Isolation, Purification and Synthesis of Procyanidin B2 and Uses Thereof

Abstract

Methods for the synthesis, isolation, and purification of procyanidin B2 are disclosed. The synthetic methods utilize epicatechin as a starting material and produce procyanidin B2 in high yields. The isolation methods extract procyanidin B2 from a sample of bark powder from plant matter of the genus Uncaria . The isolated and/or synthesized procyanidin B2 is used to treat amyloid disease, such as Alzheimer's disease and Parkinson's disease as well as improve cognitive performance and increase learning and memory in Alzheimer's patients. Pharmaceutical compositions containing the synthesized and/or isolated procyanidin B2 are also disclosed.

1 . A method for synthesizing procyanidin B2 comprising the steps of:
 a) esterification of an epicatechin to form a 5,7,3′,4′,3″-penta-O-acyl-epicatechin;   b) protecting the 5,7,3′,4′,3″-penta-O-acyl-epicatechin to form a 5,7,3′,4′-tetra-O-protected epicatechin;   c) coupling an adduct precursor with a first portion of the 5,7,3′,4′-tetra-O-protected epicatechin to form a 4″-substituted, 5,7,3′,4′-tetra-O-protected epicatechin;   d) dimerizing a second portion of the 5,7,3′,4′-tetra-O-protected epicatechin with the 4″-substituted, 5,7,3′,4′-tetra-O-protected epicatechin to form a 5,7,3′,4′-tetra-O-protected epicatechin-4β→8-5,7,3′,4′-tetra-O-protected epicatechin; and   e) deprotecting the 5,7,3′,4′-tetra-O-protected epicatechin-4β→8-5,7,3′,4′-tetra-O-protected epicatechin to form procyanidin B2.   
   
   
       2 . The method of  claim 1 , wherein the esterification step comprises the step of acetylation of an epicatechin to form a 5,7,3′,4′,3″-penta-O-acetyl-epicatechin. 
   
   
       3 . The method of  claim 1 , wherein the protection step comprises the step of protecting the 5,7,3′,4′,3″-penta-O-acyl-epicatechin to form a 5,7,3′,4′-tetra-O-benzylepicatechin. 
   
   
       4 . The method of  claim 3 , wherein the protecting step comprises the step of reacting the 5,7,3′,4′,3″-penta-O-acyl-epicatechin with benzylchloride in the presence of water to form the 5,7,3′,4′-tetra-O-benzylepicatechin. 
   
   
       5 . The method of  claim 4 , wherein the protecting step comprises the step of reacting the 5,7,3′,4′, 3″-penta-O-acyl-epicatechin with between about 4.0 and about 4.2 equivalents of benzylchloride for between about 24 hours and about 72 hours to form a reaction mixture comprising the 5,7,3′,4′-tetra-O-benzylepicatechin. 
   
   
       6 . The method of  claim 5 , wherein the protecting step comprises the step of combining the reaction mixture with an acid. 
   
   
       7 . The method of  claim 6 , wherein the acid is hydrochloric acid. 
   
   
       8 . The method of  claim 7 , wherein the hydrochloric acid is between about 0.01 M and about 3 M. 
   
   
       9 . The method of  claim 7 , wherein the hydrochloric acid is between about −10° C. and about +10° C. 
   
   
       10 . The method of  claim 4 , wherein the protecting step comprises the steps of:
 a) reacting the 5,7,3′,4,3″-penta-O-acyl-epicatechin with between about 4.0 and about 4.2 equivalents of benzylchloride for between about 24 hours and about 72 hours in the presence of water to form a reaction mixture comprising 5,7,3′,4′-tetra-O-benzylepicatechin; and   b) combining the reaction mixture with hydrochloric acid, wherein the hydrochloric acid is between about 0.01 M and about 3 M, and between about −10° C. and about +10° C.   
   
   
       11 . The method of  claim 1 , wherein the coupling step comprises the step of coupling an ethylene glycol with the first portion of the 5,7,3′,4′-tetra-O-protected epicatechin to form a 5,7,3′,4′-tetra-O-protected 4-(2-hydroxyethoxy)epicatechin. 
   
   
       12 . The method of  claim 1 , wherein the dimerization step comprises the steps of:
 a) crystallizing the 5,7,3′,4′-tetra-O-protected epicatechin-4β→8-5,7,3′,4′-tetra-O-protected epicatechin; and   b) isolating the 5,7,3′,4′-tetra-O-protected epicatechin-4β→8-5,7,3′,4′-tetra-O-protected epicatechin using column chromatography.   
   
   
       13 . A method for synthesizing procyanidin B2 comprising the steps of:
 a) acetylation of an epicatechin to form a 5,7,3′,4′,3″-penta-O-acetyl-epicatechin;   b) reacting the 5,7,3′,4′,3″-penta-O-acetyl-epicatechin with between about 4.0 and about 4.2 equivalents of benzylchloride for between about 24 hours and about 72 hours in the presence of water to form a reaction mixture comprising a 5,7,3′,4′-tetra-O-benzylepicatechin;   c) combining the reaction mixture with hydrochloric acid to form purified 5,7,3′,4′-tetra-O-benzylepicatechin, wherein the hydrochloric acid is between about 0.01 M and about 3 M, and between about −10° C. and about +10° C.;   d) coupling an ethylene glycol with a first portion of the purified 5,7,3′,4′-tetra-O-benzylepicatechin to form a 5,7,3′,4′-tetra-O-benzyl 4-(2-hydroxyethoxy)epicatechin;   e) dimerizing a second portion of the 5,7,3′,4′-tetra-O-benzylepicatechin with the 5,7,3′,4′-tetra-O-benzyl 4-(2-hydroxyethoxy)epicatechin to form a 5,7,3′,4′-tetra-O-benzylepicatechin-4β→8-5,7,3′,4′-tetra-O-benzylepicatechin;   f) crystallizing the 5,7,3′,4′-tetra-O-benzylepicatechin-4β→8-5,7,3′,4′-tetra-O-benzylepicatechin;   g) isolating the 5,7,3′,4′-tetra-O-benzylepicatechin-4β→8-5,7,3′,4′-tetra-O-benzylepicatechin using column chromatography; and   h) deprotecting the 5,7,3′,4′-tetra-O-benzylepicatechin-4β→8-5,7,3′,4′-tetra-O-benzylepicatechin to form procyanidin B2.   
   
   
       14 . A method for synthesizing a 5,7,3′,4′-tetra-O-protected epicatechin comprising the steps of:
 a) esterification of an epicatechin to form a 5,7,3′,4′,3″-penta-O-acyl-epicatechin; and   b) protecting the 5,7,3′,4′,3″-penta-O-acyl-epicatechin to form a 5,7,3′,4′-tetra-O-protected epicatechin.   
   
   
       15 . The method of  claim 2  where suitable acyl groups for acetylation of epicatechin are selected from acetyl, other short chain acyl groups (C 1 -C 6 ), benzoyl and substituted benzoyl groups. 
   
   
       16 . The method of  claim 1  where the adduct precursors are selected from C 1 -C 6  alkanols, ethylene glycol, other alcohols, water and thiols. 
   
   
       17 . The method of  claim 1  further including the steps of crystallization and isolation using column chromatography.