Flower of Abelmoschus manihot (FAM) is clinically effective to treat chronic kidney disease (CKD) with a relatively high dosage. To improve the efficacy and the compliance of patients, macroporous resins were adopted to enrich and purify flavonoids from FAM, which are thought to be the major renal protective constituents in FAM. After screening six different kinds of macroporous resins, HPD-100 was selected for its great adsorption and desorption capacity. Then, orthogonal design tests were used to optimize parameters in the processes of impurity removal and flavonoids of FAM desorption on column chromatogram. Moreover, process scale-up was performed, and purification effects maintained after amplification. After purification, the content of seven main flavonoids in the product increased from 8.29% to 51.43%. Protective and anti-inflammatory effects of crude extract and the flavonoid component of FAM after purification were investigated on the adriamycin-damaged HK-2 cells and lipopolysaccharide-stimulated Raw 264.7 cells models. Both bioactivities were improved greatly after purification for these two cell models. Therefore, the purification process had enriched the main bioactive constituents with potential alleviating kidney injury activities. The flavonoid component of FAM is worthy of being developed as an improved remedy for CKD with better patients’ compliance.

Metabonomics is a useful tool for studying mechanisms of drug treatment using systematic metabolite profiles. Ginsenosides Rg1 and Rb1, ophiopogonin D, and schizandrin are the main bioactive components of a traditional Chinese formula (Sheng-Mai San) widely used for the treatment of coronary heart disease. It remains unknown the effect of individual bioactive component and how the multi-components in combination affect the treating acute myocardial infarction (AMI). Rats were divided into 7 groups and dosed consecutively for 7 days with mono and combined-therapy administrations. Serum samples were analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy. Partial least squares discriminate analysis (PLS-DA) was employed to distinguish the metabolic profile of rats in different groups and identify potential biomarkers. Score plots of PLS-DA exhibited that combined-therapy groups were significantly different from AMI group, whereas no differences were observed for mono-therapy groups. We found that AMI caused comprehensive metabolic changes involving stimulation of glycolysis, suppression of fatty acid oxidation, together with disturbed metabolism of arachidonic acid, linoleate, leukotriene, glycerophospholipid, phosphatidylinositol phosphate, and some amino acids. β-hydroxybutyrate, cholines and glucose were regulated by mono-therapy of schizandrin and ginsenosides respectively. Besides these metabolites, combined-therapy ameliorated more of the AMI-induced metabolic changes including glycerol, and O-acetyl glycoprotein. A remarkable reduction of lactate suggested the therapeutic effect of combined-therapy through improving myocardial energy metabolism. This study provided novel metabonomic insights on the mechanism of synergistic cardioprotection of combined-therapy with ginsenosides, schizandrin, and ophiopogonin D, and demonstrated the potential of discovering new drugs by combining bioactive components from traditional Chinese formula.

Mitochondrial quality control, regulated by mitochondrial dynamics and mitophagy, has been regarded as pivotal process to induce segregation of mitochondria during myocardial ischemia/reperfusion (I/R) injury. However, few works revealed the regulation of mitochondrial quality control by therapeutic agents. Tongmai formula (TM) is a clinically used botanical drug for treating cardiovascular diseases, which mechanism is unveiled. Thus, in this study, we investigated the pharmacological effects of TM on modulating mitochondrial quality control during cardiac injury. Rats subjected to myocardial I/R injury and neonatal rat ventricular myocytes (NRVMs) exposed to hypoxia/reoxygenation (H/R) were used to simulate cardiac injury during myocardial ischemia/reperfusion process. Morphological examination, histopathological examination, echocardiography, and immunohistochemistry were used to determine the cardiac injury after I/R injury. Biochemical indices in serum were estimated by the enzyme-linked immunosorbent assays (ELISA). 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used for mitochondrial membrane potential (ΔΨm) evaluation. 2',7'-dichlorofluorescin diacetate (DCFH-DA) was used for intracellular reactive oxygen species (ROS) evaluation. Mitochondria in NRVMs were labeled by tetramethylrhodamine methyl ester (TMRM) for mitochondrial morphosis imaging and estimation. Western blotting was used for cytochrome c (CYCS), apoptosis inducing factor (AIF) and mitofusin 2 (Mfn2) contents evaluation. Immunochemistry fluorescence was used for dynamin related protein 1 (Drp1) expression measurement. TM treatment markedly decreased myocardium infarct size. It also significantly improved left ventricular contractile function and alleviated cardiomyocytes apoptosis, as well as reduced the production of cardiac troponin T, creatine kinase, lactate dehydrogenase, malondialdehyde and elevated glutathione and superoxide dismutase. Intriguingly, we found that mitochondrial membrane potential loss and mitochondrial permeability transition pore (mPTP) opening were recovered after TM treatment. It also down-regulated cytochrome c and apoptosis inducing factor contents after myocardial I/R injury. In vitro study showed that TM treatment reduced intracellular ROS content and recovered ΔΨm in NRVMs after H/R injury. We also observed that TM could reduce the expression level of Drp1, while increased Mfn2 in NRVMs after H/R injury, which indicates that TM may regulate mitochondrial dynamics during H/R injury of NRVMs. TM exhibited cardiac protective effect on ischemic myocardium of rats after reperfusion and improved mitochondrial quality control through mitochondrial dynamics in NRVMs after H/R injury.

Natural products are great treasure troves for the discovery of bioactive components. Current bioassay guided fractionation for identification of bioactive components is time- and workload-consuming. In this study, we proposed a robust and convenient strategy for deciphering the bioactive profile of natural products by mass spectral molecular networking combined with rapid bioassay. As a proof-of-concept, the strategy was applied to identify angiotensin converting enzyme (ACE) inhibitors of Fangjihuangqi decoction (FJHQD), a traditional medicine clinically used for the treatment of heart failure. The chemical profile of FJHQD was comprehensively revealed with the assistance of tandem mass spectral molecular networking, and a total of 165 compounds were identified. With characterized constituents, potential clinical applications of FJHQD were predicted by Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, and a range of cardiovascular related diseases were significantly enriched. ACE inhibitory activities of FJHQD and its constituents were then investigated with an aggregation-induced emission based fluorescent probe. FJHQD exhibited excellent ACE inhibitory effects, and a bioactive molecular network was established to elucidate the ACE inhibitory profile of constituents in FJHQD. This bioactive molecular network provided a panoramic view of FJHQD's ACE inhibitory activities, which demonstrated that flavones from Astragali Radix and Glycyrrhizae Radix et Rhizoma, saponins from Astragali Radix, and sesquiterpenoids from Atractylodis Macrocephalae Rhizoma were principal components responsible for this effect of FJHQD. Among them, four novel ACE inhibitors were the first to be reported. Our study indicated that the proposed strategy offers a useful approach to uncover the bioactive profile of traditional medicines and provides a pragmatic workflow for exploring bioactive components.