PAF-induced Platelet Aggregation Research Articles

Biosynthesis of Phomactin Platelet Activating Factor AntagonistRequires a Two-Enzyme Cascade.

Phomactin diterpenoids possess a unique bicyclo[9.3.1]-pentadecane skeleton with multiple oxidative modifications, and are good platelet-activating factor (PAF) antagonists that can inhibit PAF-induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from amarine fungus,Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate-analogue FGGPP and elucidated the novel metal ion binding mode and enzymatic mechanism of PhmA through site-directed mutagenesis. This work provides the first insights into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Consumption of yogurt enriched with polar lipids from olive oil by-products reduces platelet sensitivity against platelet activating factor and inflammatory indices: A randomized, double-blind clinical trial

Several studies have reported the positive cardio-metabolic effects of yogurt consumption. The addition of olive oil’s bioactive extracts into yogurt can result to a functional food with pleiotropic properties against cardiovascular diseases. A polar lipid extract of olive pomace (OOPLE) that contains platelet activating factor receptor (PAF-R) antagonists, has been stated not only to inhibit the development of atherosclerotic plaques in hypercholesterolemic rabbits but also to regress the already formed plaques. The present study aims to investigate the effect of the daily intake of an OOPLE-enriched low-fat yogurt, on the metabolic profile, ex vivo platelet aggregation and markers of thrombosis and inflammation, in healthy, mainly overweight volunteers. A randomized, three-arm, double-blind, placebo-controlled, parallel-group, 8 weeks duration, clinical trial was performed in apparently healthy adults (35–65 years old). Group A (control group) consumed at most one yogurt every two weeks, Group B (plain yogurt) consumed one serving of plain yogurt every day and Group C (enriched yogurt) consumed one serving of yogurt, enriched with OOPLE, daily. Enriched yogurt intake resulted in lower levels of IL-10 and to lower platelet sensitivity against PAF, compared to the other two groups (p-values<0.05), while no impact on energy intake, body weight, glucose and lipid metabolism was detected. Lower levels of IL-6 were observed only at 4 weeks enriched yogurt intake (p = 0.03) compared to plain yogurt. Intake of the enriched yogurt resulted in reduced PAF-induced platelet aggregation and suppression of IL-10 levels. NCT02259205.

Hawanoids A‒E, unprecedented diterpenoids with PAF-induced platelet aggregation inhibitory activities from the deep-sea-derived fungus Paraconiothyrium hawaiiense

Hawanoids A‒E (1‒5), five highly cyclized diterpenoids were isolated from the deep-sea-derived fungus Paraconiothyrium hawaiiense FS482. Compounds 1 and 2 possessed an unprecedented tetracyclo[6.6.2.02,7.011,15]cetane carbon skeleton while 3 and 4 possessed an unusual 11,14-macrocyclic ether moiety in phomactin family. Their structures including the stereo-chemistry were determined through spectroscopic analysis, X-ray diffractions and computational calculations. The plausible biosynthetic pathway was proposed based on the predicted biosynthetic gene cluster. All of the isolated compounds exhibited inhibitory activities against PAF-induced platelet aggregation. The molecular docking study was carried out understand the interaction between the PAF receptor and hawanoids with different skeletons.

Amide Derivatives of Ginkgolide B and Their Inhibitory Effects on PAF-Induced Platelet Aggregation.

Ginkgolides are the most important components of Ginkgo biloba extracts, whose lactone can be hydrolyzed in the aqueous environment. Although the hydrolyzed products have complex structures and their functions are not well-understood, opening the lactone ring is an important strategy in producing novel derivatives of ginkgolide. The preparation of a single pure aminolyzed ginkgolide for the study of its bioactivity and understanding of the process of aminolysis are challenging. To obtain stable aminolyzed products, four amide derivatives (2–5) of ginkgolide B (GB, 1) were prepared via the ring-opening reaction of its lactone with propylamine. These products were purified and fully identified by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy and were further evaluated for their ability to inhibit the PAF-induced platelet aggregation of rabbit platelets in vitro. Compound 2, which was obtained by selective aminolysis of the lactone ring C of GB, showed a much better inhibitory activity of platelet aggregation (IC50, 15 nM) than the parent compound GB (IC50, 442 nM). The other three products (3–5), which were obtained by the aminolysis of lactone rings C and F of GB, did not show platelet aggregation inhibitory activity. The results greatly extended our understanding of the chemistry of GB and provided important structural information for the exploration and development of new drugs based on ginkgolides in G. biloba.

Inhibition of platelet-activating factor (PAF)-induced platelet aggregation by fatty acids from human saliva

Experiments were undertaken to identify the nature of a previously identified inhibitor of PAF-induced platelet aggregation (PA) in human saliva. Human saliva fractionated by preparative thin layer chromatography (TLC) yielded a fraction that co-migrated with fatty acids (FAs) and inhibited PAF-induced aggregation of platelets. Synthetic FAs tested for their capacities to inhibit 0.1 nM PAF-induced PA showed that only the cis-unsaturated compounds were inhibitory with activities of some of the polyunsaturated FAs (PUFA) reaching almost 100% at 20 μM. Eicosapentanoic acid EPA and 8,11,14-eicosatrienoic acid also deaggregated the PAF-induced aggregates. With the exception of oleic acid (OLA), cis-monounsaturated FAs, and elaidic acid, the trans isomer of OLA, were poor inhibitors. In a direct comparison with other platelet agonists, ADP, thrombin, and ionophore A23187, the active saliva fraction and selected individual FA inhibited, to greater or lesser extent, PA induced by each of the agonists. EPA, OLA, linoleic acid (LNA), and the active saliva fraction were potent inhibitors of ADP-induced PA, EPA completely inhibited thrombin-induced PA and the saliva fraction showed only weak – moderate inhibitory activity to both thrombin- and ionophore A23187-induced PA. Other reports of endogenous PAF inhibitors in mammalian tissues are compared to the present results. PAF can trigger and amplify inflammatory cascades suggesting a possible modulation role for cis-unsaturated FAs in some diseases.

Anti-Inflammatory and Anti-Platelet Properties of Lipid Bioactives from Apple Cider By-Products.

The valorization of food industry by-products as sources of bioactive compounds is at the forefront of research in functional foods and nutraceuticals. This study focuses on bioactives of apple cider by-products (ACBPs) with putative cardio-protective properties. Total lipids (TLs) were extracted from ACBPs of apple varieties that are low (ACBP1), medium (ACBP2), and high (ACBP3) in tannins and were further separated into polar lipids (PLs) and neutral lipids (NLs). The functionality of these lipid extracts and of their HPLC-derived lipid fractions/PL subclasses were assessed in vitro against human platelet aggregation induced by the thrombotic and inflammatory platelet agonists platelet-activating factor (PAF) and adenosine diphosphate (ADP). The fatty acid profile of PLs and their most bioactive lipid fractions were evaluated by GC–MS analysis. The PL extracts exhibited higher specificity against the PAF-induced platelet aggregation compared to their anti-ADP effects, while TL and NL showed lower bioactivities in all ACBPs. HPLC analysis unveiled that the most bioactive PL from all ACBPs were those in PL fraction 3 containing phosphatidylcholines (PCs). PLs from all ACBPs and their PC bioactives were rich in polyunsaturated fatty acids (PUFAs) and especially in the essential omega-6 (n-6) linoleic acid (LA) and omega-3 (n-3) alpha linolenic acid (ALA), with favorably low values of the n-6/n-3 PUFA ratio, thus providing a rationale for their higher anti-inflammatory bioactivities. Within this study, highly bioactive PL compounds with strong anti-inflammatory and anti-platelet properties were identified in ACBPs, which can be potentially utilized for producing cardio-protective functional foods and/or nutraceuticals.

Beneficial Anti-Platelet and Anti-Inflammatory Properties of Irish Apple Juice and Cider Bioactives.

Several bioactives from fruit juices and beverages like phenolics, nucleotides and polar lipids (PL) have exhibited anti-platelet cardio-protective properties. However, apple juice and cider lipid bioactives have not been evaluated so far. The aim of this study was to investigate the anti-platelet and anti-inflammatory effects and structure activity relationships of Irish apple juice and Real Irish cider lipid bioactives against the platelet-activating factor (PAF)- and adenosine diphosphate (ADP)-related thrombotic and inflammatory manifestations in human platelets. Total Lipids (TL) were extracted from low, moderate and high in tannins apple juices and from their derived-through-fermentation cider products, as well as from commercial apple juice and cider. These were separated into neutral lipids (NL) and PL, while all lipid extracts were further assessed for their ability to inhibit aggregation of human platelets induced by PAF and ADP. In all cases, PL exhibited the strongest anti-platelet bioactivities and were further separated by high-performance liquid chromatography (HPLC) analysis into PL subclasses/fractions that were also assessed for their antiplatelet potency. The PL from low in tannins apple juice exhibited the strongest antiplatelet effects against PAF and ADP, while PL from its fermented cider product were less active. Moreover, the phosphatidylcholines (PC) in apple juices and the phosphatidylethanolamines (PE) in apple ciders were the most bioactive HPLC-derived PL subclasses against PAF-induced platelet aggregation. Structural elucidation of the fatty acid composition by gas chromatography mass spectra (GCMS) analysis showed that PL from all samples are rich in beneficial monounsaturated fatty acids (MUFA) and omega 3 (n-3) polyunsaturated fatty acids (PUFA), providing a possible explanation for their strong anti-platelet properties, while the favorable low levels of their omega-6/omega-3 (n-6/n-3) PUFA ratio, especially for the bioactive PC and PE subclasses, further support an anti-inflammatory cardio-protective potency for these apple products. In conclusion, Irish apple juice and Real Irish cider were found to possess bioactive PL compounds with strong antiplatelet and anti-inflammatory properties, while fermentation seems to be an important modulating factor on their lipid content, structures and bioactivities. However, further studies are needed to evaluate these effects.

Reversible cross-tolerance to platelet-activating factor signaling by bacterial toxins

Bacterial toxins signaling through Toll-like receptors (TLRs) are implicated in the pathogenesis of many inflammatory diseases. Among the toxins, lipopolysaccharide (LPS) exerts its action via TLR-4 while lipoteichoic acid (LTA) and bacterial lipoproteins such as Braun lipoprotein (BLP) or its synthetic analogue Pam3CSK4 act through TLR-2. Part of the TLR mediated pathogenicity is believed to stem from endogenously biosynthesized platelet-activating factor (PAF)- a potent inflammatory phospholipid acting through PAF-receptor (PAF-R). However, the role of PAF in inflammatory diseases like endotoxemia is controversial. In order to test the direct contribution of PAF in TLR-mediated pathogenicity, we intraperitoneally injected PAF to Wistar albino mice in the presence or absence of bacterial toxins. Intraperitoneal injection of PAF (5 μg/mouse) causes sudden death of mice, that can be delayed by simultaneously or pre-treating the animals with high doses of bacterial toxins- a phenomenon known as endotoxin cross-tolerance. The bacterial toxins- induced tolerance to PAF can be reversed by increasing the concentration of PAF suggesting the reversibility of cross-tolerance. We did similar experiments using human platelets that express both canonical PAF-R and TLRs. Although bacterial toxins did not induce human platelet aggregation, they inhibited PAF-induced platelet aggregation in a reversible manner. Using rabbit platelets that are ultrasensitive to PAF, we found bacterial toxins (LPS and LTA) and Pam3CSK4 causing rabbit platelet aggregation via PAF-R dependent way. The physical interaction of PAF-R and bacterial toxins is also demonstrated in a human epidermal cell line having stable PAF-R expression. Thus, we suggest the possibility of direct physical interaction of bacterial toxins with PAF-R leading to cross-tolerance.

P38 MAP-kinase inhibitor protects against platelet-activating factor-induced death in mice

Platelet-activating factor (PAF) is a potent inflammatory agonist. In Swiss albino mice, intraperitoneal injection of PAF causes sudden death with oxidative stress and disseminated intravascular coagulation (DIC), characterized by prolonged prothrombin time, thrombocytopenia, reduced fibrinogen content, and increased levels of fibrinogen degradation products. However, the underlying mechanism(s) is unknown. The PAF-R antagonist WEB-2086 protected mice against PAF-induced death by reducing DIC and oxidative stress. Accordingly, general antioxidants such as ascorbic acid, α-tocopherol, gallic acid, and N-acetylcysteine partially protected mice from PAF-induced death. N-acetylcysteine, a clinically used antioxidant, prevented death in 67% of mice, ameliorated DIC characteristics and histological alterations in the liver, and reduced oxidative stress. WEB-2086 suppressed H2O2-mediated oxidative stress in isolated mouse peritoneal macrophages, suggesting that PAF signaling may be a downstream effector of reactive oxygen species generation. PAF stimulated all three (ERK, JNK, and p38) of the MAP-kinases, which were also inhibited by N-acetylcysteine. Furthermore, a JNK inhibitor (SP600125) and ERK inhibitor (SCH772984) partially protected mice against PAF-induced death, whereas a p38 MAP-kinase inhibitor (SB203580) provided complete protection against DIC and death. In human platelets, which have the canonical PAF-R and functional MAP-kinases, JNK and p38 inhibitors abolished PAF-induced platelet aggregation, but the ERK inhibitor was ineffective. Our studies identify p38 MAP-kinase as a critical, but unrecognized component in PAF-induced mortality in mice. These findings suggest an alternative therapeutic strategy to address PAF-mediated pathogenicity, which plays a role in a broad range of inflammatory diseases.

Total, Neutral, and Polar Lipids of Brewing Ingredients, By-Products and Beer: Evaluation of Antithrombotic Activities.

The in vitro antithrombotic properties of polar lipid constituents of malted grain (MG), pelleted hops (PH), brewer’s spent grain (BSG), spent hops (SH), wort, and bottled beer from the same production line were assessed in human platelets. The total lipids (TL) were extracted according to the Bligh and Dyer method and further separated into the total neutral lipids (TNL) and total polar lipids (TPL) extracts by counter-current distribution. The TL, TNL, and TPL extracts of all samples were assessed for their ability to inhibit platelet-activating factor (PAF) and thrombin-induced human platelet aggregation. The raw materials, by-products, wort, and beer lipid extracts all exhibited antithrombotic properties against PAF and thrombin. However, the beer TPL exhibited the lowest IC50 values against PAF-induced (7.8 ± 3.9 µg) and thrombin-induced (4.3 ± 3.0 µg) platelet aggregation indicating that these polar lipids were the most antithrombotic. The lipid extracts tended to be more bioactive against the thrombin pathway. The fatty acid content of all the TPL extracts were assessed using GC-MS. The fatty acid composition of the most bioactive TPL extracts, the wort and the beer, shared similar fatty acid profiles. Indeed, it was noted that fermentation seems to play a role in increasing the antithrombotic properties of polar lipids against PAF and thrombin by moderately altering the polar lipid fatty acid composition. Furthermore, the use of brewing by-products as a source of functional cardioprotective lipids warrants further investigation and valorisation.

Chemical profiling and anticoagulant activity of Ginkgo Biloba leaves under the influence of harvesting time

Gingko biloba, family Gink, is used as a source of food and in traditional medicine for treatment of cough and promoting blood circulation, etc. The aim of the present work is to determine the chemical variation of G. biloba leaves collected from different harvesting time and in vitro anti-platelet aggregation effects, respectively. Methanol extract of G. biloba leaves was subjected to ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry analysis and triple quadrupole mass spectrometry. The anti-platelet aggregation effects induced by platelet-activating factor (PAF) and adenosine diphosphate (ADP) was measured by Born’s method. UHPLC-QTOF-MS analysis confirmed the presence of flavonoids, phenolic acid and terpene lactones in different sample. Partial least square discriminant analysis based on chemical profiling conducted to differentiate the samples according to their harvest time. All samples found highly effective against PAF-induced platelet aggregation with IC50 of 98.87 μg/mL (summer sample) and 51.55 μg/mL (autumn sample). However, on ADP-induced platelet aggregation, IC50 of these samples were greater than 200 μg/mL. Both total contents of flavonoids and terpene lactones in autumn sample were greater than that in summer sample. Qualitative and quantitative analysis showed that the distribution of chemicals was variation in different harvesting time.

In Vitro Antithrombotic Properties of Salmon (Salmo salar) Phospholipids in a Novel Food-Grade Extract

Marine and salmon polar lipids (PLs) extracted by conventional extractions with non-food-grade solvents (CE-salmon-PLs) possess antithrombotic bioactivities against platelet-activating factor (PAF) and thrombin. Similar effects of food-grade-extracted (FGE) marine PLs have not yet been reported. In this study, food-grade solvents were used to extract PLs from Irish organic farmed salmon (Salmo salar) fillets (FGE-salmon-PLs), while their antithrombotic bioactivities were assessed in human platelets induced by platelet aggregation agonists (PAF/thrombin). FGE-salmon-PLs were further separated by thin layer chromatography (TLC) into lipid subclasses, and the antithrombotic bioactivities of each subclass were also assessed. LC-MS was utilized to elucidate the structure-activity relationships. FGE-salmon-PLs strongly inhibited PAF-induced platelet aggregation, while their relevant anti-thrombin effects were at least three times more potent than the previously reported activities of CE-salmon-PLs. TLC-derived lipid fractions corresponding to phosphatidylcholines (PC) and phosphatidylethanolamines (PE) were the most bioactive lipid subclasses obtained, especially against thrombin. Their LC-MS analysis elucidated that they are diacyl- or alkyl-acyl- PC and PE moieties baring ω3 polyunsaturated fatty acids (PUFA) at their sn-2 position, such as eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Our results concerning the potent antithrombotic effects of FGE-salmon-PLs against both PAF and thrombin pathways strongly suggest that such food-grade extracts are putative candidates for the development of novel cardioprotective supplements and nutraceuticals.

Novel breviscapine nanocrystals modified by panax notoginseng saponins for enhancing bioavailability and synergistic anti-platelet aggregation effect.

Breviscapine (BVP) is a flavonoid compound with strong neuroprotective and anti-platelet aggregation effect. The objective of this study is to design novel BVP nanocrystals modified by natural panax notoginseng saponins (PNS) for enhancing dissolution and anti-platelet aggregation effect of BVP. BVP nanocrystals modified by PNS (BVP-NC/PNS) were firstly prepared by coupling homogenization technology and freeze-drying technology, and BVP nanocrystals modified by RH40 (BVP-NC/RH40) as reference for comparison. The morphology, crystals characterization, dissolution behavior and anti-platelet aggregation effect of BVP-NC/PNS was systemically evaluated. The results demonstrated that the PNS could effectively maintain stability of BVP-NC at suspensions state dependent of its surface activity and the electrostatic repulsion effect. Combination of PNS and trehalose could prevent the aggregation of BVP-NC/PNS during freeze-drying. The PXRD and DSC results demonstrated that the BVP crystal state in BVP-NC/PNS was not changed owing to PNS modification and homogenization treatment. And the freeze-dried BVP-NC could easily recover back to BVP-NS and significantly improve the dissolution of BVP. The AUC(0-∞) of the BVP-NC/PNS was 4.54 times as high as that of the coarse BVP, but not significantly different compared to that of BVP-NC/RH40 (p < 0.05). The anti-platelet aggregation results demonstrated that, BVP-NC/PNS group showed more effective inhibition on PAF-induced platelet aggregation compared with corresponding control groups, which might attribute to the enhanced bioavailability of BVP and synergistic effect of PNS with BVP. In conclusion, PNS could be used as an alternative stabilizer for preparation of BVP-NC, and BVP-NC modified by PNS is a promising formulation strategy for enhancing oral bioavailability and anti-platelet aggregation of BVP.

Structural Elucidation of Irish Organic Farmed Salmon (Salmo salar) Polar Lipids with Antithrombotic Activities.

While several marine polar lipids (PL) have exhibited cardioprotective properties through their effects on the platelet-activating factor (PAF) pathways, salmon PL have not been tested so far. In this study, the antithrombotic activities of salmon PL were assessed in human platelets and the structural characterisation of bioactive salmon PL was performed by GC-MS and LC-MS analyses. PL from fillets of Irish organic farmed salmon (Salmo salar) were extracted and separated into several lipid subclasses by thin-layer chromatography (TLC), while their fatty acid profile was fully characterised by GC-MS. Salmon total lipids (TL), total neutral lipids (TNL), total polar lipids (TPL), and each PL subclass obtained by TLC were further assessed for their in vitro effects towards PAF-induced and thrombin-induced platelet aggregation in human platelets. Salmon PL exhibited antithrombotic effects on human platelet aggregation, mostly through their strong inhibitory effects against the PAF pathway with IC50 values comparable to other marine PL, but with lower effects towards the thrombin pathway. PL fractions corresponding to phosphatidylcholine and phosphatidylethanolamine derivatives exhibited the most potent anti-PAF effects, while LC-MS analysis putatively elucidated their structure/function relationship. Several diacyl-PC/PE and alkyl-acyl-PC/PE species containing mostly docosahexaenoic acid at their sn-2 glycerol-backbone may be responsible for the bioactivity. The data presented suggests that salmon contains PL with strong antithrombotic bioactivities.

Effect of compatibility of ginkgolide A, ginkgolide B and ginkgolide K

To investigate the best active compatibility of ginkgolide A, B and K (GA，GB，GK). The effects of GA, GB, GK alone, combinations of each two of them, and combinations of these three components on platelet-activating factor (PAF)-induced platelet aggregation activity and rat cerebral ischemia reperfusion model (tMCAO) were compared in this study. Different compatibilities of GA, GB and GK could significantly reduce the maximum aggregation rate of PAF-induced platelet aggregation, and the effect was most obvious in combination of the three. Different compatibilities of GA, GB and GK could alleviate the neural function, cerebral infarction volume and cerebral edema in the tMCAO model of rats to different degrees, and the effect of combinations of the three was stronger than those of combinations of two and single use. The combination of all of GA, GB and GK had the strongest effect on nerve injury caused by anti-platelet aggregation in tMCAO rats.

Antagonistic effect of ginkgolide homologues on PAF-induced platelet aggregation and neuroprotective effect

To study the antagonistic effect of ginkgolide homologues on platelet-activating factor (PAF)-induced platelet aggregation and investigate its neuroprotective effect. PAF was used as a coagulant, and ginkgolides were added to the rabbit blood samples respectively. The inhibitory effect of each compound on platelet aggregation was detected by turbidimetry. In L-glutamate induced primary cortical neuron cell injury model, MTT assay was used to detect cell viability. Intracellular free Ca2+ concentration in neurons was measured by using the fluorescent Ca2+ indicator Fura-2 AM. Morphological observation and Hoechst 33258 staining were used to detect the inhibitory effect of ginkgolide on neuronal apoptosis. The results showed that the inhibitory effect on PAF-induced platelet aggregation activity in ginkgolide homologues was ginkgolide K (GK), ginkgolide B (GB), ginkgolide A (GA), ginkgolide C (GC), ginkgolide M (GM), ginkgolide J (GJ) and ginkgolide (GL) from high to low. GB and GK (1-100 μmol•L ⁻¹) could significantly reduce the cell damage caused by L-glutamate, with survival rate increasing, intracellular calcium concentration reducing and cell morphology restoring. This paper has identified the activities and characteristics of various compounds of ginkgolide homologues on PAF-induced platelet aggregation as well as its neuroprotective effect.

Effects of ginkgolide K on platelet aggregation activity and neuroprotection

To investigate the antagonism effects of different concentrations of ginkgolide K(GK) on platelet activating factor (PAF)-induced platelet aggregation and neuroprotective effect on cells and animal models of ischemia-reperfusion injury. GK-containing serum in rabbit was prepared, and the effects of GK-containing serum on PAF-induced platelet aggregation was observed by platelet aggregation assay. The effect of different concentrations of GK on apoptosis of SH-SY5Y cells injured by oxygen-glucose deprivation/reoxygenation (OGD/R) was investigated by Hoechst 33342/PI double staining in OGD/R cell model. The focal cerebral ischemia-reperfusion model (I/R)was established in rats to detect the effects of GK on neurobehavioral scores and cerebral infarction volume. GK could inhibit PAF-induced platelet aggregation, reverse the apoptosis induced by OGD/R injury and improve the neurobehavioral score and cerebral infarction volume after cerebral ischemia-reperfusion injury in rats in a dose-dependent manner. GK can inhibit PAF-induced platelet aggregation and improve nerve injury after cerebral ischemia-reperfusion.

Effects of extracts and isolated compounds from safflower on some index of promoting blood circulation and regulating menstruation

Ethnopharmacological relevanceCarthamus tinctorius is used as one of the Traditional Chinese Medicine (TCM) materials in prescriptions and composite to promote blood circulation to remove blood stasis, regulate menstruation and alleviate pain for over 2500 years. Modern pharmacological experiments have demonstrated that safflower has wide-reaching biological activities, including dilating coronary artery, modulating immune system, improving myocardial ischemia, anticoagulation and thromboprophylaxis, antioxidation, antihypoxic, antiaging, antifatigue, antiinflammation, anti-hepatic fibrosis, antitumor, analgesia, etc. Materials and methodsPlatelet aggregation of safflower extract and main constituents in safflower were determined by PAF-induced or ADP-induced platelet aggregation in vitro. Anticoagulation activity was measured by clotting assay of thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) according to the methods provided by the biological reagents provider (Sun Biochemical). Antioxidant effects of safflower were assessed using DPPH radical-scavenging activity test, ABTS radical-scavenging activity test and ferric reducing antioxidant power test. In addition, rats ovary granulosa cell proliferation activity was used for the bio-activity index on regulate menstruation of safflower. ResultsSafflower extract at the concentration of 0.7g/mL (P<0.001) and 0.5g/mL (P<0.01) had significantly antagonistic effect on PAF-induced platelet aggregation, compared with negative control. And the anti-platelet aggregation of 0.7g/mL safflower extract was significantly stronger than that of positive control (P<0.001). 0.7g/mL of hydroxysafflor yellow A (P<0.01), anhydrosafflor yellow B (P<0.05), 6-hydroxykaempferol-3-O-rutinoside (P<0.05), keampferol-3-O-β-rutinoside (P<0.01) had significant effect on platelet aggregation compared with negative control. Safflower extract at the concentration of 0.5g/mL (P<0.001) and 0.125g/mL (P<0.01) could significantly inhibit ADP-induced platelet aggregation, compared with negative control. And antagonistic effect of safflower extract was significantly stronger than the effect of positive control (P<0.001). Adenosine (P<0.001), anhydrosafflor yellow B (P<0.01) and 6-hydroxykaempferol-3-O-rutinoside (P<0.01) at the concentration of 0.5g/mL had significant effect on ADP-induced platelet aggregation compared with negative control. 0.125g/mL of adenosine (P<0.05) had significant effect on ADP-induced platelet aggregation compared with negative control. The effect of 0.5g/mL adenosine (P<0.01) and 6-hydroxykaempferol-3-O-rutinoside (P<0.05) was significantly stronger than that of positive control. Safflower extract at the concentration of 0.7mg/mL (P<0.001) and 0.5mg/mL (P<0.001) had significantly anticoagulation activity in PT, TT and APTT, compared with negative control. However, the respective compound didn’t have significant effect on PT and TT at experiment concentration. At the concentration of 0.7mg/mL, hydroxysafflor yellow A (P<0.01), 6-hydroxykaempferol-3,6,7-tri-O-β-d-glucoside (P<0.05), 6-hydroxyapigenin-6-O-glucoside-7-O-glucuronide (P<0.01), anhydrosafflor yellow B (P<0.001), 6-hydroxykaempferol-3-O-rutinoside (P<0.05) and keampferol-3-O-β-rutinoside (P<0.05) significantly prolonged APTT, compared with negative control. At the concentration of 0.5mg/mL, hydroxysafflor yellow A (P<0.05), 6-hydroxyapigenin-6-O-glucoside-7-O-glucuronide (P<0.05), anhydrosafflor yellow B (P<0.001), 6-hydroxykaempferol-3-O-rutinoside (P<0.05) and keampferol-3-O-β-rutinoside (P<0.05) could significantly prolong APTT, compared with negative control. From the results of DPPH, ABTS radical scavenging activity test and Fe3+ reduction power test, 5mg/mL, 2.5mg/mL and 1.25mg/mL safflower extract had antioxidant effects. Every compound with each concentration (5mg/mL, 2.5mg/mL and 1.25mg/mL) had significant effect on Fe3+ reduction power (P<0.001 vs. negative control). Safflower extract, cytidine, 6-hydroxy-kaempferol-3,6-di-O-β-d-glucoside-7-O-β-d-glucuronide, 6-hydroxykaemp-ferol-3,6,7-tri-O-β-D-glucoside and keampferol-3-O-β-rutinoside significantly promoted ovarian granulosa cell proliferation. ConclusionBased on previous researches, the activities of safflower extract and pure compounds isolated from safflower were studied in this paper. This study found some compounds with the effects of anti-platelet aggregation, anticoagulation, antioxidation and ovarian granulosa cell proliferation, and further revealed the possible pharmacological mechanism of safflower.

In Vitro Effects of Anti-Glaucomatous Eye Drops on Platelet-Activating Factor and its Metabolism

Purpose: The purpose of this study is to determine the effect of various commonly used antiglaucoma eye drops on inflammatory mediators such as the platelet activating factor (PAF). Methods: Various intraocular pressure (IOP) lowering drops were tested to examine their inhibitory effect on PAF. Multiple eye drops were tested in washed rabbit platelets (WRPs) in order to determine the interaction between these eye drops and the inhibition of PAF in the PAF-induced platelet aggregation model. In addition, we examined the eyedrops’ effect on PAF-metabolism, through in vitro analysis on PAF basic metabolic enzymes (PAF-CPT, lyso PAF-AT, and PAF-AH). Results: Latanoprost (Xalatan) was found to be the most potent in inhibiting PAF, suggesting that it is the most effective in decreasing IOP amongst the eye drops tested. Conversely, dorzolamide hydrochloride-timolol (Cosopt) exhibited the least anti-PAF action. Conclusions: This is the first study examine the relationship between PAF activity and glaucoma medication. Potency in PAF inhibition may be related to drop efficacy.

In vitro anti-atherogenic properties of traditional Greek cheese lipid fractions

Given that platelet activating factor (PAF) is a crucial inflammatory phospholipid mediator that is implicated in the mechanism of atherogenesis, the presence of PAF inhibitors in food reinforces their nutritional value in terms of protection against cardiovascular diseases. The aim of the present study was to evaluate the anti-atherogenic (anti-inflammatory) properties of two different types of Greek cheese: Kefalotyri and Ladotyri. Total lipids (TL) of both types of cheese samples were extracted by the method of Bligh and Dyer and separated into total polar lipids (TPL) and total neutral lipids (TNL) by countercurrent distribution. TPL were further separated by preparative thin-layer chromatography (TLC). TL, TPL, TNL and the obtained polar lipid fractions after TLC separation were tested to determine their biological activity towards atherosclerosis based on the in vitro inhibition of PAF-induced platelet aggregation. Both types of cheese samples exhibited strong biological activity, and their lipids were potent PAF inhibitors. Comparing the two types of cheese samples, Ladotyri cheese polar lipid fractions were found to exhibit stronger inhibitory properties than those of Kefalotyri cheese. The fact that both types of cheese were found to contain PAF inhibitors highlights their nutritional value in terms of cardio-protection.