Liwei Sun

Salidroside and tyrosol from Rhodiola protect H9c2 cells from ischemia/reperfusion-induced apoptosis.

AIMS Heart disease is the leading cause of death worldwide. Ischemia-reperfusion injury can lead to apoptotic death of heart cells and subsequently heart failure. Rhodiola is an herbal medicine with two main bioactive compounds--salidroside (SAL) and tyrosol (TYR). This study aimed to investigate whether these two compounds can prevent ischemia/reperfusion-induced apoptosis in H9c2 cells. MAIN METHODS Assays for total phenolics assay and Oxygen Radical Absorbance Capacity showed high antioxidant capacity of SAL and TYR. H9c2 cells were subjected to simulated ischemia/reperfusion (IR) in the presence and absence of SAL and/or TYR, and nuclei condensation, caspase-3 activity, cytochrome c release and JNK phosphorylation were determined. KEY FINDINGS In H9c2 cells, IR can lead to a 5-fold increase in p-JNK level. Apoptotic nuclei condensation, caspase-3 activity and cytochrome c release were markedly elevated, indicating the occurrence of apoptosis. SAL and TYR caused a dose-dependent inhibition of nuclear condensation. Furthermore, SAL and TYR, separately and in combination, significantly reduced caspase-3 activity, cytochrome c release and JNK activation. The anti-apoptotic effect of the combination was markedly higher than that of SAL or TYR alone. SIGNIFICANCE The inhibition of the JNK signaling pathway is the key mechanism for the cytoprotective effect of SAL and TYR in IR-induced apoptosis.

Tyrosol Prevents Ischemia/Reperfusion-Induced Cardiac Injury in H9c2 Cells: Involvement of ROS, Hsp70, JNK and ERK, and Apoptosis

Ischemia-Reperfusion (I/R) injury causes ROS overproduction, creating oxidative stress, and can trigger myocyte death, resulting in heart failure. Tyrosol is an antioxidant abounded in diets and medicine. Our objective was to investigate the protective effect of tyrosol on I/R-caused mortality in H9c2 cardiomyocytes through its influence on ROS, Hsp70, ERK, JNK, Bcl-2, Bax and caspase-8. A simulated I/R model was used, myocytes loss was examined by MTT, and ROS levels were measured using DCFH-DA. Nuclear condensation and caspase-3 activity were assessed by DAPI staining and fluorometric assay. Phosphorylated ERK and JNK were determined by electrochemiluminescent ELISA, and Hsp70, Bcl-2, Bax and caspase-8 were examined by Western blotting. Results show that tyrosol salvaged myocyte loss, inhibited nuclear condensation and caspase-3 activity dose-dependently, indicating its protection against I/R-caused myocyte loss. Furthermore, tyrosol significantly inhibited ROS accumulation and activation of ERK and JNK, augmenting Hsp70 expression. Besides, tyrosol inhibited I/R-induced apoptosis, associated with retained anti-apoptotic Bcl-2 protein, and attenuated pro-apoptotic Bax protein, resulting in a preservation of Bcl-2/Bax ratio. Finally, tyrosol notably decreased cleaved caspase-8 levels. In conclusion, cytoprotection of tyrosol in I/R-caused myocyte mortality was involved with the mitigation of ROS, prohibition of the activation of ERK, JNK and caspase-8, and elevation of Hsp70 and Bcl-2/Bax ratio.

Response Surface Methodology Optimization of Ultrasonic-Assisted Extraction of Acer Truncatum Leaves for Maximal Phenolic Yield and Antioxidant Activity

This study is the first to report the use of response surface methodology to improve phenolic yield and antioxidant activity of Acer truncatum leaves extracts (ATLs) obtained by ultrasonic-assisted extraction. The phenolic composition in ATLs extracted under the optimized conditions were characterized by UPLC-QTOF-MS/MS. Solvent and extraction time were selected based on preliminary experiments, and a four-factors-three-levels central composite design was conducted to optimize solvent concentration (X1), material-to-liquid ratio (X2), ultrasonic temperature (X3) and power (X4) for an optimal total phenol yield (Y1) and DPPH• antioxidant activity (Y2). The results showed that the optimal combination was ethanol:water (v:v) 66.21%, material-to-liquid ratio 1:15.31 g/mL, ultrasonic bath temperature 60 °C, power 267.30 W, and time 30 min with three extractions, giving a maximal total phenol yield of 7593.62 mg gallic acid equivalent/100 g d.w. and a maximal DPPH• antioxidant activity of 74,241.61 μmol Trolox equivalent/100 g d.w. Furthermore, 22 phenolics were first identified in ATL extract obtained under the optimized conditions, indicating that gallates, gallotannins, quercetin, myricetin and chlorogenic acid derivatives were the main phenolic components in ATL. What’s more, a gallotannins pathway existing in ATL from gallic acid to penta-O-galloylglucoside was proposed. All these results provide practical information aiming at full utilization of phenolics in ATL, together with fundamental knowledge for further research.

Changes in oxidative patterns during dormancy break by warm and cold stratification in seeds of an edible fruit tree

The transition from seed dormancy to germination is triggered by environmental factors, and in pomegranate (Punica granatum) seeds higher germination percentages are achieved by warm + cold stratification rather than by cold stratification alone. Our objective was to define the pattern of internal oxidative changes in pomegranate seeds as dormancy was being broken by warm + cold stratification and by cold stratification alone. Embryos isolated from seeds after 1–42 days of warm stratification, after 56 days of warm stratification + 7, 28 or 56 days of cold stratification, and after 1–84 days of cold stratification alone, were used in biochemical tests. Hydrogen peroxide (H2O2), nitric oxide (NO), proline, lipid peroxidation, protein carbonylation, and activities of the scavenging enzymes superoxide dismutase (SOD), hydrogen peroxide enzyme and peroxidase in the embryos were assessed by colorimetric methods. Our results indicated that warm + cold stratification had a stronger dormancy-breaking effect than cold stratification (85% versus 50% germination), which may be attributed to a higher yield of H2O2, NO, lipid peroxidation and protein carbonylation in warm + cold stratification. Furthermore, warm + cold stratification-induced H2O2 change led to greater changes (elevation followed by attenuation) in activities of the scavenging enzymes than that induced by cold stratification alone. These results indicated that restriction of the level of reactive oxygen species change within a positive and safe range by such enzymes promoted seed germination. In addition, a relatively strong elevation of proline during warm + cold stratification also contributed to dormancy breakage and subsequent germination. In conclusion, the strong dormancy alleviating effect of warm + cold stratification on pomegranate seeds may be attributed to the corresponding active oxidative change via H2O2, NO, proline, malondialdehyde, protein carbonylation and scavenging enzymes.

Primary identifications and palynological observations of Perilla in China.

Abstract  Unresolved controversies concerning the classification of the monotypic genus Perilla L. have hindered the complete understanding and subsequent sustainable use of these vital food, oil, and medicinal plants to their full potential. In the present study, we used scanning electron microscopy to obtain palynological evidence from 21 samples of Perilla plants from seven provinces in China as a potential further attribute for classification. The findings showed that pollen grains from plants of 11 samples were oblate, whereas those of the other 10 were suboblate; there were no prolate pollen grains observed. Pollen grains from all samples had diverse exine ornamentations. Based on whether they had continuous tecta on the ornamentations, the pollen grains from the 21 samples were classified into two categories: (i) 14 with irregular reticulates; and (ii) seven with continuous tecta with no perforations. None of the samples was bireticulate. The ornamentation pattern and size of pollen grains jointly provided evidence that it is appropriate for use in classifying the genus Perilla into five varieties of one species. Furthermore, by comparison, it is concluded that the shapes and exine ornamentations of Perilla are unique among those of the seven genera already investigated in the subfamily Lamioideae. Using these pollen features, Perilla can be easily distinguished from the two other subtribes (Menthinae Briq. and Thyminae Briq.) in the same tribe, supporting the view that Perilla and the other four related genera can be classified into one subtribe (Perillinae).

Urolithin C, a gut metabolite of ellagic acid, induces apoptosis in PC12 cells through a mitochondria-mediated pathway

Urolithins (Uros), metabolites of ellagitannins (ET) and ellagic acid (EA) produced by gut microbiota, showed better bioavailability and extensive bioactivity, and were considered as the active compounds responsible for the health benefits exerted by ET-containing foodstuffs. Here, we chemically synthesized three Uros including Uros A, B, and C and compared their anti-proliferative activities with that of EA in PC12 cells. MTT assay showed that EA significantly promoted, while Uros significantly inhibited the proliferation of PC12 cells, among which UroC showed the strongest anti-proliferation. UroC treatment actively increased the lactate dehydrogenase (LDH) release and lipid peroxidation malondialdehyde (MDA), stimulated reactive oxygen species (ROS) formation and mitochondrial membrane depolarization, and caused calcium dyshomeostasis. Furthermore, flow cytometry analysis showed that UroC treatment induced apoptosis and S phase cell cycle arrest with increasing UroC concentrations. Consequently, UroC also induced imbalance in the Bcl-2/Bax ratio, which triggered the caspase cascade, thereby shifting the balance in favor of apoptosis, as evidenced by western blotting and real-time PCR. These observations indicated that UroC possessed significantly different anti-proliferation activities from EA, and actively induced cell apoptosis through a mitochondria-mediated pathway.

Comparative evaluations on phenolic antioxidants of nine adulterants and anti-inflammation of four alternatives with their original herb Erycibe schmidtii

Erycibe schmidtii is widely used as folk medicine in China for treatments of various inflammations. Recently, with reduction of its wild sources, various adulterants have been misused as substitutes. To distinguish reliable alternatives from various adulterants, total phenolics and antioxidant activities of E. schmidtii and its nine adulterants, as well as three marker compounds of E. schmidtii defined by Chinese Pharmacopoeia were simultaneously quantified. And HPLC fingerprints of these ten herbs were established. Compared with E. schmidtii (S1), Porana sinensis (S2), Porana sinensis var. delavayi (S3), Celastrus hindsii (S4), and Morinda umbellata (S5) exhibited similar or higher total phenols, flavonoids and tannins along with similar or greater capacities scavenging DPPH˙, ABTS+˙ and AAPH˙, contained similar or higher total amounts of the three marker compounds, and possessed higher similarities in HPLC profiles. The other five adulterants (S6–S10, i.e., Illigera parviflora, Morinda parvifolia, Piper puberulum, Piper kadsura and Iodes seguini in sequence) were identified as absolute fakes, thus were excluded from alternatives of S1. Further anti-inflammatory experiments with LPS-induced RAW264.7 macrophages cells on NO release and transcriptions of inflammatory factors iNOS, COX-2, IL-6 and IL-1β showed that S2 and S3 possessed higher anti-inflammation activities than and similar mechanisms to those of S1. Taken together, S2 and S3 could be the best potentially alternatives of E. schmidtii among the nine adulterants. S4 and S5 might be also considered as alternatives for they contained similar fundamental compounds and equally impressive anti-inflammatory potential with E. schmidtii concerning iNOS and COX-2.

Simultaneous Preparation of Salidroside and p-Tyrosol from Rhodiola crenulata by DIAION HP-20 Macroporous Resin Chromatography Combined with Silica Gel Chromatography

The Rhodiola species have a long history of utilization in traditional medicine and have been considered as a source of adaptation to environmental challenges; salidroside and p-tyrosol are the major responsible compounds. Here we propose a novel UPLC-guided two-step method consisting of a DIAION HP-20 adsorption and silica gel column chromatographies, which can simultaneously prepare high purities of salidroside and p-tyrosol with noticeable yields from the rhizome of Rhodiola crenulata. Results demonstrated that DIAION HP-20 could successfully remove all impurities except crenulatin during a gradient elution with 5–20% ethanol, which could achieve an optimal purification of salidroside and p-tyrosol with increasing rates of 29.19% and 33.44%, respectively. Furthermore, chloroform was selected as an ideal solvent for separating p-tyrosol with salidroside, and thus crenulatin was subsequently applied in the silica gel chromatography, and the separation of salidroside with crenulatin could be achieved using silica gel chromatography with a mixture of chloroform and methanol at a volume ratio of 4:1. High purity rates of 94.17% and 97.29% and overall yields of 39.09% and 43.73% for salidroside and p-tyrosol were simultaneously achieved. Our method provides a new way to simultaneously obtain salidroside and p-tyrosol from R. Crenulata, as well as other related plant species.

Effects of thermal treatments on 10 major phenolics and their antioxidant contributions in Acer truncatum leaves and flowers

This study aimed to investigate effects of thermal treatments on major phenolics and their antioxidant contributions in Acer truncatum leaves and flowers (ATL and ATF, respectively). With ultra performance liquid chromatography-diode array detector-quadrupole time-of-flight-mass spectrometer/mass spectrometer, phenolic compositions of ATF were first characterized and compared with those of ATL. An optimized high performance liquid chromatography fingerprint was then established, and 10 major phenolics existing in both ATL and ATF were quantified. Gallic acid derivatives and flavonol-3-O-glycosides were found to be their dominant phenolic constituents, with the former being key constituents which was affected by thermal treatments and further influencing the variations of total phenols. Moreover, the mechanism underlining the changes of phenolics in ATL and ATF by the treatments was characterized as a thermolhydrolysis process. During thermal treatments, polymerized gallotannins were hydrolysed to 1,2,3,4,6-pentakis-O-galloyl-β-d-glucose, ethyl gallate and gallic acid, resulting in more than fivefold and twofold increase of their contents in ATL and ATF, respectively. By contrast, contents and antioxidant contributions of flavonol-3-O-glycosides gradually decreased during the process.\absbreak Overall, this is, to our knowledge, the first report on the effects of thermal treatments on phenolics and their antioxidant contributions in ATL and ATF, and the three gallic acid derivatives with potentially higher bioactivity could be efficiently achieved by thermal treatments.

Intraconversion of Polar Ginsenosides, Their Transformation into Less-Polar Ginsenosides, and Ginsenoside Acetylation in Ginseng Flowers upon Baking and Steaming

Heating is a traditional method used in ginseng root processing, however, there aren’t reports on differences resulting from baking and steaming. Moreover, ginseng flowers, with 5.06 times more total saponins than ginseng root, are not fully taken advantage of for their ginsenosides. Transformation mechanisms of ginsenosides in ginseng flowers upon baking and steaming were thus explored. HPLC using authentic standards of 20 ginsenosides and UPLC-QTOF-MS/MS were used to quantify and identify ginsenosides, respectively, in ginseng flowers baked or steamed at different temperatures and durations. Results show that baking and steaming caused a 3.2-fold increase in ginsenoside species existed in unheated ginseng flowers (20/64 ginsenosides) and transformation of a certain amount of polar ginsenosides into numerous less polar ginsenosides. Among the 20 ginsenosides with standards, polar ginsenosides were abundant in ginseng flowers baked or steamed at lower temperatures, whereas less polar ginsenosides occurred and were enriched at higher temperatures. Furthermore, the two types of heating treatments could generate mostly similar ginsenosides, but steaming was much efficient than baking in transforming polar- into less polar ginsenosides, with steaming at 120 °C being comparably equivalent to baking at 150 °C. Moreover, both the two heating methods triggered ginsenoside acetylation and thus caused formation of 16 acetylginsenosides. Finally, a new transformation mechanism concerning acetyl-ginsenosides formation was proposed.