| 1 Background Coronary heart disease(CHD), which has another name called asischemic heart disease(IHD), is one of the world’s most threatening diseases because of its high rates of attack, disability and mortality. Traditional Chinese medicines(TCMs) and their preparations have been used as the most important therapeutic agents in curing CHD in China for more than 2000 years. Shuang Dan prescription(SD), composed of two traditional Chinese medicinal herbs(Cortex Moutan and Radix Salvia Miltiorrhizae), has been effectively used for the treatment of angina pectoris, myocardial infarction and other CHD symptoms in clinical practice. Despite its famous clinical application, the research on the pharmacy and pharmacology of SD is far from sufficient until now. Shuang Dan prescription actully consisted of dozens of chemical components from Cortex Moutan and Radix Salvia Miltiorrhizae. It invokes the conception of system-based intervention strategies which use multi-inputs to solve the complexity of CHD state. However, essential components have not been identified in SD, whereas precise mechanisms of SD remain to be addressed by using molecular approaches, thereby impeding the modernization of SD.In this study, the therapeutic bioactive components of SD were screened by serum medicinal chemistry based approach and cardioprotective effects and mechanisms were investigated through ISO-induced myocardial infarction rat model.2 Methods 2.1 In vitro analysis of Shuang Dan prescription: Separation was performed at 30 °C on ZORBAX SB C18(250 mm×4.6 mm 5 μm, Agilent) with a mobile phase gradient(flow rate: 1 m L·min-1) prepared from methanol and 2% aqueous glacial acetic acid(v/v). The detection wavelength was 280 nm. The method was applied for the identification of major components in chemical fingerprint of SD lyophilized powder. 2.2 In vivo analysis of Shuang Dan prescription: The aforesaid HPLC system was applied for detecting the compounds in dosed serum after oral administration of SD lyophilized powder. By comparative analysis of the chemical profiles of lyophilized powder, blank serum and dosed serum, potential bioactive compounds in SD prescription could then be discovered. 2.3 Influence of co-administered danshensu on pharmacokinetic fate and tissue distribution of paeonol in rats: HPLC analysis was performed on a Sino Chrom ODS?BP C18 column(250 mm×4.6 mm, 5 μm, Yilite). The mobile phase consisted of methanol-2% glacial acetic acid solution(70:30, v/v) at a flow rate of 1 m L·min-1. The temperature of the column was maintained at 30 °C. The detection wavelength was 274 nm. For pharmacokinetic and distribution study, rats were orally administrated with the dose of 40 mg·kg-1 paeonol and 40 mg·kg-1 paeonol coupled with 80 mg·kg-1 danshensu, respectively. Biosamples were collected at scheduled timepoints and then analyzed by HPLC systems. 2.4 Cardioprotective effect of paeonol and danshensu combination on isoproterenolinduced myocardial injury in rats: Paeonol(80 mg·kg-1) and danshensu(160 mg·kg-1) were administered orally to Sprague Dawley rats in individual or in combination for 21 days. At the end of this period, rats were administered isoproterenol(85 mg·kg-1)subcutaneously to induce myocardial injury. After induction, rats were anaesthetized with pentobarbital sodium(35 mg·kg-1) to record electrocardiogram, then sacrificed and biochemical assays of the heart tissues were performed.3 Results 3.1 In vitro analysis of Shuang Dan prescription: Under the optimum chromatographic condition, 11 main peaks exist in ten sample profiles were selected as the common peaks. The 11 compounds were simultaneously identified by UV spectrum and relative retention time compared with the reference standards. They are gallic acid, paeoniflorin and paeonol from Cortex Moutan and danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, lithospermic acid, rosmarinic acid, salvianolic acid B and salvianolic acid A from Radix Salvia Miltiorrhizae. The 11 compounds could be the main compoents in SD prescription. 3.2 In vivo analysis of Shuang Dan prescription: By comparing the retention time and UV spectra with those of reference standard, 8 prototype compounds from SD lyophilized powder were discovered as potential bioactive components in rat serum. They are gallic acid, paeoniflorin and paeonol from Cortex Moutan and danshensu, protocatechuic acid, caffeic acid, rosmarinic acid and salvianolic acid B from Radix Salvia Miltiorrhizae. 3.3 Influence of co-administered danshensu on pharmacokinetic fate and tissue distribution of paeonol in rats: When co-administrated danshensu, the peak plasma concentration of paeonol was decreased(P<0.01), the mean residence time(MRT) was prolonged(P<0.001), the volume of distribution(Vd/F) was increased(P<0.001) and the concentration of paeonol in heart, brain and lung were dramatically increased(P<0.001), compared with the rats administrated paeonol alone. The results showed that the co-administration of danshensu could alter pharmacokinetic fate and tissue distribution of paeonol in rats, especially in lung, heart and brain. 3.4 Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats: Induction of rats with isoproterenol resulted in a marked elevation(P<0.001) in ST-segment, infarct size, level of serum marker enzymes(c Tn I, CK-MB and LDH), c Tn I, TBARS, activity of caspase-3 and protein expression of Bax and a significant decrease(P<0.001) in the activities of endogenous antioxidants(SOD and CAT), phase II detoxification(GPx, GR, GST and NQO1) and protein expression of Bcl-2. When compared with individual treated groups, pretreatment with combination of paeonol and danshensu showed a significant(P<0.001) decrease in ST-segment elevation, infarct size, c Tn I, TBARS, activity of caspase-3 and protein expression of Bax and a significant increase in the activities of endogenous antioxidants and protein expression of Bcl-2 and Nrf2. Thus, paeonol and danshensu significantly counteracted the pronounced oxidative stress effect of ISO by inhibition of lipid peroxidation, restoration of antioxidant status, and decrease levels of myocardial marker enzymes.4 Conclusions In the present study, the therapeutic materials(bioactive components) of SD prescription were screened by serum serum medicinal chemistry based approach, gallic acid, paeoniflorin and paeonol from Cortex Moutan and danshensu, protocatechuic acid, caffeic acid, rosmarinic acid and salvianolic acid B from Radix Salvia Miltiorrhizae have demonstrated to be possible bioactive components of SD. After investigated the pharmacokinetitic actions of danshensu combined with paeonol, we comfirmed the combination use could exerted pharmacokinetitic synergy in rats. Cardioprotective effects and mechanisms of SD were also investigated by danshensu and paeonol combination through ISO-induced myocardial infarction rat model. The cardioprotective mechanism might be associated with the enhancement of antioxidant defense system through activating of Nrf2 signaling and anti-apoptosis through regulating Bax and Bcl-2. The results of present study could provide experimental evidence to support the rationality of combinatorial use of TCM in clinical practice, and furthermore givepossible scientific foundations for the redevelopment of Shuang Dan prescription. |
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