Biosci. Biotechnol. Biochem., 70 (7), 1681–1687, 2006

Urinary Excretion of in Humans after Cranberry Juice Ingestion

y Ryoko OHNISHI,1 Hideyuki ITO,1; Naoki KASAJIMA,2 Miyuki KANEDA,2 Reiko KARIYAMA,3 Hiromi KUMON,3 Tsutomu HATANO,1 and Takashi YOSHIDA4

1Department of Pharmacognosy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan 2School of Pharmacy, Shujitsu University, Nishigawara, Okayama 703-8516, Japan 3Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata, Okayama 700-8558, Japan 4Matsuyama University, Bunkyo-cho, Matsuyama 790-8578, Japan

Received January 16, 2006; Accepted February 25, 2006; Online Publication, July 23, 2006 [doi:10.1271/bbb.60023]

Cranberry, which is rich in polyphenols, including protection against lipoprotein,2) and in vitro anticancer anthocyanins and proanthocyanidins, has been found to activity.3) Reduction of urinary tract infections in have various effects beneficial to human health, includ- women by drinking cranberry juice was also proved in ing prevention of urinary tract infections. These effects randomized, double-blind placebo-controlled trials.4,5) have been associated with polyphenols in the fruit. We Among the reported ingredients of cranberry, including investigated the excretion of anthocyanins in human proanthocyanidins,6,7) anthocyanins,8–10) flavonoids,11,12) urine after ingestion of cranberry juice. Eleven healthy triterpenoids,13) iridoids,14) and organic acids,15,16) the volunteers consumed 200 ml of cranberry juice contain- proanthocyanidins, fructose, and macromolecules17) ing 650.8 g total anthocyanins. Urine samples were have been found to inhibit the adherence of uropatho- collected within 24 h before and after consumption. Six genic P-fimbriated Escherichia coli to eucaryotic cells, of 12 anthocyanins identified in cranberry were quanti- and this might be associated with the prevention of fied in human urine by HPLC coupled with electrospray urinary infection by cranberry.6,7) On the other hand, the ionization and tandem mass spectrometry (HPLC–ESI– preventive effect on urinary infection has also been MS–MS). Among these, 3-O-galactoside, the accounted for by the acidification of urine with hippuric second most plentiful in the juice, was acid (glycine-conjugate of benzoic acid), produced by found most abundantly in urine within 24 h, corre- the metabolism of quinic acid through benzoic acid.18,19) sponding to 41.5 nmol (56.1% of total anthocyanins). Thus the in vivo urinary metabolites of the other The urinary levels of anthocyanins reached a maximum polyphenolic ingredients, such as proanthocyanidins between 3 and 6 h after ingestion, and the recovery of and anthocyanins, might also play an important role in total anthocyanins in the urine over 24 h was estimated the prevention of urinary tract infections. to be 5.0% of the amount consumed. This study found Anthocyanins are widely distributed in fruits and high absorption and excretion of cranberry anthocya- vegetables such as blueberries, strawberries, cherries, nins in human urine. plums, grapes, and red cabbage. The daily consumption of anthocyanins by humans has been estimated to be Key words: anthocyanins; cranberry juice; human urine; 180–215 mg/d in the United States,20) much higher than HPLC–ESI/MS; MS–MS that of other flavonoids such as quercetin, kaempferol, and myricetin in the Dutch diet (23 mg/d).21) Never- Cranberry (Vaccinium macrocarpon Ait., Ericaceae), theless, information about the rate and extent of distributed in North America, has traditionally been used absorption, metabolism, and excretion of cranberry in the treatment and prevention of urinary tract anthocyanins in the human body is to date quite limited. infections, and has been reported to exhibit various The aim of the present study was to investigate the biological properties, including inhibition of Helico- urinary excretion of anthocyanins in cranberry juice bacter pylori adhesion to human gastric mucus,1) through the identification of metabolites in human urine

y To whom correspondence should be addressed. Fax: +81-86-251-7926; E-mail: [email protected] Abbreviations: HPLC–ESI–MS–MS, HPLC coupled with electrospray ionization and tandem mass spectrometry; DAD, diode array detector; TFA, trifluoroacetic acid 1682 R. OHNISHI et al. using the HPLC–ESI–MS–MS technique as described triple-stage quadrupole mass spectrometer, API 4000 for various polyphenols.22–25) (Applied Biosystems, Tokyo, Japan). The chromato- graphic column was a Nucleosil 100-5C18 (4.6 mm Materials and Methods i.d. 150 mm, GL Sciences, Tokyo, Japan) maintained 40 C, and the mobile phase consisted of acetonitrile– Materials and reagents. The cranberry juice (Cran- water–TFA (20:80:0.5, by vol.) (solvent A) and aceto- berry UR-100, Kikkoman Corporation, Chiba, Japan) nitrile–water–TFA (95:5:0.5 by vol.) (solvent B). A used in this study was prepared by concentration of fresh gradient system was applied as follows: The proportion juice to about 1/5 volume, followed by adjustment with of solvent B in the eluent initialized at 0% (t ¼ 10 min), water and tasting agents (sugar and organic acid) to increased from 0% to 100% (t ¼ 15 min) and 100% contain 100% solid body of the fresh juice. 3- (t ¼ 20 min), and decreased back to 0% (t ¼ 20:1 min) O-galactoside chloride (ideain chloride), 3-O- until the next injection (t ¼ 30 min). The flow rate was glucoside chloride (kuromanine chloride), and 6-hy- 0.8 ml/min with 0.2 ml/min split directed to the mass droxyflavone were purchased from Extrasynthese (Lyon, spectrometer. Mass detection was carried out using France). All solvents used for HPLC analysis were of an electrospray interface operating in positive-ion mode HPLC grade. at 450 C, with nebulizer pressure of 90 pounds per square inch, a drying nitrogen gas flow of 11 l/min, a Study design. After the study was approved by the fragmentor voltage of 20 V, and capillary voltage of ethical committee for human experimentation of Faculty 4,000 V. Ionization and fragmentation were optimized of Pharmaceutical Sciences, Okayama University (ethics for authentic anthocyanins by direct infusion of a reference no. 1), all subjects provided written informed standard solution (0.2 mM in 50% aqueous acetonitrile consent prior to participation. Eleven healthy volunteers containing 0.5% TFA solution). The mass data were in total (nine men and two women) aged 25:5 4:9 collected in multiple reaction monitoring (MRM) mode years (mean SD) followed an anthocyanin-free diet by the transition of parent and product ions specific for containing no fruits, vegetables, coffee, or tea, and were each anthocyanin at a dwell time of 80 ms. HPLC–DAD allowed to drink water from half a day before test analysis was performed on a Hitachi diode array ingestion to the next morning. All subjects consumed detector L-7455 monitoring absorbance at 524 nm, and 200 ml cranberry juice at 9 AM. Blank urine samples a Hitachi L-2130 pump. Other HPLC methodology was were collected before dosing, along with individual as mentioned above. urine over the next 24 h, in plastic bottles containing Identification of the anthocyanins was based on the 1 ml 2% aqueous ascorbic acid and 1 M HCl, and were matching of molecular weight (parent and product ions) immediately stored in a freezer until analysis. The and retention time with those of available anthocyanin subjects were under their own control except for standards and a comparison of those reported HPLC– restriction of diet and drink throughout the study MS–MS data.8–10) Anthocyanins were quantified by periods. They did not use any medications. comparison with a standard curve obtained using known concentrations of an available standard, cyanidin 3-O- Analysis of anthocyanins in cranberry juice and urine. galactoside. The urinary concentrations of anthocyanins Anthocyanins in cranberry juice (Cranberry UR-100) were determined based on the corrected peak area and urine were quantified by HPLC–ESI–MS–MS. divided by the peak area ratios shown in Table 1. Urine samples (500 ml) acidified with 1 M HCl (20 ml) Calibration curves were prepared by spiking blank urine and 25% trifluoroacetic acid (TFA) (10 ml), and spiked with solution at deferent concentrations (0.01–100 mM), with 6-hydroxyflavone (50 mM) in 0.58 M acetic acid with duplicate injections at each level. (40 ml) as an internal standard, were extracted with solid- phase extraction (SPE) cartridges (Bond-Elut C18, Statistical analysis. Values are given as means with 50 mg/1 ml; Varian, CA, USA), which were washed standard errors. with 0.5% TFA–methanol and equilibrated with 0.5% aqueous TFA before use. Urine samples loaded onto the Results and Discussion cartridge were washed with 0.5% aqueous TFA, and anthocyanins were eluted with 0.5% TFA–methanol. Anthocyanins in cranberry juice The methanolic eluate was evaporated to dryness under Anthocyanins constitute a large group of plant pig- nitrogen gas at ambient temperature. The residue was ments distributed mainly in flowers, fruits, and vegeta- dissolved in 25% aqueous methanol containing 0.5% bles. Cranberry contains large amounts of sugars, TFA (200 ml), and filtered. An aliquot (20 ml) of this proanthocyanidins, flavonoids, and organic acids as well solution was injected into an HPLC–ESI–MS–MS as anthocyanins. The structures of aglycone of 12 system and an HPLC equipped with diode array detector analyzed anthocyanins are depicted in Fig. 1. The ion (DAD). chromatograms of cranberry juice analyzed by HPLC– HPLC–ESI–MS–MS analysis was performed on a ESI–MS–MS are shown in Fig. 2. By the HPLC–ESI– Hewlett-Packard 1100 HPLC system equipped with a MS–MS method, 12 anthocyanins were identified by Urinary Excretion of Cranberry Anthocyanins 1683

Table 1. Parent and Product Ions, Retention Times, and Peak Area R Ratios of in Cranberry and Internal Standard OH Retention Parent ion Product ion Peak area Anthocyanin time (m=z) (m=z) ratioa HO O (min) R' Cy 3-O-gal 449 287 4.53 1.0 Cy 3-O-glc 449 287 4.81 1.0 OH Cy 3-O-ara 419 287 5.79 1.6 Pn 3-O-gal 463 301 6.35 1.6 OH Pn 3-O-glc 463 301 7.07 2.6 Pn 3-O-ara 433 301 8.75 3.8 b Dp 3-O-ara 435 303 4.60 — R R' Pg 3-O-gal 433 271 5.58 — Pg 3-O-ara 403 271 7.52 — Pt 3-O-gal 479 317 4.98 — (Pg) H H Mv 3-O-gal 493 331 7.16 — Cyanidin (Cy) OH H Mv 3-O-ara 463 331 10.03 — (Dp) OH OH 6-Hydroxyflavone 239 137 16.99 — Peonidin (Pn) OCH3 H (Internal standard) (Pt) OCH3 OH a Peak area ratio calculated by HPLC–ESI–MS–MS versus HPLC–DAD at (Mv) OCH3 OCH3 the same concentration of individual anthocyanins. bNot detected by HPLC–DAD. Cy, cyanidin; Pn, peonidin; Dp, delphinidin; Pg, pelargonidin; Pt, petunidin; Fig. 1. Chemical Structures of Aglycones of Anthocyanins Used in Mv, malvidin; gal, galactoside; glc, glucoside; ara, arabinoside the HPLC–ESI–MS–MS Assay.

TIC Cy 3-O-gal Cy 3-O-glc 449 / 287a

Cy 3-O-ara 419 / 287 Pn 3-O-gal Pn 3-O-glc 463 / 301 Pn 3-O-ara 433 / 301 Dp 3-O-ara 435 / 303

Pg 3-O-gal 433 / 271 Pg 3-O-ara 403 / 271

Pt 3-O-gal 479 / 317 Mv 3-O-gal 493 / 331 Mv 3-O-ara 463 / 331

0 10 20 30 (min)

Fig. 2. Total Ion Chromatogram (TIC) and Extracted Single Ion Chromatograms of Cranberry Juice Obtained by the HPLC–ESI–MS–MS Method in Multiple Reaction Monitoring Mode with Positive Ionization. aThe m=z values of parent/product ions. 1684 R. OHNISHI et al. comparing respective retention times and parent/prod- cranberry anthocyanins in urine over 24 h reached 5.0% uct ion pairs with those of known standards, or by of the amount consumed. Most studies have reported reference to the literature (Table 1).8–10,26) A significant that relative urinary excretion of anthocyanins was very difference was observed in the peak areas calculated by low, less than 0.1% of ingested pure anthocyanin or the MS and UV for individual anthocyanins. In order to foods containing it,28–33) indicating poor absorption and calculate a more accurate quantity value (Table 1), we excretion of these compounds compared with other took the peak area ratio of ionization by MS and UV polyphenols,27) but there are a few reports indicating absorbance at 524 nm by DAD of individual detectable higher anthocyanin levels in urine (up to a few anthocyanins into consideration. The concentrations of percentage points) after red wine34) or strawberry quantifiable anthocyanins in cranberry juice and urine, consumption.23) The above data suggest that the absorp- which were defined as cyanidin 3-O-galactoside equiv- tion of anthocyanins is accelerated by other components alents, were determined by relating their peak areas to in cranberry. It might also be due to a sufficient that of 6-hydroxyflavone as internal standard. The extraction procedure as well as high selectivity and calibration curves was nicely linear over the concen- sensitivity by the HPLC–ESI–MS–MS method. Follow- tration range (0.01–100 mM) studied, with correlation ing cranberry juice ingestion, almost all the urinary coefficients r2 > 0:995. The limit of quantification anthocyanins over 24 h were excreted as respective (S=N > 10) was determined for all samples. galactoside and arabinoside of cyanidin and peonidin, The total amount of quantifiable anthocyanins in accounting for more than 90% of total anthocyanin cranberry juice was estimated at 650.8 mg/200 ml in- excretion. Among these, the urinary concentration of gested in the present study. Among these, peonidin 3-O- peonidin 3-O-galactoside reached a level of 41:5 arabinoside, at a concentration of 230.0 mg/200 ml juice, 6:2 nmol/24 h (56.1% of total anthocyanin excretion). was the major anthocyanin in the juice, accounting for Up to 80% of the total anthocyanin dose and the main 35.3% of total quantifiable anthocyanins. Subsequently, urinary anthocyanin, peonidin 3-O-galactoside, were the other anthocyanins, cyanidins 3-O-galactoside and excreted within 6 h post-consumption. The urinary 3-O-arabinoside, and peonidin 3-O-galactoside, at 19.7, excretion of all anthocyanins was maximal between 3 16.1, and 26.8% in total respectively, were predominant and 6 h after ingestion, and they were mostly exhausted in the juice. The cranberry juice contained six types of in urine within 12 h, indicating that a major proportion anthocyanidin (cyanidin, peonidin, pelargonidin, malvi- of anthocyanins was quickly excreted in the urine din, delphinidin, and petunidin), with two types of sugar (Fig. 4). Additionally, the urinary level (up to 11% of form (galactoside and arabinoside). Furthermore, gluco- the dose) of peonidin glycosides over 24 h was remark- sides of cyanidin and peonidin were also present in the ably higher than that of other glycosides (a few juice. percentage points), except for peonidin arabinoside. The methylation of cyanidin glycosides into peonidin Human urine glycosides by catechol O-methyltransferase in the liver The purpose of the present study was to identify has been suggested in human and animal studies.35–41) potential metabolites of anthocyanins in human urine for The high excretion of peonidin glycosides in urine might a better understanding of the active principles in the have resulted partly from methylation of cyanidin prevention of urinary tract infection. Several human glycosides. Although there is insufficient evidence for studies have reported that anthocyanins are recovered in a metabolic difference among cyanidin glycosides, urine in intact or conjugated forms.27) In the present metabolic methylation for anthocyanins might occur study, we found a higher excretion of several anthocya- preferably in the galactosides and glucosides over the nin metabolites in human urine after a single dose of arabinosides. cranberry juice. Moreover, some researchers have reported that the Figure 3 presents typical chromatograms of urine major metabolites of anthocyanins were recovered as samples after consumption of cranberry juice, obtained their glucuronide conjugated forms in human urine by the HPLC–ESI–MS–MS method. The representative following anthocyanin-rich food consumption.23,31,42) anthocyanins in the juice, peonidin 3-O-arabinoside, We evaluated for glucuronide conjugations by detection peonidin 3-O-galactoside, cyanidin 3-O-galactoside, and of their parent and product ion pairs due to the cyanidin 3-O-arabinoside, were observed in the urine of anthocyanidin glucuronides and anthocyanidins respec- all subjects. Excretion of peonidin 3-O-glucoside, tively. The peaks corresponding to the glucuronides of pelargonidin 3-O-arabinoside, and petunidin 3-O-galac- cyanidin and peonidin were not detectable in the toside among the minor pigments in the juice was also extended single-ion chromatograms of human urine detected and identified in the urine of most of the samples after cranberry juice intake, suggesting an subjects. Malvidin 3-O-arabinoside was not detected in absence of these glucuronide in the urine samples or any sample. degradation of conjugates during sample processing Table 2 gives the urinary excretion of individual owing to instability.23) cranberry anthocyanins after cranberry juice intake in In conclusion, 12 anthocyanins, including cyanidin, the present study. The total excretion of six main peonidin, delphinidins, pelargonidin, malvidin, and Urinary Excretion of Cranberry Anthocyanins 1685

TIC

Cy 3-O-gal Cy 3-O-glc 449 / 287a Cy 3-O-ara 419 / 287 Pn 3-O-gal Pn 3-O-glc 463 / 301 Pn 3-O-ara 433 / 301 Dp 3-O-ara 435 / 303

433 / 271

403 / 271

Pt 3-O-gal 479 / 317

493 / 331

463 / 331 IS 239 / 137

0 10 20 30 (min)

Fig. 3. Typical Extracted Single Ion Chromatograms of Human Urine Samples after Consumption of Cranberry Juice Obtained by the HPLC–ESI– MS–MS Method in Multiple Reaction Monitoring Mode with Positive Ionization. IS, internal standard. aThe m=z values of parent/product ions.

Table 2. Concentrations of Anthocyanins in Cranberry Juice and in Human Urine after Juice Consumption by 11 Healthy Volunteers

Cranberry juice Urinary excretion Concentration % of total Concentrationb,c % of total Anthocyanin % of doseb,e (mg/200 ml) anthocyaninsa (nmol/24 h) anthocyaninsd Cy 3-O-gal 128.4 19.7 10:5 1:7 14.3 3:7 0:6 Cy 3-O-glc 3.6 0.6 0:11 0:06 0.2 1:4 0:7 Cy 3-O-ara 104.6 16.1 8:7 1:2 12.0 3:6 0:5 Pn 3-O-gal 174.3 26.8 41:5 6:2 56.1 11:0 1:6 Pn 3-O-glc 9.9 1.5 2:4 0:6 3.3 11:3 2:6 Pn 3-O-ara 230.0 35.3 10:4 1:7 14.1 2:0 0:3 total 650.8 100 73:6 11:0 100 5:0 0:8

aPercentages of total quantifiable anthocyanins in cranberry juice. bValues are means SEM. cUrinary concentrations during 24 h following ingestion were determined using HPLC–ESI–MS–MS and HPLC–DAD, and were defined as cyanidin 3-O-galactoside equivalents. dPercentages of the total anthocyanins excreted in urine. ePercentages of ingested individual anthocyanin amounts. 1686 R. OHNISHI et al.

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