Chemical Stability and Bioactivity of tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone in in vitro test systems.

Danshen Si Wu is a Traditional Chinese Medicine used for menopausal complains. Beside tanshinone IIA (Tan IIA), Danshen also contains tanshinone I (Tan I), cryptotanshinone (CT) and dihydrotanshinone (DT). The aim of this study was to compare the biological activity of these tanshinones and to determine their cytotoxicity and genotoxicity. Purities and stabilities of the substances were analyzed by LC-DAD and LC-MS analyses. DT and CT concentrations decreased rapidly in dimethylsulfoxide and were converted to Tan I and Tan IIA, respectively. In aqueous solution concentration of all tanshinones decreased after 24 h. Tan I and Tan IIA showed dose-dependent bioactivity mediated by ERα and ERß. No cytotoxic and genotoxic effects for Tan I and Tan IIA were detected. In a yeast transactivation assay Tan I and Tan IIA showed antiandrogenic activity. A significant anabolic activity in C2C12 cells could be detected for Tan I and Tan IIA. In conclusion our data provide evidence that Tan I and Tan IIA are the most relevant bioactive tanshinones in Danshen. Our finding that all tanshinones display a certain instability in aqueous solutions is relevant when discussing their potential therapeutic benefits in humans.

Tanshinone IIA derivatives induced S-phase arrest through stabilizing c-myc G-quadruplex DNA to regulate ROS-mediated PI3K/Akt/mTOR pathway.

Herein, a derivate from tanshinone IIA, 1,6,6-trimethyl-11-phenyl-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]phenanthro[3,4-d]imidazole (TA25), has been synthesized and investigated as potential inhibitor against the proliferation, migration and invasion of lung cancer cells. MTT assay and cell colony formation assay results showed that TA25 exhibits acceptable inhibitory effect against the proliferation of lung cancer A549 cells, and the value of IC50 was about 17.9 µM. This result was further confirmed by the inhibition of TA25 against the growth of xenograft lung cancer cells on zebrafish bearing tumor (A549 lung cancer cells). The results of wound-healing assay and FITC-gelatin invasion assay displayed that TA25 could inhibit the migration and invasion of lung cancer A549 cells. Moreover, the studies on the binding properties of TA25 interact with c-myc G-quadruplex DNA suggested that TA25 can bind in the G-quarter plane formed from G7, G11, G16 and G20 with c-myc G-quadruplex DNA through π-π stacking. Further study of the potential anti-cancer mechanism indicated that TA25 can induce S-phase arrest in lung cancer A549 cells, and this phenomenon resulted from the promotion of the production of reactive oxygen species and DNA damage in A549 cells under the action of TA25. Further research revealed that TA25 could inhibit the PI3K/Akt/mTOR signal pathway and increase the expression of p53 protein. Overall, TA25 can be developed into a promising inhibitor against the proliferation, migration and invasion of lung cancer cells and has potential clinical application in the near future.

Inflammatory and Cytotoxic Activities of Abietane Terpenoids from Nepeta bracteata Benth.

Nepeta bracteata Benth. is used clinically to treat tracheal inflammation, coughs, asthma, colds, fevers, adverse urination, and other symptoms, along with functions in clearing heat and removing dampness. However, there have been few studies characterizing the material basis of its efficacy. Therefore, the aim of this study was to screen for compounds with anti-inflammatory activities in N. bracteata Benth. Using silica gel, ODS C18, and Sephadex LH-20 column chromatography, as well as semipreparative HPLC, 10 compounds were separated from N. bracteata Benth. extract, including four new diterpenoids (1-4), one amide alkaloid (5), and five known diterpenoids (6-10). The structures of all the isolates were elucidated by HR-ESI-MS, NMR, and CD analyses. Using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, we investigated the anti-inflammatory activities of compounds 1-10. It is worth noting that all were able to inhibit nitric oxide (NO) production with IC50 values < 50 µM and little effect on RAW 264.7 macrophage viability. Compounds 2 and 4 displayed remarkable inhibition with IC50 values of 19.2 and 18.8 µM, respectively. Meanwhile, screening on HCT-8 cells demonstrated that compounds 2 and 4 also had moderate cytotoxic activities with IC50 values of 36.3 and 41.4 µM, respectively, which is related to their anti-inflammatory effects.

A network-based method for mechanistic investigation and neuroprotective effect on treatment of tanshinone â against ischemic stroke in mouse.

ETHNOPHARMACOLOGICAL RELEVANCE: Tanshinone-Ⅰ (TSNⅠ), a member of the mainly active components of Salvia miltiorrhiza Bunge (Dan Shen), which is widely used for the treatment for modern clinical diseases including cardiovascular and cerebrovascular diseases, has been reported to show the properties of anti-oxidation, anti-inflammation, neuroprotection and other pharmacological actions. However, whether TSNⅠ can improve neuron survival and neurological function against transient focal cerebral ischemia (tMCAO) in mice is still a blank field. AIM OF THE STUDY: This study aims to investigate the neuroprotective effects of TSNⅠ on ischemic stroke (IS) induced by tMCAO in mice and explore the potential mechanism of TSNⅠ against IS by combining network pharmacology approach and experimental verification. MATERIALS AND METHODS: In this study, the pivotal candidate targets of TSNⅠ against IS were screened by network pharmacology firstly. Enrichment analysis and molecular docking of those targets were performed to identify the possible mechanism of TSNⅠ against IS. Afterwards, experiments were carried out to further verify the mechanism of TSNⅠ against IS. The infarct volume and neurological deficit were evaluated by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Longa respectively. Immunohistochemistry was used to observe neuronal death in the hippocampus and cortical regions by detecting the change of NeuN. The predicting pathways of signaling-related proteins were assessed by Western blot in vitro and in vivo experiments. RESULTS: In vivo, TSNⅠ was found to dose-dependently decrease mice's cerebral infarct volume induced by tMCAO. In vitro, pretreatment with TSNⅠ could increase cell viability of HT-22 cell following oxygen-glucose deprivation (OGD/R). Moreover, the results showed that 125 candidate targets were identified, Protein kinase B (AKT) signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and mitogen-activated protein kinases 1 (MAPK1) and AKT1 could be bound to TSNⅠ more firmly by molecular docking analysis, which implies that TSNⅠ may play a role in neuroprotection through activating AKT and MAPK signaling pathways. Meanwhile, TSNⅠ was confirmed to significantly protect neurons from injury induced by IS through activating AKT and MAPK signaling pathways. CONCLUSION: In conclusion, our study clarifies that the mechanism of TSNⅠ against IS might be related to AKT and MAPK signaling pathways, which may provide the basic evidence for further development and utilization of TSNⅠ.

Critical roles of tyrosyl-DNA phosphodiesterases in cell tolerance to carnosol-induced DNA damage.

Carnosol is a natural compound with pharmacological action due to its anti-cancer properties. However, the precise mechanism for its anti-carcinogenic effect remains elusive. In this study, we used lymphoblastoid TK6 cell lines to identify the DNA damage and repair mechanisms of carnosol. Our results showed that carnosol induced DNA double-strand breaks (DSBs). We also found that cells lacking tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme related to topoisomerase 1 (TOP1), and tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme related to topoisomerase 2 (TOP2), were supersensitive to carnosol. Carnosol was found to induce the formation of the TOP1-DNA cleavage complex (TOP1cc) and TOP2-DNA cleavage complex (TOP2cc). When comparing the accumulation of γ-H2AX foci and the number of chromosomal aberrations (CAs) with wild-type (WT) cells, the susceptivity of the TDP1-/- and TDP2-/- cells were associated with an increased DNA damage. Our results provided evidence of carnosol inducing DNA lesions in TK6 cells and demonstrated that the damage induced by carnosol was associated with abnormal topoisomerase activity. We conclude that TDP1 and TDP2 play important roles in the anti-cancer effect of carnosol.

Synthesis and structure-activity relationships of novel abietane diterpenoids with activity against Staphylococcus aureus.

Aim: To find alternative compounds against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA), novel derivatives from dehydroabietic acid were synthesized. Methods & results: Compound 12 was the most effective against 15 MRSA and 11 MSSA with minimum inhibitory concentration values ranging from 3.9 to 15.6 µg/ml. Although less active than 12, compound 11, followed by 25 and 13, also exhibited anti-staphylococcal activity. Additional studies showed that compound 12 is devoid of toxic effect on non-target cells. A structure-activity relationship study revealed that an oxime at C-13 together with a hydroxyl at C-12 could play a key role in the activity. Conclusion: These structures, in particular compound 12, could arise as templates for the development of agents against MRSA and MSSA.

Tanshinone IIA attenuates high glucose induced human VSMC proliferation and migration through miR-21-5p-mediated tropomyosin 1 downregulation.

The proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in the development and progression of diabetes-related vascular complications. Recently, microRNAs (miRNAs) have been suggested to be involved in the pathogenesis of vascular diseases. This study was designed to investigate the influences of tanshinone IIA, an active compound extracted from Chinese herb Salvia miltiorrhiza, on the proliferation and migration of human aortic VSMCs (HASMCs). cultured in a high glucose medium and the underlying mechanisms related miRNAs. Using a miRNA microarray method, we profiled the miRNA expression signature in human aortic VSMCs (HASMCs) exposed to normal glucose, high glucose with and without Tanshinone IIA. Cell proliferation was measured with 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Cell migration was evaluated using transwell migration assay and wound scratch assay. Western blot was used to examine the expression of tropomyosin 1 (TPM1) and miRNA level was quantified by real-time PCR. The results showed that several miRNAs that were highly expressed in the high glucose group were significantly decreased in the high glucose with Tanshinone IIA group compared with the normal glucose group (P < 0.05). Among these miRNAs, miR-21-5p was significantly upregulated in the high glucose group and downregulated after Tanshinone IIA treatment (P < 0.05). The depletion of miR-21-5p in HASMCs resulted in decreased cell proliferation and migration (P < 0.05). Moreover, we found that Tanshinone IIA inhibited proliferation and migration partly through miR-21-5p-mediated TPM1 downregulation (P < 0.05). In conclusion, the present study demonstrates that Tanshinone IIA is able to protect HASMCs from high glucose-induced proliferation and migration through regulating expression of miRNAs.

Antioxidant activity evaluation of rosemary ethanol extract and their cellular antioxidant activity toward HeLa cells.

Rosemary ethanol extract (REE) from Rosmarinus officinalis was identified by LC-ESI-MS/MS and 12 compounds were found. Among them, rosmarinic acid (389.78 µg/mg in REE), luteolin-3'-O-glucuronide (325.58 µg/mg), luteolin-5-O-glucuronide (120.92 µg/mg), and geniposide (120.83 µg/mg) are the major components. The antioxidant activity evaluation of REE by off-line HPLC methods indicated that among the 12 compounds, rosmarinic acid had the strongest scavenging activities in both DPPH· and ·OH. The cytotoxicity experiment showed that REE with the concentration ranges from 1 to 100 µg/ml did not significantly affect the cell viability of HeLa, while inhibitory rate reduced to 62.3% when the concentration was increased to 1,000 µg/ml. The results of intracellular antioxidation assay showed that the ability of REE in reducing the reactive oxygen species (ROS) in HeLa cells was higher than rosmanol, and lower than rosmarinic acid without cell toxicity. PRACTICAL APPLICATIONS: Plant polyphenols are essential components of functional foods, due to their antioxidant and enzyme inhibition activities. This paper is the first study about the quantification of antioxidant compounds, antioxidant activity evaluation, and their cellular antioxidant activity of polyphenols extract from R. officinalis toward HeLa cells. We aimed to elucidate the chemical composition and recognition of antioxidant components with DPPH and OH free radicals scavenging activity. In addition, the polyphenols dose-response correlations with cellular antioxidant activity were also determined. These results indicated that off-line HPLC method with DPPH and OH free radicals as markers is available for screening antioxidant activity of polyphenols from the mixture.

Cytotoxic Tolerance of Healthy and Cancerous Bone Cells to Anti-microbial Phenolic Compounds Depend on Culture Conditions.

Carnosol and carnosic acid are polyphenolic compounds found in rosemary and sage with known anti-oxidant, anti-inflammatory, and anti-microbial properties. Here, we addressed the potential use of carnosol and carnosic acid for in vitro bone tissue engineering applications, specifically depending on their cytotoxic effects on bone marrow stromal and stem cells, and osteosarcoma cells in monolayer and 3D cultures. Carnosol and carnosic acid displayed a bacteriostatic effect on Gram-positive bacteria, especially on S. aureus. The viability results indicated that bone marrow stromal cells and bone marrow stem cells were more tolerant to the presence of carnosol compared to osteosarcoma cells. 3D culture conditions increased this tolerance further for healthy cells, while not affecting the cytotoxic potential of carnosol for osteosarcoma cells. Carnosic acid was found to be more cytotoxic for all cell types used in the study. Results suggest that phenolic compounds might have potential use as anti-microbial and anti-carcinogenic agents for bone tissue engineering with further optimization for controlled release.

Dual-mixed/CMC model for screening target components from traditional Chinese medicines simultaneously acting on EGFR & FGFR4 receptors.

Radix Salviae Miltiorrhiae (also known as DanShen (DS) in China), a popular herbal drug in traditional Chinese medicine (TCM) for promoting blood circulation and treating blood stasis, has been reported to possess potential anti-tumor effects. The aim of the study was to develop an effective and practical method for screening and identifying bioactive compounds from Radix Salviae Miltiorrhiae. In this work, the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptors 4 (FGFR4) dual-mixed/cell membrane chromatography (CMC) coupled with high performance liquid chromatography-electrospray ionization-ion trap-time of flight-multistage mass spectrum (HPLC-ESI-IT-TOF-MSn) was established and successfully used to identify the active components from Radix Salviae Miltiorrhiae. Salvianolic acid C (SAC), tanshinone I (Tan-I), tanshinone IIA (Tan-IIA), and cryptotanshinone (C-Tan) were identified as bioactive components with EGFR and FGFR4 activities. MTT and kinase assay were performed to investigate inhibitory effects of these compounds against EGFR and FGFR4 cells growth in vitro. Both cell viability and kinase activity showed that cryptotanshinone acting on EGFR receptor and tanshinone IIA acting on FGFR4 receptor. In conclusion, the EGFR & FGFR4 dual-mixed/CMC can simultaneously screen the bioactive components from TCMs that act on both EGFR and FGFR4 receptors, which significantly improve the efficiency of specific bioactive components identification from a complex system.

Ferruginol exhibits anticancer effects in OVCAR­3 human ovary cancer cells by inducing apoptosis, inhibition of cancer cell migration and G2/M phase cell cycle arrest.

The primary aim of the current study was to investigate the antitumor effects of ferruginol in OVCAR­3 human ovary cancer cells. The effects of ferruginol on cell apoptosis, cell migration and cell cycle phase distribution were also evaluated. Cell cytotoxicity induced by ferruginol was determined by an MTT assay, while fluorescence microscopy and transmission electron microscopy (TEM) were performed to investigate apoptotic effects. Flow cytometry was employed to determine the effects of ferruginol on the cell cycle and an in vitro wound healing assay was performed to investigate effects on cancer cell migration. The results indicated that ferruginol inhibited the growth rate of OVACR­3 cells in a dose­ and time­dependent manner. When cells were treated with 20, 80 and 300 µM ferruginol, cells began to exhibit yellow fluorescence, which indicated the onset of apoptosis. TEM results demonstrated that untreated control cells exhibited intact nuclei and nucleolus. However, on treating cells with various doses of ferruginol, chromatin condensation occurred and disappearance of the nuclear envelope and formation of apoptotic bodies were also observed. The percentage of migrated cells, determined by the wound healing assay, decreased from 98.7% in control to 68.2% and 45.3 in 80 and 300 µM ferruginol­treated cells, respectively. Flow cytometry results demonstrated that ferruginol induced G2/M cell cycle arrest in OVCAR­3 cells. In conclusion, ferruginol may exhibit anticancer effects in OVCAR­3 human ovary cancer cells by inducing apoptosis, inhibiting cancer cell migration and inducing G2/M cell and may therefore prove beneficial in the treatment and management of ovarian cancer.

Tanshinone IIA Inhibits VEGF Secretion and HIF-1α Expression in Cultured Human Retinal Pigment Epithelial Cells under Hypoxia.

PURPOSE: The current work intends to study the activity of tanshinone IIA on secretion of vascular endothelial growth factor (VEGF) and expression of hypoxia inducible factor 1α (HIF-1α) in human retinal pigment epithelial cells (ARPE-19 cells) under hypoxic condition. METHODS: The cytotoxicity of tanshinone IIA was tested in ARPE-19 cells by MTT assay. ARPE-19 cells were incubated with different concentrations of cobalt chloride (100, 150, and 200 µM) for 12 h, and levels of expressed HIF-1α and secreted VEGF were quantified through Western blot and ELISA, respectively. Further, ARPE-19 cells were pretreated for 1 h with different concentrations of tanshinone IIA (5, 10, 15, and 18 µM). After 1 h, the cells were subjected to hypoxic condition using 150 µM cobalt chloride for 12 h in the presence and absence of tanshinone IIA. The cells were then harvested, and the secreted VEGF and expressed HIF-1α was studied. RESULTS: Tanshinone IIA at concentrations of 5, 10, 15, and 18 µM did not show cytotoxicity in ARPE-19 cells. Chemical hypoxia induced by cobalt chloride caused a significant increase in VEGF level in a dose-dependent manner, and HIF-1α expression peaked at 150 µM. Based on the data, cobalt chloride concentration was maintained at 150 µM for further studies. Tanshinone IIA decreased the level of HIF-1α and VEGF secretion in a dose-dependent manner under hypoxic condition. CONCLUSION: Tanshinone IIA could be explored as a new potential candidate for treating wet AMD.

Evaluation of Tanshinone IIA Developmental Toxicity in Zebrafish Embryos.

Tanshinone IIA (Tan-IIA) is derived from the dried roots of Salvia miltiorrhiza Bunge, a traditional Chinese medicine. Although Salvia miltiorrhiza has been applied for many years, the toxicity of the mono-constituent of Salvia miltiorrhiza, tanshinone IIA, is still understudied. This study evaluated the cardiotoxicity and developmental malformations of Tan-IIA by using zebrafish normal embryos and dechorionated embryos. After treatment with Tan-IIA in different concentrations for four-day periods, obvious pericardial edema, spinal curvature, and even missing tails were observed in zebrafish embryos. The LC50 values in the dechorionated embryo group at 72 h post-fertilization (hpf) and 96 hpf were 18.5 µM and 12.8 µM, respectively, and the teratogenicity was manifested at a concentration of about 1 µM. The main endpoints of teratogenicity were scoliosis, malformation of tail, and pericardium edema. Our findings displayed the potential cardiotoxicity and severe impact on the abnormal development of Tan-IIA in zebrafish embryo at high concentrations, which may help avoid the risk of its clinical application.

Tanshinone IIA increases protein expression levels of PERK, ATF6, IRE1α, CHOP, caspase­3 and caspase­12 in pancreatic cancer BxPC­3 cell­derived xenograft tumors.

Tanshinone (Tan)-IIA is a derivative of phenanthrenequinone and the main active ingredient isolated from Salviae miltiorrhizae radix (Danshen). Previous studies have demonstrated that Tan­IIA increased the protein expressions levels of protein kinase RNA­like endoplasmic reticulum kinase (PERK), activating transcription factor (ATF) 6, caspase­12 and CCAAT­enhancer­binding protein homologous protein (CHOP), to induce endoplasmic reticulum (ER) stress and apoptosis in human pancreatic cancer BxPC­3 cells. However, to the best of our knowledge, the effects of Tan­IIA on pancreatic cancer cells have not been investigated in vivo. Further studies are required to elucidate the therapeutic potential of Tan­IIA in inducing ER stress in cancer cells in vivo. The present study aimed to investigate the effects of Tan­IIA on the expression of ER stress­related proteins in BxPC­3­derived xenograft tumors. A total of 30 male severe combined immunodeficiency mice (age, 4 weeks) were implanted with BxPC­3 cells (2x106/0.2 ml) and subsequently treated with various doses of Tan­IIA (0, 30 and 90 mg/kg) for 4 weeks. After mice were sacrificed on day 33, the xenograft tumors were dissected and total protein was extracted for western blot analysis. The results of the present study demonstrated that Tan­IIA inhibited the growth of BxPC­3­derived xenograft tumors. In addition, Tan­IIA increased the protein expression levels of PERK, ATF6, caspase­12, inositol­requiring enzyme (IRE) 1α, eukaryotic initiation factor (eIF) 2α, phosphorylated (p)­c­Jun N­terminal kinase (JNK), CHOP and caspase­3 in a dose­dependent manner. These results indicated that Tan­IIA induced ER stress via increasing the protein expression levels of PERK, ATF6, caspase­12, IRE1α, eIF2α, p­JNK, CHOP and caspase­3 in BxPC­3 cells in vivo. Therefore, it may be hypothesized that Tan­IIA has potential for the development of novel therapeutic strategies for the treatment of patients with pancreatic cancer.

Dehydroabietic acid cytotoxicity in goldfish radial glial cells in vitro.

Dehydroabietic acid (DHAA) is a resin acid present in aquatic environments shown to induce cellular and molecular damage in aquatic animals. In this study, the cytotoxicity of DHAA on primary cultured goldfish radial glial cells (RGCs), an important component of the central nervous system, was evaluated. Here, it is reported that a concentration of 20mg/L DHAA affected cellular morphology and expression of genes involved in RGC steroidogenesis and metabolism. Higher concentration exposures of DHAA (40mg/L) lead to RGC death based on a lactate dehydrogenase leakage assay. Together, these data have implications in understanding the effects of DHAA on an integral central nervous system cell type important for neurogenesis, steroidogenesis and structural support. Due to the continuous presence of DHAA into water systems, results from this study provide indications as to the potential impacts of DHAA and demonstrate the importance of this class of chemicals on aquatic organisms.

Evaluation of the anti-inflammatory activities of tanshinones isolated from Salvia miltiorrhiza var. alba roots in THP-1 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza var. alba roots are used as the Chinese traditional medicine Danshen for the treatment of cardiovascular diseases in local clinical practice. Tanshinones are the major effective constituents of S. miltiorrhiza var. alba roots, but only tanshinone IIA, tanshinone I, cryptotanshinone, and 15,16-dihydrotanshinone have been investigated for their anti-inflammatory activities. MATERIALS AND METHODS: Eleven known compounds were isolated from S. miltiorrhiza var. alba roots, and the structures of all compounds were elucidated by spectroscopic analysis and comparisons with reported data. Immune anti-inflammatory activities were assessed by the ability to inhibit the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and interleukin (IL)-8 using enzyme-linked immunosorbent assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was also used to compare the inhibitory effects of the compounds on TNF-α, IL-1ß, and IL-8 mRNA expression with that of tanshinone IIA in lipopolysaccharide-stimulated THP-1 macrophages. RESULTS: All tanshinones, except for compound 5, significantly inhibited the mRNA and protein expression of TNF-α, IL-1ß, and IL-8, and their anti-inflammatory activities were stronger than that of tanshinone IIA. Compound 9 (5µM) showed the highest inhibitory effects for TNF-α, IL-1ß, and IL-8, at 56.3%, 67.6%, and 51.7%, respectively. CONCLUSIONS: Ten of the 11 tanshinones were shown to have anti-inflammatory properties superior to those of TSIIA, and which significantly inhibited the expression of TNF-α, IL-1ß, and IL-8. The present results provided a referential basis for explaining the use of S. miltiorrhiza var. alba root as a Chinese folk medicine for treating cardiovascular diseases associated with inflammation, and show the importance of trace constituents of this herb.

Enhanced Eryptosis Following Exposure to Carnosic Acid.

BACKGROUND/AIMS: The phenolic abietane diterpene component of rosemary and sage, carnosic acid, may either induce or inhibit apoptosis of nucleated cells. The mechanisms involved in the effects of carnosic acid include altered mitochondrial function and gene expression. Human erythrocytes lack mitochondria and nuclei but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how carnosic acid induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to carnosic acid significantly increased the percentage of annexin-V-binding cells (2.5 µg/ml), significantly decreased forward scatter (10 µg/ml), significantly increased Fluo3 fluorescence (10 µg/ml), significantly increased ceramide abundance (10 µg/ml), significantly increased hemolysis (10 µg/ml), but significantly decreased DCFDA fluorescence (10 µg/ml). The effect of carnosic acid on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSION: Carnosic acid triggers cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane, an effect paralleled by and/or in part due to Ca2+ entry and increased ceramide abundance.

Enhanced hepatic targeting, biodistribution and antifibrotic efficacy of tanshinone IIA loaded globin nanoparticles.

Tanshinone IIA (TA) has been recently used to treat liver diseases. However, the poor water solubility and fast metabolism obstruct TA in to be used for the treatment of liver diseases. To overcome this, TA was encapsulated into globin to form nanoparticles (TA-Gb-NPs) by our self-assembling method. We evaluated their biodistribution, pharmacokinetics, targeting ability to liver and antifibrotic effects. As a result, TA-Gb-NPs had a good hepatic targeting ability and achieved higher concentration and longer retention in liver than tanshinone IIA suspension (TA-S). Compared with TA-S, TA-Gb-NPs significantly improved serum biochemical parameters in thioacetamide (TAA) induced liver fibrosis mouse model. Furthermore, histological analysis of mouse liver slices revealed that TA-Gb-NPs could markedly reduce the fibrosis scores and attenuate the progression of the hepatic fibrosis. In conclusion, the TA-Gb-NPs may be a good candidate for the treatment of hepatic fibrosis.

Suaveolic acid: a potent phytotoxic substance of Hyptis suaveolens.

Hyptis suaveolens (Lamiaceae) is an exotic invasive plant in many countries. Earlier studies reported that the aqueous, methanol, and aqueous methanol extract of H. suaveolens and its residues have phytotoxic properties. However, to date, the phytotoxic substances of this plant have not been reported. Therefore, the objectives of this study were isolation and identification of phytotoxic substances of H. suaveolens. Aqueous methanol extract of this plant was purified by several chromatographic runs through bioassay guided fractionation using garden cress (Lepidium sativum) as a test plant. Final purification of a phytotoxic substance was achieved by reverse phase HPLC and characterized as 14α-hydroxy-13ß-abiet-8-en-18-oic acid (suaveolic acid) by high-resolution ESI-MS, (1)H-,(13)C-NMR, CD, and specific rotation. Suaveolic acid inhibited the shoot growth of garden cress, lettuce (Lactuca sativa), Italian ryegrass (Lolium multiflorum), and barnyard grass (Echinochloa crus-galli) at concentrations greater than 30 µM. Root growth of all but lettuce was also inhibited at concentrations greater than 30 µM. The inhibitory activities were concentration dependent. Concentrations required for 50% growth inhibition of suaveolic acid for those test plant species were ranged from 76 to 1155 µM. Therefore, suaveolic acid is phytotoxic and may be responsible for the phytotoxicity of H. suaveolens plant extracts.

Synergistic combinations of tanshinone IIA and trans-resveratrol toward cisplatin-comparable cytotoxicity in HepG2 human hepatocellular carcinoma cells.

AIM: To determine the combinative effects of tanshinone IIA (Tan IIA) and trans-resveratrol (Resv) on cytotoxicity, apoptosis, cell-cycle arrest and DNA fragmentation in HepG2 human liver cancer cells. MATERIALS AND METHODS: Cytotoxicity was detected by the cell proliferation and cytotoxicity WST-1 assay. Cell-cycle arrest and apoptosis were determined using flow cytometry analysis. DNA fragments were separated by gel electrophoresis. RESULTS: Tan IIA and Resv at mixture ratios of 1/2:1/2 and 1/3:2/3 exerted synergistic cytotoxicity comparable to that of cisplatin. Elevated proportions of sub-G1 and apoptotic cells were respectively found in the combinative treatments in comparison with hypothetic values of additive effects. Moreover, a more intensive pattern of apoptotic DNA fragmentation was visualized in combined treatments than in individual ones. CONCLUSION: Combining Tan IIA and Resv causes synergistic cisplatin-comparable, cytotoxicity and robustly induces apoptosis, sub-G1 cell cycle arrest and DNA fragmentation. This study provides evidence supporting further pre-clinical investigations of the combinational synergism.