Optimization of the extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc produced during traditional wine processing and characterization of the extracts

The extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc generated during traditional wine processing was optimized using response surface methodology (RSM). A face-centered cube design (FCD) consisting of 17 experimental runs, including five replicates at the center point, was used to investigate the effects of the three variables (solid-liquid ratio, time, and temperature) on anthocyanin extraction, and the results showed that the relationship between the three variables and the total anthocyanin content followed a quadratic model (R2=0.8853). In addition, the RSM analysis predicted that the optimum conditions for extraction consisted of a solid-liquid ratio of 20, a time of 60min, and a temperature of 60 degrees C. Verification tests performed under these optimum conditions gave 34.7+/-1.4mg/100g of anthocyanin, which was close to predicted value of 37.2mg/100g. Additionally, analysis of water extracts prepared using the predicted optimum conditions revealed that the carbohydrates (sugar and pectin) in Bokbunja marc underwent significant variation toward the formation of by-products (glycerol and uronic acids) during yeast fermentation, and that the amount of anthocyanin produced was reduced 10-fold when compared to the original extraction. Further, the results of HPLC-PDA-MS/MS analysis of the anthocyanins extracted from Bokbunja marc revealed the presence of six anthocyanin components, which were tentatively identified as cyanidin 3-O-sambubioside, cyanidin 3-O-xylosylrutinoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-rutinoside, delphinidin 3-O-rutinoside-?, and delphinidin 3-O-glucuronide.     

Anthocyanins of grasses

The anthocyanin content of 23 grass species (Poaceae) belonging to five subfamilies has been determined. Altogether 11 anthocyanins were identified; the 3-(6″-malonylglucosides) and 3-glucosides of cyanidin, peonidin and delphinidin, the 3-(3″,6″-dimalonylglucoside), 3-(6″-rhamnosylglucoside) and 3-(6″-glucosylglucoside) of cyanidin, in addition to peonidin 3-(dimalonylglucoside) and delphinidin 3-(6″-rhamnosylglucoside). Anthocyanins acylated with one and/or two malonic acid moieties dominated the anthocyanin profiles of all the species in the subfamilies Pooideae and Panicoideae. On the other hand, the 3-glucoside and 3-rutinoside of cyanidin were the major anthocyanins in Sinarundinaria murielae (subfamily Bambusoideae) and Molinia caerulea (subfamily Arundinoideae), while the 3-glucosides of cyanidin and peonidin were the principal anthocyanins in rice, Oryza sativum (subfamily Oryzoideae). Pelargonidin derivatives and free anthocyanidins have previously been reported to occur in several Poaceae species, however, not identified in any of the species included in this survey.     

Anthocyanins in fruits of Vaccinium Japonicum

Cyanidin-3-arabinoside (54%) and pelargonidin-3-arabinoside (39%) were the main anthocyanins isolated from berries of Vaccinium japonicum. In addition smaller amounts of 3-galactosides of cyanidin (5%) and pelargonidin (2%) were found. The total anthocyanin content in the fruit averaged 113 mg/100 g fresh fruit. This is the first report of pelargonidin derivatives in the genus Vaccinium.     

Acylated cyanidin 3-sambubioside-5-glucosides in three garden plants of the Cruciferae

Seven acylated cyanidin 3-sambubioside-5-glucosides were isolated from the flowers of three garden plants in the Cruciferae. Specifically, four pigments were isolated from Lobularia maritima (L.) Desv., together with a known pigment, as well as, three pigments from Lunaria annua L., and two known pigments from Cheiranthus cheiri L. These pigments were determined to be cyanidin 3-O-[2-O-((acyl-II)-(beta-d-xylopyranosyl))-6-O-(acyl-I)-beta-d-glucopyranoside]-5-O-[6-O-(acyl-III)-beta-d-glucopyranoside], in which the acyl-I group is represented by glucosyl-p-coumaric acid, p-coumaric acid and ferulic acid, acyl-II by caffeic acid and ferulic acid, and acyl-III by malonic acid, respectively. The distribution and biosynthesis of acylated cyanidin 3-sambubioside-5-glucosides are discussed according to the variations of acylation and glucosylation at their 3-sambubiose residues.     

Acylated anthocyanins from the violet-blue flowers of Orychophragonus violaceus

Three acylated cyanidin 3-sambubioside-5-glucosides (1-3) were isolated from the violet-blue flowers of Orychophragonus violaceus, and their structures were determined by chemical and spectroscopic methods. Two of those acylated anthocyanins (1 and 3) were cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-trans-acyl)-beta-D-glucopyranoside]-5-O-(6-O-malonyl-beta-D-glucopyranoside)s, in which the acyl groups were p-coumaric acid for 1, and sinapic acid for 3, respectively. The last anthocyanin 2 was cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-trans-feruloyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside. In these flowers, the anthocyanins 2 and 3 were present as dominant pigments, and 1 was obtained in rather small amounts.     

Flavonoid composition related to petal color in different lines of Clitoria ternatea

Flavonoids in the petals of several C. ternatea lines with different petal colors were investigated with LC/MS/MS. Delphinidin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside was newly isolated from the petals of a mauve line (wm) together with three known anthocyanins. They were identified structurally using UV, MS, and NMR spectroscopy. Although ternatins, a group of 15 (poly)acylated delphinidin glucosides, were identified in all the blue petal lines (WB, BM-1, 'Double Blue' and 'Albiflora'), WM accumulated delphinidin 3-O-(6"-O-malonyl)-beta-glucoside instead. The white petal line (WW) did not contain anthocyanins. Quantitative data showed that the total anthocyanin contents in WB and 'Double Blue' were ca. 8- and 10-fold higher than that in BM-1, a bud mutant of 'Double Blue', respectively. The total anthocyanin content in 'Albiflora' was less than 2 x 10(-3) times those in WB or 'Double Blue'. While all the lines contained the same set of 15 flavonol glycosides in similar relative ratios, the relative ratio of myricetin glycosides in ww and 'Albiflora' was ca. 30-70 times greater than those in the other lines. The change in flower color from blue to mauve was not due to a change in the structure of an anthocyanidin from delphinidin, but to the lack of (polyacylated) glucosyl group substitutions at both the 3'- and 5'-positions of ternatins. This implies that glucosylation at the 3'- and 5'-positions of anthocyanin is a critical step in producing blue petals in C. ternatea.     

Cyanidin 3-[6-(p-coumaroyl)-2-(xylosyl)-glucoside]-5-glucoside and other anthocyanins from fruits of Sambucus canadensis.

From the fruits of Sambucus canadensis four anthocyanin glycosides have been isolated by successive application of an ion-exchange resin, droplet-counter chromatography and gel filtration. The structure of the novel, major (69.8%) pigment, cyanidin 3-O-[6-O-(E-p-coumaroyl-2-O-(beta-D-xylopyranosyl)-beta-D- glucopyranoside]-5-O-beta-D-glucopyranoside, was determined by means of chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques. The other anthocyanins were identified as cyanidin 3-sambubioside-5-glucoside (22.7%), cyanidin 3-sambubioside (2.3%) and cyanidin 3-glucoside (2.1%).     

Effect of barbitone sodium and thiopental sodium on brain dopamine, noradrenaline, serotonin and 5-hydroxyindoleacetic acid content in Arvicanthis niloticus

The quantitative estimation of total dopamine (DA), noradrenaline (NE), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content in the whole brain tissue of normal Nile grass rat, Arvicanthis niloticus, gives and average of 631 +/- 12 ng DA/g, 366 +/- 12 ng NE/g, 617 +/- 15 ng 5-HT/g and 431 +/- 10 ng 5-HIAA/g fresh brain tissue. The effect of barbitone sodium and thiopental sodium on the total DA, NE, 5-HT and 5-HIAA content in the brain tissue of the Nile grass rat, Arvicanthis niloticus, was studied. The total DA, NE, 5-HT and 5-HIAA contents were determined 5 hr after i.p. injection of different doses of barbitone sodium (20, 40 and 80 mg/ml/100 g body wt) and thiopental sodium (5, 10 and 20 mg/ml/100 g body wt). The effect of different time intervals (1, 10, 30 min, 1, 2.5, 5, 8, 16, 24 and 48 hr) on the total brain DA, NE, 5-HT and 5-HIAA content was investigated after i.p. injection of 40 mg of barbitone sodium and 10 mg of thiopental sodium/ml/100 g body wt. Both barbitone sodium and thiopental sodium caused an increase in DA, NE and 5-HT content and a decrease in 5-HIAA content in the brain tissue of Arvicanthis niloticus. The increase in the whole brain contents of DA, NE and 5-HT after the administration of barbitone sodium and thiopental sodium may be due either to inhibition of transmitter release by an action at the monoamine nerve terminal or to effects causing a decrease in nerve impulse flow. On the other hand, the decrease in 5-HIAA may be due to the decrease in the turnover of 5-HT.     

Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae)

Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of various ailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production. The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time on cell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of different concentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50 mg/L) had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30 g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45 g/L sucrose without MeJA showed the highest pigment content (0.69 +/- 0.22 CV/g-FCM). The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5 mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40 CV/g-FCM). This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures.     

桃果肉花色苷遗传多样性及红肉桃判定指标的探讨

运用比色法(UV)和高效液相色谱法(HPLC)测定68份桃品种果肉花色苷含量,比较2种测定结果的一致性,分析桃果肉花色苷的遗传多样性,初步提出红肉桃的判定标准。结果表明,2种方法测定白肉品种花色苷含量接近,可根据实际情况进行选择分析方法,而黄肉品种应尽可能选用高效液相色谱法(HPLC)。HPLC定性定量检测结果表明,48份材料检测出矢车菊-3-葡萄糖苷,是桃果肉中主要的花色苷种类,乌黑鸡肉桃,大果黑桃果肉中同时检测到矢车菊-3-芸香糖苷。根据高效液相色谱法测得花色苷含量将桃划分为5个等级,含量20 mg/100 g定为红肉桃的划分临界点。本研究中共鉴定出5份红肉桃品种,感官判定且风味为甜的红肉桃不符合上述判定标准,需要进一步研究完善。     

Characterization and inheritance of the Anthocyanin fruit (Aft) tomato

Tomato (Lycopersicon esculentum) accession LA1996 with the Anthocyanin fruit (Aft) gene has dark green foliage, elevated anthocyanin expression in the hypocotyls of seedlings, and anthocyanin in the skin and outer pericarp tissues of the fruit. Interest in the health benefits and antioxidant capacity of anthocyanins led to this study of the genetic potential for increased levels of this important class of phytonutrients in tomato fruit. In order to conform to tomato gene nomenclature rules, we propose changing the symbol Af for Anthocyanin fruit to Aft. Segregation ratios of anthocyanin expression in F(2) and BC(1) populations of a cross between the processing tomato UC82B and LA1996 were consistent with a single dominant gene hypothesis. Anthocyanin expression was reduced in backcross populations compared to F(2 )populations. Anthocyanin concentration, as measured by the pH differential method, of pigment-rich pericarp and skin tissues from LA1996 was estimated to be 20.6 mg/100 g and 66.5 mg/100 g, respectively. Anthocyanidin composition was characterized by high-performance liquid chromatography (HPLC). Fruit of accession LA1996 contained predominantly petunidin, followed by malvidin and delphinidinin. Lycopene, beta-carotene, phytoene, and phytofluene levels were similar to those of normal tomatoes and lower than those found in high pigment tomatoes.     

Anthocyanin composition of the fruit of Coriaria myrtifolia L

The anthocyanin composition of the fruit of Coriaria myrtifolia L. and the changes which occur during ripening were studied using HPLC-PAD and LC-MS. Ten anthocyanins were detected and identified by their absorption and mass spectra as the 3-glucoside and 3-galactoside derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin. Fruit ripening was accompanied by substantial changes in the anthocyanin profile, with methoxylated anthocyanins, i.e. malvidin and peonidin, predominating in the final stages of ripening, and the trihydroxylated anthocyanin, delphinidin, during the earlier stages. Furthermore, galactoside derivatives were more abundant than glucosides in the ripe fruit. At full maturity, the fruits of C. myrtifolia were very rich in anthocyanins with a content of 10.7% (on a dry weight basis), a level which is higher than that found in most fruits usually considered to be anthocyanin-rich. The ability to grow C. myrtifolia in damaged and nitrogen poor soils, together with the possibility of using this plant for the extraction of anthocyanin, makes it ideal for consolidating soils and repopulating semi-desert or fire-damaged areas.     

Genetic control of UDP-glucose: anthocyanin 5-O-glucosyltransferase from flowers of Matthiola incana R.Br.

In flower extracts of defined genotypes of Matthiola incana, an enzyme was demonstrated which catalyzes the transfer of the glucosyl moiety of uridine 5-diphosphoglucose (UDPGlc) to the 5-hydroxyl group of pelargonidin and cyanidin 3-glycosides and acylated derivatives. The best substrate for 5-glucosylation is the 3-xylosylglucoside acylated with p-coumarate, followed by the 3-xylosylglucoside and by the acylated (p-coumarate) 3-glucoside. The 3-glucoside itself is a very poor substrate. Besides UDPGlc, thymine 5-diphosphoglucose is a suitable glucosyl-donor, but with a reduced reaction rate (42%). The anthocyanin 5-O-glucosyltransferase exhibits a pH optimum at 7.5 and is generally inhibited by divalent ions and by ethylenediaminetetraacetic acid and p-chloromercuribenzoate. Investigations on different genotypes showed that the 5-O-glucosyltransferase activity is clearly controlled by the gene l. In confirmation of earlier chemogenetic work, enzyme activity is only present in lines with the wild-type allele l⁺. The anthocyanin 5-O-glucosyltransferase activity is strictly correlated with the formation of 5-glucosylated anthocyanins during bud development.Abbreviations Cg 3,5-T-cyanidin 3-sambubioside-5-glucoside - EDTA ethylene diaminetetraacetic acid - 5GT UDP-glucose: anthocyanin 5-O-glucosyltransferase - 3GT UDP-glucose: anthocyanidin/flavonol 3-O-glucosyltransferase - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - UDPGlc uridine 5-diphospho-glucose     

MODEL DEVELOPMENT AND PROCESS OPTIMIZATION FOR SOLVENT EXTRACTION OF POLYPHENOLS FROM RED GRAPES USING BOX–BEHNKEN DESIGN

The objective of the present study is to find out the optimum extraction conditions for extraction of polyphenols from red grapes using Box–Behnken design. Red grapes polyphenols were extracted using acid–ethanol solvent at various extraction temperature (40–60°C), extraction time (20–100 min) and different solid–liquid ratio (1:5–1:15 g:ml). The effect (main and interactive) of extraction conditions on total anthocyanin, phenolic and flavonoid content were studied using Box–Behnken design (three factors at three levels). The results showed that the contribution of the quadratic model was significant for all the responses. Second-order mathematical regression models were developed and were found to fit well with observed data. Derringer's desirability function methodology was performed to find out the optimal conditions based on both individual and combinations of all responses (extraction temperature: 57°C, time: 61 min, and solid–liquid ratio: 1:8.7 g:ml) were established. At this optimal condition, the anthocyanin yield, total phenolic and flavonoid content were 73.92 mg/100 g, 221.4 mg GAE/100 g, and 79.08 mg CE/100 g, respectively. A desirability value of 0.902 was achieved at this point.     

Blueing of sepal colour of Hydrangea macrophylla

Blue and red sepals of Hydrangea macrophylla were quantitatively analyzed for aluminium, anthocyanin (delphinidin 3-glucoside) and copigments (caffeoyl- and p-coumaroylquinic acids). All the blue sepals examined contained both Al and copigments (especially 3-caffeoylquinic acid) in considerable amounts. In in vitro experiments using 3- and 5-caffeoylquinic acids, Al and delphinidin 3-glucoside, it was shown that 3-caffeoylquinic acid and Al formed a blue complex with the anthocyanin. Absorption spectra of the blue complex were practically identical with those of the blue solutions obtained from blue hydrangea sepals by extraction with 4 M NACl. In contrast, 5-caffeoylquinic acid (chlorogenic acid) which was also present in hydrangea sepals gave only a red-purple colour with Al and the anthocyanin. Neither 3-caffeoylquinic acid nor Al independently produced blue colour when mixed with the anthocyanin in the mole ratios of 1–30, this being the range that the compounds were found in blue sepals. These results suggest that blue colour of hydrangea sepals is due mainly to the blue complex of delphinidin 3-glucoside-aluminium-3-caffeoylquinic acid. The role of aluminium may be to stabilize an interaction between the quinic ester and the anthocyanin.     

Glutathione correlates with lipid peroxidation in liver mitochondria of triiodothyronine-injected hypophysectomized rats

The temporal relationship of changes in state 3 respiration, lipid peroxidation, and glutathione (GSH) content was investigated in liver mitochondria of hypophysectomized rats after an injection of 3,3',5-triiodo-L-thyronine (T3). Lipid peroxidation induced by ADP/Fe3+/NADPH was determined by the amount of malondialdehyde formed. Hypophysectomy decreased respiration and lipid peroxidation (from 19.88 +/- 3.04 to 14.19 +/- 1.14 nmol malondialdehyde.mg protein-1.10 min-1) but increased GSH content (from 7.06 +/- 2.08 to 12.46 +/- 3.58 nmol/mg protein). Daily injections of a low dose (5 micrograms/100 g) of T3 for 7 days restored the parameters. Time course (up to 96 h) of these changes was followed after one injection of a moderate (100 micrograms/100 g) and high (1000 micrograms/100 g) dose of the hormone. Respiration showed a significant increase at 24 h and declined slightly at 96 h. There was a slow loss of respiratory control ratio after 24 h. Lipid peroxidation remained unchanged at 24 h and showed a gradual increase, becoming significantly higher at 72-96 h depending on the hormone dosage. Changes in GSH content followed a time course similar to that of lipid peroxidation except that it showed a decrease instead of an increase. There was a high degree of inverse linear correlation between lipid peroxidation and GSH (correlation coefficient = 0.95). Because GSH is required for detoxification of hydroperoxides generated by the respiratory chain, it is suggested that lipid peroxidation may play a major role in the modulation of intramitochondrial GSH.     

Effects of anthocyanin and carotenoid combinations on foliage and immature fruit color of Capsicum annuum L

Shades ranging from violet to black pigmentation in pepper (Capsicum annuum L.) are attributed to anthocyanin accumulation. High-performance liquid chromatography and mass spectrometry analysis of violet and black fruit tissue identified a single anthocyanin that was determined to be delphinidin-3-p-coumaroyl-rutinoside-5-glucoside. Leaf tissue of a black-pigmented foliage genotype contained the same anthocyanin found in fruit but at a considerably higher concentration in comparison to violet and black fruit tissue. Fruit chlorophyll concentration was approximately 14-fold higher in black fruit in comparison to violet fruit that contained relatively little chlorophyll. Beta-carotene, lutein, violaxanthin, and neoxanthin carotenoid concentrations in black fruit were also significantly greater in comparison to violet fruit. High concentrations of delphinidin in combination with chlorophyll and accessory carotenoid pigments produced the characteristic black pigmentation observed in fruits and leaves of selected genotypes. Anthocyanins were accumulated in the outer mesocarp of violet and black fruit and in the palisade and mesophyll cells of black leaves. Consistent with chlorophyll content of respective genotypes, chloroplast density was greater in cells of black fruits. Utilizing Capsicum pigment variants, we determine the biochemical factors responsible for violet versus black-pigmented pepper tissue in the context of described pepper color genes.     

Survey of bioactive components in Western Canadian berries

Berries native to Western Canada were analyzed for total anthocyanins, total phenolics, and trolox equivalent antioxidant activity (TEAC). Values ranged from 1.60 to 9.55 mmol trolox equivalent per 100 g fresh mass. Anthocyanin content ranged from 41.6 (in red twinberries) to 1081 mg cyanidin-3-glucoside equivalents per 100 g fresh mass (in honeysuckle fruits). Honeysuckle fruits contained the highest amount of total polyphenols, 1111 mg gallic acid equivalents per 100 g, among analyzed fruits. Additionally, anthocyanins in the investigated berries were identified and characterized by HPLC - electrospray ionization - tandem mass spectrometric method coupled with diode array detection. The number of anthocyanins varied from 4 in saskatoon berries (Amelanchier alnifolia Nutt.) to 20 in bilberries (Vaccinum myrtilloides Michx.). In all the samples analyzed, 6 common anthocyanidins:, cyanidin, delphinidin, pelargonidin, petunidin, peonidin, and malvidin, were found. Half the analyzed berries contained acylated anthocyanins, but a significant amount was found only in bilberries. The analyzed berry seed oils contained high amounts of unsaturated fatty acids (over 90%), but only the golden currant seed oil contained gamma-linolenic acid.     

Acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers of Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (Brassicaceae)

A novel acylated cyanidin 3-sambubioside-5-glucoside was isolated from the purple-violet flowers of Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (family: Brassicaceae), and determined to be cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-β-xylopyranosyl)-6-O-(trans-feruloyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside] by chemical and spectroscopic methods. In addition, two known acylated cyanidin 3-sambubioside-5-glucosides, cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-β-xylopyranosyl)-6-O-(trans-feruloyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside] and cyanidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(trans-feruloyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside] were identified in the flowers.     

Determination of acylated anthocyanin in human urine after ingesting a purple-fleshed sweet potato beverage with various contents of anthocyanin by LC-ESI-MS/MS

Eighty-seven healthy volunteers ingested a purple-fleshed sweet potato beverage with various contents of anthocyanin (beverage A; 22.1 mg/250 ml, B; 107.8, C; 84.9). An acylated anthocyanin, peonidin 3-caffeoylsophoroside-5-glucoside, was detected in the urine 2 h after ingestion. The concentrations were 15.1+/-2.2 microg/l of urine (mean+/-SEM), 46.6+/-5.3, and 53.3+/-2.2 for beverages A, B, and C respectively.