Analysis of Anthocyanins in Dietary Supplements Containing Blueberry Extract

October 2008 339 20 3 21 Analysis of Anthocyanins in Dietary Supplements Containing Blueberry Extract Fusako IH=>@6L6, Mitsuo O>H=>, Tetsuya S=>C9D, Masao HDG>:, Akiko Y6HJ> and Mitsuo N6@6O6ID Tokyo Metropolitan Institute of Public Health: 3 24 1 Hyakunin-cho, Shinjuku-ku, Tokyo 169 0073, Japan; Corresponding author An extraction and analytical method was developed for determination of the content and profiles of anthocyanins in commercial dietary supplements containing blueberry extract. Dietary supplements were refluxed with hydrochloric acid-methanol solution, and spectrophotometric assay was performed to evaluate the total anthocyanidin content in extracted solutions as delphinidin, which is one of the anthocyanidins in blueberry extract. Twenty compounds (15 anthocyanins and 5 anthocyanidins) in extracted solutions were separated by HPLC analysis with gradient elution using formic acid and methanol-acetonitrile as the mobile phase, and each peak was confirmed by LC/MS analysis. The proposed method was applied to 25 kinds of commercial dietary supplements, and one supplement whose profile was di#erent from that of the fresh bilberry extract was found. (Received March 21, 2008) Key words: blueberry; bilberry; anthocyanins anthocyanidins; HPLC; / LC/MS; dietary supplements 1 Vaccinium myrtillus, 1). 2), 3). 169 0073 3 24 1 Wu 27 4). 1 15 2), 5), 6). malvidin, cyanidin, delphinidin, peonidin, petunidin 5 3 glucose, galactose arabinose 15 Fig. 1 HPLC, LC/MS

340 Vol. 49, No. 5 Fig. 1. Structures of anthocyanidins in bilberry extract 1. 13 18 25 22 1 1 1 2. -25 (Bilberry, Vaccinium myrtillus) 25.0 50.0 mg 1 50 ml (1000 mg/ml) 1 malvidin chloride, cyanidin chloride, delphinidin chloride, peonidin chloride, petunidin chloride, oenin chloride (malvidin 3 - glucoside chloride), malvidin 3 -galactoside chloride, peonidin 3-glucoside chloride, kuromanin chloride (cyanidin 3-glucoside chloride), ideanin chloride (cyanidin 3-galactoside chloride) EXTRASYNTHESE 2.0 mg 1 20 ml 100 mg/ml 1 HPLC TFA HPLC (GA-55,125 mm): ADVANTEC 1 35 57.14 g 2L 5 5mL 100 ml 3. HPLC PU-1580, MD-1515, AS-1555 10, CO-965, LG-1580 02, DG-980 50 LC/MS: LC Separations Module Waters-2695 Waters MS Quattro-Ultima-PT TM MICRO- MASS 4. 1 2 0.5 g 1 60 ml 30 (80 90 ) 1 100 ml

October 2008 341 5. HPLC ZORBAX SB-C18 4.6 mm i.d. 250 mm, Agilent Technologies A 5 B (1 : 1) B 10 0 15 30 15 40 35 55 35 60 540 nm; 30 1.0 ml/min 20 ml 6. LC/MS LC ZORBAX SB-C18 2.1 mm i.d. 150 mm A 5 B (1 : 1), B 10 0 18 30 18 40 45 55 45 60 30 0.2 ml/min; 2 ml MS ESI ( ) 60 V; 4.00 kv; 500 140 m/z 250 550 1. TFA 7), 8) 9) 10) 11) 12) (No. 13) (No. 15) 1-25 0.5 g, 0.1 g 0.1 TFA 60 ml 20 0.1 TFA 20 ml 0.1 TFA 0.1 TFA 100 ml 1 TFA (70 : 30), 2 (70 : 30) 12) (350 600 nm) (mg/g) A 10/1020 (ml) 1/W 1020 (E 1 1cm); A: W (g) 1 TFA 40 70 0.1 60 ml 30 0.1 100 ml 1 2-25 1-25 1 TFA 1.5 1 2 1 30 2. 25 Table 1 25 (No. 2) 1 delphinidin 3 - glucoside (243.4 mg/280 g) 25 23 80 6 (No. 1, 3, 4, 17, 20, 25). 2 1.2 mg/6 0.9 mg/6 80 480 mg/day, 13 No. 13, 18, 23

342 Vol. 49, No. 5 Table 1. Amounts of anthocyanidins in dietary supplements, evaluated by spectrophotometry Sample No. Indication Capsule/day Anthocyanidins a) (mg) Blueberry extract b) (mg) Detected value/ indicated value ( ) 1 Blueberry extract, 180 mg/3 cap. 3 cap. 28.8/3 cap. 115.2/3 cap. 64.0 2 Conc. blueberry extract, total anthocyanins (ca. 280 mg/280 g) 158.6/280 g 634.4/280 g 86.9 c) 3 Blueberry extract, 120 mg/2 cap. 2 cap. 22.5/2 cap. 90.0/2 cap. 75.0 4 Blueberry extract, 160 mg/2 cap. (anthocyanidin glycosides 25 ) 2 cap. 29.4/2 cap. 117.6/2 cap. 73.5 5 Blueberry extract, 120 mg/4 cap. (anthocyanins 30 mg) 4 cap. 24.5/4 cap. 98.0/4 cap. 81.7 6 Blueberry extract 3 6 cap. 1.2/6 cap. 4.8/6 cap. 7 Blueberry extract, 250 mg/3 cap. (anthocyanidins 62.5 mg/3 cap.) 2 3 cap. 56.3/3 cap. 225.2/3 cap. 90.1 8 Blueberry extract, 120 mg/2 cap. (anthocyanidin glycosides 25 ) 2 cap. 24.8/2 cap. 99.2/2 cap. 82.7 9 Bilberry extract, 200 mg/4 cap. (anthocyanidin glycosides 36 ) 2 4 cap. 52.1/4 cap. 208.4/4 cap. 104.2 10 Bilberry extract, 11.25 mg/cap. 3 cap. 2.7/cap. 10.8/cap. 96.0 11 Bilberry extract, 140 mg/cap. (anthocyanidins 35 mg/cap.) 1 cap. 35.1/cap. 140.4/cap. 100.3 12 Blueberry extract, 85 mg/cap. (anthocyanins 36 ) 2 cap. 20.9/cap. 83.6/cap. 98.4 13 Anthocyanins, 25 mg/2 cap. 2 4 cap. 25.1/2 cap. 100.4/2 cap. 100.4 d) 14 Blueberry extract, 12 mg/cap. 10 20 cap. 3.3/cap. 13.2/cap. 110.0 15 Blueberry extract, 100 mg/2sack 2 3 sack 24.5/2 sack 98.0/2 sack 98.0 16 Bilberry extract 6 cap. 0.9/6 cap. 3.6/6 cap. 17 Blueberry extract, 60 mg/cap. 2 3 cap. 4.8/cap. 19.2/cap. 32.0 18 Whortleberry, 120 mg (anthocyanins 31.2 mg)/12tab. 8 12 tab. 31.2/12 tab. 124.8/12 tab 100.0 d) 19 Blueberry extract, 160 mg/4 cap. 3 4 cap. 37.2/4 cap. 148.8/4 cap. 93.0 20 Blueberry extract, 30 mg/3 cap. (anthocyanidins 25 ) 3 5 cap. 5.9/3 cap. 23.6/3 cap. 78.7 21 Blueberry extract, 35 mg/cap. (anthocyanidins 25 ) 3 4 cap. 8.3/cap. 33.2/cap. 94.9 22 Blueberry extract, 100 mg/2 cap. 2 cap. 24.1/2 cap. 96.4/2 cap. 96.4 23 Bilberry extract (anthocyanins 11.4 mg)/cap. 2 4 cap. 11.9/cap. 47.6/cap. 104.4 d) 24 Blueberry extract, 40 mg/cap. 3 cap. 10.4/cap. 41.6/cap. 104.0 25 Blueberry extract, 60 mg/6 cap. (anthocyanin glycosides 25 ) 3 6 cap. 11.4/6 cap. 45.6/6 cap. 76.0 a) The mean value (n 3). b) The amount of blueberry extract was calculated from the content of anthocyanidins detected in the samples on the assumption that blueberry extracts contain 25 anthocyanidins. c) The ratio was calculated from the detected anthocyanidin content expressed as delphinidin 3-glucoside (detected value) and the total anthocyanins content labeled on the supplement package (indicated value, shown in the column Indication ). d) The ratio was calculated from the detected anthocyanidin content (detected value) and labeled anthocyanins content on the supplement package (indicated value, shown in the column Indication ). 100.4, 100.0, 104.0 No. 13, 18, 23 No. 5 No. 25 3. HPLC HPLC 15 5 20 HPLC 14, 15) 8 10) 16) TFA 17) TFA HPLC 18). 15 5 HPLC ZORBAX SB-C18 (4.6 mm i.d. 250 mm) 540 nm A TFA, B A 5 B (1 : 1) B 10 0 15 30 15 40 35 55 35 60 20 LC/

October 2008 343 Fig. 2. HPLC chromatogram of bilberry extract HPLC conditions were as follows: Column: ZORBAX SB-C18 (4.6 mm i.d. 250 mm); mobile phase: A, 5 formic acid, B, acetonitrile methanol (1 : 1), gradient profile, B 10 (0 min) 15 (30 min) 15 (40 min) 35 (55 min) 35 (60 min); detection wavelength: 350 650 nm; column temperature: 30 ; flow rate: 1 ml/min; injection volume: 20 ml No. of peaks: 1. delphinidin 3-galactoside; 2. delphinidin 3-glucoside; 3. cyanidin 3-galactoside; 4. delphinidin 3-arabinoside; 5. cyanidin 3-glucoside; 6. petunidin 3-galactoside; 7. cyanidin 3-arabinoside; 8. petunidin 3-glucoside; 9. peonidin 3-galactoside; 10. petunidin 3-arabinoside; 11. delphinidin; 12. peonidin 3-glucoside; 13. malvidin 3-galactoside; 14. peonidin 3-arabinoside; 15. malvidin 3-glucoside; 16. malvidin 3-arabinoside; 17. cyanidin; 18. petunidin; 19. peonidin; 20. malvidin The peak drawn with boldface were identical with standards purchased from Funakoshi Co., Ltd. MS LC/MS COSMOSIL 5C18-AR INERTSIL ODS-2 GL INERTSIL ODS-3 GL DEVEROSIL ODS-T-5 DEVER- OSIL ODS-HG-5 DEVEROSIL ODS- UG-5 L-column ODS ZORBAX SB-C18 Fig. 2-25 1 HPLC No. 4. LC/MS HPLC 20 10 LC/MS Malvidin 3-galactoside MS Fig. 3. TIC and mass chromatograms of refluxed bilberry extract LC conditions were as follows: Column: ZORBAX SB-C18 (2.1 mm 150 mm); mobile phase: A, 5 formic acid, B, acetonitrile methanol (1 : 1), gradient profile, B 10 (0 min) 18 (30 min) 18 (40 min) 45 (55 min) 45 (60 min); column temperature: 30 ; flow rate: 0.2 ml/min; injection volume: 2 ml MS conditions were as follows: ESI ( ); capillary voltage: 4.00 kv; corn voltage: 60 V; source temperature: 140 ; desolvation temperature: 500 ; scan range: m/z 250 550 Peak numbers were the same as described in Fig. 2. (ESI ), 60 V, 4.00 kv, 600 V, 500 140 Malvidin 3-galactoside (m/z 331) (m/z 493) MS LC HPLC Fig. 3-25 (TIC) 3 4 Fig. 4 m/z 303 (delphinidin) m/z 287 (cyanidin)

344 Fig. 4. Mass chromatograms of m/z 303 and m/z 287, and mass spectra of the main peaks. Peak numbers were the same as described in Fig. 2 m/z 303 1, 2, 4 (m/z 465, m/z 435) (m/z 303) 4 delphinidin 3 cyanidin m/z 331 (malvidin), m/z 301 (peonidin), m/z 317 (petunidin) HPLC LC/MS Fig. 2 5. -25 HPLC 100 Table 2 1 20 15 5 Vol. 49, No. 5 No. 2, 6, 18 delphinidin 3-arabinoside, cyanidin 3-arabinoside No. 15, 18, 21, 23-25 delphinidin 3-galactoside, delphinidin 3 -glusoside, cyanidin 3 -galacyoside, cyanidin 3 - glucoside, petunidin 3-glucoside peonidin No. 16 3 100 Table 2 21 25-25 p 26, 27 No. 6, 16, 20, 25 21 0.960-25 No. 6, 20, 25 delphinidin (27 29 ) cyanidin (36 38 ) 0.933, 0.910, 0.915 p 0.04 5 20 No. 16 petunidin 3-glucoside malvidin 3-glucoside 15 malvidin 19) HPLC, LC/MS 1

Table 2. The anthocyanin profiles of commercial dietary supplements Peak Sample No. a) No. b) Anthocyanin A B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1 Delphinidin 3-galactoside 12.7 9.1 9.7 6.0 10.5 10.7 10.4 7.3 7.5 8.9 7.2 7.9 8.0 8.9 6.9 7.8 5.6 0.8 12.2 4.9 9.8 7.5 10.2 6.5 9.2 10.9 7.1 2 Delphinidin 3-glucoside 13.5 10.9 10.9 7.1 12.3 11.1 11.9 8.4 9.3 10.2 9.8 9.9 10.6 11.6 8.7 10.6 9.0 12.6 14.2 7.9 13.6 9.2 13.6 8.9 13.1 11.7 9.2 3 Cyanidin 3-galactoside 9.9 7.8 7.4 5.7 8.0 7.6 7.0 7.0 6.8 7.7 6.2 6.3 6.1 6.9 5.8 5.5 4.4 1.5 11.7 4.9 7.2 7.1 6.0 5.6 7.4 9.4 7.0 4 Delphinidin 3-arabinoside 11.5 6.4 4.4 1.9 6.6 6.0 4.6 2.4 3.2 4.5 2.5 3.1 4.1 5.9 2.4 2.6 1.2 0.8 7.8 1.0 1.9 2.5 2.2 1.8 1.8 4.9 4.1 5 Cyanidin 3-glucoside 10.9 9.5 8.3 5.3 9.1 7.9 7.4 8.1 8.4 8.8 8.3 7.8 7.9 8.7 7.2 7.8 7.3 1.7 8.7 7.5 10.0 7.6 7.6 7.6 10.5 8.2 7.4 6 Petunidin 3-galactoside 4.1 3.1 3.6 3.8 3.7 4.0 3.1 2.8 3.0 3.3 2.8 2.9 2.8 3.6 2.6 2.7 2.1 0.7 5.4 2.1 3.6 3.7 3.3 2.3 3.3 4.5 3.5 7 Cyanidin 3-arabinoside 7.6 4.6 2.9 2.5 4.3 3.7 2.5 2.3 2.5 3.3 1.8 2.1 2.5 3.8 1.5 1.6 0.8 1.0 3.5 0.7 1.1 1.9 0.9 1.3 1.1 3.2 2.9 8 Petunidin 3-glucoside 8.6 7.2 7.6 5.4 8.1 7.9 7.1 6.8 6.8 7.2 6.9 7.1 7.2 8.0 6.5 7.2 6.0 15.9 6.9 6.0 9.2 7.5 8.4 6.2 9.3 8.8 7.6 9 Peonidin 3-galactoside 1.0 0.8 0.6 1.1 0.7 0.8 0.3 0.8 0.8 0.8 0.5 0.6 0.5 1.1 0.8 0.8 0.7 0.0 1.2 0.7 0.9 0.9 0.0 0.6 0.9 1.2 0.5 10 Petunidin 3-arabinoside 2.4 1.6 0.7 1.2 1.4 1.5 0.4 1.3 0.8 1.1 0.5 0.9 0.8 1.9 0.9 1.0 0.4 0.0 0.6 0.6 0.8 1.3 0.0 0.6 0.8 1.8 1.3 11 Delphinidin 0.0 8.5 13.4 19.8 11.3 9.5 16.1 10.2 15.8 13.9 15.1 14.4 17.1 10.8 21.2 18.5 21.4 1.6 3.2 22.5 8.0 8.2 18.7 21.6 15.1 7.9 8.1 12 Peonidin 3-glucoside 3.8 3.4 2.8 1.3 0.5 2.5 0.4 5.0 2.8 2.7 2.6 1.7 2.2 3.1 1.0 2.5 2.6 1.8 0.0 2.2 3.6 4.2 2.2 2.2 3.2 2.1 3.0 13 Malvidin 3-galactoside 2.8 2.6 2.2 0.7 2.4 3.2 1.3 0.3 2.6 2.4 2.4 2.2 1.9 2.6 1.5 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 1.1 0.2 14 Peonidin 3-arabinoside 0.3 0.3 0.2 0.0 0.2 0.1 0.1 0.2 0.2 0.1 0.0 0.2 0.1 0.4 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.2 0.1 0.2 15 Malvidin 3-glucoside 8.7 7.9 7.7 1.7 7.4 9.1 7.1 8.6 8.0 7.4 8.1 8.1 7.1 7.2 6.9 7.0 5.3 44.0 7.0 6.7 8.2 8.8 5.8 6.6 8.4 6.3 7.4 16 Malvidin 3-arabinoside 1.9 1.5 0.8 0.0 1.2 1.4 0.7 1.3 0.9 1.1 0.8 1.0 1.0 1.4 0.8 0.9 0.9 0.0 2.2 0.7 0.9 0.7 0.0 0.7 0.8 1.3 0.5 17 Cyanidin 0.0 9.0 10.4 17.9 7.9 7.6 13.1 18.8 12.2 10.3 13.7 15.5 12.2 8.2 14.5 13.5 18.6 10.9 11.9 18.7 15.5 21.1 14.2 16.2 10.7 12.3 19.8 18 Petunidin 0.1 2.9 3.2 2.6 2.3 2.7 3.6 5.1 3.9 3.0 4.9 4.1 4.2 2.9 5.3 5.3 6.7 2.8 2.6 6.3 2.9 4.0 3.8 5.5 2.7 2.0 6.3 19 Peonidin 0.1 1.0 1.0 0.0 0.6 0.8 0.9 1.3 1.4 1.1 1.7 1.1 1.0 0.8 1.5 1.3 2.1 0.0 0.0 1.9 1.1 1.2 1.1 1.5 0.6 1.2 2.0 20 Malvidin 0.1 2.1 2.3 15.9 1.5 1.9 2.2 2.1 3.0 2.2 4.0 3.0 2.7 2.0 3.9 3.4 4.1 4.0 0.8 4.6 1.7 1.6 2.0 4.1 0.7 1.3 2.0 Delphinidin 37.7 34.9 38.4 34.8 40.7 37.2 42.9 28.2 35.8 37.5 34.7 35.4 39.8 37.3 39.2 39.5 37.2 15.8 37.5 36.4 33.3 27.3 44.7 38.9 39.3 35.4 28.5 Cyanidin 28.4 30.8 29.0 31.4 29.3 26.9 30.0 36.2 29.9 30.2 30.1 31.7 28.7 27.7 29.0 28.3 31.1 15.0 35.8 31.8 33.9 37.7 28.7 30.7 29.8 33.1 37.1 Petunidin 15.1 14.7 15.1 13.1 15.6 16.1 14.1 16.0 14.6 14.6 15.1 14.9 14.9 16.4 15.2 16.2 15.2 19.3 15.5 15.0 16.4 16.5 15.6 14.6 16.1 17.0 18.6 Peonidin 5.3 5.5 4.6 2.4 2.0 4.1 1.7 7.3 5.2 4.7 4.9 3.6 3.8 5.4 3.3 4.6 6.1 1.8 1.2 4.8 5.6 6.4 3.3 4.5 4.8 4.6 5.7 Malvidin 13.4 14.0 13.0 18.3 12.4 15.6 11.3 12.3 14.6 13.0 15.3 14.3 12.8 13.2 13.2 11.3 10.3 48.0 10.1 12.0 10.8 12.1 7.8 11.4 9.9 10.0 10.1 Correlation coe$cient c) 0.991 0.979 0.986 0.978 0.985 0.933 0.998 0.997 0.998 0.999 0.986 0.985 0.989 0.980 0.989 0.020 0.992 0.998 0.982 0.910 0.960 0.993 0.982 0.985 0.915 P-value c) 0.001 0.004 0.002 0.004 0.002 0.021 0.000 0.000 0.000 0.000 0.002 0.002 0.001 0.004 0.001 0.975 0.001 0.000 0.003 0.032 0.009 0.001 0.003 0.002 0.029 The values (upper lines 1 20) show the percent of the peak area of each anthocyanin or anthocyanidin to the total peak area of anthocyanins and anthocyanidins in each sample. The values (under lines 21 25) show the percent of the total peak area of each antocyanidin and its three glycosides to the total peak area of anthocyanins and anthocyanidins in each sample. a) Sample A is the solution of fresh bilberry extract. Sample B is the refluxed A. Sample numbers are the same as described in Table 1. b) Peak numbers are the same as described in Fig. 2. c) The composition of anthocyanin aglycons in samples were compared with sample B, using the Pearson test for the correlation coe$cient and regression analysis for the p-value. 345 October 2008

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