrease in the total polyphenols and free amino acids and increase in the total flavonoids. In addition, HPLC analysis showed that the catechins, caffeine, theanine and some free amino acids in C. sinensis leaves were significantly reduced in response to drought stress, implying that drought stress severely decreased the quality of C. sinensis leaves. Furthermore, differentially expressed genes (DEGs) related to amino acid metabolism and secondary metabolism were identified and quantified in C. sinensis leaves under drought stress using high-throughput Illumina RNA-Seq technology, especially the key regulatory genes of the catechins, caffeine, and theanine biosynthesis pathways. The expression levels of key regulatory genes were consistent with the results from the HPLC analysis, which indicate a potential molecular mechanism for the above results. Taken together, these data provide further insights into the mechanisms underlying the change in the quality of C. sinensis leaves under environmental stress, which involve changes in the accumulation of major bioactive ingredients, especially catechins, caffeine, theanine and other free amino acids.

Oral intake of a combination of glucosyl hesperidin and caffeine elicits an anti-obesity effect in healthy, moderately obese subjects: a randomized double-blind placebo-controlled trial.

BackgroundWe have previously shown that a combination of glucosyl hesperidin (G-hesperidin) plus caffeine reduces accumulation of body fat, whereas G-hesperidin or caffeine alone shows little effect on high-fat diet-induced obesity in mice. The aim of this study is to evaluate the anti-obesity effect of G-hesperidin plus caffeine on body fat and serum TG in healthy subjects with moderately high body mass index (BMI) and serum TG. Since we considered that there are individual differences in caffeine sensitivity, we conducted dose-finding study of caffeine combined with G-hesperidin.MethodsSeventy-five healthy subjects with moderately high BMI (24–30 kg/m2) and serum TG (100–250 mg/dl) were divided and assigned to 12-week intervention with daily intakes of 500 mg of G-hesperidin with or without 25, 50, or 75 mg of caffeine, or placebo in a randomized double-blind placebo-controlled design .ResultsAfter intervention, decreases in abdominal fat area (AFA), especially subcutaneous fat area (SFA), were significantly greater in the G-hesperidin with 50-mg caffeine group (AFA:-8.4 ± 21.9 v.s. 16.3 ± 34.1 cm2; p < 0.05, SFA: -9.3 ± 17.1 v.s. 11.2 ± 18.3 cm2; p < 0.01) and in the G-hesperidin with 75-mg caffeine group (AFA:-17.0 ± 31.4 v.s. 16.3 ± 34.1 cm2; p < 0.01, SFA: -12.4 ± 18.7 v.s. 11.2 ± 18.3 cm2; p < 0.01) than in the placebo group. Fat-decreasing effects of G-hesperidin were enhanced dose-dependently by caffeine addition. BMI decreases were significantly greater in the G-hesperidin with 75-mg caffeine group than in the placebo group (-0.56 ± 0.74 v.s. -0.02 ± 0.58 kg/m2; p < 0.05). G-hesperidin with/without caffeine had no effect on serum TG (p > 0.05 v.s. placebo).ConclusionsThese data suggested that a combination of 500-mg G-hesperidin with 50- or 75-mg caffeine may be useful for the prevention or treatment of obesity.Trial registrationUMIN Clinical Trials Registry 000019241.

Salicylic acid and methyljasmonate restore the transcription of caffeine biosynthetic N-methyltransferases from a transcription inhibition noticed during late endosperm maturation in coffee

Transcripts of the three N-methyltransferase (NMT) genes involved in the core caffeine biosynthesis of coffee are repressed during the early stages of endosperm maturation, coincident with the onset of dry weight accumulation and seed desiccation. Previously, we noticed that the exogenous application of salicylic acid (SA) and methyljasmonate (MeJ) to Coffea canephora var. robusta cv. S-274 overexpresses transcripts of the three NMTs in young leaves. Thus, transcriptional regulation of caffeine biosynthetic genes may play a vital role in caffeine accumulation. We carried out this study to find if salicylic acid (SA) (50μM and 500μM concentrations) and MeJ treatment of the maturing fruits is capable of restoring the transcription activity of NMT genes. MeJ leads to overexpression of the first two NMTs (XMTs and MXMTs) whereas the effect of SA appeared to be concentration-dependent. Though SA (500μM) de-repressed all three NMTs, SA (50μM) restored transcription of only the second NMT. Neither SA (50μM) nor MeJ could restore transcription of the third and final NMT (DXMTs) of the pathway. Biochemical estimation of methylxanthines indicates a slight but significant increase in theobromine content (11.8% increase) in endosperms treated with 50μM SA. Caffeine showed a statistically significant increase in both MeJ (14.4% increase) and SA (50μM) (14.8% increase) treatments. Since de-repression in DXMTs (caffeine synthase) was not observed, theobromine to caffeine conversion in MeJ and SA (50μM) treated fruits is probably catalyzed by the basal or the extant DXMT gene product. Caffeine levels remained constant in SA (500μM) treatments indicating a possible post-transcriptional regulation. These results are indicative of cross talk between the cascades induced by SA and MeJ and the maturation-induced regulation of caffeine accumulation and may be helpful in studying the interaction between these pathways.