绿咖啡豆中低咖啡因绿原酸的提取分离研究

建立高效液相色谱—双波长同时检测咖啡豆提取物中绿原酸和咖啡因含量的方法。咖啡因和绿原酸在0.2～1.0 mg·mL-1范围内,峰面积与浓度呈现良好的线性关系。咖啡因：Y＝44077X＋227.78,R＝0.9997,RSD＝0.78%;绿原酸：Y＝34896X＋289.96,R＝0.9995, RSD＝0.93%。以绿原酸含量为指标,在单因素试验基础上建立正交试验,确定从绿咖啡豆中提取绿原酸的最佳条件为乙醇浓度20%,液料比6∶1,提取时间60 min,提取温度70 ℃。以咖啡因和绿原酸含量为指标,通过萃取试验,确定最佳脱咖啡因萃取剂为环己烷-三氯甲烷-丙酮混合溶剂,萃取工艺为1.5倍体积萃取剂萃取3次,产物中咖啡因含量由(3.52±0.08)%下降至(0.47±0.11)%。     

Biosynthesis of Caffeine in Flower Buds of Camellia sinensis

The biosynthesis of purine alkaloids in flower buds of tea plantswas investigated. More than 25% of total radioactivity of [8-¹⁴C]adeninetaken up by stamens isolated from tea flower buds was foundto have been incorporated into purine alkaloids, namely, theobromineand caffeine, 24 h after administration of the labelled compound.Pulse-chase experiments indicated that [8-¹⁴C]adenine takenup by the stamens was converted to adenine nucleotides and subsequentlyincorporated into theobromine and caffeine. Since 5 µMcoformycin, an inhibitor of AMP deaminase, inhibited the incorporationof radioactivity into the purine alkaloids, synthesis of caffeinefrom adenine nucleotides seems to be initiated by the reactionof AMP deaminase. Although most of the radioactivity from [8-¹⁴C]inosinewas recovered as CO₂ and ureides, considerable amounts of radioactivitywere recovered as purine alkaloids. The incorporation of radioactivityfrom [8-¹⁴C]inosine into the purine alkaloids was not affectedby coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphatefrom glucose were present in the stamens and petals of tea flowerbuds. From present and previous results, the pathway for thebiosynthesis of caffeine from adenine nucleotides in flowerbuds of tea is discussed.Copyright 1993, 1999 Academic Press Camellia sinensis, tea, stamen, flower, biosynthesis, purine alkaloids, caffeine, theobromine, adenine nucleotides, nucleotide biosynthesis     

Methylation of Xanthosine by Tea-leaf Extracts and Caffeine Biosynthesis

N-Methyltransferase catalyzing the transfer of methyl groups from [¹⁴CH₃]SAM to xanthosine, producing 7- methylxanthosine, was extracted in cell-free systems from tea leaves. The purine nucleotides, nucleosides, and bases, except for xanthosine, were all inactive substrates in the methylation at the N-7 position of their purine rings. This confirmed that the methylation of xanthosine to 7- methylxanthosine is the first step of methylation in the pathway for caffeine biosynthesis. The optimum pH for the methyltransferase is 7.5 to 8.0. PCMB (0.5 mM), Zn^{2 +} (1 mM) and Cu²⁺ (1 mM) strongly inhibited the enzyme activity. The Km values for xanthosine and SAM are 0.25 mM at 3.9µM of SAM and 3.3µM at 0.40 mM of xanthosine, respectively.     

Separate de Novo and Salvage Purine Pools Are Involved in the Biosynthesis of Theobromine but Not Caffeine in Leaves of Coffea arabica L

We used a video digitizer system to (a) measure changes in the pattern of longitudinal surface extension in primary roots of maize (Zea mays L.) upon application and withdrawal of auxin and (b) compare these patterns during gravitropism in control roots and roots pretreated with auxin. Special attention was paid to the distal elongation zone (DEZ), arbitrarily defined as the region between the meristem and the point within the elongation zone at which the rate of elongation reaches 0.3 of the peak rate. For roots in aqueous solution, the basal limit of the DEZ is about 2.5 mm behind the tip of the root cap. Auxin suppressed elongation throughout the elongation zone, but, after 1 to 3 h, elongation resumed, primarily as a result of induction of rapid elongation in the DEZ. Withdrawal of auxin during the period of strong inhibition resulted in exceptionally rapid elongation attributable to the initiation of rapid elongation in the DEZ plus recovery in the main elongation zone. Gravistimulation of auxin-inhibited roots induced rapid elongation in the DEZ along the top of the root. This resulted in rapid gravitropism even though the elongation rate of the root was zero before gravistimulation. The results indicate that cells of the DEZ differ from cells in the bulk of the elongation zone with respect to auxin sensitivity and that DEZ cells play an important role in gravitropism.     

Diagrammatic Scale for Assessment of Bacterial Blight in Coffee Leaves

Due to the lack of a standardized visual method for assessing bacterial blight (Pseudomonas syringae pv. garcae) in coffee leaves, a diagrammatic scale was developed and validated to quantify the disease. Leaves were collected in crops and nursery with different intensity of symptoms, and the true severity was determined electronically. Based on the frequency distribution of severity values and according to the Weber–Fechner's law of visual stimulus, the minimum and maximum limits and the intermediate levels in the scale were determined. Validation was performed by ten evaluators who estimated the severity of 50 leaves with different intensity of symptoms. One evaluation was performed without diagrammatic scale and two evaluations with the scale at 7‐day intervals. The accuracy, precision, repeatability and reproducibility of the estimates were evaluated. The scale had nine levels: 0 (0%), 1 (0.1–0.99%), 2 (1–2%), 3 (2.01–4%), 4 (4.01–8%), 5 (8.01–16%), 6 (16.01–25%), 7 (25.01–45%) and 8 (≥45.1%). Using the scale, the evaluators were able to improve accuracy, precision, reproducibility and repeatability of estimates, compared to evaluators without scale. The scale was appropriate to visual estimation of severity of bacterial blight in coffee leaves.     

Site of Monoterpene Biosynthesis in Majorana hortensis Leaves

Excised epidermis of Majorana hortensis Moench (sweet marjoram) leaves incorporates label from [U-¹⁴C]sucrose into monoterpenes as efficiently as do leaf discs, while mesophyll tissue has only a very limited capacity to synthesize monoterpenes from exogenous sucrose. These results strongly suggest that epidermal cells, presumably the epidermal oil glands, are the primary site of monoterpene biosynthesis in marjoram. Using a leaf disc assay, it was demonstrated that label from [U-¹⁴C]sucrose is incorporated into monoterpenes most efficiently in very young leaves.     

The Metabolic and Performance Effects of Caffeine Compared to Coffee during Endurance Exercise

There is consistent evidence supporting the ergogenic effects of caffeine for endurance based exercise. However, whether caffeine ingested through coffee has the same effects is still subject to debate. The primary aim of the study was to investigate the performance enhancing effects of caffeine and coffee using a time trial performance test, while also investigating the metabolic effects of caffeine and coffee. In a single-blind, crossover, randomised counter-balanced study design, eight trained male cyclists/triathletes (Mean±SD: Age 41±7y, Height 1.80±0.04 m, Weight 78.9±4.1 kg, VO₂ max 58±3 ml•kg⁻¹•min⁻¹) completed 30 min of steady-state (SS) cycling at approximately 55% VO₂max followed by a 45 min energy based target time trial (TT). One hour prior to exercise each athlete consumed drinks consisting of caffeine (5 mg CAF/kg BW), instant coffee (5 mg CAF/kg BW), instant decaffeinated coffee or placebo. The set workloads produced similar relative exercise intensities during the SS for all drinks, with no observed difference in carbohydrate or fat oxidation. Performance times during the TT were significantly faster (∼5.0%) for both caffeine and coffee when compared to placebo and decaf (38.35±1.53, 38.27±1.80, 40.23±1.98, 40.31±1.22 min respectively, p<0.05). The significantly faster performance times were similar for both caffeine and coffee. Average power for caffeine and coffee during the TT was significantly greater when compared to placebo and decaf (294±21 W, 291±22 W, 277±14 W, 276±23 W respectively, p<0.05). No significant differences were observed between placebo and decaf during the TT. The present study illustrates that both caffeine (5 mg/kg/BW) and coffee (5 mg/kg/BW) consumed 1 h prior to exercise can improve endurance exercise performance.     

Spectral Dependence of Chlorophyll Biosynthesis Pathways in Plant Leaves

This review covers studies on the dependence of chlorophyll photobiosynthesis reactions from protochlorophyllide on the spectral composition of actinic light. A general scheme of the reaction sequence for the photochemical stage in chlorophyll biosynthesis for etiolated plant leaves is presented. Comparative analysis of the data shows that the use of light with varied wavelengths for etiolated plant illumination reveals parallel transformation pathways of different protochloro-phyllide forms into chlorophyllide, including a pathway for early photosystem II reaction center P-680 pigment formation.     

Exaggeration of Postprandial Hyperglycemia in Patients with type 2 Diabetes by Administration of Caffeine in Coffee

ObjectiveTo test whether caffeine administered in coffee increases postprandial hyperglycemia in patients with type 2 diabetes who are habitual coffee drinkers.MethodsThe study used a within-subject, double-blind, placebo-controlled experimental design. Twenty adult coffee drinkers (11 women and 9 men) with type 2 diabetes treated with diet, exercise, orally administered antidiabetic agents, or some combination of these factors completed two mixed-meal tolerance tests (MMTT) after an overnight fast. Before the MMTT, each study participant received 250 mg of caffeine in 16 oz (475 mL) of decaffeinated coffee or decaffeinated coffee alone, with the treatment order counterbalanced in the group. Fasting and 1-hour and 2-hour postprandial blood samples were collected for measurement of plasma glucose and insulin concentrations.ResultsGlucose and insulin responses to the MMTT were quantified by the incremental areas under the 2-hour concentration-time curves (AUC_2h). Administration of caffeine in decaffeinated coffee increased postprandial glucose and insulin responses (both P = 0.02). The mean plasma glucose AUC_2h was 28% larger and the mean plasma insulin AUC_2h was 19% larger after administration of caffeine than after administration of placebo.ConclusionOther constituents in coffee did not prevent the exaggeration of postprandial hyperglycemia by caffeine in these patients with type 2 diabetes, who were habitual coffee drinkers. Repeated on a daily basis, such effects could impair long-term glucose control in those patients with type 2 diabetes who habitually drink coffee or other caffeinated beverages. (Endocr Pract. 2007;13: 239-243)     

Polyamines Promote the Biosynthesis of Ethylene in Detached Rice Leaves

The effects of polyamines (putrescine, spermidine, spermineand diaminopropane) on the production of ethylene in detachedrice leaves were investigated. Polyamines effectively promotedthe production of ethylene in detached rice leaves under bothlight and dark conditions. Putrescine stimulated the productionof ethylene within 4 hours of its application, a result suggeststhat putrescine enhances the production of ethylene directly.Putrescine also stimulated the production of ethylene in detachedleaves that had been aged for 2 and 4 days. The stimulatoryeffect of putrescine resulted from the enhancement of the synthesisof 1-aminocyclopropane-l-carboxylic acid (ACC) and the conversionof ACC to ethylene. The activity of S-adenosylmethio-nine decarboxylasein segments of rice leaves was inhibited by the applicationof putrescine. Thus, the enhancement of the synthesis of ACCby putrescine seems to be mediated by increases in the activityof ACC synthase and in the level of the substrate (S-adenosylmethionine)for ACC synthase. (Received February 27, 1991; Accepted June 5, 1991)     

Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

Fungal Epiphytes and Endophytes of Coffee Leaves (Coffea arabica)

Plants harbor diverse communities of fungi and other microorganisms. Fungi are known to occur both on plant surfaces (epiphytes) and inside plant tissues (endophytes), but the two communities have rarely been compared. We compared epiphytic and endophytic fungal communities associated with leaves of coffee (Coffea arabica) in Puerto Rico. We asked whether the dominant fungi are the same in both communities, whether endophyte and epiphyte communities are equally diverse, and whether epiphytes and endophytes exhibit similar patterns of spatial heterogeneity among sites. Leaves of naturalized coffee plants were collected from six sites in Puerto Rico. Epiphytic and endophytic fungi were isolated by placing leaf pieces on potato dextrose agar without and with surface sterilization, respectively. A total of 821 colonies were isolated and grouped into 131 morphospecies. The taxonomic affinities of the four most common nonsporulating fungi were determined by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region: two grouped with Xylaria and one each with Botryosphaeria and Guignardia. Of the most common genera, Pestalotia and Botryosphaeria were significantly more common as epiphytes; Colletotrichum, Xylaria, and Guignardia were significantly more common as endophytes. Suprisingly, more morphospecies occurred as endophytes than as epiphytes. Differences among sites in number of fungi per plant were significant. Thus epiphytic and endophytic communities differed greatly on a single leaf, despite living only millimeters apart, and both communities differed from site to site. Significant correlations between occurrence of fungal morphospecies suggested that fungi may have positive or negative effects on their neighbors. This is the first quantitative comparison of epiphytic and endophytic fungal floras in any plant, and the first to examine endophytic fungi or epiphytic fungi in leaves of coffee, one of the world’s most valuable crops.     

Effects of Calcium and Lanthanum on ABA Biosynthesis in Cucumber Leaves

Cucumber (Cucumis sativus L.) leaves were treated with CaCl₂ and LaCl₃ at concentrations of 0.002, 0.02, 0.04, 0.2, 0.4, and 2.0 mM to study the effects of calcium and lanthanum on plants, especially on the pathway of abscisic acid biosynthesis. The activity of lipoxygenase (Lox) was measured, and the contents of violaxanthin (Vio, a precursor to ABA) and ABA were estimated. In addition, the soluble proteins in treated leaves were analyzed. The results suggest that La³⁺ influences Lox activity and the contents of Vio and ABA. Their changes are correlated to one another, thus indicating that ABA can be derived from a carotenoid in the presence of lanthanum. After leaf treatments with ions, the content of soluble proteins changed and a 41 kD polypeptide appeared confirming responsive gene expression. The effects of both ions on ABA and protein biosyntheses suggest that the appearance of this protein is not related to the endogenous ABA levels.     

TMV-RNA Biosynthesis in the Light-Green and Dark-Green Regions of Tobacco Leaves

Changes in the proteins, chlorophyll, virus content and activity of key enzymes of viral RNA biosynthesis were investigated in the light- and dark-green regions of tobacco leaves systemically infected with tobacco mosaic virus. The protein content was increased to 118 % in the dark-green islands in contrast to 60 % in the light-green regions when compared with the control healthy leaves. The comparative analysis of soluble proteins from healthy and light- or dark-green regions of leaves by means of SDS-PAGE revealed that the main soluble proteins are equal in pattern but differ in quantity. The contents of chlorophylls did not differ from healthy tissues in the dark-green islands but were considerably lower in the light-green regions. The content of virus in light-green tissues was about 10 times higher than in the dark-green islands. The activities of key enzymes of oxidative pentosephosphate cycle -- glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase -- did not differ from healthy tissues in the dark-green islands but were considerably higher in the light-green tissues when compared with healthy control. Similar relationships were observed for ribonuclease, phosphomonoesterase and phosphodiesterase activities. The biosynthesis of viral RNA in the dark-green islands is probably restricted by the steady (or reduced) activities of key enzymes of these metabolic pathways.     

Biosynthesis of Resin Terpenes in Leaves and Glandular Hairs of Newcastelia viscida

Label from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid was incorporatedinto resin components of Newcastelia viscida. About 25% of thelabel from [2-¹⁴C]MVA was incorporated into resin componentsafter 4 h. Approximately 80% of the label in the resin was ina TLC band coinciding with the major terpenoid components, with60% associated with the major tricyclic diterpene, a pimaradienewhich contributes 15% to the leaf resin dry weight. Experimentswith detached trichomes showed about 0?01% incorporation oflabel from [2-¹⁴C]MVA into pimaradiene, with most of the [abelbeing associated with ß-sitosterol. It is proposedthat the glandular hairs are the sites of terpene resin synthesis,as well as secretion, in this species.     

Purine Alkaloid Biosynthesis in Young Leaves of Camellia sinensis in Light and Darkness

14 C] adenine to young leaves of tea (Camellia sinensis L.). Light did not have any significant influence on the levels of radioactivity associated with the purine alkaloids. The long-term effects of light on caffeine production were studied using young shoots from plants that were maintained in almost complete darkness (1% full sunlight) by being covered with black lawn cloth. In the control shoots of the naturally-grown plants there were net increases in the total purine alkaloid contents of 2,430 nmoles shoot⁻¹, while in shoots that had been in darkness for 7 days much lower increases, 564 nmoles shoot⁻¹, were observed. Caffeine synthase (CS) activity was 332±55 pkat shoot⁻¹ in light which is ca. 40% higher than the 237±37 pkat shoot⁻¹ in plants kept darkness for 7-days. However, a similar pattern of the metabolism of [8-¹⁴C] adenine was observed in naturally-grown and dark-grown shoots. These findings indicate light is not essential for the biosynthesis of caffeine in young tea shoots. The lower net formation of caffeine in shoots maintained in darkness is an indirect consequence of the reduced growth rate of the young shoots in the absence of light. Received 17 January 2000/ Accepted in revised form 13 March 2000     

Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea (Camellia sinensis L.)

Purine alkaloid catabolism pathways in young, mature and agedleaves of tea (Camellia sinensis L.) were investigated by incubatingleaf sections with ¹⁴C-labelled theobromine, caffeine, theophyllineand xanthine. Incorporation of label into CO₂ was determinedand methanol-soluble metabolites were analysed by high-performanceliquid chromatography-radiocounting and thin layer chro-matography.The data obtained demonstrate that theobromine is the immediateprecursor of caffeine, which accumulates in tea leaves becauseits conversion to theophylline is the rate limiting step inthe purine alkaloid catabolism pathway. The main fate of [8-¹⁴C]theophyllineincubated with mature and aged leaves, and to a lesser extentyoung leaves, is conversion to 3-methylxanthine and onto xanthinewhich is degraded to ¹⁴CO₂ via the purine catabolism pathway.However, with young leaves, sizable amounts of [8-¹⁴C]-theophyllinewere salvaged for the synthesis of caffeine via a 3-methylxanthine