A network pharmacology-based study on the quality control markers of antithrombotic herbs: Using Salvia miltiorrhiza - Ligusticum chuanxiong as an example.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza (Danshen, DS), the dried root and rhizome of Salvia miltiorrhiza Bunge and Ligusticum chuanxiong (Chuanxiong, CX), the dried rhizomes of Ligusticum striatum DC are effective in invigorating blood circulation and eliminating stasis which is highly related with cardiovascular disease (CVD). AIM OF STUDY: The identification of activity-based chemical markers is very important, but the complex mechanism of "multi-component, multi-target, and multi-effect" within traditional Chinese medicine (TCM) poses a great challenge to this work. In this study, we combined network pharmacological prediction with experimental validation of the DS and CX to explore an effective method for discovering quality control (QC) of antithrombotic herbs by clarifying the intermediate layer "module/cluster" between the whole complex system and a single component. MATERIALS AND METHODS: Based on structural similarity analysis of compound and the thrombosis network published before, we firstly modularized two layers called chemical cluster (CC) network and functional module (FM) network respectively and linked them into one bilayer modularized compound target (BMCT) network. "Two-step" calculation was applied on identifying the significant compounds as the potential QC markers from CC. The in vitro inhibitory activity of selected QC marker compounds on thrombin was evaluated to partially verify their pharmacological activities. HPLC was used to determine contents. RESULTS: According to the network-based analysis, nine compounds with great importance in the BMCT network were identified as QC markers of DS-CX, including tanshinone I, tanshinone IIA, cryptotanshinone, salvianolic acid B, ferulic acid, salvianolic acid A, rosmarinic acid, chlorogenic acid, and coniferyl ferulate. Enzyme inhibitory test partially verified the activity of tanshinone I and tanshinone IIA. Chemical profiling indicated that the nine marker compounds are the main components in the herbal pair. CONCLUSIONS: This study identified activity-based QC markers of DS-CX herbal pair and provided a new methodology that can be used in the QC of other herbs, herbal pairs, or formulas.

Effect of Qizhitongluo capsule on lower limb rehabilitation after stroke: A randomized clinical trial.

BACKGROUND: An individual's level of lower limb motor function is associated with his or her disability level after stroke, and motor improvement may lead to a better prognosis and quality of life. Data from animal models show that Qizhitongluo (QZTL) capsule facilitates recovery after focal brain injury. We aimed to validate the efficacy and safety of the QZTL capsule for promoting lower limb motor recovery in poststroke patients. METHODS: In this randomized, multicenter, double-blind, placebo- and active-controlled trial from 13 sites in China, participants with ischemic stroke and Fugl-Meyer motor scale (FMMS) scores of <95 were eligible for inclusion. Patients were randomly assigned in a 2:1:1 ratio to the QZTL group, Naoxintong (NXT) group or placebo group for 12 weeks at 15-28 days after the onset of stroke. The primary outcome was the change in the Lower Limb FMMS (FMMS-LL) score from baseline over the 12-week intervention period. RESULTS: 622 participants were randomly assigned to the QZTL group (309), NXT group (159), or placebo group (154). The FMMS-LL score increased by 4.81 points (95 % CI, 4.27-5.35) in the QZTL group, by 3.77 points (95 % CI, 3.03-4.51) in the NXT group and by 3.00 points (95 % CI, 3.03-4.51) in the placebo group at week 12. The QZTL group showed significantly larger improvements compared with the placebo group at each interview from weeks 4-12 (difference, 0.89 [0.30,1.49] at week 4, P = 0.0032; difference, 1.83[1.01,2.66] at 90 days poststroke, P < 0.0001; difference, 1.81[0.88,2.74] at week 12, P = 0.0001). CONCLUSION: The QZTL capsule is an effective treatment for lower limb motor impairment. The finding indicates that the QZTL capsule may be used as a potential new strategy for stroke rehabilitation.

Improved phenolic acid content and bioactivities of Salvia miltiorrhiza hairy roots by genetic manipulation of RAS and CYP98A14.

Phenolic acids from Salvia miltiorrhiza have been widely used in nutritious, health-promoting products with an increasing demand. In the current study, two biosynthetic genes RAS (rosmarinic acid synthase) and CYP98A14 (a cytochrome P450-dependent monooxygenase) were successfully introduced into S. miltiorrhiza hairy roots. Overexpression of RAS and CYP98A14 resulted in higher content of phenolic acids (up to over 3-fold) in transgenic lines compared to the control. Meanwhile, DPPH results revealed that engineered S. miltiorrhiza hairy roots had stronger antioxidant activities than the control. In addition, phenolic acid crude extracts of the engineered hairy root lines overexpressing RAS or CYP98A14 showed improved antibacterial activities compared to the control lines. Our work exhibits a useful strategy for enhancement of phenolic acid production and bioactivities of S. miltiorrhiza hairy roots by genetic manipulation of RAS and CYP98A14, and also provides a new resource material to obtain active phenolic acids for food and healthy products.

Nose to brain drug delivery - A promising strategy for active components from herbal medicine for treating cerebral ischemia reperfusion.

Cerebral ischemia reperfusion injury (CIRI), one of the major causes of death from stroke in the world, not only causes tremendous damage to human health, but also brings heavy economic burden to society. Current available treatments for CIRI, including mechanical therapies and drug therapies, are often accompanied by significant side-effects. Therefore, it is necessary to discovery new strategies for treating CIRI. Many studies have confirmed that the herbal medicine has the advantages of abundant resources, good curative effect and little side effects, which can be used as potential drug for treatment of CIRI through multiple targets. It's known that oral administration commonly has low bioavailability, and injection administration is inconvenient and unsafe. Many drugs can't delivery to brain through routine pathways due to the blood-brain-barrier (BBB). Interestingly, increasing evidences have suggested the nasal administration is a potential direct route to transport drug into brain avoiding the BBB and has the characteristics of high bioavailability for treating brain diseases. Therefore, intranasal administration can be treated as an alternative way to treat brain diseases. In the present review, effective methods to treat CIRI by using active ingredients derived from herbal medicine through nose to brain drug delivery (NBDD) are updated and discussed, and some related pharmacological mechanisms have also been emphasized. Our present study would be beneficial for the further drug development of natural agents from herbal medicines via NBDD.

Pharmacodynamics and pharmacokinetics of Danshen in isoproterenol-induced acute myocardial ischemic injury combined with Honghua.

ETHNOPHARMACOLOGICAL RELEVANCE: Herb pair, the most fundamental and simplest form of herb compatibility, serves as the basic building block of traditional Chinese medicine formulae. The Danshen-Honghua herb pair (DH), composed of Salviae Miltiorrhizae Radix et Rhizoma (Danshen in Chinese) and Carthami Flos (Honghua in Chinese), has remarkable clinical efficacy to cure cardio-cerebrovascular diseases. This study was designed to investigate the pharmacodynamics of DH in comparison with single herbs and pharmacokinetics of DH relative to Danshen in acute myocardial ischemic injury. MATERIALS AND METHODS: Sixty male Wistar rats were divided into control, model and drug treated groups. The acute myocardial ischemia rat model was induced by administering 85 mg/kg/d isoproterenol (ISO) subcutaneously for two consecutive days. For pharmacodynamic study, histopathological and biochemical analysis were performed to assess the anti-myocardial ischemic effects. While for pharmacokinetic study, a UPLC-MS/MS method was developed for determination of nine main active ingredients, namely danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, lithospermic acid, rosmarinic acid, salvianolic acid B, salvianolic acid A and salvianolic acid C in rat plasma. RESULTS: The histopathological and biochemical analysis revealed that DH exerted enhanced anti-myocardial ischemic effects against the ISO-induced myocardial ischemia compared with single herbs. The pharmacokinetic study indicated that DH could significantly increase the t1/2z of danshensu, Tmax, AUC0-∞ and MRT0-t of protocatechuic acid in comparison with Danshen alone in normal rats, but more importantly elevate systemic exposure level and prolong t1/2z of protocatechualdehyde, caffeic acid, Tmax of danshensu in acute myocardial ischemia rats. CONCLUSIONS: Our findings demonstrated the greater effects of DH after the compatibility in ISO-induced acute myocardial ischemia rats at pharmacodynamic and pharmacokinetic levels and provided valuable information for clinical application of herb pairs.

Ethanol extracts of Danlou tablet attenuate atherosclerosis via inhibiting inflammation and promoting lipid effluent.

As a chronic inflammatory disease, atherosclerosis is characterized by accumulation of lipid-rich macrophages on the inner walls of arteries. Deposited macrophages promote atherosclerotic lesion progression; therefore they are viewed as the main targets in order to alleviate atherosclerosis. Danlou tablet, a patented Chinese Medicine, has long been used to treat cardiovascular diseases. In the present study, we used Apolipoprotein E-deficient (ApoE-/-) mice model and in vitro cell line of RAW264.7 to explore the mechanisms of ethanol extracts of Danlou tablet (EEDT) in treating atherosclerosis. The potential targets that EEDT works to treat atherosclerosis were predicted by "Network pharmacology analysis", based on which we designed mRNA array of 93 genes. Then mRNA array and oil red O staining were performed in aortic extracted from the cohorts of Control (C57BL/6 mice, chow fed), Model (ApoE-/- C57BL/6 mice, 20 weeks of high-fat diet) and EEDT intervening (ApoE-/- mice, 20 weeks of high-fat diet with 12 weeks of EEDT treatment) group. Furthermore, mRNA array, inflammation cytokines and lipid content were examined in RAW264.7 cell line. It was showed that EEDT decreased the expressions of inflammation cytokines by down regulating NF-κB singling pathway and accelerated cholesterol effluent through activating PPARα/ABCA1 signaling pathway. On the other hand, activation of NF-κB pathway or suppression of PPARα/ABCA1 signaling pathway both abolished the therapeutic effect of EEDT. In conclusion, EEDT played a key role in anti-inflammation and preventing lipid deposition in macrophages of atherosclerosis via suppressing NF-κB signaling and triggering PPARα/ABCA1 signaling pathway.

Danhong injection in cardiovascular and cerebrovascular diseases: Pharmacological actions, molecular mechanisms, and therapeutic potential.

Cardiovascular and cerebrovascular diseases are the main cause of mortality worldwide, currently with less than optimum therapeutic options. Danhong injection (DHI) is a medicinal preparation based on two eminent Chinese herbal medicines, Salviae Miltiorrhizae (Dan Shen; family: Lamiaceae) and Flos Carthami (Hong Hua; family: Compositae/Asteraceae). DHI has been mainly used in the clinical therapy of cardiovascular (such as acute coronary syndrome and angina pectoris) and cerebrovascular diseases (such as stroke) in China for many years. The pharmacological properties of DHI include anti-inflammatory, anti-oxidant, anti-coagulatory, hypolipidemic, anti-apoptotic, vasodilatory, and angiogenesis-promoting actions. DHI offers a safe and effective therapeutic agent against cardiovascular and cerebrovascular diseases by modulating multiple disease-relevant signaling pathways and molecular targets. Herein, we provide a comprehensive review of the phytochemistry, therapeutic effects, molecular mechanisms, and adverse reactions of DHI in cardiovascular and cerebrovascular diseases. We also highlight the latest pharmacological advances and therapeutic potential of this promising herb-derived cardiovascular drug preparation.

The AP2/ERF transcription factor SmERF1L1 regulates the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza.

Tanshinones and phenolic acids are two important metabolites synthesized by the traditional Chinese medicinal plant Salvia miltiorrhiza. There is increasing market demand for these compounds. Here, we isolated and functionally characterized SmERF1L1, a novel JA (Jasmonic acid)-responsive gene encoding AP2/ERF transcription factor, from Salvia miltiorrhiza. SmERF1L1 was responsive to methyl jasmonate (MJ), yeast extraction (YE), salicylic acid (SA) and ethylene treatments. Subcellular localization assay indicated that SmERF1L1 located in the nucleus. Overexpression of SmERF1L1 significantly increased tanshinones production in transgenic S. miltiorrhiza hairy roots by comprehensively upregulating tanshinone biosynthetic pathway genes, especially SmDXR. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that SmERF1L1 binds to the GCC-box of SmDXR promoter while dual luciferase (Dual-LUC) assay showed that SmERF1L1 positively regulated the expression of SmDXR. Our study suggested that the SmERF1L1 may be a good potential target for further metabolic engineering of bioactive component biosynthesis in S. miltiorrhiza.

Network pharmacology-based study on the mechanism of action for herbal medicines in Alzheimer treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Alzheimer's disease (AD), as the most common type of dementia, has brought a heavy economic burden to healthcare system around the world. However, currently there is still lack of effective treatment for AD patients. Herbal medicines, featured as multiple herbs, ingredients and targets, have accumulated a great deal of valuable experience in treating AD although the exact molecular mechanisms are still unclear. MATERIALS AND METHODS: In this investigation, we proposed a network pharmacology-based method, which combined large-scale text-mining, drug-likeness filtering, target prediction and network analysis to decipher the mechanisms of action for the most widely studied medicinal herbs in AD treatment. RESULTS: The text mining of PubMed resulted in 10 herbs exhibiting significant correlations with AD. Subsequently, after drug-likeness filtering, 1016 compounds were remaining for 10 herbs, followed by structure clustering to sum up chemical scaffolds of herb ingredients. Based on target prediction results performed by our in-house protocol named AlzhCPI, compound-target (C-T) and target-pathway (T-P) networks were constructed to decipher the mechanism of action for anti-AD herbs. CONCLUSIONS: Overall, this approach provided a novel strategy to explore the mechanisms of herbal medicine from a holistic perspective.

In vivo distribution and pharmacokinetics of multiple active components from Danshen and Sanqi and their combination via inner ear administration.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge (Labiatae sp. plant, Chinese name Danshen) and Panax notoginseng (Burk.) F. H. Chen (Araliaceae plant, Chinese name Sanqi) have a long history in treating coronary heart disease, cerebrovascular disease and inner ear disorders in traditional Chinese medicine. To provide a rational basis for the use of these herbs in clinical practice, we investigated the in vivo distribution and pharmacokinetics of marker agents in Danshen and Sanqi via intravenous and inner ear administration and explored the potential interactions of these agents in compound prescription. MATERIALS AND METHODS: Guinea pigs were given Danshen extracts (salvianolic acid B, tanshinone IIA), Sanqi extracts (Panax notoginseng saponins) and combination of the two extracts via intravenous and intratympanic administration (IT). Samples from the brain, inner ear perilymph (PL), cerebrospinal fluid (CSF) and plasma were collected at different time points. The concentration of salvianolic acid B (Sal B), tanshinone IIA (Ts IIA), notoginsenoside R1 (R1), ginsenoside Rg1 (Rg1) and ginsenoside Rb1 (Rb1) was determined by high-performance liquid chromatography coupled with a diode array detector (DAD). Pharmacokinetic parameters were estimated using non-compartmental methods. RESULTS: Local drug application via inner ear greatly improved drug distribution within the PL, CSF and brain tissues compared with intravenous administration (IV). The values of Cmax and AUC(0-t) after IT were significantly higher than IV. In comparison with IT of Danshen and Sanqi alone, the pharmacokinetic parameters for R1, Rg1, Rb1, Sal B and Ts IIA were markedly different in the compound prescription. The compound compatibility enhanced the transport of Danshen components into the brain through the inner ear and apparently prolonged the retention time in CSF while decreasing the distribution of Sanqi components in the inner ear and brain. CONCLUSIONS: The results indicated that local drug application to the inner ear was a more effective delivery route than systemic administration. Co-administration of Danshen and Sanqi could cause significant pharmacokinetic herb-herb interactions in guinea pigs. The multiple active components via inner ear administration might be promising candidates for the treatment of inner ear and brain diseases.

Expression of miR-106b-25 induced by salvianolic acid B inhibits epithelial-to-mesenchymal transition in HK-2 cells.

Epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in renal fibrosis. Previous studies have shown that transforming growth factor (TGF)-ß1 induces EMT in human kidney proximal tubular epithelial cells (HK-2 cells), whereas salvianolic acid B (Sal B) has a protective effect against EMT. The molecular pathogenesis of such processes is currently not well understood. In this study, a miRCURYTM LNA Array was used to screen HK-2 cells for expression changes of microRNAs (miRNAs) implicated in EMT. After validation by real-time PCR, all three members of the miR-106b-25 cluster (miR-106b, miR-93, and miR-25) were found to be markedly down-regulated during EMT in response to TGF-ß1, whereas these miRNAs were up-regulated by Sal B treatment in a dose-dependent manner. Moreover, enhanced expression of miR-106b attenuated EMT by retaining the epithelial morphology of HK-2 cells, reducing the levels of α-smooth muscle actin (α-SMA), and increasing the levels of E-cadherin. To explore the molecular basis underlying the inhibitive effect of the miR-106b-25 cluster against EMT, bioinformatics analysis revealed that TGF-ß type II receptor, a regulator of TGF-ß signaling, might be a direct target of the miR-106b-25 cluster. In turn, low levels of TGF-ß type II receptor in EMT of HK-2 cells were shown under the increase of miR-106b. In conclusion, our data suggest that the miR-106b-25 cluster may contribute to EMT in the kidney, and is involved in the protective effect of Sal B. Targeting of specific miRNAs may be a novel therapeutic approach to treat renal fibrosis.

Network pharmacology analyses of the antithrombotic pharmacological mechanism of Fufang Xueshuantong Capsule with experimental support using disseminated intravascular coagulation rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Xueshuantong (FXST) Capsule is developed on a traditional Chinese medicine remedy, with a four-herb formula of Panax notoginseng, Radix astragali, Salvia miltiorrhizae and Radix scrophulariaceae. It has been used for treatment of the clinic cardiovascular disease for many years. MATERIALS AND METHODS: Due to its complexity of compositions and polypharmacological effects, it often complicates understanding of the mechanisms of action. In the present work, we have constructed an integrated model of system pharmacology to investigate the polypharmacological mechanisms of FXST formulation for treatment of thrombosis disease. RESULTS: The predicted results showed that 22 ingredients in FXST were closely associated with 41 protein targets related to blood coagulation, fibrinolysis and platelet aggregation. Through analysis of the compound-protein target association, significant cross-targets between each herb indicated the multiple active chemical ingredients might interact with the same target simultaneously and thus explained the synergistic mechanisms of the principle of Traditional Chinese medicines (TCMs) as ''Jun (emperor) - Chen (minister) - Zuo (adjuvant) - Shi (courier)''. To validate the polypharmacological effects predicted by our network pharmacology (NetPharm) analysis, we have carried out experimental investigation the effects of FXST on the disorders of the blood coagulation system in a lipopolysaccharide-induced disseminated intravascular coagulation (DIC) rat model. The results showed that FXST could significantly ameliorate the activation of coagulation system, which is congruent with the cross-target prediction by NetPharm approach. CONCLUSIONS: The combined investigations provide more insight into better understanding of the pharmacological mechanisms of FXST, and may also offer an alternative avenue to further explore the chemical and pharmacological basis of TCMs.