Assign Of Antioxidant Activity And Protective Effects On Tissue Damage Of Salvianolic Acid

Salvianolic acid are a group of phenolic compounds which naturallypresent abundant in Labiatae Salvia species (Salvia L.). Many studies haveindicated that salvianolic acid are effective in eliminating blood stasis,promoting blood flow, increasing coronary flow, resisting artherosclerosis,protecting the tissue injury induced by ischemic and ischemic reperfusion aswell. Therefore, much attention has been paid to the pharmacological actionand efficiency of salvianolic acid. However, it is difficult to screening theactivity of these phenolic compounds high-flux and study the mechanism ofaction, due to the species of salvianolic acid spread multitude, the structuralsimilar and complicated, as well as the growth and decline of quantity lackof regularity.Oxidative damage is one of common mechanism of actions of the bloodvessel disease and related diseases. In view of all salvianolic acid havestructural requirements of free radical scavengers, we presume that the antioxidant activity may be one of the mechanisms of pharmacologicalaction possessed by salvianolic acid. The aim of this study was to investigatesalvianolic acid systematically, employing methods including extract andsuperficial syndrome of salvianolic acid, research the antioxidant acitivity invitro and vivo, submitted to HPLC analysis to establish a rapid evaluationsystem of antioxidant activity of salvianolic acid, and preliminary discussthe protective effects of salvianolic scid on tissue oxidative damage. Webelieved that such findings might provide a new method to high-fluxscreening the activity of polyphenols compounds, and establish a foundationto study the mechanism of the blood vessel disease and related diseases ofsalvianolic acid.Objective: To study on the relationship between the antioxidantacitivity and protective effects on tissue damage of salvianolic acid and itsmechanism of action, construct a rapid evaluation system of antioxidantactivity of salvianolic acid, in order to establish a foundation to study themechanism of the blood vessel disease and related diseases of salvianolicacid, and expand a new method to analyze natural plant materials.Methods: Salviae miltiorrhizae (The dry root of Salvia rniltiorrhizaBge, commonly known as Danshen) were used as raw material, differentsamples of salvianolic acids were extracted by different solvents and amacro-pore absorption resin, then some main polyphenolic constituents insamples were compared with standard phenolic compounds and superficialsyndrome submitted to HPLC analysis. We employed various in vitro assaysystems, such as DPPH radical scavenging assay, reducing power, ferrousmetal ions chelating ability, 13-carotene bleaching method, and the protectionagainst DNA damage induced by hydroxyl free radicals to evaluation the antioxidant acitivity and the protection against DNA antioxidative aamage ofsalvianolic acid systematically. Besides these vitro models, we alsoestablished a acute hepatic injury model in rats, in order to study theprotctive effect of oxidative damage tissue by salvianolic acid in vivo.Moreover, a rapid evaluation system of antioxidant activity of salvianolicacid was established by using an on-line HPLC-DPPH method. A HPLCmethod to determine angiotensin-â… converting enzyme inhibitor (ACEI)activity of salvianolic acid in vitro was established also, so as to study therelationship of reactive oxygen species (ROS) and the bioactive ofsalvianolic acid.Results:1. The extraction yields based on the Danshen plant dry weight of theAE, EE, and RE were 73.2%, 3.6%, 2.2%, respectively. The colour of AEand EE was dark brown, and that of RE was yellow. The character ofsalvianolic acids were stable and had good powdery. The HPLC analysisshowed that there were 8 common peaks in the chromatogram of AE, EE,and RE. The qualitation of danshensu (DSS), caffeic acid (CA) andsalvianolic acid B (Sal B) in AE, EE, and RE were determined by standardphenolic acid. In addition, the quantify of SalB in AE, EE, RE were about5.60%, 74.38%, 90.45%, respectively. Great significance (p＜0.05) wasobserved of the content of Sal B under the temperature about 80℃and 90℃compared with 60℃after heating 120 minutes. However, the content of SalB was showed no significant difference under the pH3.2. Rresults suggested that salvianolic acid AE, EE and RE exhibitedpositive but different levels of antioxidant activity by all the testingprotocols employed. When the concentration at 100μg/mL, the DPPH radical scavenging activity of AE,EE,RE were 35.87±0.34, 88.55±0.12, 89.48±0.12 respectively, the reducing power (expressed as absorbance)were 0.106±0.0015, 0.7624±0.0053, 0.8016±0.0076 respectively, theferrous metal ions chelating ability were 2.08±0.05,1.28±0.22,0.39±0.03 respectively, and the antioxidant activity in theβ-carotene bleachingsystem were 2.63±0.09,8.73±1.74,14.47±2.19 respectively. In addition, The IC50 value of chemiluminescence of AE, EE and RE were 1293.6, 62.0, and 49.2μg/mL. The results showed that the extracts exhibited adose-dependent antioxidant and free radical scavenging activities by theDPPH radical scavenging, reducing power models, the antioxidant activityin theβ-carotene bleaching system and the protect against DNA damage. Astatistically analysis of the datas sets revealed that there was a strongcorrelation between the Sal B content and the antioxidant activity asmeasured by the DPPH radical scavenging, reducing power models andPhen-Cu-AscA-H₂O₂-DNA reaction system. The highest correlationcoefficient(r) as measured by reducing power models models was 0.9644, and "r" as measured by Phen-Cu-AscA-H₂O₂-DNA reaction system was0.9012.3. Salvianolic acid RE had the protective effects on acute hepatic injuryin rats, the results showed that the antioxidative activity in model group werereducd. Compared with model group, RE reduced the increase of ALT andAST induced by CCl₄ (p＜0.05), the activities of SOD and GSH in REgroup were increased and the concentrations of MDA were descreasedsignificantly (p＜0.05). Degeneration and necrosis of liver tissues wererelieved.4. This new on-line HPLC-DPPH method was applied for a screening of several radical scavenging components in this salvianolic acid extract aswell as for quantitative analysis. The results suggested that there were 13different salvianolic acids had quenching ability towards to DPPH radical atthe highest concentration altogether, and the DPPH radical-scavengingactivity increased with increasing concentrations of the salvianolic acidextract. Salvianolic acid B which the appearance time at 55.8 minute showedthe highest DPPH radical-scavenging activity, and inferior activity ofsalvianolic acid were the appearance time at SA8＞SA6＞SA12＞SA5＞SA1 in sequence.5. A HPLC method to determine angiotensin-â… converting enzymeinhibitor (ACEI) activity of salvianolic acid in vitro was established, by usingN-hippuryl-His-Leu tetrahhydrate as the reaction substrate and hippuric acidas the reaction product. XiangDan injection and salvianolic acid EE hadeffective inhibitory action to ACE. The inhibitory activity of XiangDaninjection was 39.95% when the concentration was 1/8 of originconcentration. The inhibitory activity of salvianolic acid EE was 31.44%when the concentration was 1mg/mL. It was found that there was adose-response curves for the ACEI activity and the concentration ofsalvianolic acid. The ACEI activity of DSS, CA, protocatechualdehyde andSal B were about 94.70%, 62.26%, 49.39%, 23.14% when the concentrationat 1mg/mL respectively.Conclusions: The above mentioned results suggested that theestablished HPLC conditions could be used to assign and superficialsyndrome the different compositions of salvianolic acid. From five testingprotocols employed, salvianolic acid exhibited positive antioxidant activityand protective effect against DNA damage. Sal B was a major factor responsible 1or the antioxidant activity. The effect of salvianolic acid onantioxidant activity could be through the hydrogen donating ability of thephenolic functionalities. Protection DNA damage by prevented breaking ofDNA chain and made the process delay. Salvianolic acid also showedprotective effect against CCl₄-induced acute liver injuries in rats, and themechanism was relevant to improving the antioxidative activity of rats.The rapid evaluation of antioxidant activity of multicomponentsalvianolic acid was determined by using an on-line HPLC-DPPH method.The HPLC-DPPH analysis revealed the presence of several radicalscavenging components in salvianolic acid extract. The dominantantioxidative composition in the testing salvianolic acid extract wasidentified as Sal B. The results of ACE inhibitory activity of salvianolic acidand its characteristic assignment suggested that multicomponent andmonomer salvianolic acid all had ACEI activity, the antioxidant ofsalvianolic acid may be permeate in the Renin-Angiotensin-System (RAS), involve in the regulation of RAS.