Biflavones from Ginkgo biloba as inhibitors of human thrombin.

Ginkgo Biloba leaf extract has been widely used for the prevention and treatment of thrombosis and cardiovascular disease in both eastern and western countries, but the bioactive constituents and the underlying mechanism of anti-thrombosis have not been fully characterized. The purpose of this study was to investigate the inhibitory effects of major constituents in Ginkgo biloba on human thrombin, a key serine protease regulating the blood coagulation cascade and the processes of thrombosis. To this end, a fluorescence-based biochemical assay was used to assay the inhibitory effects of sixteen major constituents from Ginkgo biloba on human thrombin. Among all tested natural compounds, four biflavones (ginkgetin, isoginkgetin, bilobetin and amentoflavone), and five flavonoids (luteolin, apigenin, quercetin, kaempferol and isorhamnetin) were found with thrombin inhibition activity, with the IC50 values ranging from 8.05 µM to 82.08 µM. Inhibition kinetic analyses demonstrated that four biflavones were mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki values ranging from 4.12 µM to 11.01 µM. Molecular docking method showed that the four biflavones could occupy the active cavity with strong interactions of salt bridges and hydrogen bonds. In addition, mass spectrometry-based lysine labeling reactivity assay suggested that the biflavones could bind on human thrombin at exosite I rather than exosite II. All these findings suggested that the biflavones in Ginkgo biloba were naturally occurring inhibitors of human thrombin, and these compounds could be used as lead compounds for the development of novel thrombin inhibitors with improved efficacy and high safety profiles.

Exercise preconditioning promotes myocardial GLUT4 translocation and induces autophagy to alleviate exhaustive exercise-induced myocardial injury in rats.

Exercise preconditioning (EP) is a line of scientific inquiry into the short-term biochemical mediators of cardioprotection in the heart. This study examined the involvement of autophagy induced by energy metabolism in myocardial remodelling by EP and myocardial protection. A total of 120 healthy male Sprague Dawley (SD) rats were randomly divided into six groups. Plasma cTnI, HBFP staining and electrocardiographic indicators were examined in the context of myocardial ischemic/hypoxic injury and protection. Western blotting and fluorescence double labelling were used to investigate the relationship between energy metabolism and autophagy in EP-resistant myocardial injury caused by exhaustive exercise. Compared with those in the C group, the levels of myocardial ischemic/hypoxic injury were significantly increased in the EE group. Compared with those in the EE group, the levels of myocardial ischemic/hypoxic injury were significantly decreased in the EEP + EE and LEP + EE groups. Compared with that in the EE group, the level of GLUT4 in the sarcolemma was significantly increased, and the colocalization of GLUT4 with the sarcolemma was significantly increased in the EEP + EE and LEP + EE groups (P < 0.05). LC3-II and LC3-II/LC3-I levels of the EEP + EE group were significantly elevated compared with those in the EE group (P < 0.05). The levels of p62 were significantly decreased in the EEP + EE and LEP + EE groups compared with the EE group (P < 0.05). EP promotes GLUT4 translocation and induced autophagy to alleviate exhaustive exercise-induced myocardial ischemic/hypoxic injury.

Comparison of myocardial ischemic/hypoxic staining techniques for evaluating the alleviation of exhaustive exercise-induced myocardial injury by exercise preconditioning.

Exercise preconditioning (EP) can alleviate myocardial ischemic/hypoxic injury by inducing endogenous cardioprotection. Hematoxylin-eosin (HE), hematoxylin-basic fuchsin-picric acid (HBFP), and chromotrope-2R brilliant green (C-2R BG) staining have been used to visualize myocardial ischemic/hypoxic changes in previous EP studies, but comprehensive evaluation and comparisons of these methods are lacking. This study evaluated ischemic/hypoxic changes in adjacent myocardial sections by HE, HBFP, and C-2R BG and compared the characteristics of sections stained by these three methods to show changes associated with exercise-induced myocardial ischemic/hypoxic injury. Rats were randomly divided into four groups: control (C), exercise preconditioning (EP), exhaustive exercise (EE), and exercise preconditioning + exhaustive exercise (EP + EE). Adjacent myocardial sections were stained as described above and compared to evaluate the effects of exercise-induced myocardial ischemic/hypoxic injury. The three staining methods revealed consistent localization patterns of myocardial ischemic/hypoxic injury in all groups. Results suggest that EP can alleviate exhaustive exercise-induced myocardial ischemic/hypoxic injury, and the three staining methods are suitable for the histological study of exercise-induced myocardial ischemic/hypoxic injury and protection. HE staining is a simple procedure but is not specific for myocardial ischemic/hypoxic injury. HBFP and C-2R BG staining can be used to specifically visualize myocardial ischemic/hypoxic injury.

Inhibition of human thrombin by the constituents of licorice: inhibition kinetics and mechanistic insights through in vitro and in silico studies.

Thrombin inhibition therapy is a practical strategy to reduce thrombotic and cardiovascular risks via blocking the formation of blood clots. This study aimed to identify naturally occurring thrombin inhibitors from licorice (one of the most popular edible herbs), as well as to investigate their inhibitory mechanisms. Among all tested licorice constituents, licochalcone A was found as the most efficacious agent against human thrombin (IC50 = 7.96 µM). Inhibition kinetic analyses demonstrated that licochalcone A was a mixed inhibitor against thrombin-mediated Z-Gly-Gly-Arg-AMC acetate hydrolysis, with a K i value of 12.23 µM. Furthermore, mass spectrometry-based chemoproteomic assays and molecular docking simulations revealed that licochalcone A could bind to human thrombin at both exosite I and the catalytic site. In summary, our findings demonstrated that the chalcones isolated from licorice were a new class of direct thrombin inhibitors, also suggesting that licochalcone A was a promising lead compound for developing novel anti-thrombotic agents.

Natural constituents of St. John's Wort inhibit the proteolytic activity of human thrombin.

Thrombin, a multifunctional serine protease responsible for the proteolytic hydrolysis of soluble fibrinogen, plays a pivotal role in the blood coagulation cascade. Currently, thrombin inhibitor therapy has been recognized as an effective therapeutic strategy for the prevention and treatment of thrombotic diseases. In this study, the inhibitory effects of natural constituents in St. John's Wort against human thrombin are carefully investigated by a fluorescence-based biochemical assay. The results clearly demonstrate that most of naphthodianthrones, flavonoids and biflavones exhibit strong to moderate inhibition on human thrombin. Among all tested compounds, hypericin shows the most potent inhibitory capability against thrombin, with the IC50 value of 3.00 µM. Further investigation on inhibition kinetics demonstrates that hypericin is a potent and reversible inhibitor against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki value of 2.58 µM. Inhibition kinetic analyses demonstrate that hypericin inhibits thrombin-mediated Z-GGRAMC acetate hydrolysis in a mixed manner, which agrees well with the results from docking simulations that hypericin can bind on both catalytic cavity and anion binding exosites. All these findings suggest that hypericin is a natural thrombin inhibitor with a unique dianthrone skeleton, which can be used as a good candidate to develop novel thrombin inhibitors with improved properties.

Anthraquinones from Cassiae semen as thrombin inhibitors: in vitro and in silico studies.

Thrombin inhibitor therapy is one of the most effective therapeutic strategies for the prevention and treatment of cardiovascular and thrombotic diseases. Although several marketed direct thrombin inhibitors (DTIs) have been widely used in clinic, the potentially serious complications of these DTIs prompted the researchers to find more DTIs with improved safety profiles. Herein, we report that natural anthraquinones in Cassiae semen (the seed of Cassia obtusifolia L. or C. tora L.), including obtusifolin, obtusin, aurantio-obtusin and chryso-obtusin, display strong to moderate inhibition on human thrombin, with the IC50 values ranging from 9.08 µM to 27.88 µM. Further investigation on the inhibition kinetics demonstrates that these anthraquinones are mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, while obtusifolin and aurantio-obtusin show strong thrombin inhibition capacity, with the Ki values of 9.63 µM and 10.30 µM, respectively. Docking simulations demonstrate that both obtusifolin and aurantio-obtusin can simultaneously bind on the catalytic cavity and the two anion binding exosites (ABE1 and ABE2), while the hydroxyl group at the C-7 site and the methoxyl group at the C-8 site can create key interactions with the amino acids surrounding the catalytic cavity via hydrogen bonding. All these findings suggest that obtusifolin and aurantio-obtusin are strong thrombin inhibitors possessing a unique anthraquinone skeleton, and could be used as lead compounds for the development of new thrombin inhibitors with improved properties.

Heavy metal pollution recorded in Porites corals from Daya Bay, northern South China Sea.

We examined metal-to-calcium ratios (Fe/Ca, Mn/Ca and Zn/Ca) in the growth bands of two Porites corals from Daya Bay, South China Sea, in order to trace long-term trends in local ambient pollution levels. Although Fe and Mn did not show any obvious increasing trends over 32 years in the period 1976-2007, peak values of Fe/Ca and Mn/Ca occurred in the mid-late 1980s, temporally-coeval with the local construction of a nuclear power station. Furthermore, both corals showed rapid increases in Zn concentrations over the past 14 years (1994-2007), most likely due to increases in domestic and industrial sewage discharge. The Daya Bay corals had higher concentrations of metals than other reported corals from both pristine and seriously polluted locations, suggesting that acute (Fe and Mn) and chronic (Zn) heavy metal contamination has occurred locally over the past approximately 32 years.

[Low water temperature tolerance and responding mode of scleractinian corals in Sanya Bay].

In an experimental temperature-regulated mesocosm, the low water temperature tolerance of five dominant scleractinian coral species Pavona decussate, Acropora pulchra, Acropora florida, Acropora valida, and Porites lutea in Sanya Bay was investigated, and their responding modes to the cold water stress were analyzed. The tolerance of test corals to low water temperature was closely related to their morphologies, with the branching corals being the most vulnerable to bleaching and death by separating the symbiotic polyps from their skeletons. The lethal low water temperature for branching Acropora corals was 14 degrees C lasting for 3 days, and that for foliose P. decussate was 12 degrees C lasting for 10 days. Massive P. lutea corals responded to low water temperature by forming mucus membrane, which helped to prevent the further losing of symbiotic algae. The corals showing strong tolerance to high water temperature also had strong tolerance to low water temperature, and had similar responding modes to both high and low water temperature, i.e., the corals didn't extend their tentacle first, followed by the continuous release of mucus and the discharge of symbiotic zooxanthellae, and finally, bleached and died.