Absorption of Anthocyanin Rutinosides After Consumption of a Blackcurrant (Ribes Nigrum L.) Extract

Absorption of Anthocyanin Rutinosides after Consumption of a Blackcurrant ( Ribes nigrum L.) Extract.

Item Type Article

Authors Röhrig, Teresa; Kirsch, Verena; Schipp, Dorothea; Galan, Jens; Richling, Elke

Citation J Agric Food Chem. 2019 Jun 19;67(24):6792-6797. doi: 10.1021/ acs.jafc.9b01567. Epub 2019 Jun 10.

DOI 10.1021/acs.jafc.9b01567

Publisher American Chemical Society

Journal Journal of agricultural and food chemistry

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Link to Item http://hdl.handle.net/10033/621879 This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Article

Cite This: J. Agric. Food Chem. 2019, 67, 6792−6797 pubs.acs.org/JAFC

Absorption of Anthocyanin Rutinosides after Consumption of a Blackcurrant (Ribes nigrum L.) Extract † ∥ ⊥ † ‡ § † Teresa Röhrig, , , Verena Kirsch, Dorothea Schipp, Jens Galan, and Elke Richling*, † Division of Food Chemistry and Toxicology, Department of Chemistry, Technische Universitaet Kaiserslautern, Erwin-Schroedinger-Strasse 52, D-67663 Kaiserslautern, Germany ‡ ds-statistik.de, Pirnaer Strasse 1, 01824 Rosenthal-Bielatal, Germany § Specialist in Inner & General Medicine, Hochgewanne 19, 67269 Gruenstadt, Germany

*S Supporting Information

ABSTRACT: The dominant anthocyanins in blackcurrant are delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside. Data on their absorption and distribution in the human body are limited. Therefore, we performed a human pilot study on five healthy male volunteers consuming a blackcurrant (Ribes nigrum L.) extract. The rutinosides and their degradation products gallic acid and protocatechuic acid were determined in plasma and urine. The rutinosides’ concentrations peaked in both plasma and urine samples within 2 h of extract ingestion. The recoveries of delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside from urine samples were 0.040 ± 0.011% and 0.048 ± 0.016%, respectively, over a 48 h period. Protocatechuic acid concentration increased significantly after ingestion of the blackcurrant extract. Our results show that after ingestion of a blackcurrant extract containing delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside, significant quantities of biologically active compounds circulated in the plasma and were excreted via urine. Furthermore, these results contribute to the understanding of anthocyanin metabolism in humans. KEYWORDS: blackcurrant, Ribes nigrum L., rutinoside, bioavailability, anthocyanin

■ INTRODUCTION tion, whereas anthocyanins linked to the disaccharide rutinose α 20 Anthocyanins are well-known red- and blue-colored flavonoids (6-O- -L-rhamnosyl-D-glucose) exhibited only minor losses. found in many different plants, especially food crops such as The greater stability of rutinoside anthocyanins during strawberries,1 blueberries,2 blackcurrant,3 and red cabbage.4 gastrointestinal passage may be due to a lower susceptibility Structurally, individual anthocyanins are distinguished by the to enzymatic deglycosylation and a higher stability toward pH presence and position of hydroxy and methoxy groups on the changes. Additionally, since the human metabolism lacks A and B rings of the flavonoid moiety, and by the identity of rhamnosidases, the major deglycosylation of rutinosides is limited to the colon where bacterial rhamnosidases catalyze the the 3-O-linked saccharide. The C ring contains a hetero- 21 aromatic oxygen that can be protonated at low pH, forming a cleavage of rhamnose. Similarly, human absorption studies flavylium cation. Anthocyanins have been associated with using blackcurrant extract or juice indicated that the absolute ff fi bioavailability expressed as urinary excretion of anthocyanin several e ects bene cial to human health, especially 22−24 cardiovascular health. They are antioxidants that can act rutinosides was higher than that of glucosides. Black- directly (e.g., radical stabilization) as well as on a cellular level currants are particularly rich in anthocyanin rutinosides, the See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. (e.g., suppression of NF-κB).5 Notably, in vitro experiments most abundant of which are delphinidin-3-O-rutinoside − Downloaded via HELMHOLTZ INST PHARM RSRCH SAARLAND on July 16, 2019 at 13:12:08 (UTC). have established that they reduce LDL oxidation,6 9 activate (Del3rut, tulipanin) and cyanidin-3-O-rutinoside (Cy3rut, the Nrf2-ARE pathway10 and inhibit 5-lipoxygenase,11 an antirrhinin; see Figure 1). The reported concentrations of − enzyme that is upregulated in atherosclerotic lesions.12 Regular Del3rut and Cy3rut in blackcurrant fruits are 180.3 311.4 and − 3 consumption of anthocyanins also correlates with reductions in 70.4 138.8 mg/100 g fresh weight, respectively, while those 13,14 15 measured in blackcurrant juices are 89−689 and 55−356 mg/ CVD risk factors and CVD occurrence. Therefore, food 25 rich in anthocyanins complement a healthy balanced diet. L, respectively. Additionally, blackcurrants contain other 16,17 fl fl 26 Anthocyanins are sparsely bioavailable because they are avonoids like avonols and hydroxycinnamic acids. To ’ degraded extensively during passage through the gut; they are better understand anthocyanins bioavailability in order to 18 fi particularly prone to microbial degradation. However, their facilitate the exploitation of their bene cial physiological relative bioavailability (which is determined by considering the effects, and to test the hypothesis that anthocyanin rutinosides uptakeofdegradationproductsaswellastheparent are readily bioavailable, the bioavailability of blackcurrant compounds) is comparable to that of other flavonoids.19 In general, their stability depends on the pH and their exposure to Received: March 10, 2019 digestive enzymes, light, oxygen, and heat. In vitro studies of Revised: May 27, 2019 gastrointestinal passage revealed a loss of 42.6% of anthocyanin Accepted: May 28, 2019 monosaccharides from chokeberries during pancreatic diges- Published: May 28, 2019

Figure 1. Structures of selected blackcurrant anthocyanins and anthocyanin degradation products. anthocyanins in humans was assessed in plasma and urine CXP 28 V, dwell time 60 ms; Del3rut, DP 71 V, EP 7.5 V, CEP 28.88 samples after ingestion of a blackcurrant extract in the frame of V, CP 31 V, CXP 26 V, dwell time 60 ms; GA, DP −35 V, EP −5V, − − − our pilot study presented here. CEP 12 V, CP 22 V, CXP 0 V, dwell time 150 ms; PCA, DP 25 V, EP −4 V, CEP −10 V, CP −22 V, CXP −2 V, dwell time 150 ms. The recovery rates of Del3rut and Cy3rut in urine were 89 ± 25% and ■ MATERIALS AND METHODS ± ± 87 29%, respectively, whereas those in plasma were 77 5% and 85 Chemicals and Standards. All standards were of at least 95% ± 10%, respectively. Delphinidin-3,5-diglucoside was used as the purity. Cy3rut-chloride and Del3rut-chloride were obtained from internal standard for anthocyanin quantification, and isoferulic acid-d3 Phytolab (Vestenbergsgreuth, Germany), GA from VWR Interna- was used as the internal standard for GA and PCA quantification.27 tional (Radnor, PA), PCA from Sigma-Aldrich (St. Louis, MO), The resulting calibration curves covered the range from 3.9 to 1000 delphinidin-3,5-diglucoside-chloride from Extrasynthese (Genay ng/mL, and the determined LOD and LOQ values were comparable Cedex, France), and isoferulic acid-d3 from Toronto Research to those reported previously.27 Chemicals Inc. (Toronto, Canada). Analytical grade solvents were Statistical Analysis. Arithmetic means of replicate measurements purchased from VWR and Sigma-Aldrich. Blackcurrant extract (Ribes for each combination of subject and time point were computed and nigrum fruit extract, 20%) was purchased from Iprona, Lana, Italy. used as the basis for subsequent analyses. The results for the five Study Design. Anthocyanin absorption after consumption of volunteers are reported as means and standard deviations. blackcurrant extract (BCE) was assessed in an open monocentric Concentrations below the LOQ but above the LOD were treated as human absorption pilot study. This study was approved by the Ethics zero when computing means, standard deviations, cmax and AUC. All Committee of the German federal state of Rhineland-Palatinate statistical hypotheses were tested at the 5% significance level against a (Mainz) on April first 2016 (case number 837.290.15 (10058)), and two-sided alternative. Paired t tests were used to compare dose effects. signed informed consent was obtained from all participants. All All statistical analyses were performed with SAS V9.0. procedures were performed according to institutional guidelines and Chemical Compounds Studied in This Article. delphinidin-3- the Declaration of Helsinki. Five male volunteers aged between 24 O-rutinoside, Tulipanin (PubChem CID: 5492231); cyanidin-3-O- and 32, with BMI values between 22.9 and 25.3 kg/m2 were recruited. rutinoside, Antirrhinin (PubChem CID: 441674); gallic acid The inclusion criteria for volunteers were: good health, Caucasian (PubChem CID: 370); protocatechuic acid (PubChem CID: 72) ethnicity, nonsmoker, nonconsumption of medication or supple- ments, not engaging in high levels of sporting activity, transit time ■ RESULTS below 24 h, absence of metabolic disorder, and having made no recent blood donations. Volunteers were advised to eat an anthocyanin-free Extract Analysis and Dosing. Measurements performed diet starting 72 h before and ending 48 h after BCE ingestion (see before the study began indicated that the combined content of Supporting Information (SI)). They were not asked to prepare diet the anthocyanins Del3rut, Cy3rut, Del3glc, and Cy3glc in the protocols and due to the lack of a suitable compliance biomarker the BCE amounted to 28.3 ± 1.4% by mass. The relative and collected samples were not tested for compliance of the advised diet. absolute abundances of these rutinosides are given in Table 1. The night before BCE administration, volunteers were asked to fast The contents of Del3glc and Cy3glc in the BCE were 3.1 ± for at least 12 h before ingesting a single oral bolus dose of 1.5 g BCE 0.1% and 1.0 ± 0.3%, respectively. with 200 mL of water (pH adjusted to 4 using citric acid). Blood samples were collected shortly before BCE ingestion and at 1, 2, 4, 6, and 8 h after ingestion. Urine samples were collected 24−0 h prior to Table 1. Anthocyanin Rutinoside Contents of BCE BCE ingestion and 0−2h,2−4h,4−6h,6−8h,8−12 h, 12−24 h, Expressed As Percentages, Absolute Doses In Mg and Nmol, and 24−48 h after ingestion. Plasma and urine samples were stored as and Mean Individual Doses In Mg/kg BW described previously.27 Volunteers received three anthocyanin-free, standardized meals 1.5, 5, and 7 h after consumption of BCE, with absolute content in absolute dose mean individual dose water available ad libitum. BCE in % dose (mg) (μmol) (μmol/kg BW) Extract Analysis. The blackcurrant extract (BCE) was analyzed ± ± ± ± by HPLC-UV/vis before the study began, as described previously.2 Del3rut 12.7 0.5 190 7 294 11 3.7 0.2 ± ± ± ± Anthocyanins were identified by comparing their retention times to Cy3rut 11.5 0.5 172 8 273 12 3.4 0.2 those of the relevant standards, and were quantified with respect to Cy3rut. Anthocyanins in Plasma Samples. The concentrations HPLC-ESI-MS/MS Analysis of Anthocyanins and Metabolites of Del3rut and Cy3rut in the plasma of the five volunteers were in Plasma and Urine Samples. Sample preparation and semi- determined shortly before (0 h) BCE ingestion and at 1, 2, 4, quantitative analysis of the anthocyanins Cy3rut and Del3rut and 6, and 8 h afterward. As expected, no anthocyanins were their metabolites GA and PCA in plasma and urine samples were performed by HPLC-ESI-MS/MS in MRM mode as described detected in plasma shortly before BCE ingestion. Glucosides 27 were only detected in traces in plasma samples and therefore previously. Urine samples were prepared in triplicate and each fi replicate was analyzed once, whereas plasma samples were prepared not quanti ed. Interestingly, Del3rut was not detected in the once only but analyzed three times. The ESI parameters used in the plasma of one volunteer at any time point after ingestion, analysis were: Cy3rut, DP 71 V, EP 8.5 V, CEP 28.35 V, CP 31 V, although Cy3rut was detectable. Both Del3rut and Cy3rut

6793 DOI: 10.1021/acs.jafc.9b01567 J. Agric. Food Chem. 2019, 67, 6792−6797 Journal of Agricultural and Food Chemistry Article were detected in the majority of the plasma samples of the other volunteers, but their levels were often below the LOQ in samples collected at later time points. The plasma anthocyanin concentrations rose during the ﬁrst hour after ingestion but then decreased continuously (Figure 2). However, detectable

Figure 2. Mean plasma concentrations of delphinidin3-rutinoside (Del3rut) and cyanidin-3-rutinoside (Cy3rut) after ingestion of BCE in nmol/L. Values are means ± SD based on data for five volunteers. levels of both anthocyanins were present in most of the plasma samples even 8 h after BCE ingestion. The maximal plasma ± concentration, cmax, was determined to be 8.6 5.8 nmol/L for Del3rut and 9.8 ± 3.1 nmol/L for Cy3rut. The time point at ± which cmax occurred (tmax) was calculated to be 1.5 0.6 h for Del3rut and 1.4 ± 0.6 h for Cy3rut, respectively. Despite the ff slight di erence in cmax, the two anthocyanins had very similar AUC values: 30.5 ± 20.6 nmol*h/L for Del3rut, and 30.8 ± 11.2 nmol·h/L for Cy3rut, respectively. The pharmacokinetic parameters of Del3rut and Cy3rut are summarized in Table 2. Figure 3. - Urinary excretion rates in nmol/h (a) and absolute urinary Table 2. Pharmacokinetic Parameters of Delphinidin-3- excretions in nmol (b) of delphinidin-3-rutinoside (Del3rut) and rutinoside (Del3rut) and Cyanidin-3-rutinoside (Cy3rut) in cyanidin-3-rutinoside (Cy3rut) before and after BCE ingestion. Values are means ± SD based on data for five volunteers. Plasma Samples Collected from Five Volunteers 8 h After BCE Ingestion ’ c (nmol/L) t (h) AUC (nmol·h/L) volunteer. This individual s Del3rut excretion rate was 56 max max pmol/h during the 24−48 h interval, so the mean excretion Del3rut 8.6 ± 5.8 1.5 ± 0.6 30.5 ± 20.6 ± ± ± ± rate for the volunteers collectively in this interval was 11.1 Cy3rut 9.8 3.1 1.4 0.6 30.8 11.2 24.8 pmol/h. Cy3rut was successfully quantified in the urine of all volunteers for all sampling intervals; the mean Cy3rut Anthocyanins in Urine Samples. Urine samples were excretion rate for the 24−48 h interval was 226.6 ± 71.4 pmol/ collected 24−0 h before blackcurrant extract (BCE) ingestion h. As shown in Figure 3b, the absolute urinary excretion of and 0−2h,2−4h,4−6h,6−8h,8−12 h, 12−24 h, and 24− Cy3rut in the 24−48 h interval was 20 times higher than that 48 h after ingestion. All samples were analyzed for Del3rut and of Del3rut. However, over the entire 48 h postingestion period, Cy3rut, yielding the excretion rates shown in Figure 3a. No the absolute urinary excretion of Cy3rut (134 ± 43 nmol) was anthocyanins were excreted before extract ingestion. Gluco- only slightly greater than that of Del3rut (120 ± 34 nmol). sides were only found in traces in the urine samples. The The total excreted amounts correspond to 0.040 ± 0.011% and urinary excretion rates of Del3rut and Cy3rut both peaked 0−2 0.048 ± 0.016% of the ingested doses of Del3rut and Cy3rut, h after ingestion, at 20.0 ± 2.6 nmol/h and 21.2 ± 3.8 nmol/h, respectively. respectively. The excretion rates for subsequent sampling Phenolic Acids in Urine and Plasma Samples. Urinary intervals decreased as expected, with the lowest rates being excretion of the phenolic acids protocatechuic acid (PCA) and observed for the 24−48 h interval. The Del3rut concentration gallic acid (GA) was monitored over the same intervals as the at the end of this interval was below the LOQ (2.7 nmol/L) anthocyanins before and after BCE ingestion. Unlike the but above the LOD (0.8 nmol/L) in the urine of four anthocyanins, phenolic acids were excreted before BCE volunteers, and so was only quantified in the urine of the fifth ingestion; their excretion rates and absolute excretion values

6794 DOI: 10.1021/acs.jafc.9b01567 J. Agric. Food Chem. 2019, 67, 6792−6797 Journal of Agricultural and Food Chemistry Article are shown in Figure 4a. Before BCE ingestion, the excretion quantified in two samples from one volunteer. Based on these ± ± rates of PCA and GA were 37.8 16.3 nmol/h and 1.2 1.1 samples, the tmax for both PCA and GA occurred 1.0 h after BCE ingestion, and the cmax values for these compounds were 25.7 ± 3.9 nmol/L and 12.9 ± 2.5 nmol/L, respectively. However, taking the samples from the other four volunteers ± into account, the mean cmax values for PCA and GA were 5.1 11.5 nmol/L and 2.6 ± 5.8 nmol/L, respectively. ■ DISCUSSION The absorption and distribution of anthocyanins in humans have been studied previously. Czank et al.19 examined volunteers who ingested 500 mg of stable isotopically labeled 13 − cyanidin-3-O-glucoside ( C5 Cy3glu), and observed that 12% of the ingested anthocyanins were excreted, with the nonexcreted material giving rise to 49 identifiable metabolites. More recently, Mueller and co-workers27 fed 10 g of a bilberry extract containing 2.4 g of 16 different anthocyanins (4.95 mmol) to volunteers with and without a colon. The tmax for anthocyanins in plasma samples from volunteers with a colon ± was 2 h (the corresponding cmax was 91.2 41.8 nmol/L), and levels of anthocyanin degradation products in the plasma of these volunteers peaked at 1494.8 ± 275.7 nmol/L 1 h after ingestion. Conversely, the tmax for anthocyanins in plasma samples from volunteers without a colon occurred 1 to 2 h after ingestion of the extract, while the tmax for the urine samples from these volunteers was around 2 h, and that for the degradation samples occurred between 2 and 4 h after ingestion. Mueller et al.27 also reported that the anthocyanin content of urine samples collected over 24 h after bilberry extract ingestion amounted to 0.03 ± 0.02% of the amount consumed. The major anthocyanins in the plasma and urine samples were the glucuronidated forms of malvidin and peonidin, while the main excreted degradation product in urine was 4-hydroxybenzoic acid (HBA), which accounted for 48.7% of all observed degradation products by concentration. Conversely, the main degradation products observed in this work were gallic acid (GA) and protocatechuic acid (PCA), which originate from delphinidin and cyanidin. Unlike Czank et al.19 and Mueller et al.,27 we did not seek to identify all Figure 4. Urinary excretion rates in nmol/h (a) and absolute urinary metabolites of the studied anthocyanins; instead, we focused excretions in nmol (b) of protocatechuic acid (PCA) and gallic acid on the parent compounds (i.e., the rutinosides) and the (GA) before and after BCE ingestion. Values are means ± SD based products of B-ring degradation. The total urinary excretion of on data for five volunteers. GA and PCA was 235 ± 180 nmol and 2492 ± 1208 nmol, respectively. Because the plasma analysis of phenolic acids nmol/h, respectively. Their excretion rates peaked during the generally yielded unquantifiable results, our discussion focuses period 0−2 h after ingestion, at 136.4 ± 64.7 nmol/h for PCA on the urinary excretion of PCA and GA. These phenolic acids and 20.5 ± 11.5 nmol/h for GA. In later intervals, the were monitored because they are metabolites of cyanidin and excretion rates declined but stabilized at 18.9 ± 9.6 nmol/h delphinidin, respectively. PCA is formed from the B-ring of (during the 8−12 h interval) in the case of PCA and 2.0 ± 1.8 cyanidin while GA is formed from delphinidin. Several studies nmol/h (during the 12−24 h interval) in the case of GA. The have detected the corresponding phenolic acids and their excretion rates of both compounds increased slightly during phase II metabolites in samples collected from human − the subsequent intervals (24−48 h). Interestingly, the PCA volunteers after anthocyanin ingestion.19,27 29 Accordingly, in excretion rate before ingestion (after having eaten a low- this study, both phenolic acids exhibited increased excretion polyphenol diet for 72 h) was twice the minimum excretion rates directly after ingestion of BCE. However, PCA and GA rate after BCE ingestion. Furthermore, as shown in Figure 4b, were present in the volunteers’ urine and plasma before PCA excretion peaked before BCE ingestion (at 907 ± 392 ingestion of the blackcurrant extract, making it difficult to nmol); a third of the total PCA excretion occurred before BCE quantify the amount of phenolic acid formed by metabolism of was consumed. The total urinary excretion of PCA was 2492 ± anthocyanins present in BCE. Similar findings have been 1208 nmol and was 10 times that for GA (235 ± 180 nmol). reported previously.27,28 The volunteers were asked to follow a The phenolic acids PCA and GA were also detected in diet plan that excluded foods containing anthocyanins and plasma samples collected from the volunteers up to 8 h after foods rich in polyphenols (e.g., coffee, tea, and chocolate). BCE ingestion. Unfortunately, the PCA and GA levels in However, PCA and GA are found in various foods that were plasma were generally below the LOQ and could only be not excluded from the diet plan, including bananas,30 apples,30

6795 DOI: 10.1021/acs.jafc.9b01567 J. Agric. Food Chem. 2019, 67, 6792−6797 Journal of Agricultural and Food Chemistry Article

31 32 33 34 rice, green beans, carrots, spices, beer, and olive oil. Instruction for volunteers during the human intervention Overall, the results presented here indicate that phenolic acids study (diet plan) (PDF) alone are not suitable markers of anthocyanin incorporation or dietary compliance. 24 AUTHOR INFORMATION In a prior study reported by Matsumoto and co-workers ■ healthy volunteers ingested 3.58 mg (6.24 μmol) of Corresponding Author anthocyanins, corresponding to doses of 2.75 μmol/kg bw *Phone. +49 631 205 4061. Fax: +49 631 205 3085. E-mail: (1.68 mg/kg bw) Del3rut, 2.08 μmol/kg bw (1.24 mg/kg bw) [email protected]. Cy3rut, 1.04 μmol/kg bw (0.488 mg/kg bw) of Del3glu, and ORCID 0.37 μmol/kg bw (0.165 mg/kg bw) of Cy3glu. Their plasma − Elke Richling: 0000-0001-5746-8032 cmax values occurred after 1.25 1.75 h, and the excreted urinary ± Present Addresses amounts were 0.11 0.05% of the ingested doses. The authors ∥ ff claimed that rutinosides are directly absorbed and circulate in Helmholtz Center for Infection Research, Inho enstrasse 7, 38124 Braunschweig, Germany. the plasma. Although the ingested amounts were much lower ⊥ Helmholtz Institute for Pharmaceutical Research Saarland, than in the present study, the tmax values for both rutinosides ̈ were similar to those reported here, and the total excreted Campus E8.1, 66123 Saarbrucken, Germany. amounts are also similar. In the present pilot study total Notes excreted anthocyanin amounts corresponded to 0.040 ± The authors declare no competing financial interest. 0.011% and 0.048 ± 0.016% of the ingested doses of Del3rut − and Cy3rut, in good agreement with literature.22 24,27 In the ■ ACKNOWLEDGMENTS present study the urinary excretion of glucoside anthocyanins We are grateful to the volunteers for their cooperation during was too low to quantify, rendering a direct comparison of the study. We also thank our assistants during the study and rutinosides and glucosides ineffective. From the aforemen- 24 the Institute of Laboratory Medicine of the Westpfalz- tioned studies by Matsumoto and co-workers, Nielsen and Klinikum Kaiserslautern, Germany, for blood work during 22 23 co-workers as well as McGhie and co-workers where the screening process. volunteers ingested blackcurrant extract or juice it was already known that rutinosides present higher bioavailability in direct ■ ABBREVIATIONS USED − comparison to their corresponding glucosides.22 24 This is in BCE, blackcurrant extract; BW, body weight; Cy3glc, cyanidin- part due to the higher stability of rutinosides during the 3-O-glucoside; Cy3rut, cyanidin-3-O-rutinoside; Del3glc, passage of the gastrointestinal tract (GIT) compared to delphinidin-3-O-glucoside; Del3rut, delphinidin-3-O-rutino- glucosides. Degradation of anthocyanins in the GIT passage is side; GA, gallic acid; GIT, gastrointestinal tract; PCA, dependent on pH and exposure to metabolic enzymes. protocatechuic acid. Deglycosylation of rutinosides is limited to bacterial rhamnosidases in the colon whereas glucosides are readily REFERENCES metabolized by unspecific glucosidases. Furthermore, from the ■ study of Mueller and co-workers27,28 it is known that the main (1) Lopes-da-Silva, F.; de Pascual-Teresa, S.; Rivas-Gonzalo, J.; metabolites of anthocyanins are the anthocyanidin glucur- Santos-Buelga, C. Identification of anthocyanin pigments in onides. In theory the rutinoside anthocyanins cannot be strawberry (cv Camarosa) by LC using DAD and ESI-MS detection. Eur. Food Res. Technol. 2002, 214 (3), 248−253. deglycosylized once they enter the bloodstream in contrast to (2) Müller, D.; Schantz, M.; Richling, E. High performance liquid glucoside anthocyanins. 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6797 DOI: 10.1021/acs.jafc.9b01567 J. Agric. Food Chem. 2019, 67, 6792−6797