Supporting Information

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1 Supporting Information Staility of Hydroxycinnamic Acid Derivatives, Flavonol Glycosides and Anthocyanins in Black Currant Juice Leenamaija Mäkilä,*, Oskar Laaksonen, Aino-Liisa Alanne, Maaria Kortesniemi, Heikki Kallio,, Baoru Yang Food Chemistry and Food Development, Department of Biochemistry, University of Turku, FI- 14 Turku, Finland Instrument Centre, Department of Chemistry, University of Turku, FI-14, Turku, Finland Department of Food Science and Engineering, Jinan University, 516 Guangzhou, China * Corresponding author (Tel: ; Fax: : leenamaija.makila@utu.fi) Tale of Contents Tales S1 S and Figures S1 S8. Reference S1

2 Tale S1. List of the Reference Compounds Used for Quantitation of the Sample Compounds. Sample compound Reference compound used for quantitation No HYDROXYCINNAMIC ACID COMPOUNDS 1 neochlorogenic acid (E)-caffeic acid (E)-caffeic acid O-glucoside and (E)-pcoumaric (E)-caffeic acid acid O-glucoside * (E)-caffeoylglucose (E)-caffeic acid 4 (E)-p-coumaroylquinic acid (E)-p-coumaric acid 5 (Z)-p-coumaric acid O-glucoside (E)-p-coumaric acid 6 (E)-p-coumaroylglucose (E)-p-coumaric acid and chlorogenic acid * 7 (Z)-p-coumaroylglucose (E)-p-coumaric acid 8 (E)-caffeic acid (E)-caffeic acid 9 (E)-feruloylglucose (E)-ferulic acid 1 (E)-p-coumaric acid (E)-p-coumaric acid 11 (Z)-p-coumaric acid (E)-p-coumaric acid 1 (E)-ferulic acid (E)-ferulic acid 1 (E)-caffeoyloxymethyleneglucosyloxyutenenitrile purified compound of (E)-coumaroyloxymethyleneglucosyloxyutenenitrile (peak 14) 14 (E)-coumaroyloxymethyleneglucosyloxyutenenitrile purified compound of (E)-coumaroyloxymethyleneglucosyloxyutenenitrile (14) 15 (Z)-coumaroyloxymethyleneglucosyloxyutenenitrile purified compound of (E)-coumaroyloxymethyleneglucosyloxyutenenitrile (14) 16 (E)-feruloyloxymethyleneglucosyloxyutenenitrile purified compound of (E)-coumaroyloxymethyleneglucosyloxyutenenitrile (14) FLAVONOL COMPOUNDS 19 myricetin rutinoside myricetin glucoside myricetin glucoside myricetin glucoside 1 myricetin arainoside myricetin glucoside myricetin malonylglucoside and aureusidin myricetin glucoside glucoside * quercetin rutinoside quercetin rutinoside 4 quercetin glucoside quercetin glucoside 5 kaempferol rutinoside kaempferol glucoside 6 quercetin malonylglucoside quercetin glucoside 7 isorhamnetin rutinoside quercetin glucoside 8 kaempferol glucoside kaempferol glucoside 9 isorhamnetin glucoside ** quercetin glucoside myricetin myricetin 1 quercetin quercetin kaempferol kaempferol isorhamnetin ** quercetin ANTHOCYANINS 7 delphinidin glucoside delphinidin glucoside 8 delphinidin rutinoside delphinidin rutinoside 9 cyanidin glucoside cyanidin glucoside 4 cyanidin rutinoside cyanidin rutinoside Anthocyanin degradation products (hydroxyenzoic acids) 4 protocatechuic acid protocatechuic acid 5 4-hydroxyenzoic acid 4-hydroxyenzoic acid 6 phloroglucinaldehyde phloroglucinaldehyde CITRIC ACID METHYL ESTERS AND FLAZIN 17 citric acid methyl esters (E)-p-coumaric acid S

3 18 flazin purified compound of flazin * ) When the sample peaks overlapped, the reference compound was chosen for the more aundant compound. ** ) Due to low content of isorhamnetin glucoside and isorhamnetin, quantification was done as quercetin glucoside and quercetin. Figure S1. Identification of anthocyanidins monitored at 5 nm. Chromatograms of (A) cyanidin and (B) delphinidin reference compounds, (C) aseline juice sample co-injected with reference compounds, (D) aseline juice sample and (E) 5 month storage sample. Juice samples (C-E) were extracted with acidified MeOH. Identification was carried out with HPLC-DAD-apparatus using previous method 16. Column used was a 1 mm 4.6 mm i.d.,.6 μm, Kinetex C18 1A, with a guard column of the same material (Phenomenex, Torrance, CA). S

4 Figure S. 1 H NMR (CD OD, 5.1 MHz) spectrum of -(Z)-p-coumaroyloxymethylene-4-β-Dglucopyranosyloxy--(Z)-utenenitrile. Figure S. 1 C NMR (CD OD, MHz) spectrum of -(Z)-p-coumaroyloxymethylene-4-β-Dglucopyranosyloxy--(Z)-utenenitrile. S4

5 Figure S4. Optimizing chromatographic separation of the peaks 14 (E)- coumaroyloxymethyleneglucosyloxyutenenitrile and 15 (Z)- coumaroyloxymethyleneglucosyloxyutenenitrile. Various inary gradients were applied (data not shown). (A) The highest separation of the isomers, approximately 1 minutes, was otained with the gradient program (lue line), with solvents A:.1% formic acid in water and B: 1% acetonitrile. The gradient for solvent B, 15 min, 18%; 15 4 min, 18 19%; 4 5 min, 19 8%; 5 min, 8%; 5 min, 8 %; 5 8 min, 69%; 8 41 min, 69 %; min, %. The flow rate was 4.9 ml / min. (B) The purity of the re-isolated (Z)-coumaroyloxymethyleneglucosyloxyutenenitrile was checked via analytical HPLC-DAD machinery. S5

6 Figure S5. Decomposition of phenolic compounds and flazin in lack currant juice at various storage conditions during 1 months of storage. RT, room temperature. (A) Total HCA compounds, (B) total HCA derivatives, (C) total free HCAs, (D) total flavonol compounds, (E) total flavonol glycosides and (F) total free flavonols, (G) total anthocyanins, (H) anthocyanin degradation products and (I) flazin. Values at -month of storage equal with 1% on y-axis, asolute values are given in Tale S. Statistically significant changes*, when compared with the aseline sample ased on Oneway ANOVA together with Tukey s post hoc and LSD test (p <.5). The error ars are relative standard deviations (%). S6

7 Figure S6. Decomposition of individual HCA compounds in lack currant juice during one year of storage. RT, room temperature. Changes in p-coumaric, caffeic and ferulic acids and their derivatives (A D) when stored at RT in light, (E H) at RT in dark and (I L) in refrigerator (+ 4 C), respectively. E-isomers of free HCAs,(Z)-p-coumaric acid, (E)-caffeoyl / coumaroyl / feruloylglucose, (E)-coumaroyl-/caffeoyl-/feruloyloxymethyleneglucosyloxyutenenitrile, (Z)-coumaroyloxymethyleneglucosyl-oxyutenenitrile, neochlorogenic acid / (E)-pcoumaroylquinic acid, (Z)-coumaroylglucose, (E)-caffeic and (E)-p-coumaric acid O- glucoside, (Z)-p-coumaric acid O-glucoside. The co-eluting O-glucosides of (E)- caffeic and (E)-p-coumaric acids are represented y the same lines. Values at -month of storage equal with 1% on y-axis, asolute values are given in Tale S. Statistically significant changes*, when compared with the aseline sample ased on One-way ANOVA together with Tukey s post hoc and LSD test (p <.5). The error ars are relative standard deviations (%). S7

8 Figure S7. Decomposition of individual flavonol compounds and anthocyanins in lack currant juice during 1 months of storage. RT, room temperature. Myricetin, quercetin and cyanidin glycosides and their aglycones / degradation products (A C) stored at RT in light, (D F) at RT in dark and (G I) in refrigerator (+ 4 C), respectively. Myricetin and quercetin, myricetin / quercetin rutinoside, myricetin / quercetin glucoside, myricetin / quercetin malonylglucoside, myricetin arainoside. Protocatechuic acid, phloroglucinaldehyde, cyanidin glucoside, cyanidin rutinoside. Values at -month of storage equal with 1% on y-axis, asolute values are given in Tale S. Statistically significant changes*, when compared with the aseline sample ased on One-way ANOVA together with Tukey s post hoc and LSD test (p <.5). The error ars are relative standard deviations (%). S8

9 Tale S. Concentrations of the Metaolites in Black Currant Juices During Storage for One Year. concentration (µg per 1 g of juice) No Compounds -mo mo 6 mo 9 mo 1 mo RT light RT dark +4 C RT light RT dark +4 C RT light RT dark +4 C RT light RT dark +4 C ph Brix HYDROXYCINNAMIC ACID COMPOUNDS 1 neochlorogenic acid 1.8±1. (E)-caffeic acid O- 1.4±1. glucoside and (E)-pcoumaric 8 acid O- glucoside (E)-caffeoylglucose 97.5± (E)-p-coumaroylquinic acid 5 (Z)-p-coumaric acid O- glucoside 6 (E)-p-coumaroylglucose chlorogenic acid and 1.464± ±.95 a 1.5± ± ±1. 6 a 1.6±. a 14.6±. 41.9±.9 a 5.87±. 8.±. 7.1± 5.5±..7±. 8.47±. 85 a ±4.6 1±1 65.9± ±. 91 c 6.1± ± ± ± ±. 79.1± ±. 95.5±. a 9.6±. a 7.66± a 55 a 7 a 7.7 a a ±7. 61.±. 11.9± 14.8±1. 6.6± ±..1±.1 5.4±.6.±7.6.4± ±.57 a 7± ±9. 6.4±1. ±14 a ± ± 6 1 a.5 a.891± 6.5±.1 6.5± ± ± ±. 15.4±4. 4 9±11 4.8±. 41.4±.4.±6. 11±6 7.8± ±. 9±18 1±9 6.1±5. 1.8±9. 11±1 4 a a 8±1.9±6. 5.7±5..± ±4. 15.±. 59.±. 4±96 a 1.6±1 88.5±1. ± 6 a 7 a. a a 7 (Z)-p-coumaroylglucose c.±7. 6.6±7. a 7.5± ± ±4.1 a.7±1..6±1.6 7±1.75±. 44±16 48.±. a 1.9±.8 c 7.± (E)-caffeic acid 48.4±. 8.1±1. 9.4±.4 a 48.7±. 18.± ± 4.8±4.1 1±11 a 18.71± 56.6± ± ±1. 5.8±5. 7 a 64 5 a.1 a.7 a 8 8 a 9 c 9 (E)-feruloylglucose 11±1 8.±. c 17.1± 94.8±. 7.± ± ± 78.4±1. 8.±1.9 1±9 69±11 9±1 1±.6 a 54 9 a.48 a 1 (E)-p-coumaric acid 7.1± ± 144.8±. 75.±5. c 17.1± ± 55.7±4.8 c 157.±..9± ±6.9 c 18.6±6. 76.± ± a 5. a 5 1 a 5 4 a 7 c 11 (Z)-p-coumaric acid 11.±4. 7.7±6.9 a 9.6±.8 16.±4.5 c 11.±4. 5.4± ±. c 1.8±. 5.1±..1±6.7 c 14±1 a 8±15.4±7. 6 a a 8 c 1 (E)-ferulic acid 4.9± ±1.6 6.±. a 9.±1.6 c 59.7± ±4..9±. c 7.7± ±. 7.6±.1 c 75.7±. a 99±14 a 6.4±5. 5 a 45 a 5 S9

10 1 (E)-caffeoyloxymethyleneglucosyloxyutenenitrile 14 (E)-coumaroyloxymethyleneglucosyloxyutenenitrile 15 (Z)-coumaroyloxymethyleneglucosyloxy utenenitrile 16 (E)-feruloyloxymethyleneglucosyloxyutene nitrile Tot. caffeic acid derivatives* Tot. (E/Z) p-coumaric acid derivatives Tot. (E)-p-coumaric acid derivatives Tot. (Z)-p-coumaric acid derivatives Tot. ferulic acid derivatives Tot. O-acylglucoses of HCAs Tot. acyloxymethyleneglucosyloxyutenenitriles of HCAs Tot. O-acylquinic acids of HCAs Tot. O-glucosides of HCAs Tot. HCAs and their derivatives* 9±6 6±1 69.4±. 11± 6 5. ±7. 6.± ±9. 1±7 a 89±55 a 5±16 9±66 4 a ±1 7± a 17.6±. 17±11 5±17 a 14±1 9 5± ± ±. 1±11 a 141.6±. ±9 6 a a S1 4±1 9±16 81.±9..±7. 7 4±66 4±8 4± 6±7 944.± ±4. 8±17 a 99±77 a 5±6 88± 94±19 9 a ±. 8±1 a 14±1 ±5 a 6±5 a 11.9±9. 17± a 6 a 6.± ± 1±1 a 6 a.47 8±8 a 1.1± ±19 a ±8 a 4±6 8± ±. 8± ±6. 8±9 4± ± ± ±1 7± 7±1 4±65 19±6 1±46 17±15 a 15±9 a 11±9 16± ± ± 14±4 a 18±7 11±81 15± 16± a.87 a.9 a 7 16±5 8±19 15±7 a 1±76 a 77±18 14± ± 74.67± 1± 16± 76±4 1±1 14± 6 5 a.5 a.98 1 a a 7±4 6±7 a ±1 ±17 4± a 19± 47.1± ±. 18±1 9±5 a 4±9 a 15±18 ±4 6 a a a 6±5 44.8±7..8±. ±1 a 14.8±4. ±4 6.9±4. 8.6±1. 9±15 a 4±57 a 19± 8± ± a 6 a a 1 a 6 61±4 4± ±6. 51±15 a 79.7±5. 5±1 597.±6. 8±19 4.8±. 67±18 ± 47±18 a 55±64 a a a 6 a 18± 1±58 17± a 15±8 a 1±41 15±1 17±16 a 1±7 14±5 a 18± 11±97 14±5 16± 8 5 a a 9 1± ±. 16.±. 18.7± ±.7 c 1.1± 118.4±1. 78.± ±. 14± 77.7±6.4 1±1 1±18 91 c 6 a 7 9. a ± ±. 61.± ± ±1.1 a 6.±. 5.5±. 65.± ± 67± 71.7± ±8.6 55±1 1 5 a 9 c. 8±8 1±97 8±7 a 4±11 1±57 7± 567.9±4 ±7 5±7 9±45 ±16 7± 5±4 8 1 a. a 1 Tot. HCA derivatives* 6±7 18±78 4±16 a ±1 17±5 ±1 4±11 a 171.4±9 ±41 7±4 16±14 1±9 ± 9 a 9 a. 8 Tot. free HCA 16±1 ±19 a 4±11 a 17±11 96.±7. 45±14 1±15 c 49±18 a 499.5±4. ±19 54± a 6±7 a ±9 1 a 1 a FLAVONOL COMPOUNDS

11 19 myricetin rutinoside 7.8±. 5 7± ±. 69 myricetin glucoside 65± 6±98 79.±. 6 1 myricetin arainoside 6.1±1..9±. 6.± myricetin 1.5± 65.5±.5 malonylglucoside and 6. aureusidin glucoside 6.±.1 7±1 8.± ±.7 78.±1.4 7.±1.1 1±5 85.±.8 7±19 74±11 54±41 59±84 68±47 6± ± ±4. 7±14 7±66 64±17 6±14.8±1..± ± 4.5±1. a.16±..±. 5.4±.1.± ±.1.6±4..91 a ±4. 1.1±. 65.7±.9 6.±. 1±17 a 5.8±7.4 56±11 96± a ±. 9.±7. a 58.4±1. 1 a 19.±. 8±1 a 9±4 5±7 ±61 quercetin rutinoside 87±14 96.±. 11.8±. 96± ±17 1.5±5. 9.7±4.6 15±1 1±11 1±19 14±9 4 a 9.8±4.6 ±.1 4 quercetin glucoside 6±1 ±4 8.8± ±1 ±5 8±8 5±1 5.4±9..1 a 5 kaempferol rutinoside 4.5±4. 5.±.8 1.5± ±.4 1.5±. 6.8± ± ±. 5.7±.6 9.4±11.±9. 7.7± ±. 9 6 quercetin 46.4±4. 1.4±.1 c.1±1.1 5.±. a 1.5±. 1.8±.±4. a 8.±1.1.8±.6.84±. 5.8±1..94±.7 4±1 a malonylglucoside 1. 4 a 7 isorhamnetin rutinoside.±. ±11 1.4± ± ± ± ±5.4.9± ±.6 19.± ±.4 1.±4.5.6±5. 8 kaempferol glucoside 59.±. 56.6±8. 68.±1. 5.9± ± ± ± ± ±. 65.1±6. 77±1 6±15 57± isorhamnetin glucoside.±7. 4±1.6± ± ± ±. 14.±. 8± ±. 14.8± ± ± ±5. 5 myricetin 1.4± 79±15 1.9± 96.18±. 9.±.5 a 11.7± 9.1±.9 1.7± ±.9 7.±. c 14±19 1±18 8± a 1 a 4 1 quercetin 1.±4. 5.± ± ±.9.6±1.4.8± ±4.1 4.±1. a 6.7±. 9.6±1. c 4±1 4.5±1.7.1±. kaempferol 1.8± ±. 1.7± ± ± ±. 9.56± ±. 11.9±1. 8.6±.4 1.4±.5 16.± ±. isorhamnetin 8.6± ±.4 9.7±.4 1.± ±. 8 1.± ± ±. 5 a 1.5±. 5 a 4.4± ±.8 a 1.81±. 97 a 4.94±. 57 Tot. myricetin and myricetin glycosides 97±9 86±1 1.±.5 81±5 8±99 97±6 9±8 91±1 8.6±6. 1± 1±11 91± 89±1 Tot. quercetin and quercetin glycosides 51±4 46± ±. 44±17 46±8 5±46 54±1 5±17 a 45.± ±51 a 55±66 5±91 5±1 Tot. kaempferol and 96.4±7. 9±1 11.1± ±. 87.8± ± 95.4± ±5. 91.±6. 1±15 1±5 11±9 95±19 kaempferol glycosides Tot. isorhamnetin and isorhamnetin 51.1± ± 54.1±1. 8.9±5. 41.± ± ±8.9 65±1 4.7± ± ±9. 4.±. 6 44±1 S11

12 glycosides Tot. FlaGly and FlaAgl 16±5 8 15± ±.8 Tot.FlaGly 15±5 1± ±6 4.5 Tot. FlaAgl 14±1 1± 154.9±. 7 14±64 14±15 16±1 16±54 16± ±9.5 1±77 1±14 15±9 15±45 14± ±6.6 1±14 14±5.4 17±1 15.4±9. 18±7 17±7 18± 15±17 16±4 14± 16± 5 14± ± ±. 16.±1. 1±7 199±5 118±9 4 a 9 6 c ANTHOCYANINS 158.± ± ± 4.4±8. 9±17 1± 11±11 1± 11± ± ±14 99±19 a 19.±1. 68.±.4 c 74±1 ±1 64±17 58±44 61±6 76.7±1.1 c 99.± ±. 9.7±5.1 47±1 15±4 1±1 1±4 1± 169.6±8. ±18 1±4 19.7± 8.4±5.8 91±1 5±16 7± 64±5 66±7 85.1±4. 54±5 a 64.1±.1 47±1 7 delphinidin glucoside 5±1 71±1 79±4 8±1 9.8±.8 ±5 a 19.9±1. 1.± a a ±.6 a 8 delphinidin rutinoside 16± 6±9 5 8 a 7 a.94 a 1 a 9 cyanidin glucoside 11± ±18 ±8 9±14 a 59.±9. 5.8±1. 1.±5.6 67± ±64 8.4±.9 1.8± ±54 16.±.1 a 4 cyanidin rutinoside 99±9 ± 57± ± 4±8 a a.81 a 97 8 a Tot. delphinidin 19± 44±51 glycosides 65 1 a 1 9 a 1 4 a.1 4 a Tot. cyanidin 11± ±4 64± 11.9±. 9.67±. glycosides 11 a a 5 1 a Tot. anthocyanins ± 11 67±9 71±7 ±7 1 a 19±17 19±6 7 19±6 1 a 54.6±4. c 4± ± 9.6 a 19.1± ±6. 14± 1 a Anthocyanin degradation products (hydroxyenzoic acids) 615.±6. 66±5 4 protocatechuic acid ±1 46±51 a 48±14 a 4.6±. 8 a a 5 4-hydroxyenzoic acid **n.d. **n.d. **n.d. **n.d. 1± ±.81 6 phloroglucinaldehyde 8.9±6. 1.6±8.9.± ±.4 6.7±. 1.1± Tot. Ant. degradation 6±1 48±59 5± ±4. 77± a 8±8 products 5 a CITRIC ACID METHYL ESTERS AND FLAZIN S1 8.1± 1±7 99±6 a 9±5 a 5.6± 19± ±. 9 a.44 a 5. **n.d ± 14±1 **n.d ±. 17.6±6. **n.d ±1 6.6±1.8.77±. 4.± ± ± ± ± ±1 99±11 a 4± 1±4 a 11±51 a 4.1± 1. a.6 8±19 5±16 17 citric acid methyl esters 97.± ±1 4±7 a 6.4±6. 8± c 7±17 48±14 a 4± 48.1±. 47±78 41±8 6.7 a 1 18 flazin 9±1 48±7 a 49±4 a ±11 94± a 9±7 ±11 16±5 a 1±48 5±19 c 1±5 a 16±9 ±87

13 a a Means and standard deviations of two replicates, aseline sample had 5 replicates, n.d., not detected. Significant differences etween storage conditions are marked with a c. Statistical significance was ased on One-way ANOVA with Turkey s HSD and LSD test (p <.5). * ) Chlorogenic acid was not taken into account in the sum of total caffeic acid and its derivatives, total caffeic acid derivatives and total quinic acid derivatives. ** ) At aseline and at 1-month storage in refrigerator (+ 4 C) 4-hydroxyenzoic acid did not show its characteristic UV asorption maximum. S1

14 Figure S8. Increase of peak 17 during pasteurization. Peak 17 contains citric acid methyl esters. Chromatograms were otained via analytical HPLC-DAD. S14

15 REFERENCE (16) Sandell, M.; Laaksonen, O.; Järvinen, R.; Rostiala, N.; Pohjanheimo, T.; Tiitinen, K.; Kallio, H. Orosensory profiles and chemical composition of lack currant (Ries nigrum) juice and fractions of press residue. J. Agric. Food Chem. 9, 57, S15