Antithrombotic components of Malus halliana Koehne flowers.

Flowers of Malus halliana (M. halliana) Koehne have been used as a Chinese traditional medicine to treat metrorrhagia and in our study, its chemical composition and anticoagulant effect were investigated. Five compounds were isolated and identified from M. halliana flowers, including limocitrin-3-O-glucoside (1), baohuoside Ⅱ (2), kaempferol-3-O-α-L-furan arabinoside (3), phloretin-4'-O-glycosidase (4) and afzeloside (5). Compound 1-3 were isolated for the first time from this genus. The anticoagulant effect of the compounds and extracts of M. halliana flowers were evaluated by APTT, PT, TT and FIB on plasma of rabbit in vitro. The results indicated that several fractions of M. halliana flowers and compounds 2-5 exhibited anticoagulant activity in vitro. Subsequently, afzeloside (5), the abundant component in M. halliana flowers, was investigated further for its antithrombotic effect in vivo and its antithrombotic mechanisms were evaluated on rats acute blood-stasis model. The antithrombotic effect was evaluated by WBV, PV, HCT, ESR, APTT, PT, TT, FIB, 6-keto-PGF1α, TXB2, ET-1 and eNOS in vivo. Afzeloside demonstrated inhibitory effect of thrombus formation, and its underlying antithrombotic mechanism was found to be related to the regulation of vascular endothelium active substance, activating blood flow and anticoagulant effect. Hence, we postulate that flavonoids may be the active ingredients of the plant.

Analysis of fatty acid samples by hydrophilic interaction liquid chromatography and charged aerosol detector.

HILIC/CAD techniques were used in analysis of samples containing fatty acids. Amine base column appeared to be the more retentive stationary phase compared to zwitterionic and BEH silica. The retention decreased with pH mobile phases changing from 3 to 5. Acetonitrile and acetone organic modifier were compared. Acetone gave higher eluotropic strength and better peak symmetry whereas acetonitrile led to higher efficiency. The retention decreased when ammonium acetate concentration increased from 5 to 20mM. The use of sub-2µm column did not show flat Van Deemter curves at high flow rates. A rapid separation of PGI2 and its main degradation product, 6-keto prostaglandin F1α was obtained in 1.6min with a Hypersil GOLD, 50mm×2.1mm, 1.9µm with; acetonitrile/acetate ammonium pH 5 at 20mM (85/15; v/v at 0.7ml/min).

Physicochemical stability study of a new Trimix formulation for treatment of erectile dysfunction.

Currently, severe erectile dysfunction can be treated by intracavernous injections of solutions containing three active ingredients: prostaglandin E1 (PGE1), papaverine and urapidil. Very few data exist on this mixture where phentolamine has been replaced by Urapidil because Phentolamine is not used for this indication in France. The aim of our study was to assess the stability of this formulation and to extend its expiration to permit preparation of batches. Three batches of the preparation containing 15µg/mL PGE1, 15mg/mL of papaverine and 2mg/mL urapidil were made aseptically and then packed in polypropylene syringes stored at 4°C. The physico-chemical stability has been tested as follows: HPLC stability-indicating method, visual observation, measurement of pH and osmolarity. We found that the limiting factor was PGE1 and we exceeded the threshold of 10% loss after 55 days. Replacement of Urapidil by Phentolamine seems to have a slight detrimental effect on stability. Nevertheless, these results allow us to consider the advance preparation of this formulation and provide quality treatment to these patients by avoiding too frequent visits to the hospital.

Dietary polyunsaturated fatty acids reduce retinal stress induced by an elevation of intraocular pressure in rats.

N-6 and n-3 polyunsaturated fatty acids (PUFAs) have been shown to prevent tissue release of inflammatory molecules. We have shown that a combination of n-6 and n-3 PUFAs is more efficient than single supplementations on the long-term consequences of intraocular pressure elevation. We hypothesized that such an association is also more effective during early retinal stress by modifying retinal proinflammatory prostaglandin and cytokine productions. Rats were supplemented for 3 months with n-6 PUFAs, n-3 PUFAs, or both n-6 and n-3 PUFAs. After 3 months, a surgical elevation of intraocular pressure was induced. Retinal morphometry and glial cell activation were evaluated 24 hours after laser treatment. The retinal levels of prostaglandin E(1) (PGE(1)) and prostaglandin E(2) (PGE(2)) and the messenger RNA levels of interleukin-1ß, interleukin-6, and tumor necrosis factor-α were measured. Retinal glial cell activation after laser treatment was partly prevented by dietary n-6, n-3, and n-6 and n-3 PUFAs. Retinal PGE(1) was unaffected by the laser treatment or by the diet. Dietary n-6 and/or n-3 PUFAs prevented the increase in PGE(2) levels observed in laser-treated retinas without affecting the induction of interleukin-1ß, interleukin-6, and tumor necrosis factor-α messenger RNAs. This study shows that not only a combination of n-6 and n-3 PUFAs but also single supplementations can preserve the retina from early glial cell activation and PGE(2) release. The protective effect is not mediated by changes in cytokine expression but may be related to modifications in retinal prostaglandin metabolism.

The occurrence of 15-keto-prostaglandins in the red alga Gracilaria verrucosa.

New 15-keto-prostaglandins (1-4) were isolated from the MeOH extract of the red alga, Gracilaria verrucosa. Their structures were determined to be prostaglandin B congeners (1-3) and a prostaglandin E congener (4) based on the NMR and MS data. Prostaglandins with a C-15 keto function are rare from natural sources. The presence of these metabolites in the alga is notable because 15-keto-prostaglandins (15-keto-PGs) are considered to be the metabolic products of regular prostaglandins in mammals. The occurrence of different prostaglandins in this alga might be due to the existence of different oxidative enzymes, as previously mentioned for oxygenated fatty acids of the red alga Gracilariopsis lemaneiformis. The antiinflammatory activity of these prostaglandins was examined by evaluating their inhibitory effects on nitric oxide production in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophage cells. These prostaglandins showed weak activity on nitric oxide production.

The application of gas chromatography/atmospheric pressure chemical ionisation time-of-flight mass spectrometry to impurity identification in Pharmaceutical Development.

Accurate mass measurement (used to determine elemental formulae) is an essential tool for impurity identification in pharmaceutical development for process understanding. Accurate mass liquid chromatography/mass spectrometry (LC/MS) is used widely for these types of analyses; however, there are still many occasions when gas chromatography (GC)/MS is the appropriate technique. Therefore, the provision of robust technology to provide accurate mass GC/MS (and GC/MS/MS) for this type of activity is essential. In this report we describe the optimisation and application of a newly available atmospheric pressure chemical ionisation (APCI) interface to couple GC to time-of-flight (TOF) MS.To fully test the potential of the new interface the APCI source conditions were optimised, using a number of standard compounds, with a variety of structures, as used in synthesis at AstraZeneca. These compounds were subsequently analysed by GC/APCI-TOF MS. This study was carried out to evaluate the range of compounds that are amenable to analysis using this technique. The range of compounds that can be detected and characterised using the technique was found to be extremely broad and include apolar hydrocarbons such as toluene. Both protonated molecules ([M + H](+)) and radical cations (M(+.)) were observed in the mass spectra produced by APCI, along with additional ion signals such as [M + H + O](+).The technique has been successfully applied to the identification of impurities in reaction mixtures from organic synthesis in process development. A typical mass accuracy of 1-2 mm/zunits (m/z 80-500) was achieved allowing the reaction impurities to be identified based on their elemental formulae. These results clearly demonstrate the potential of the technique as a tool for problem solving and process understanding in pharmaceutical development. The reaction mixtures were also analysed by GC/electron ionisation (EI)-MS and GC/chemical ionisation (CI)-MS to understand the capability of GC/APCI-MS relative to these two firmly established techniques.

Dietary n-3 and n-6 PUFA enhance DHA incorporation in retinal phospholipids without affecting PGE(1) and PGE (2) levels.

The purpose of this study was to determine whether dietary n-3 and n-6 PUFA may affect retinal PUFA composition and PGE(1) and PGE(2) production. Male Wistar rats were fed for 3 months with diets containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% gamma-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA, or (4) a balanced diet deprived of EPA, DHA, and GLA. The fatty acid composition of retinal phospholipids was determined by gas chromatography. Prostaglandin production was measured by enzyme immunoassay. When compared to rats fed the control diet, the retinal levels of DHA were increased in rats fed both diets enriched with n-3 PUFA (EPA + DHA and EPA + DHA + GLA diets) and decreased in those supplemented with n-6 PUFA only (GLA diet). The diet enriched with both n-6 and n-3 PUFA resulted in the greatest increase in retinal DHA. The levels of PGE(1) and PGE(2) were significantly increased in retinal homogenates of rats fed with the GLA-rich diet when compared with those of animals fed the control diet. These higher PGE(1) and PGE(2) levels were not observed in animals fed with EPA + DHA + GLA. In summary, GLA added to EPA + DHA resulted in the highest retinal DHA content but without increasing retinal PGE(2) as seen in animals supplemented with GLA only.

Determination of prostaglandin E(2) by on-line solid-phase extraction-liquid chromatography with ultraviolet detection for microsomal prostaglandin E(2) synthase-1 inhibitor screening.

A rapid, robust and selective on-line solid-phase extraction-liquid chromatographic method with ultra-violet detection (on-line SPE-LC-UV) for microsomal prostaglandin E(2) synthase-1 (mPGES-1) inhibitor screening was developed and validated. Disrupted A549 cells were used as mPGES-1 source and the formation of prostaglandin E(2) (PGE(2)) out of the substrate prostaglandin H(2) (PGH(2)) was determined at 195 nm. Direct on-line sample clean up was achieved by automated column switch (C18 trap column) prior isocratic separation using a C18 analytical column. The on-line SPE-LC-UV method was accurate, precise and reproducible in the range of 71-1763 ng/ml for PGE(2) and met the generally accepted criteria for bioanalytical methods. The method was successfully applied to determine the IC(50) value of the known mPGES-1 inhibitor NS-398.

Jet nebulization of prostaglandin E1 during neonatal mechanical ventilation: stability, emitted dose and aerosol particle size.

BACKGROUND: We have previously reported the safety of aerosolized PGE1 in neonatal hypoxemic respiratory failure. The aim of this study is to characterize the physicochemical properties of PGE1 solution, stability, emitted dose and the aerodynamic particle size distribution (APSD) of PGE1 aerosol in a neonatal ventilator circuit. METHODS: PGE1 was diluted in normal saline and physicochemical properties of the solution characterized. Chemical stability and emitted dose were evaluated during jet nebulization in a neonatal conventional (CMV) or high frequency (HFV) ventilator circuit by a high performance liquid chromatography-mass spectrometry method. The APSD of the PGE1 aerosol was evaluated with a 6-stage cascade impactor during CMV. RESULTS: PGE1 solution in normal saline had a low viscosity (0.9818 cP) and surface tension (60.8 mN/m) making it suitable for aerosolization. Little or no degradation of PGE1 was observed in samples from aerosol condensates, the PGE1 solution infused over 24h, or the residual solution in the nebulizer. The emitted dose of PGE1 following jet nebulization was 32-40% during CMV and 0.1% during HFV. The PGE1 aerosol had a mass median aerodynamic diameter of 1.4 microm and geometric S.D. of 2.9 with 90% of particles being <4.0 microm in size. CONCLUSION: Nebulization of PGE1 during neonatal CMV or HFV is efficient and results in rapid nebulization without altering the chemical structure. On the basis of the physicochemical properties of PGE1 solution and the APSD of the PGE1 aerosol, one can predict predominantly alveolar deposition of aerosolized PGE1.

[Determination of apomorphine, sildenafil and alprostadil in medicines for erectile dysfunction by high performance liquid chromatography-mass spectrometry].

A high performance liquid chromatography-mass spectrometry (LC-MS) analytical method for illicit drugs, apomorphine, sildenafil and alprostadil, in medicines for erectile dysfunction has been developed. The samples were extracted with methanol using ultrasound-assisted extraction. The chromatographic separation was performed on a Zorbax Eclipse XDB-C18 column using acetonitrile-0.5% formic acid aqueous solution as mobile phase. The three compounds were identified by retention time and m/z and quantified by peak area. The results demonstrated that the linear ranges were 50.0 - 5 000.0 microg/L, 10.0 - 1 000.0 microg/L, 40.0 - 4 000.0 microg/L, with detection limits of 20.0, 4.0, 10.0 microg/L for apomorphine, sildenafil and alprostadil, respectively. The average recoveries and the relative standard deviations were 89% - 95% and 9.5% - 11%. The method is simple, rapid, accurate and suitable for the simultaneous determination of these drugs in medicines for erectile dysfunction.

Prostaglandin levels in seminal plasma and sperm extracts of the domestic turkey, and the effects of cyclooxygenase inhibitors on sperm mobility.

BACKGROUND: Turkey reproduction is by artificial insemination using pooled semen so there is interest in storing semen. Fertilizing capacity declines after six hours storage, possibly due to poor sperm mobility. Prostaglandins (PG) affect mammalian sperm motility, but avian sperm has not been widely studied. For this study, levels of PG E1, E2, and F2 alpha in turkey seminal plasma and sperm extract, and effects of cyclooxygenase (COX) inhibitors on sperm mobility were determined. METHODS: Seminal Plasma and sperm extract PG E1, E2, and F2 alpha, from 1.0 mL pooled semen, were measured by ELISA. In Trial 1, PG were determined from 122 wk old toms (n = 4). Trial 2 used 36 wk old toms (n = 7). For Trial 3, PGE2 only was measured from 48 wk (n = 6) and 154 wk old toms (n = 3). The effects of non-specific COX inhibitors indomethacin, diclofenac, tolmetin, or aspirin (n = 10), or specific COX-1 or COX-2 inhibitors (n = 3) on sperm mobility were measured (Accudenz swim-down test). RESULTS: Seminal plasma PG (pg/mL) in Trials 1 and 2, respectively, were 185.2 +/- 88.4 and 187.2 +/- 33.7 for PGE1; 141.4 +/- 43.1 and 100.4 +/- 14.6 for PGF2 alpha; and 431.0 +/- 155.1 for PGE2 (Trial 1 only). Sperm extract PG (pg/10 billion cells) in Trials 1 and 2, respectively, were 215.1 +/- 38.1 and 208.9 +/- 41.5 for PGE1; 133.7 +/- 51.7 and 49.8 +/- 8.3 for PGF2 alpha; and 52.3 +/- 8.6 for PGE2 (Trial 1 only). In Trial 3, seminal plasma PGE2 (pg/mL) in older versus younger males was 1097.9 +/- 99.3 versus 853.2 +/- 144.6 and sperm extract PGE2 (pg/10 billion cells) was 208.0 +/- 56.1 versus 102.4 +/- 14.8. Cyclooxygenase inhibitors (0.001 to 10 mM) decreased sperm mobility: indomethacin 15 to 100%; diclofenac 4 to 100%; tolmetin 27 to 74%; aspirin (tested at 0.01 to 15 mM) 22 to 42%; resveratrol (COX-1) and NS-398 (COX-2), both tested at 0.1 to 10 mM, 38 to 98% and 44 to 85%, respectively. CONCLUSION: These results indicate that PG are present in turkey seminal plasma and sperm, and COX inhibitors decrease turkey sperm mobility.

Cooperative induction of 15-lipoxygenase in rheumatoid synovial cells by IL-4 and proinflammatory cytokines.

OBJECTIVE: To clarify the role of interleukin-4 (IL-4) in the expression of 15-lipoxygenase (15-LOX), whose metabolities are known to suppress the inflammatory reaction, in freshly prepared rheumatoid synovial cells. METHODS: Adherent synovial cells were prepared by enzymatic digestion of synovia obtained from patients with rheumatoid arthritis (RA). Protein expression of 15-LOX was determined by Western blot analysis. The messenger RNAs of 15-LOX were determined by reverse transcription and the polymerase chain reaction (RT-PCR). RESULTS: Freshly prepared rheumatoid synovial cells did not express 15-LOX at either the mRNA or protein levels. IL-4 induced the protein expression of 15-LOX after 24 hours of culture. Although interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF alpha), major inflammatory cytokines in rheumatoid synovia, did not induce the expression of 15-LOX, IL-4 and these inflammatory cytokines synergistically enhanced the protein expression of 15-LOX. The synergistic effect was also observed at the level of mRNA. CONCLUSIONS: We demonstrate that IL-4 cooperated with the inflammatory cytokines IL-1 alpha and TNF alpha to enhance the expression of 15-LOX in rheumatoid synovial cells. Since 15-LOX metabolites have potent anti-inflammatory actions, our data suggest that IL-4 might downregulate rheumatoid inflammation via the induction of 15-LOX and its metabolites.

A chromatographic method for the quantification of prostaglandin E(1) and prostaglandin A(1) encapsulated in an intravenous lipid formulation.

Peripheral vascular disease is a common ailment of the aged and diabetic communities. As the numbers of these individuals increase, the need for therapeutic interventions will continue to grow. One of the possible therapies is the use of prostaglandins (PGE(1), prostacyclin and Iloprost) to decrease the vascular tone and increase vascular blood flow. Due to the hydrophobicity of the prostaglandins and prostaglandin analogues, various vehicles have been utilized to maintain the active pharmaceutical ingredient in a stable solution, e.g. alpha-cyclodextrin (Alprostadil, Edex) or emulsified lipid vehicles. In our laboratory, we designed a method for separating and assaying lipid-encapsulated PGE(1). Utilizing organic extraction, automated solid-phase extraction and precipitation techniques, we validated the measurement of the PGE(1) and PGA(1) content of the clinical drug formulation in the microgram per milliliter concentration range with an high performance liquid chromatography (HPLC) assay.

[Alprostadil Reference Standard (Control 001) of National Institute of Health Sciences].

The raw material of Alprostadil was examined for the preparation of "Alprostadil Reference Standard (Control 001)". Analytical data obtained were: IR spectrum, same as that of the Alprostadil Reference Standard (Control 923); thin-layer chromatography, no impurities were detected until 20 micrograms; high-performance liquid chromatography (HPLC), total amount of impurities estimated to be less than 0.2%. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Alprostadil Reference Standard (Control 001).

[Determination of prostaglandinum E1 by fluorescence extinction method].

In this paper, we have studied the fluorescence extinction effect of methyl alcohol by prostaglandinum E1 (PG E1) and found the linear relation between the fluorescence extinction intensity and prostaglandinum E1 concentration. Its linear equation is deltaF = 0.222c + 9.33 x 10(-2)(deltaF: fluorescence extinction value; c: PG E1 concentration microg/mL), correlation coeficient r = 0.99930. The results derived from fluorescence extinction method correspond to the results from violet spectrophotometric method.

Effects of dietary gamma-linolenic acid on the tissue phospholipid fatty acid composition and the synthesis of eicosanoids in rats.

The purpose of this study was to evaluate the effects of different doses of dietary gamma-linolenic acid (GLA) on the tissue phospholipid fatty acid composition and the synthesis of eicosanoids in growing rats. The supplementation with different oils rich in GLA (borage oil, evening primrose oil, or Spirulina oil) and poor in n-3 polyunsaturated fatty acids or biomass of Spirulina results in a significant dose-related increase of GLA and dihomo-GLA in liver, erythrocyte, and aorta phospholipids in rats fed during 6 weeks different levels of GLA. The arachidonic acid (AA)/dihomo-GLA ratios decreased with increasing intake of dietary GLA, but the AA proportions remained stable. The dietary administration of GLA increased the in vitro production by the aorta of prostaglandin E1 derived from dihomo-GLA, but did not significantly influence the production of prostaglandin E2 derived from AA by the aorta and the thromboxane B2 level in serum.

Naproxen concentrations in plasma and synovial fluid and effects on prostanoid concentrations.

OBJECTIVE: To test the hypothesis that unbound concentrations of naproxen in synovial fluid (SF) and plasma (P) are equal over a drug dosage interval at steady state or after a single dose of drug. The relationship between plasma and SF concentrations of naproxen, respectively, and prostaglandin concentrations were also examined. METHODS: Paired, sequential, total, and unbound naproxen concentrations were determined in plasma and SF in 2 groups of 6 patients. A single dose group was given naproxen 500 mg. The chronic dose group was given 500 mg bd for 7 days before collection of blood and SF samples. The effect of naproxen on prostanoid production by clotting whole blood (thromboxane B2, TXB2) and in SF (PGE2, 6-keto-PGF1 alpha) was determined by radioimmunoassay. RESULTS: Average area under the curve (AUC) of unbound (U) naproxen concentrations against time in plasma and SF were the same over a dosage interval at steady state (ratio AUCU,SF/AUCU,P, 1.12 +/- 0.18; p = 0.108), but not after a single acute dose (AUCU,SF/AUCU,P, 1.34 +/- 0.32; p = 0.044). Data from the single dose study revealed that the mean (+/- SD) of the concentrations required for 50% inhibition (EC50) of platelet derived TXB2 by total naproxen was 7.7 +/- 4.4 micrograms/ml (n = 5) and for unbound drug 25.4 +/- 22.0 ng/ml (n = 5). SF prostanoid concentrations after both acute and chronic dosing were low, as expected, but temporal and dose relationships of prostanoid concentrations with SF naproxen could not be discerned. However, this may reflect study design. CONCLUSIONS: The AUC of unbound naproxen in SF and plasma were similar at steady state. Plasma concentrations correlated with inhibition of TXB2 generation by platelets. There was sustained depression of PG concentrations in SF beyond the time suggested by plasma drug concentrations.