Cinnamaldehyde accelerates wound healing by promoting angiogenesis via up-regulation of PI3K and MAPK signaling pathways.

The bark of Cinnamomum cassia (C. cassia) has been used for the management of coronary heart disease (CHD) and diabetes mellitus. C. cassia may target the vasculature, as it stimulates angiogenesis, promotes blood circulation and wound healing. However, the active components and working mechanisms of C. cassia are not fully elucidated. The Shexiang Baoxin pill (SBP), which consists of seven medicinal materials, including C. cassia etc., is widely used as a traditional Chinese patent medicine for the treatment of CHD. Here, 22 single effective components of SBP were evaluated against the human umbilical vein endothelial cells (HUVECs). We demonstrated that in HUVECs, cinnamaldehyde (CA) stimulated proliferation, migration, and tube formation. CA also activated the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Furthermore, the secretion of vascular endothelial growth factor (VEGF) from HUVECs was increased by CA. In vivo, CA partially restored intersegmental vessels in zebrafish pretreated with PTK787, which is a selective inhibitor for vascular endothelial growth factor receptor (VEGFR). CA also showed pro-angiogenic efficacy in the Matrigel plug assay. Additionally, CA attenuated wound sizes in a cutaneous wound model, and elevated VEGF protein and CD31-positive vascular density at the margin of these wounds. These results illustrate that CA accelerates wound healing by inducing angiogenesis in the wound area. The potential mechanism involves activation of the PI3K/AKT and MAPK signaling pathways. Such a small non-peptide molecule may have clinical applications for promoting therapeutic angiogenesis in chronic diabetic wounds and myocardial infarction.

Protective effects of (E)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeine against hydrogen peroxide-induced injury in PC12 cells.

(E)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeine (HOEC), a naturally caffeic ester isolated from Incarvillea mairei, has been reported to possess anti-inflammatory activity by targeting 5-lipoxygenase. However, its other potential activities have yet to be explored. In this study, we measured antioxidant activity of HOEC using the DPPH free radical-scavenging assay. Then, we exposed rat pheochromocytoma (PC12) cells to hydrogen peroxide (H2O2)-induced damage and investigated the antioxidant activity of HOEC. Cell viability, lactate dehydrogenase (LDH) release, cellular morphology, Hoechst 33342 fluorescent staining, and apoptosis of the PC12 cells were assessed after treatment with 0.3-10 µM HOEC for 2 h and exposure to 600 µM H2O2. Additionally, glutathione reductase (GR), superoxide dismutase (SOD), lipid peroxidation malondialdehyde (MDA), and intracellular reactive oxygen species (ROS) accumulation were assayed after the PC12 cells were exposed to H2O2. To investigate mechanism, apoptosis-related protein were evaluated, including cleaved caspase 3/7, cleaved PARP, Bcl-2, Bcl-XL, and cytochrome c. The results showed that HOEC possessed potent antioxidant activity and pre-treatment with HOEC prior to H2O2 exposure significantly increased cell viability, reduced the release of LDH, ameliorated changes in cell morphology, and inhibited apoptosis. Further, HOEC did the following: reduced intracellular accumulation of ROS and MDA; rescued loss of SOD and GR activities; inhibited activated caspase-3 and caspase-7, cleaved PARP, and cytochrome c release; up-regulated the antiapoptosis-related protein Bcl-2 and Bcl-XL; and down-regulated the apoptosis-related proteins Bax and Bad. These findings suggested that HOEC may be a therapeutic agent for treating oxidative stress-derived neurodegenerative disorders.

Pharmacokinetic study of five ginsenosides using a sensitive and rapid liquid chromatography-tandem mass spectrometry method following single and multiple oral administration of Shexiang Baoxin pills to rats.

Shexiang Baoxin pills (SBP) are a traditional Chinese medicine that are used for treating coronary heart disease. Ginsenosides are the main effective components of SBP, but a comprehensive and deep pharmacokinetic study of ginsenosides in SBP, including multiple dosing and linear or nonlinear properties, is lacking. This study was designed to investigate and compare the pharmacokinetic characteristics of ginsenosides in SBP at a single dose and in multiple doses. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of the ginsenosides Rg1, Re, Rb3, Rc and Rb1 in rat plasma. Rats were randomly assigned to receive a single dose of 4, 8 or 12 g/kg and multiple doses (4 g/kg) of SBP for 8, 15 or 22 consecutive days. The results revealed that ginsenosides, following a single oral dose of 4 or 8 g/kg, were absorbed rapidly, with a Tmax ranging from 0.250 to 1.08 h. The AUC0-t and Cmax of the ppd-type ginsenosides Rb3, Rc and Rb1 were greater than those of the ppt-type ginsenosides Rg1 and Re. Nondose-dependent exposure was observed at doses of 4-12 g/kg for all of the ginsenosides. After multiple dosing, the plasma levels of the ppt-type ginsenosides decreased, whereas those of the ppd-type ginsenosides did not change significantly. In conclusion, the LC-MS/MS method was successfully applied to investigate the pharmacokinetics of ginsenosides after single and multiple oral administrations of SBP. The ginsenosides did not accumulate after multiple dosing. The ppd-type ginsenosides displayed more favorable pharmacokinetic properties compared with the ppt-type ginsenosides.

Comparison of the apoptotic effects of supercritical fluid extracts of Antrodia cinnamomea mycelia on hepatocellular carcinoma cells.

Antrodia cinnamomea (AC) has been widely used as a folk medicine in the prevention and treatment of liver diseases, such as hepatitis, hepatic fibrosis, and hepatocellular carcinoma. Previous studies have indicated that triterpenoids and benzenoids show selective cytotoxicity against human hepatoma cell lines. The aim of the study was to compare the triterpenoid content of extract and the extract-induced cytotoxicity in HepG2 cells from mycelia extracts of solid state cultured AC obtained by supercritical fluid extraction (SFE) and the conventional solvent extraction method. SFE with CO2 mixed with a constant amount of ethanol co-solvent (10% of CO2 volume) applied at different temperatures and pressures (40, 60 and 80 °C and, 20.7, 27.6 and 34.5 Mpa) was also compared in the study. Although the extraction yield of triterpenoids (59.7 mg/g) under the optimal extraction conditions of 34.5 MPa (5000 psi)/60 °C (designated as sample S-5000-60) was equivalent to the extraction yield using conventional liquid solvent extraction with ethanol (ETOH-E) at room temperature (60.33 mg/g), the cytotoxicity of the former against the proliferation of HepG2 cell line measured as the inhibition of 50% of cell growth activity (IC50) at dosages of 116.15, 57.82 and 43.96 µg/mL was superior to that of EtOH-E at 131.09, 80.04 and 48.30 µg/mL at 24, 48 and 72 h, respectively. Additionally, we further proved that the apoptotic effect of S-5000-60 presented a higher apoptosis ratio (21.5%) than ETOH-E (10.5%) according to annexin V-FITC and propidium iodide double staining assay results. The high affinity and selectivity of SFE on bioactive components resulted in a higher extraction efficiency than conventional solvent extraction. The chemical profile of the obtained extracts from solid state cultivated mycelium of AC was also determined by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS), whereby three benzenoids and four triterpenoids were found for the first time in SFE extracts with 4,7-dimethoxy-5-methyl-l,3-benzodioxole (5.78 mg/g) being the most abundant component, followed by 2,4-dimethoxy-6-methylbenzene-1,3-diol (3.03 mg/g) and dehydroeburicoic acid (0.40 mg/g).

Physicochemical characteristics and anti-inflammatory activities of antrodan, a novel glycoprotein isolated from Antrodia cinnamomea mycelia.

Antrodia cinnamomea (AC) is a unique fungus found inhabiting the rotten wood of Cinnamomum kanehirai. A submerged liquid culture of AC has been developed and its bioproducts have been used to meet the market demand for natural fruiting bodies. AC exhibits anti-inflammatory, antitumor, antioxidant, and immunomodulatory effects. Previously, we isolated polysaccharide AC-2 from AC mycelia by means of alkali extraction with subsequent acid precipitation and found it had a pronounced anti-inflammatory effect. In this study, a novel polysaccharide named "antrodan" was obtained by further purification of AC-2 using Sepharose CL-6B column chromatography. Antrodan exhibited a molecular weight of 442 kD and contained a particularly high content of uronic acid (152.6±0.8 mg/g). The protein content was 71.0%, apparently, higher than the carbohydrate content (14.1%), and thus antrodan was characterized as a glycoprotein. Its total glucan content was 15.65%, in which ß-glucan (14.20%) was prominently higher than α-glucan (1.45%). Its FTIR confirmed the presence of ß-linkages between sugars, and intramolecular amide bonds between sugars and amino acids. Its 1H-NMR spectrum showed that antrodan was a complex union of α- and ß-glucans, which had (1→4)-linked α-Glcp and (1→3)-linked ß-Glcp linkages to the carbohydrate chains via asparagine linked to protein site. Biologically, antrodan was confirmed to be totally non-detrimental to RAW 264.7 cell line even at dose as high as 400 µg/mL. It showed potent suppressing effect on the lipopolysaccharide-induced inflammatory responses in RAW 264.7 cell line. Moreover, antrodan significantly reduced the nitrogen oxide production at doses as low as 18.75 µg/mL.

Prelinguistic gesture and developmental abilities: A multi-ethnic comparative study.

The present study examined the frequency of gesture use and the relationship between frequency of gesture use and developmental abilities in typically developing 9- to 15-month-old, prelinguistic Hispanic and non-Hispanic White children. Data was collected through parent questionnaires, the Mullen Scales of Early Learning (MSEL), and two, 15-min video samples for each participant (semi-structured and structured settings). All video samples were coded for the frequency of the following gestures: total frequency, behavior regulation, social interaction, and joint attention. Results showed that children from both ethnicities used fewer gestures in a semi-structured setting in comparison to a structured setting and non-Hispanic White children produced higher frequencies of behavior regulation gestures and joint attention gestures, but lower frequencies of social interaction gestures. When controlling for ethnicity, gender, and age total frequency of gesture and frequencies of behavior regulation and social interaction were predictive of various developmental abilities. Furthermore, participant gender, age, and ethnicity were significantly related to various developmental abilities explored. These relationships were dependent upon setting. An understanding of the use of gesture and the relationship between gesture use and developmental abilities in prelinguistic children from different ethnic backgrounds has implication for early identification of delays and differences and is important to consider when exploring the connection between gesture and language and whether there are gesture-language, gesture-motor, and/or gesture-cognition integrated systems.

The anti-melanogenic effects of ellagic acid through induction of autophagy in melanocytes and suppression of UVA-activated α-MSH pathways via Nrf2 activation in keratinocytes.

Ellagic acid (EA) is a natural phenol antioxidant in different fruits, vegetables, and nuts. As a copper iron chelator from the tyrosinase enzyme's active site, EA was reported to inhibit melanogenesis in melanocytes. Here, we demonstrated the anti-melanogenic mechanisms of EA through autophagy induction in melanoma B16F10 cells and the role of Nrf2 and UVA (3 J/cm2)-activated α-melanocyte stimulating hormone (α-MSH) pathways in keratinocyte HaCaT cells. In vitro data showed that EA suppressed the tyrosinase activity and melanogenesis by suppressing cAMP-mediated CREB and MITF signaling mechanisms in α-MSH-stimulated B16F10 cells. ERK, JNK, and AKT pathways were involved in this EA-regulated MITF downregulation. Notably, EA induced autophagy in B16F10 cells was evidenced from increased LC3-II accumulation, p62/SQSTM1 activation, ATG4B downregulation, acidic vesicular organelle (AVO) formation, PI3K/AKT/mTOR inhibition, and Beclin-1/Bcl-2 dysregulation. Interestingly, 3-MA (an autophagy inhibitor) pretreatment or LC3 silencing (siRNA transfection) of B16F10 cells significantly reduced EA-induced anti-melanogenic activity. Besides this, in UVA-irradiated keratinocyte HaCaT cells, EA suppressed ROS production and α-MSH generation. Moreover, EA mediated the activation and nuclear translocation of Nrf2, leading to antioxidant γ-GCLC, HO-1, and NQO-1 protein expression in HaCaT cells. However, Nrf2 knockdown has significantly impaired this effect, and there was an uncontrolled ROS generation following UVA irradiation. JNK, PKC, and ROS pathways were involved in the activation of Nrf2 in HaCaT cells. In vivo experiments using the zebrafish model confirmed that EA inhibited tyrosinase activity and endogenous pigmentation. In conclusion, ellagic acid is an effective skin-whitening agent and might be used as a topical applicant.

Synthesis and evaluation of new α-methylene-γ-lactone carbamates with NO production inhibitory effects in lipopolysaccharide-induced RAW 264.7 macrophages.

A series of new α-methylene-γ-lactone carbamates were synthesized by an asymmetric synthetic route. The activities on inhibiting nitric oxide (NO) release of these compounds were evaluated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. The results indicated that most of the compounds except one exhibited potent NO inhibitory effect with IC50 value more than 2 µΜ. The cytotoxicities of these compounds were estimated via MTT assays. The results suggested that six compounds were accompanied by low cytotoxicity. The structure-activity relationships were also discussed. The S configuration of C3 on lactones ring would be more helpful to NO inhibitory effect.

Pharmacokinetics and tissue distribution of five bufadienolides from the Shexiang Baoxin Pill following oral administration to mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Shexiang Baoxin Pill (SBP) is a well-known composite formula of traditional Chinese medicine (TCM), widely used to treat cardiovascular diseases such as angina pectoris and myocardial infarction. Bufadienolides are major active compounds of Venenum Bufonis, which is one of the seven materiamedicas that comprise the Shexiang Baoxin Pill. Previous pharmacokinetics studies of bufadienolides have typically used a single medicinal material delivered to rats. In this study, we have chosen the mouse, a more proper animal model than the rat, to investigate the in vivo pharmacokinetics and tissue distribution of bufadienolides from the Shexiang Baoxin Pill. MATERIALS AND METHODS: The concentrations of bufadienolides in plasma and tissues were identified using high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS). The samples were prepared by liquid-liquid extraction with ethyl acetate, and the separation of bufadienolides was achieved using an ACQUITY HSS T3 column by gradient elution using water (containing 0.1% formic acid) and acetonitrile as the mobile phase at a flow rate of 0.3 mL/min. The pharmacokinetic parameters were determined using non-compartmental analysis. RESULTS: The results showed that the five bufadienolides were rapidly absorbed and distributed into the body. The pharmacokinetic curve showed double peaks after oral administration. The major tissue depots for resibufogenin, bufalin, and bufotalin in mice were the intestines, lung and kidney, whereas the major tissue depots of gamabufotalin and arenobufagin were the intestines, liver and kidney. CONCLUSION: The information gained from this research provides a meaningful insight for the clinical applications of the Shexiang Baoxin Pill.

Chemical constituents of Abies fabri.

Systematic phytochemical investigations on Abies fabri resulted in the isolation of 94 compounds, consisting of 68 terpenoids, six lignans, seven flavonoids, and 13 other miscellaneous chemical constituents. Their structures were elucidated on the basis of spectroscopic methods, and the absolute configurations of three of these previously unknown compounds were determined by Cu-Kα X-ray crystallographic analysis. Twelve previously unreported compounds, one artifact, and one potential artifact were identified, including six triterpenoids, four diterpenoids, two sesquiterpenoids, one lignan, and one phenol. 23-Hydroxy-3-oxolanosta-8,24-dien-26,23-olide showed weak cytotoxic activity against A549 and THP-1 cells with the IC50 values of 5.3 and 5.1 µM, respectively.

Simultaneous determination of four volatile compounds in rat plasma after oral administration of Shexiang Baoxin Pill (SBP) by HS-SPDE-GC-MS/MS and its application to pharmacokinetic studies.

In this study, a headspace, solid-phase dynamic extraction method coupled to gas chromatography-tandem mass spectrometry (HS-SPDE-GC-MS/MS) method was developed for the simultaneous determination of four volatile compounds, namely, isoborneol, borneol, muscone and cinnamaldehyde, in rat plasma after oral administration of Shexiang Baoxin Pill (SBP) using naphthalene as an internal standard (IS). The target compounds were extracted using an SPDE needle device coated with a poly (dimethylsiloxane) (PDMS) phase. The detection was achieved by GC-MS/MS in multiple reaction monitoring (MRM) mode. The optimised mass transition ion pairs (m/z) for quantitation were 95.1/67.1 for isoborneol and borneol, 85.0/67.0 for muscone, 131.0/77.0 for cinnamaldehyde and 128.1/102.1 for the IS. The parameters that affect the extraction ratio, such as the pre-incubation time, extraction temperature, number of extraction cycles, desorption volume and pH, were also optimised. The method was thoroughly validated with respect to specificity, linearity, precision, accuracy, recovery and stability. A sufficiently sensitive HS-SPDE-GC-MS/MS method was first developed in this study to determine the pharmacokinetics of volatile compounds found in rat plasma following oral administration of SBP. The method developed uses a simple procedure for plasma sample preparation and could be a promising tool for the analysis of complex volatile samples, such as traditional Chinese medicine (TCM).

Hepatoprotective bioactivity of the glycoprotein, antrodan, isolated from Antrodia cinnamomea mycelia.

Antrodan, a protein-bound polysaccharide isolated from Antrodia cinnamomea mycelia, was demonstrated to exhibit significant anti-inflammatory bioactivity in vitro. However, its role in hepatic injury in vivo still remains unclear. We hypothesized that antrodan may have beneficial hepatoprotective effects. To verify this, a lipopolysaccharide (LPS)-Sprague-Dawley rat model was used. Antrodan protected against liver damage by suppressing LPS-stimulated serum glutamine-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), interleukin (IL)-6, hepatic thiobarbituric acid reactive substances (TBARS), nitric oxide (NO), inducible NO synthase (iNOS) and nuclear factor (NF)-κB, and by effectively alleviating the downregulated hepatic superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px). Hematoxylin-eosin staining revealed that antrodan at a dosage of 40 mg/kg was able to alleviate LPS-induced liver damage to a normal status. In addition, we identified the partial main architectural backbone of antrodan to have a 1 → 3 linear ß-glycosidic backbone of mannan linked by ß-1 → 3 glucosidic branches. Conclusively, antrodan can potentially ameliorate liver damage in vivo by suppressing oxidative stress induced by LPS.

Simultaneous determination of seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill by liquid chromatography-electrospray ionization-tandem mass spectrometry: application to a pharmacokinetic study.

A liquid chromatography-electrospray ionization-tandem mass spectrometry method was described for the simultaneous determination of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin and bufotalin in rat plasma. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate. Chromatographic separation was carried out on an ACQUITY HSS T3 column with gradient elution using mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.3 mL/min. All analytes showed good linearity over a wide concentration range (r>0.99). The lower limit of quantification was in the range of 0.5-10 ng/mL for seven bufadienolides. The mean recovery of the analytes ranged from 94.36 to 104.18%. The intra- and inter-day precisions were in the range of 1.74-13.78% and the accuracies were between 89.37 and 101.38%. The validated method was successfully applied to a pharmacokinetic (PK) study of the seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill (SBP). The selected PK marker compounds with typical efficacy/toxicity may provide a practical solution for marker compound selection and dosage design for the therapeutic drug monitoring and PK study of SBP in its clinical applications.