US 20080255226A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0255226A1 Eidenberger (43) Pub. Date: Oct. 16, 2008 (54) STABILIZED ANTHOCYANIN filed on Jul. 26, 2007, provisional application No. COMPOSITIONS 60/985,603, filed on Nov. 5, 2007. (76) Inventor: Thomas Eidenberger, Steyr (AT) Publication Classification (51) Int. Cl. Correspondence Address: A63L/352 (2006.01) DORSEY & WHITNEY LLP A6IP 43/00 (2006.01) INTELLECTUAL PROPERTY DEPARTMENT (52) U.S. Cl. ........................................................ 514/.456 SUITE 1500, 50 SOUTH SIXTH STREET MINNEAPOLIS, MN 55402-1498 (US) (57) ABSTRACT Appl. No.: 12/047,993 The invention describes stabile anthocyanin compositions, (21) methods to prepare Such compositions and also methods of use of Such compositions to treat various afflictions. The (22) Filed: Mar. 13, 2008 present invention describes unique compositions of an antho cyanin and a stabilizing compound Such that the combination Related U.S. Application Data of the two components provides that the anthocyanin does not (60) Provisional application No. 60/895,034, filed on Mar. readily undergo degradation, such as oxidation, pH instabil 15, 2007, provisional application No. 60/952,113, ity, etc. Lack of bathochromic shift for cy-3-O-glucoside (1 mMolar solution) lamda max of 10-3 M solutions at different pH ... ... NOne - - - DHA - Gutath Patent Application Publication Oct. 16, 2008 Sheet 1 of 27 US 2008/0255226A1 Figure 1 Lack of bathochromic shift for cy-3-O-glucoside (1 mMolar solution) lamdamax of 10-3 M solutions at different pH o so - - - - None - - - DHLA - Glutath Figure 2 Lack of hyperchromic shift for cy-3-O-glucoside (1 mMolar solution) Absorption of 10-3 M solutions at different pH a- - - - - - - None - - - DHA - Glutath Patent Application Publication Oct. 16, 2008 Sheet 2 of 27 US 2008/0255226 A1 Figure 3 % residual anthocyanosides of bilberry extract (unprotected) 60 minutes at room temperature (None) O I i I l O 2 4. 6 8 1.O • e o O.O. M. m. m OOO1 M. m. O.OOOM Figure 4 % residual anthocyanosides of bilberry extract (DHLA protected) 60 minutes at room temperature (DHLA) 120 9. re 100 sid th 60 ---------------- m ya 40 -------- - - - - - - - - ---------------------------------------- • e o O.O M as OOOM In OOOO. M. Patent Application Publication Oct. 16, 2008 Sheet 3 of 27 US 2008/0255226A1 Figure 5 % residual anthocyanosides of bilberry extract (GSH protected) 60 minutes at room temperature (Glutathione) 120 9/o re 100 | th 60 -- - - - - - - - - O - I --- O 2 4. 6 8 10 0.01 M - - 0.001 M - 0.0001 M Figure 6A through 6D Comparative degradation kinetics of bilberry extract (DHLA-protected) Figure 6A Figure 6B pH = 1.0 pH=3. 3.5 3.5 3 y 3 s2.5Ab s b 2.5 51. 2 - 52 2 1.5 - 1.5 rt i - m i : 0.5 0.5i C - t t r t - O ------------------ --- - -- O 50 100 150 200 250 300 O 50 100 150 200 250 300 Time (min) Time (min) i DHLA) None DHLAo None Patent Application Publication Oct. 16, 2008 Sheet 4 of 27 US 2008/0255226 A1 Figure 6C Figure 6D pH=7.0 pH=9.0 1.5 3.5 y = 2.4789.000: Ab y = 1.1842a00028x Ab. R = 0.9713 S 1 R = 0.9972 s 56 56 2 O 0 15 0.5 y s -0.0037x + 1,2888 y = -0.0025x + 1.977 r R = 0.9933 r R = 0.9566 OS 0. -- - - ---- -- O - I T O 50 00 SO 200 250 300 O 50 OO 50 2OO 2SO 300 time (min) Time (min) DHA. O. None - Linear (None)-Exponentiell (DHLA) : DHLA 8 None re-linear (None)-Exponentiel) (DHLA) Figure 7 Comparative degradation kinetics of delphinidin-3-O-galactoside Delp.-Galac. y = 695.02e.92* R = 0.8142 y = -1.6123x + 575.14 100 R = 0.857. O -- - - - - - - I t — 0.00 50.00 100.00 5000 200.00 250,00 300.00 Time (h) O None O Glutathione - Exponentiel (Glutathione) -Linear (None) Patent Application Publication Oct. 16, 2008 Sheet 5 of 27 US 2008/0255226 A1 Figure 8 Comparative degradation kinetics of petunidin-3-O-galactoside Petu.-Galac. 30 y = 226.45e'* as 2 6 O O R = 0.7124 2 O 1. 6 O 1. 1. O 6 O y = -0.441.4x + 192.02 O - R = 0.7558 0.00 50.00 100.00 5O.OO 200.00 250.00 300.00 Time (h) O None O Glutathione -Linear (None) - Exponentiell (Glutathione) Figure 9 Comparative degradation kinetics of delphinidin-3-O-galactoside Cyan.-Galac. 600 y - 474.77e-0.0006x - 500 R2 -is 0.5561 c 400 D 300 200 y = - 0.6286x + 416.46 100 R = 0.6427 O - --- g---------------------------- - 0.00 50.00 100.00 150.00 200.00 250.00 300.00 Time (h) t O None e Glutathione - Exponentiell (Glutathione) -tinear (None) Patent Application Publication Oct. 16, 2008 Sheet 6 of 27 US 2008/0255226A1 Figure 10 Comparative degradation kinetics of cyanidin-3-O-galactoside (red line... unprotected, blue line. GSH protected) Degradation of Cyanidin-3-O-Galactoside 20 - O - -- O.O1 0.1 1. 10 100 Time log (h) Patent Application Publication Oct. 16, 2008 Sheet 7 of 27 US 2008/0255226 A1 Figure 11 Comparative degradation of 15 bilberry anthocyanosides (DHLA protected) 80 - is i a 5 s is a 5 O 5 Unprotected DHLA-protected Figure 12 Comparative degradation of 15 bilberry anthocyanosides (GSH protected) 00 80 - 60 - r III i. : : g g g Unprotected Glutathione-protected Patent Application Publication Oct. 16, 2008 Sheet 8 of 27 US 2008/0255226A1 Figure 13 Stability in buffered solution - Stability of lead-anthocyanosides (Black Currant) in presence of glutahione at pH=7.0 and 37 °C (buffer solution) O I -- - - - - - - T - O 20 40 60 80 100 120 Time (h) - - - - - - Dp-Glc-Dp-Ruta - Cn-Rut Figure 14 Stability in incubation medium Stability of lead-anthocyanosides (Black Currant) in presence of glutahione at pH=7.0 and 37 °C (CaCO2-test incubation medium) O ------- - st- ---------, --------- - - - - - - - --- --------. O 20 40 60 8O 100 120 Time (h) ....... Dp-Glc-Dp-Rut - - Cn-Rut Patent Application Publication Oct. 16, 2008 Sheet 9 of 27 US 2008/0255226A1 Figure 15 Cellular uptake of lead-anthocyanosides into CaCo-2 cells Cellular Uptake of lead-anthocyanosides (Black Currant) 60 Timh (h) - - - - - - - Dp-Gic - Dp-Rut - - , Cn-Rut Patent Application Publication Oct. 16, 2008 Sheet 10 of 27 US 2008/0255226A1 Figure 16 Degradation of bilberry anthocyanosides with/without glutathione (1 hour at 37 °C, pH=7.0) CaCO2-Test: Incubation Media (1 h at 37 °C) 1.23ai5 OOOOOO i g i ; 60 minutes without glutathione is 60 minutes with glutathione Legend: Dp... Delphinidin, Cn. Cyanidin, Pt. Petunidin, Pe... Peonidin, Mv. Malvidin; Gal... Galactose, Glc... Glucose, Ara... Arabinose (Anthocyanosides are listed in accordance with elution from the HPLC) Patent Application Publication Oct. 16, 2008 Sheet 11 of 27 US 2008/0255226A1 Figure 17 Cellular uptake of bilberry anthocyanosides with/without glutathione into CaCo-2 cells CaCO2-Test: Cellular uptake of bilberry anthocyanosides (1 h at 37 °C) 250 - ! 200 É150 100 w O -- w is s t -- -- f t ?t p p ps --p ? t- - g t & --- > b f 5 5 . al ?y A. > ce 60 minutes without glutathione 60 minutes with glutahione Legend: Dp... Delphinidin, Cn. Cyanidin, Pt... Petunidin, Pe... Peonidin, Mv. Malvidin; Gal. Galactose, Glc... Glucose, Ara. Arabinose (Anthocyanosides are listed in accordance with elution from the HPLC) Figure 18 Gradient Profile 41; 30 30 --O 45; 30 O 10 20 30 40 50 Patent Application Publication Oct. 16, 2008 Sheet 12 of 27 US 2008/0255226A1 Figure 19 Residual Ratio and pH value 100 -- Blank Sample -O-Protected Sample 80 SS d 60 s 1. S 40 9. ?: 20 O Figure 20 The Protection Effect and pH value l: .9 5 92 S l pH value Patent Application Publication Oct. 16, 2008 Sheet 13 of 27 US 2008/0255226A1 Figure 21 HPLC graph of fresh solution of 20070601 Figure 22 HPLC graph of final solution of 20070601 Patent Application Publication Oct. 16, 2008 Sheet 14 of 27 US 2008/0255226A1 Figure 23 HPLC graph of fresh solution of 20070602 g s: 8 8. g 8 s ; : | A.3w N5 A: 5 is Figure 24 HPLC graph of final solution of 20070602 Patent Application Publication Oct. 16, 2008 Sheet 15 of 27 US 2008/0255226A1 Figure 25 Peak Comparison of 20070601 800 Fresh SOlution of 2007060 Final Solution of 20070601 700 600 UD < E 500 9 400 k Ss 300 n 200 OO O V 2 3 4 5 6 7 8 9 0 1 12 13 14 5 Peak Number Figure 26 Peak Comparison of 20070602 800 Fresh Solution of 20070602 700 XXXX Final Solution of 20070602 600 Cc) sool X is n & & C) 400 & X& & & X -CS 300 KX &X &X &S. S&& &X X C) X X X & S. RS S& di &X X& X& &X X.& S&X S& 200 - X X XX XX X.X KKX& & S&X O 8 Xx X Xx X X& &g KXX SX SX SS &S S KX, &X &X. X S&SX 100 - SX Xx XSS & XS &X &X. &X x &X 8x S&X oxx& S. xxxx,S. & RS & KKKKKKXXKS &S & & & S& 2 3 4 5 6 7 8 9 10 1 2 3 4 5 Peak Number Patent Application Publication Oct. 16, 2008 Sheet 16 of 27 US 2008/0255226A1 Figure 27 Comparison of anthocyanidins observed in human plasma after treatment with bilberry or Bilberry/Cysteine combo Total Anthocyanidins (Bilberry) 200, ......... C wn new Pt 160 ...B. Pe 120 Time 200 ........