Synthesis and antithrombogenicity assessment of tetramethylpyrazine-capped poly(ethylene glycol)

Tetramethylpyrazine(TMPZ) is an active ingredient of a Chinese herbal medicine Chuanxiong (Liqusticum wallichii Franchat). In order to enhance its stability and delivery in vivo TMPZ-capped poly(ethylene glycol) conjugate was designed and synthesized efficiently by the condensation reaction of 2-cardoxyl-3, 5, 6-trimethylpyrazine (TMPZCOOH) with PEG(M=2000) in the presence of dicyclohexylcarbodiimide (DCCI). The TMPZ-PEG conjugate obtained was then purified by gel filtration chromatography (SephadexG-15 column,1.6X100cm; eluent: distilled water) and characterized by FTIR, UV spectra. FTIR (KBr): 2868cm(-1)(CH2), 1718cm(-1) (C=O), 1455cm(-1) (-C=N-), 1113cm(-1)(O-CH2-CH2). The degree of end-capping of TMPZ per PEG molecules was estimated to be 87% from UV absorbance at 294nm. The anticoagulant activity of the conjugate was evaluated by in vitro coagulation time test. The result showed that the activated partial thromboplastin time (APTT) of the TMPZ-PEG conjugate is nearly equal to that of blank plasma in our experiment conditions, this behavior is similar to that of TMPZ. But TMPZCOOH, one of the main metabolic products of TMPZ in vivo, exhibited more potent anticoagulant activity than TMPZ and TMPZ-PEG, its APTT is even larger than the maximum clotting time set by the instrument. It is also found that APTT of the conjugate increased as the time of the sample stayed in water bath at 37 degrees C. This is probably because the ester bond between TMPZCOOH and PEG hydrolyzed during the stay time and released the free TMPZCOOH.