Vasorelaxation Effects And Their Mechanisms Of Hawthorn Flavanols On Isolated Thoracic Aortic Rings From Rats

Objective：This study was designed to systematacially investigate the vasorelaxationefficacy of hawthorn flavanol extracts, and pure flavanols including monomer, dimmers(B2and B5), trimers (C1, C2and C3), and tetramer (D1) on isolated thoracic aortas fromnormal rats. The aim was to find the specific compositions which can cause endothelium-dependent vasodilatation, and explore the structure-efficacy relationships and the possiblemechanisms.Methods: Using in vitro rat aortic ring tensile test method, the vasodilatory effects ofhawthorn flavanol extracts, and pure flavanols for the L-phenylephrine hydrochloride (PE)pre-contracted thoracic aortic rings were evaluated by cumulative dosing way. Theresearch content included:1. Using rat thoracic aortic rings with or without endothelium, the endothelium-dependent vasodilatation of hawthorn extracts from two different process levels (hawthornsyrup (HS) and hawthorn flavanols (HF) crude extract) was investigated;2. Combining with HPLC methods, two series of fractions of the HF crude extractsseparated by normal phase silica gel and Sephadex LH-20gel chromatography wereinvestigated and analysed about the vasodilatation effects and potential active components;3. The vasodilator effects of pure hawthorn flavanols EC, B2, B5, C1, C2C3, D1，andC1, D1fractions which were not completely purified by prepared HPLC were investigated;4. The influences of endothelium denuded treatment, or NG-nitro-L-arginine methylester (L-NAME), methylene blue (MB) and indomethacin (Indo) pretreatment onendothelium-dependent relaxing effects of unpurifed D1were observed.Results:1. HS and crude extractings of HF (the scope of flavanols concentration was about14~140mg/L) showed concentration-dependent vasodilator function on PE pre-contractedisolated thoracic aortic rings. Endothelium denuding made the vasodilatation efficiency ofHS obviously reduced, and the effect of HF crude extracting almost completely abolished.2Among the6fractions of HF crude extract separated by normal phase silica gel, the4th,5th, and6th fractions, which were latter eluated, revealed excellent vasodilator effects(3~20mg/L). And among the Sephadex LH-20isolated fractions, the4th fraction, whichwas latter eluated after middle stages, produced relatively excellent vasodilator effects (3~ 100mg/L). Combining with the HPLC analysis results, the effective ingredients may behawthorn flavanol polymers or some special unknown compounds exsiting during theprocesses of chromatographic separations.3. Hawthorn flavanols, including EC, B2, B5, C1, C2, C3, and D1had mildvasodilator efficiency. The major components EC, B2and B5had no effect within theconcentration1~100mg/L. Effects of trimers and tetramer, namely C1, C2, C3, and D1,are more outstanding, which showed vasodilator function within the concentration of30~300mg/L. Vasodilator efficiency of these7HF was affected by their degree ofpolymerization and the way of bonding. C1, D1were more effective. D1was the mostsensitive compound causing vasodilation reaction, which started to show significantdifferences at17.3mg/L compared with the control group. The biggest relaxation ratecaused by it was (65.1±2.8)%.4. Unpurified C1, D1had endothelium-dependent vasodilatation function, andcompared with pure compounds of C1, D1, the effective concentrations were decreasedobviously. The efficiency of Unpurified D1was almost7times higher than purecompounds D1(EC50(Unpurified D1)=11.8mg/L, EC50(D1)=94.5mg/L). The relaxationeffect of Unpurified D1could abolished by L-NAME or MB pretreatment, but nosignificant impact was observed after Indo pretreated. L-NAME, MB, Indo showed thesame impacts on the relaxant effect of pure D1(datas were not given).Conclusions:1. In vitro, crude extract of HF does have endothelium-dependent vasodilationfunction. The mechanism of the active ingredients is likely to be acting on eNOS, thentriggering the NO-cGMP pathway mediated endothelium-dependent vasodilatation, andhas nothing to do with the PGI2synthesis.2. Hawthorn flavanols have mild endothelium-dependent vasodilation function, whichare affected by their degree of polymerization and bonding way. The trimers and tetramerhave lower effective concentrations, and D1is the most utility. The most effectiveconstituents of HF crude extract, which can cause endothelium-dependent vasodilation, areunlikely to be EC, B2, B5, C1, C2, C3, D1, or chlorogenic acid.3. Some other unknown components in the HF crude extract attributed to flavanolclasses exsiting during the processes of chromatographic separations may be moreeffective to cause endothelium-dependent vasodilation.