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Ten new scarce dihydrobenzophenanthridine alkaloids, including seven monomers, ambidalmines A1/2, B1/2–E (1a/b, 2a/b–5), and three dimers, ambidimerines F1 (6a), F2 (6b) and F3 (6c), were isolated from the tubers of Corydalis ambigua var. amurensis. All of these compounds were discovered in the forms of enantiomers. The structures were elucidated based on extensive spectroscopic analysis, with absolute configurations of the enantiomeric compounds assigned by single-crystal X-ray diffraction analysis, circular dichroism (CD) and optical rotations. Bioactivity evaluation showed that compounds 1a, 2a, 2b and 6b exhibit comparable protective effects on hypoxic H9C2 cells.

Pharmacology as a modern science was introduced in China approximately 150 years ago, and has been used since then to study traditional Chinese medicine (TCM). Pharmacology has experienced its own development over this time and continues to provide new tools for the study of TCM. In the present review, three models for the pharmacological study of TCM are considered: (i) chemistry-focused study; (ii) target-directed study; and (iii) systems-biology-based study. These approaches correspond to recent developments in pharmacology, and in particular to new tools available to the field. Representative achievements and the pharmacological tools used to study TCM are reviewed. Pharmacology has played, and will continue to play, an indispensable role in elucidating the chemical basis, biological targets, and mechanisms of action of TCM medicines, and in developing a scientific basis for the theory of TCM.

Kansui radix is a famous poisonous traditional Chinese medicine. However, due to its different types of constituents with broad polarity, a variety of UV absorptions and lack of the reference standards, it was difficult to simultaneously determine the main component in kanui radix. A single, multi-faceted, enhanced strategy, exogenous reference standard - single standard to determine multi-components method (ERS-SSDMC), was proposed. Thirteen major components of kansui radix, including three jatrophane diterpenoids, eight ingenane diterpenoids and two triterpenes, among which there were three pairs of isomers, were simultaneously assayed. A C8 column, packed with 2.7μm core-shell particles, was optimized to separate these constituents in 25min on HPLC instrument detected at a program wavelength. Ethyl benzoate employed as single exogenous reference standard. The method was fully validated with respect to linearity (r(2)>0.9995), LOQs (0.1-0.4μg/mL), precision, accuracy (92-114%, RSD

Licorice (Gancao) is derived from the dried roots and rhizomes of Glycyrrhiza species (Leguminosae) and appears as a component herb in about 60% of traditional Chinese medicine (TCM) prescriptions. Modern pharmacological studies have shown that flavonoids are one class of the major components responsible for the bioactivities of licorice. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/QTOF MS) has proven to be a powerful tool for rapid profiling and identification of natural products in complex herbal medicines.A UPLC/QTOF MS method was established for the first time for profiling and structural characterization of the phenolic compounds (most of them flavonoids) in licorice. The combined use of data-independent acquisition (MS(E) ) and data-dependent acquisition (DDA) was illustrated.Fifteen flavonoid reference compounds were used to explore the fragmentation pathways. Compound identification was based upon the exact mass, general fragmentation behaviors, retention times, UV absorption, and the related botanical biogenesis. As a result, a total of 51 compounds were characterized, three of which were reported for the first time.The LC/MS analysis for each injection took less than 9 min. The developed method is fast, accurate and reliable due to its high resolution and high efficiency characteristics as a result of combining both UPLC separation and QTOF exact mass measurement.

It is a challenging task to simultaneously and quantitatively analyze multiple components in DFF [Da-Fu-Fang, namely, complex traditional Chinese medicine (TCM) preparations containing more than ten TCMs] due to their numerous and extreme complex chemical compositions possessing a wide variety of chemical and physical features, and their very low content. Rather than using a conventional mass spectrometry (MS) method with multiple reaction monitoring (MRM), in the current study, this challenge was addressed by using dynamic multiple reaction monitoring (DMRM). Using a DFF, Niuhuang Shangqing pill, which is composed of 19 TCMs, as a model, a rapid (one run in 20min), sensitive [lower limit of detection (LOD) and limit of quantitation (LOQ) were achieved comparable with MRM] and accessible (a standard HPLC/MS/MS instrumentation was employed) MS method was successfully developed for the simultaneous quantification of 41 bioactive components which represented 15 of the 19 medicinal plants. A comparison of LOD and LOQ using MRM and DMRM was made to quantitatively reveal that the latter demonstrated advantages over the former. Meanwhile, a standard operating procedure concerning the development of a new DMRM method was recommended. The MS data were obtained in the positive ion mode with electrospray ionization as the ion source, acetonitrile and water as mobile phase and a Kinetex C18 core-shell column (100mm×2.10mm, 2.6μm, Phenomenex Inc.) as the analytical column. This method was then applied to 32 batches of samples. It transpired, through principal component analysis and orthogonal partial least squares discriminant analysis, that the consistency of the products was relatively good within one company, but poor among different companies among the 32 samples; one failed to qualify in terms of the Chinese Pharmacopeia. This work illustrated that the proposed DMRM method was particularly suitable for quantifying the trace components in DFF and capable of ensuring the quality of DFF.

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase.

Aim of the study To investigate the chemical differences between Ganoderma lucidum (G. lucidum, Chizhi) and Ganoderma sinense (G. sinense, Zizhi). Materials and methods Thirty two batches of commercial Ganoderma samples were collected, including 20 batches of G. lucidum and 12 batches of G. sinense cultivated in different geographical regions. Chemical substances in aqueous extract and alcoholic extract, mainly polysaccharides and triterpenes respectively, were investigated. Determination of polysaccharides was carried out with a high performance liquid chromatography with an variable wavelength detector. Meanwhile, analysis of triterpenes were performed on an ultraviolet spectrophotometer, an ultra performance liquid chromatography and a rapid resolution liquid chromatograph combined with an electrospray ionization mass spectrometer. Chromatograms and spectra for all batches and reference standards of main components were obtained and used for direct comparison. Further discussion was made on the basis of the result of principal component analysis (PCA). Results Significant difference of triterpenes was shown between G. lucidum and G. sinense. In 20 batches of G. lucidum, 12 main components, including eight ganoderic acids and four ganoderenic acids were identified and ten of them were quantitatively determined, with the total content from 0.249% to 0.690%. However, none of those triterpenes was found in either batch of G. sinense. As for constituents of polysaccharides, seven monosaccharides were identified and four main components among them were quantitatively determined. Difference of polysaccharides was not directly observed, but latent information was revealed by PCA and the discrimination became feasible. Conclusions G. lucidum and G. sinense were chemically different, which might result in pharmacological distinction. Preparations of traditional Chinese medicine (TCM) from Ganoderma should make accurate specification on the origin of species.

Ethnopharmacological relevance Luan-Pao-Prescription (LPP) has been clinically proven to be effective on infertility. In the present study we explored the improvement and underlying mechanism of LPP on ovarian dysfunction in rats. Materials and methods 13 month old female rats were randomly divided into 4 groups: the Saline group, the LPP groups treated by low (1.67 g/kg), and high-dose (5 g/kg) LPP respectively, and the hormone group treated by pregnant mare gonadotrophin serum and chorionic gonadotrophin (PMSG/hCG). The estrous cycle was determined by daily observation of vaginal smears; serum estradiol and testosterone were estimated by enzyme-linked immunosorbent assay; ovarian morphology, ovary volume and fertility of female rats were all detected during the study. Results During 21 days of LPP treatment, about 20% increase of rats with regular estrous cycle of 4–6 days was found, but no change was detected on serum estradiol and testosterone at the dose of 1.67 g/kg and 5 g/kg LPP. Both ovary index and uterus index were up-regulated significantly at the dose of 5 g/kg LPP, but no regulation on oviduct index, adrenal gland index, pancreatic gland index and spleen index was observed at the two LPP groups. 5 g/kg LPP increased total number of pregnant mothers and the offspring; however there are no offspring in PMSG/hCG group. The offspring exhibited similar body weight in each treatment, and no apparent malformation was found for the cubs. While PMSG/hCG treatment increased the ovary index, serum estradiol and testosterone concentration considerably, but no improvement was found on estrous cycle, oviduct index, uterus index, and reproduction. Conclusion Administration of LPP may have comparable benefits for ovarian dysfunction, but with fewer side effects. Oral LPP have a better overall influence on rats than PSMG/hCG; it may be more effective in improvement of estrous cycle, ovary function and reproduction.

Ethnopharmacological relevance Salvia miltiorrhiza and Panax notoginseng are popularly used traditional Chinese medicine for cardiovascular disorders and they are often used in the form of combination. However, mechanisms of their cardioprotective effects were still not clear. In the present study, the protective effects of salvianolic acids (SA), notoginsengnosides (NG) and combination of SA and NG (CSN) against rat cardiac ischemia–reperfusion injury were checked and the protein expression profiles of heart tissues were examined to search their possible protein targets. Materials and methods The cardioprotective effects of SA, NG and CSN were checked in a rat model of ischemia–reperfusion (IR) by temporarily occluding coronary artery for 20 min followed by reperfusion. Rats were grouped into sham-operation group, IR group, IR + SA group, IR + NG group and IR + CSN group. The plasma creatine kinase (CK) activities were measured using commercial kit and the percentages of infarcted area in total ventricle tissue were calculated after nitroblue-tetrazolium (N-BT) staining of heart tissue slices. Two-dimensional protein electrophoresis (2-DE) was used to check the protein expression profiles of heart tissues. Then, proteins differentially expressed between IR group and sham-operation group were identified using matrix assisted laser desorption ionization-time of flight-mass spectrometry/mass spectrometry (MALDI-TOF MS/MS). The regulative effects of SA, NG and CSN on these IR-related proteins were analyzed. Results Treatments including SA, NG and CSN all showed cardioprotective effects against ischemia–reperfusion injury and CSN exhibited to be the best. Eighteen proteins involved in IR injury were found. These proteins are involved in pathways including energy metabolism, lipid metabolism, muscle contraction, heat shock stress, cell survival and proliferation. The regulation of these proteins by SA, NG or CSN suggested possible protein targets in their cardioprotective effects. Conclusions SA and NG showed both similarity and difference in their protein targets involved in cardioprotective effects. The capability of CSN to regulate both protein targets of SA and NG might be the basis of CSN to show cardioprotective effects better than that of SA or NG.

Targeting cellular function as a system rather than on the level of the single target significantly increases therapeutic potency. In the present study, we detect the target pathway of salvianolic acid B (SalB) in vivo. Acute myocardial infarction (AMI) was induced in rats followed by the treatment with 10 mg/kg SalB. Hemodynamic detection and pathological stain, 2-dimensional electrophoresis, MALDI-TOF MS/MS, Western blot, pathway identification, apoptosis assay and transmission electron microscope were used to elucidate the effects and mechanism of SalB on cardioprotection. Higher SalB concentration was found in ischemic area compared to no-ischemic area of heart, correlating with improved heart function and histological structure. Thirty-three proteins regulated by SalB in AMI rats were identified by biochemical analysis and were classified as the components of metabolism and apoptosis networks. SalB protected cardiomyocytes from apoptosis, inhibited poly (ADP-ribose) polymerase-1 pathway, and improved the integrity of mitochondrial and nucleus of heart tissue during AMI. Furthermore, the protective effects of SalB against apoptosis were verified in H9c2 cells. Our results provide evidence that SalB regulates multi-targets involved in the apoptosis pathway during AMI and therefore may be a candidate for novel therapeutics of heart diseases.

Cardiac fibroblasts play the key role in cardiac function and matrix metalloproteinases-9 (MMP-9) is a well known contributor to the development of myocardial remodeling. However, the direct regulation of MMP-9 on the function of cardiac fibroblasts and the underlying mechanism are far from elucidation. In the present research, recombinant protein encoding catalytic domain of MMP-9 (MMP-9 CD) was constructed and the function of neonatal cardiac fibroblasts was investigated by cell proliferation assay, migration assay, picrosirius red assay, multiplex cytokine assay and fibroblast phenotype detection. 200 nM MMP-9 CD stimulated cardiac fibroblasts migration (169.4±22.5% versus 100±0%, p

Tanshinone IIA (TSNIIA) is the major active component of Salvia miltorrhiza, which is used in treatment of cardiovascular and cerebrovascular diseases traditionally in China. However, the low bioavailability of TSNIIA limits its clinical usage. To reveal the mechanism relating the absorption of TSNIIA, we detected the expression of efflux transporters in Caco-2 cells under the stimulation of TSNIIA. TSNIIA was purified and above 98% purity was confirmed by high-performance liquid chromatography. The expression of nine efflux transporters including BCRP, hPepT1, MCT and MRP1-6 in Caco-2 were verified by reverse transcription polymerase chain reaction, and the up-regulation of TSNIIA on the expression of these transporters was found by reverse transcription real-time polymerase chain reaction. These results indicate that the low bioavailability of TSNIIA is partially due to its up-regulation on efflux transporters.

Salvianolic acid B (SB) is a natural compound with protective effect against ischemia-reperfusion heart injury. However, the signal network of SB including both direct target proteins and downstream signal-related proteins has not been clarified. In the present study, epidermal growth factor receptor (EGFR) was predicted to be the most possible direct protein target of SB by INVDOCK, a ligand-protein inverse-docking algorithm. Possible signal-related proteins of SB in H9C2 cells, including both under normal condition and under ischemia-reperfusion injury, were searched using 2-DE analysis. Totally, 14 signal-related proteins were found. Finally, signal network from EGFR to the signal-related proteins was established using bioinformatic analysis. Interestingly, 9 of the 14 signal-related proteins could be included in a network together with EGFR through direct interaction or only one intermediate partner. The signal cascade from EGFR to heat shock protein 27 (HSP27) and mitofilin (IMMT, inner membrane mitochondrial protein) might be the most important cascade. The signal network was certified by measuring the binding affinity of SB to EGFR in vitro, the effect of SB on internalization and phosphorylation of EGFR, the effect of SB on viability and proliferation of H9C2 cells, and the expression of inner membrane mitochondrial protein in the presence of EGFR inhibitor AG 1478.

Acute myocardial infarction (AMI) remains the leading cause of mortality in the world. Early intervention using salvianolic acids (SA) can substantially improve clinical outcomes. However, in spite of the great achievements that have been made in elucidating the protective effects of SA on AMI, the effects of SA on the contractile performance of the left ventricle (LV) and the underlying mechanism are still not so clear. In the present study, AMI was introduced by ligation of the left anterior descending coronary artery near the main pulmonary artery. Administration of SA significantly decreased infarct size, improved LV function and appearance of the myocardium and decreased myocardial malondialdehyde levels compared with the AMI group. Furthermore, treatment with SA significantly downregulated the mRNA expression level and activity of matrix metalloproteinase-9 (MMP-9), but did not regulate the tissue inhibitor of metalloproteinase-1 (TIMP-1) expression level at the infarct area. Lisinopril (an angiotensin converting enzyme inhibitor), which holds potential effects on cardioprotection, was chosen as the positive control in this study. Lisinopril elevated LV function and appearance of the myocardium, decreased malondialdehyde levels without an influence on infarct size, and regulated the MMP-9 enzyme level but not the MMP-9 mRNA and TIMP-1 protein levels. These findings suggest that early SA treatment is effective to improve LV function; and SA may exert preventative effects against myocardial remodeling after infarction.

Triterpenes are the main components with cytotoxicity in Ganoderma lucidum, which is used popularly as a complementary treatment for cancer therapy in traditional Chinese medicine. To investigate the possible interaction between chemotherapeutic agents and triterpenes extracted from G. lucidum, the cytotoxicity of doxorubicin (DOX) combined with Ganoderma triterpenes (GTS) or lucidenic acid N (LCN), a purified compound, was examined in HeLa cells. The combinations targeting DOX with GTS or LCN resulted in a synergistic interaction in HeLa cells. Moreover, to identify the molecular targets of GTS, two-dimensional gel electrophoresis-based comparative proteomics was carried out and proteins with altered expression levels after GTS treatment in HeLa cells were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. The results of our proteomic study indicated that the GTS treatment caused regulated expression of 14 proteins, which play important roles in cell proliferation, the cell cycle, apoptosis, and oxidative stress. Flow cytometric analysis confirmed that GTS could induce weak G(0)-G(1) phase arrest and combined use of GTS with DOX could induce apoptosis in cells. Furthermore, GTS enhanced the reactive oxygen species (ROS)-producing effect of DOX, and a ROS scavenger could affect the synergism between GTS and DOX. In cells with high Ku80 protein expression, the synergism between GTS and DOX was also partly affected. Importantly, in cells with high Ku80 expression that were treated with a ROS scavenger, the synergism between GTS and DOX totally disappeared. These results suggest that the synergism between GTS and DOX might be based on GTS-induced sensitization of cells to chemotherapeutics through enhanced oxidative stress, DNA damage, and apoptosis.

Although doxorubicin is an effective antitumor agent, the serious cardiotoxicity mediated by the production of reactive oxygen species has remained a considerable clinical problem. In China, salvianolic acids has been widely used for cardioprotection. To test whether salvianolic acids holds the potential to be protective against cardiotoxicity of doxorubicin, we created an acute cardiac injury mice model. Therapeutic treatment with salvianolic acids (40 mg/kg for 3 connective days) significantly reduced doxorubicin-induced (15 mg/kg) toxicity, including elevation of body weight and heart weight/tibia length ratio, decrease of creatine kinase, improvement of electrocardiography and heart vacuolation. Furthermore, the antioxidative effects of salvianolic acids were verified by oxygen radicals absorbance capacities assay in vitro and malondialdehyde detection in vivo, suggesting one possible mechanism of salvianolic acids on cardioprotection through blocking oxidative stress.

s / European Journal of Integrative Medicine 6 (2014) 686–745 731 Salvianolic acid A, a novel matrix metalloproteinase-9 inhibitor, reduces microvascular damage induced by hypertension Fukang Teng, Yanping Deng, Dengfang Li, Li Zhang, Tingting Zhang, Jun Yin, Baohong Jiang, De-an Guo ∗ Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China E-mail address: daguo@simm.ac.cn (D.-a. Guo). Introduction: Despite plenty of pharmacological agents available for hypertension therapy, hypertension-related damage to themicrovascular remodeling is not resolved. The present work aimed to investigate the protection of Salvianolic acid A (SalA), a novel matrix metalloproteinase-9 inhibitor, against microvascular remodeling induced by hypertension. Methods: Spontaneously hypertensive rats (SHR) were administered with 2.5, 5 or 10mg/kg SalA by intraperitoneal injection once a day for 4 weeks. Tail-cuff method was applied to measure blood pressure; microvascular structure of kidney and retina was detected by hematoxylin-eosin and/or immunohistochemical staining; collagen deposition around cardiac microvessels was determined by picric sirius red staining; isometric tensions of small mesenteric arteries were measured by DMTwiremyograph. SalA attenuated themicrovascular inward remodeling on retina and kidney, protected against collagen deposition around cardiac microvessels. Results: Besides the detection on microvascular structure, the improvement of SalA on microvascular vasodilatation was also confirmed based on wire myograph. Even though SalA had significant protection on microvessels, no antihypertensive effect of SalA was found. Conclusions: These results proved that SalA, a novel matrixmetalloproteinase-9 inhibitor, holds significant therapeutic potential for microvascular damage induced by hypertension. http://dx.doi.org/10.1016/j.eujim.2014.09.100 Fangchinoline targets proteasome 1 subunit to exert its anti-cancer effects Dong Li, Xiaobo Qu, Xuan Liu, Dean Guo ∗ Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China E-mail address: daguo@simm.ac.cn (D. Guo). Introduction: In our study screening natural proteasome inhibitors with compounds isolated from traditional Chinese medicines, fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore (known as Fangji in China), were identified as potent inhibitors for C-L activity of cellular proteasome in PC-3 prostate cancer cells. Fangji is a traditional Chinese medicine used for long time as an antirheumatic, anti-inflammatory and anticancer agent. In the present study, we characterized the inhibiting effects of fangchinoline on proteasome and the involvement of proteasome inhibition in its anti-tumor effects. Methods: The enzymatic activities of cellular proteasome in cell lysate were measured as reported in our previous paper. Fangchinoline dose-dependently inhibit the catalytic activities, especially the caspase-like activity mediated by catalytic 1 subunit, of cellular proteasome. The binding affinities of fangchinoline to recombinant proteasome catalytic subunits, 1, 2 and 5 subunit, were assayed in vitro using a SPR-based Biacore 3000 instrument as reported before. Results: Fangchinoline showed direct binding affinity with recombinant proteasome 1 subunit and also inhibited its enzyme activity in vitro. Inhibition of the proteasome activity by fangchinoline in PC-3 cells resulted in accumulation of ubiquitinated proteins including important proteasome substrates accompanied by cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts and accumulation of proteasome substrates. Conclusions: In conclusion, fangchinoline exhibited proteasome activity through direct binding with 1 subunit. Proteasome inhibition induced by fangchinoline contributed to its anti-cancer effects. http://dx.doi.org/10.1016/j.eujim.2014.09.101 Anti-cancer activities of BF211, a derivative of bufalin Lixing Feng, Lihong Hu, Xuan Liu, Dean Guo ∗ Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China E-mail address: daguo@simm.ac.cn (D. Guo). Introduction: Epidemiological data as well as in vitro and in vivo studies indicated that cardiac glycosides have anti-cancer activities and also emphasize the possibility of developing this class of compound as anti-cancer agents. Promising compounds such as Anvirzel and UNBS1450 are now in clinical trials in USA and Belgium, respectively. In China, Chansu, a traditional medicine containing bufadienolides, has long been used for treatment of cancer. Among the bufadienolides, bufalin appears to be the most active. However, its narrow therapeutic window limits its therapeutic application. Methods: To find possible new drug candidates for cancer therapy,BF211, a derivative of bufalin,was designed and synthesized. The patent protection for the preparation and anti-tumor usage of BF211 had been authorized (CN 102532235B). Results: Compared with bufalin, BF211 exhibited stronger anti-tumor activity and lower toxicity. The IC50 values of bufalin and BF211 in inhibiting the proliferation of human A549 lung carcinoma cells (72 h treatment) are 8.74± 2.52 and 0.77± 0.28 nM, respectively. The LD50 values of bufalin and BF211 inmice (i.p.) are 2.20 and 14.75mg/kg formalemice and 2.64 and 18.21mg/kg for female mice, respectively. BF211 at 2, 732 Abstracts / European Journal of Integrative Medicine 6 (2014) 686–745 4 and 6mg/kg doses (i.p.) exhibited dose-dependent anti-cancer effects in nude mice innoculating with A549 cells. The binding affinities of BF211 to the alpha subunits of Na,K-ATPase and the inhibiting effects of BF211 on the activity of Na,K-ATPase were similar to that of bufalin. Both bufalin and BF211 could activate caspases and induce apoptosis of A549 cells. There was difference in the regulation of phosphorylation of Src kinase by bufalin or BF211. Conclusion: These results suggest an important role for Src kinase in the anti-cancer mechanism of cardiac glycosides. http://dx.doi.org/10.1016/j.eujim.2014.09.102 Therapeutic effects of Benincasa hispida seed extracts on chronic bacterial prostatitis models in rats Jing Liu , Pinglin Li , Jie Yan , Bin Li , Xuli Tang , Guoqiang Li 1,∗ 1 Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China 2 Dezhou Institute for Drug Control, Dezhou 253000, China 3 Dezhou People’s Hospital, Dezhou 253000, China 4 College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, PR China E-mail address: liguoqiang@ouc.edu.cn (G. Li). Introduction: The effects of three different extracts of B. hispida seeds including water extract (WE), methanol extract (ME), and petroleum extract (PE) in a rat model of CBP, and the underlying mechanisms were studied in this research. Methods: Six randomly divided groups of male Sprague–Dawley rats including control, model, ciprofloxacin, AE, ME and PE groups were studied. Morphological changes of prostate and histopathological analyses were conducted, and the changes of several CBP-related factors including TNF, IL-6, ICAM-1, SOD, and MDA in the prostate were analyzed. Results: All three extracts displayed significant therapeutic efficacy with ME being the best comparable to antibiotic ciprofloxacin. The therapeutic effect was suggested to be by down-regulating the pro-inflammatory TNF, IL-6 and intercellular adhesion molecule ICAM-1, as well as up-regulating SOD level. Conclusions: The result provides a potential therapeutic agent for CBP disease, especially for patients with antibiotic resistance. Acknowledgements: Hi-tech Research and Development Program of China (No. 2013AA093002). http://dx.doi.org/10.1016/j.eujim.2014.09.103 Toxic and detoxic principles of compatibility of Euphorbia kansui and Zizyphus jujuba in vitro and in vivo Yu-Mei Liu , Cui-Cui He , Rong-Rong Hui , Yue Huai , Jin-Ao Duan , Yu-Ping Tang , Jian-Xin Li 1,∗ 1 State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China 2 Jiangsu Key Lab of TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China E-mail address: lijxnju@nju.edu.cn (J.-X. Li). Introduction: The roots of Euphorbia kansui (EK) and the fruits of Zizyphus jujuba (ZJ) have been clinically used in traditional Chinese medicine (TCM) for a long time. This work is to clarify the toxic and detoxic principles that are responsible for the compatibility of EK and ZJ. Methods: The water extracts of EK and ZJ were partitioned. The primary rat hepatocytes, SD rats and NMR-based metabonomic approach were applied for toxicity assay. Results:Assays in vitro revealed that EK2was the toxic principle of EK and ZJ2 exerted detox actions. In vivo studies in rats showed that EK2 possessed liver and kidney toxicities, disturbed gut microbial activities, while, ZJ2 not only relieved the EK2induced toxicity, but also made the toxicity appear ahead in the experiment period Conclusions: EK2 had some accumulative hepatotoxicity, nephrotoxicity and resulted in an imbalance of gut microbial activities. ZJ2 not only lowered EK2-induced toxicity, but also disintegrated the toxic accumulation of EK2. The results proved the excellence of the compatibility of EK and ZJ. Acknowledgements: The study was supported by National Natural Science Fund for Creative Research Groups of China (21121091) and 973 Program (2011CB505300-03). http://dx.doi.org/10.1016/j.eujim.2014.09.104

ANCOVA for normally distributed continuous variables, Mann–Whitney U test and Kruskal–Wallis ANOVA for non-normal distributions. The clinical characteristics of the patients enrolled in the study are presented in Supplementary Table 1. Higher body mass index (P = 0.002), systolic and diastolic blood pressure (P b 0.05), and antihypertensive treatment (P b 0.001) were found in AD patients comparedwith CHDpatients.There are nosignificantdifferencesin age, sex, blood urea, and creatinine between CHD and AD patients. Spatiotemporal changes of leukocytes and chemokines accompanying the course and types of AD are shown in Table 1. Leukocyte number and chemokine level between CHD and AD patients were compared firstly. Higher value of white blood cells (P b 0.001), neutrophils (P b 0.001), monocytes (P =0 .019) and CCL13 (P b 0.01), with lower value of lymphocytes (P = 0.004), basophils (P = 0.009), eosinophils (P = 0.011)and CXCL11 (P b 0.001)wasfound in ADpatients compared with CHD patients. Then, temporal change of leukocytes and chemokines with the course of AD was found. White blood cells and neutrophils maintained hi gher level during the three time points of detection in AD patients compared with CHD patients, with the highest level during the first week.Andalso,thehighestnumberofmonocytesandlowestnumberof eosinophils were detected in AD patients of the 1-week group. CXCL11 level remained low during the three time points of detection for AD patients compared with CHD patients, with the lowest level during the first week.

ClC-5 is a chloride channel known to be expressed in the kidney. We previously reported that ClC-5 mRNA was also strongly expressed in immature human myeloid cell line (HL-60), but weakly expressed in mature neutrophils. To clarify the underlying mechanisms, we examined the relationship between ClC-5 expression and cell cycle. Dimethyl sulfoxide treatment of HL-60 that causes differentiation with G0/G1 cell cycle arrest decreased the expression of ClC-5 mRNA. Cell sorting and synchronization experiments revealed that ClC-5 mRNA expression was high in S and G2/M phases and low in G0/G1 phase. ClC-5 antisense oligonucleotide suppressed proliferation of HL-60 cells with a decrease in ClC-5 protein expression, probably due to G2 arrest. These results suggest that cell cycle-dependent expression of ClC-5 has a role in cell cycle progression in myeloid cells.

Chloride channels on immune cells reportedly play important roles in cell volume regulation, cell proliferation and immune functions, but they are not well characterized at the molecular level. We examined the expression of swelling-and/or pH-regulated chloride channels (ClC-2, 3, 4 and 5) in human leukemic cell lines [Jurkat and Hut-78 (T cells), Raji and Daudi (B cells), K-562 and HL-60 (myeloid cells)] and T cells, B cells and neutrophils from 8 normal subjects to clarify the difference of their expression among different cell types and maturity. Semi-quantitative RT-PCR and Northern blot analysis showed that ClC-3 was most abundantly expressed in all cells regardless of the cell types and maturity, while expression of ClC-2 was weak in these cells. Expression of ClC-4 was observed mainly in leukemic B cell lines, and in B cells and neutrophils from normal subjects. ClC-5 was expressed in all cell lines, while it was observed in only T and B cells but not in neutrophils from normal subjects. Thus, these chloride channels (ClC-2, 3, 4 and 5) showed distinct distribution among human immune cells, suggesting that they have specific roles in these cells. Molecular identification of chloride channels in leukocytes of different types and maturity may provide a new approach for the treatment of leukemia.

We examined GH and GH receptor expression in human leukemic cell lines and leukocytes of normal subjects to elucidate the cell types expressing GH and GH receptor, the individual variations of their expressions, their correlation and the relationships with serum IgG and IGF-I concentrations. In addition, the expression of GH secretagogue receptor, which enhances GH secretion from the anterior pituitary by synthetic GH secretagogues and that of its endogenous ligand, ghrelin, were also examined in these immune cells. GH expression in human leukemic cell lines was observed mainly in B cell lines at both the mRNA and protein level [3.8 +/- 0.2 pg/10(6) cells in Raji and 19.9 +/- 3.3 pg/10(6) cells in Daudi vs. negligible in T cell lines (Jurkat and Hut-78) and in myeloid cell lines (K-562 and HL-60)]. B cells in normal subjects were also found to be the major immune cells expressing GH mRNA, with significant individual variation. GH receptor mRNA expression was detectable in all human leukemic cell lines, although the expression level varied widely among the cell lines and was weaker than that in the liver. On the other hand, GH receptor mRNA expression was mainly found in B cells, with marked individual variation in normal subjects. There was a positive correlation between the mRNA expressions of GH and GH receptor in B cells of normal subjects (r = 0.89; P < 0.001). Single cell RT-PCR revealed that some B cells expressed both GH and GH receptor transcripts, and others expressed only GH. GH/GH receptor expression levels in B cells did not show any correlation with serum IgG and IGF-I levels in normal subjects. Expression of GH secretagogue receptor and ghrelin was detectable in all immune cells regardless of the maturity and cell types with great individual variations. In summary, GH secreted from B cells may act locally on their own receptors, and their variable expressions may be related to individual immune functions. Widespread distribution of ghrelin and GH secretagogue receptor in human immune cells may indicate unknown biological functions other than enhancing GH secretion in the immune system.

Background Although a number of medicines are available for the management of hypertension, the organ damage induced by hypertension is not resolved. The aim of this study was to investigate the protection of ginsenoside Rg1 (Rg1) against vascular remodeling and organ damage in spontaneously hypertensive rats (SHR). Methods Male SHR were treated with 5, 10 or 20 mg/kg Rg1 through intraperitoneal injection per day for 1 month. SHR or Wistar-Kyoto rats (WKY) receiving vehicle (saline) was used as control. Blood pressure detection and pathological stain, transmission electron microscope, immunohistochemical assay were used to elucidate the protection of Rg1. Results Blood pressures were not different between control SHR rats and Rg1 treated SHR rats, but Rg1 improved the aortic outward remodeling by lowering the lumen diameter and reducing the media thickness according the histopathological and ultrastructural detections. Rg1 also protected the retinal vessels against inward remodeling detected by immunohistochemical assay. Furthermore, Rg1 attenuated the target heart and kidney damage with improvement on cardiac and glomerular structure. Conclusions These results suggested that Rg1 held beneficial effects on vascular structure and further protected against the organ-damage induced by hypertension. These findings also paved a novel and promising approach to the treatment of hypertensive complications.

Luan-Pao-Prescription is a famous Chinese herbal formula, which is commonly used for the treatment of female sterility in clinical practice in China. In the present paper, a reliable method based on liquid chromatography coupled with electrospray ionization tandem mass spectrometry in both positive and negative ion modes has been established for the analysis of major chemical constituents in Luan-Pao-Prescription. A total of 34 compounds were either identified or tentatively characterized. These compounds include flavonoids, anthraquinones, iridoids, xanthones and organic acids. Flavonoids were the major constituents of the formula. The results profiled the chemical composition of Luan-Pao-Prescription comprehensively for the first time.

Background Infarct-induced left ventricular (LV) remodeling is a deleterious consequence after acute myocardial infarction (MI) which may further advance to congestive heart failure. Therefore, new therapeutic strategies to attenuate the effects of LV remodeling are urgently needed. Salvianolic acid B (SalB) from Salviae mitiorrhizae, which has been widely used in China for the treatment of cardiovascular diseases, is a potential candidate for therapeutic intervention of LV remodeling targeting matrix metalloproteinase-9 (MMP-9). Results Molecular modeling and LIGPLOT analysis revealed in silico docking of SalB at the catalytic site of MMP-9. Following this lead, we expressed truncated MMP-9 which contains only the catalytic domain, and used this active protein for in-gel gelatin zymography, enzymatic analysis, and SalB binding by Biacore. Data generated from these assays indicated that SalB functioned as a competitive inhibitor of MMP-9. In our rat model for cardiac remodeling, western blot, echocardiography, hemodynamic measurement and histopathological detection were used to detect the effects and mechanism of SalB on cardio-protection. Our results showed that in MI rat, SalB selectively inhibited MMP-9 activities without affecting MMP-9 expression while no effect of SalB was seen on MMP-2. Moreover, SalB treatment in MI rat could efficiently increase left ventricle wall thickness, improve heart contractility, and decrease heart fibrosis. Conclusions As a competitive inhibitor of MMP-9, SalB presents significant effects on preventing LV structural damage and preserving cardiac function. Further studies to develop SalB and its analogues for their potential for cardioprotection in clinic are warranted.