Analysis of Citrus Bioflavonoid Content and Dipeptidyl Peptidase-4 Inhibitory Potential of Commercially Available Supplements.

Citrus bioflavonoids are polyphenolic plant-derived pigments found in high levels in oranges, lemons, grapefruits and other citrus fruits. The three most abundant types of citrus bioflavonoids are hesperidin, naringenin and eriocitrin. Citrus bioflavonoids have long been known to possess powerful free radical-scavenging properties and cardioprotective effects. The study involved the analysis of 10 commercially available citrus bioflavonoid supplements from three different countries: Australia, the United States and Canada. The supplements were tested for their citrus bioflavonoid content which varied from 0.8 to 33.3% w/w. The daily bioflavonoid dose varied from 19 mg to 560 mg. Hesperidin was the major citrus bioflavonoid in nine out of ten supplements. One supplement was found to contain less than 10% of the quantity of rutin claimed to have been added. The DPP-4 inhibitory potential, compared through an estimation of rutin equivalence, ranged from 1.9 mg to 400 mg per day. This data highlights the variability between the supplements in their potential to inhibit DPP-4 for subsequent health benefits.

Pharmacokinetic and pharmacodynamic effects of 2 registered omeprazole preparations and varying dose rates in horses.

BACKGROUND: Omeprazole preparations vary in bioavailability in horses. HYPOTHESIS/OBJECTIVES: To characterize the pharmacokinetics and pharmacodynamics of an existing enteric-coated oral omeprazole paste (REF) and a novel, in-feed, enteric-coated dry granule preparation (NOV). ANIMALS: Twelve Standardbred/Thoroughbred mares free from clinical disease. METHODS: A prospective, blinded randomized interventional study was trial, conducted in 3 parts: (a) bioavailability study, (b) dose titration study, and (c) comparative clinical pharmacodynamic study, each using a blocked crossover design. RESULTS: Consistent with the larger dose administered, Cmax (median, 1032 ng/mL; range, 576-1766) and AUC0-24 (median, 63.9 µg/mL*min; range, 42.4-152.4) were greater after single oral administration of NOV than REF (282.7 ng/mL; range, 94.8-390.2, and 319 23.8 µg/mL*min; range, 8.2-42.3, respectively; both P = .004). No differences were observed between products for absolute oral bioavailability (NOV 55% range, 15-88; REF 17% range, 10-77; P = .25). Treatment with both preparations was associated with reduced gastric squamous ulcer scores and increased pH of gastric fluid. Bioequivalence was demonstrated for pharmacodynamic measures with the exception of % time pH <4, despite differences in dose rate and subsequent plasma omeprazole concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: The findings of this study indicate that the NOV product would be a suitable alternative to the reference product, and confirm that plasma concentrations of omeprazole and omeprazole dose do not predict drug pharmacodynamics in horses.

Phytosterol supplements do not inhibit dipeptidyl peptidase-4.

BACKGROUND AND AIMS: Several commercially available phytosterol supplements are promoted for their cholesterol-lowering effects. However, limited information is available about their potential anti-hyperglycaemic effects. This study aimed to evaluate the dipeptidyl peptidase-4 (DPP-4) inhibitory effects of phytosterol supplements in silico and in vitro to determine their potential for anti-diabetic activity. METHODS: Docking studies were carried out in silico to evaluate the potential for interactions between three major phytosterol compounds (stigmasterol, ß-sitosterol, campesterol) and the DPP-4 enzyme, the enzyme that is inhibited by the anti-diabetic gliptins. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to analyse three different supplements for phytosterol content. DPP-4 inhibitory activity was tested in vitro for these phytosterol supplements and two major phytosterol standards. RESULTS: In silico calculations predicted free binding energies for DPP-4 with the phytosterols to be: stigmasterol -8.78 kcal/mol; ß-sitosterol -8.70 kcal/mol; campesterol -8.40 kcal/mol. These binding energies indicated a potential for significant DPP-4 inhibition. However, these results were not supported by the in vitro studies. Stigmasterol and ß-sitosterol had an IC50 > 50 mg/ml (maximum tested concentration) and the Thompson's Cholesterol Manager® and Mega Strength Beta Sitosterol® supplements gave an IC50 > 100 mg/ml (maximum tested concentration). Blackmores Cholesterol Health® gave an IC50 value of 40 mg/ml which was attributed to ß-carotene content. CONCLUSIONS: Phytosterol supplements do not appear to offer any anti-diabetic activity potential via pathways that involve the inhibition of DPP-4.

Enantioselective disposition of (R,R)-formoterol, (S,S)-formoterol and their respective glucuronides in urine following single inhaled dosing and application to doping control.

Formoterol is a long-acting beta2-adrenoceptor agonist (LABA) used for the treatment of asthma and exercise-induced bronchoconstriction. Formoterol is usually administered as a racemic (rac-) mixture of (R,R)- and (S,S)-enantiomers. While formoterol is restricted by the World Anti-Doping Agency (WADA), inhalation of formoterol is permitted to a predetermined dose (54 µg/24 hours) and a urine threshold of 40 ng/mL. However, chiral switch enantiopure (R,R)-formoterol is available, effectively doubling the therapeutic advantage for the same threshold. The aim of this study was to investigate whether formoterol exhibits enantioselective urinary pharmacokinetics following inhalation. Six healthy volunteers were administered a 12 µg inhaled dose of rac-formoterol. Urine was collected over 24 hours and analyzed by enantioselective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay. Total (free drug plus conjugated metabolite) median (min-max) rac-formoterol urine levels following inhalation were 1.96 (1.05-13.4) ng/mL, 1.67 (0.16-9.67) ng/mL, 0.45 (0.16-1.51) ng/mL, 0.61 (0.33-0.78) ng/mL, and 0.17 (0.08-1.06) ng/mL at 2, 4, 8, 12, and 24 hours, respectively, well below the 2019 urine threshold. The proportion of conjugation differed between enantiomers with glucuronide conjugation much greater for (R,R)-formoterol (around 30%-60% of total) compared to (S,S)-formoterol (0%-30%). There was clear evidence of inter-individual enantioselectivity observed in the ratios of (R,R):(S,S)-formoterol, where (S,S)- was predominant in free formoterol, and (R,R)- predominant in the conjugated metabolite. In conclusion, rac-formoterol delivered by inhalation exhibits enantioselective elimination in urine following single-dose administration. Enantioselective assays should be employed in doping control to screen for banned beta2-agonist chiral switch products such as (R,R)-formoterol, and total hydrolyzed rac-formoterol is warranted to account for inter-individual differences in enantioselective glucuronidation.

Beta2 -adrenergic ligand racemic formoterol exhibits enantioselective disposition in blood and skeletal muscle of humans, and elicits myocellular PKA signaling at therapeutic inhaled doses.

While studies have demonstrated substantial differences in beta2 -adrenergic agonist enantiomer pharmacology, enantioselective disposition of long-acting beta2 -adrenergic ligand racemic (rac)-formoterol in blood is inadequately explored after inhaled therapy given analytical challenges. Furthermore, information on enantioselective disposition and partitioning of beta2 -adrenergic agonist in skeletal muscle is absent despite its promising data on muscle anabolism in humans. Using a sensitive ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) assay, we determined disposition of (R,R)-formoterol and (S,S)-formoterol in plasma and skeletal muscle samples from 11 non-asthmatic men who had inhaled rac-formoterol at therapeutic doses (2 × 27 µg). Mean (SD) concentrations of (R,R)- and (S,S)-formoterol in plasma and in muscle biopsies of the vastus lateralis 1 hour after inhalation of formoterol were 31 (15) and 45 (18) pg × mL-1 for (R,R)-formoterol and (S,S)-formoterol, respectively, in plasma, and 0.56 (0.32) and 0.51 (0.29) pg × mgwet wt -1 , respectively, in muscle. Formoterol exhibited different enantioselective disposition in plasma and muscle (p < 0.0001). In plasma, mean log (R,R):(S,S)-formoterol ratio was lower than 0 [-0.17(0.07), p < 0.0001], whereas in muscle, mean log (R,R):(S,S)-formoterol ratio was slightly higher than 0 [0.04(0.07), p < 0.05]. Log (R,R):(S,S)-formoterol ratio in muscle was related to muscle fiber-type composition. Furthermore, formoterol induced an approximately two-fold increase in muscle p-PKASer/thr phosphorylation (p < 0.01), indicating a substantial beta2 -adrenergic response. Collectively, these findings suggest that formoterol exhibits modest enantioselective disposition in plasma after inhaled therapy in humans, which appear related to a greater (R,R)-enantiomer disposition in skeletal muscle that may be dependent on fiber-type composition.

Citrus bioflavonoids dipeptidyl peptidase-4 inhibition compared with gliptin antidiabetic medications.

This study compared dipeptidyl peptidase-4 (DPP-4) inhibitory activity of citrus bioflavonoid nutraceuticals compared with three gliptins. Citrus bioflavonoid standards and three commercially available citrus bioflavonoid supplements (Thompson's Super Bioflavonoid Complex®(SB), Ethical Nutrients Bioflavonoids Plus Vitamin C®(EN), and Country Life Citrus Bioflavonoids and Rutin®(CB)) were considered in this study. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was undertaken to identify and quantitate the citrus bioflavonoids present in each supplement. The DPP-4 inhibitory activity was determined by fluorometric assay. All of the tested individual citrus flavonoids demonstrated DPP-4 inhibitory activity, with IC50 values ranging from 485 µM (rutin) to 5700 µM (hesperitin and eriodictyol). Similarly, the flavonoid supplements had IC50 values of 16.9 mg/mL (EN), 3.44 mg/mL (SB) and 2.72 mg/mL (CB). These values compare with gliptin IC50 values of 0.684 µM (sitagliptin), 0.707 µM (saxagliptin) and 2.286 µM (vildagliptin). The supplement flavonoid content varied from 11.98% (CB) to 5.26% (EN) and 14.51% (SB) of tablet mass, corresponding to daily flavonoid doses of around 300, 150 and 400 mg, respectively, with CB and SB containing rutin at levels of 7.0% and 7.5% of tablet mass, respectively. While our data demonstrated that citrus bioflavonoid based supplements do possess DPP-4 inhibitory activity, they are several orders of magnitude less potent than gliptins. Further studies using higher concentrations of citrus bioflavonoids, as well as investigations into antioxidant properties which may add additional benefit are warranted.

Salmeterol undergoes enantioselective bronchopulmonary distribution with receptor localisation a likely determinant of duration of action.

BACKGROUND: Salmeterol (a long acting beta2-agonist) is a chiral molecule. (RR)-salmeterol is responsible for pharmacological effect, but basic knowledge of enantioselective pulmonary pharmacodynamics and pharmacokinetics of salmeterol remains unknown. There are safety concerns with (S)-enantiomers of beta2-agonists, with suggestions that these enantiomers may increase bronchial hyperresponsivneness in asthma patients. METHODOLOGY: Horses (n = 12) received racemic (rac-) salmeterol 250 µg via inhalation. Enantioselective UPLC-MS/MS was used to determine (R)- and (S)-salmeterol concentrations in pulmonary epithelial lining fluid (PELF) sampled 2, 5, 10 and 15 min after administration, in central lung (endoscopic bronchial biopsy) and peripheral lung (percutaneous pulmonary biopsy) tissues (at 20 and 25 min respectively), and in plasma samples. RESULTS: Physiologically relevant tissue concentrations were found for both enantiomers, with median levels greater in central than peripheral lung (equivalent to 32 and 5 mM (R)-salmeterol for central and peripheral lung respectively). Levels in PELF decreased around 50% over 15 min and enantioselective distribution was observed in the central lung with levels of (R)-salmeterol around 30% higher than (S)-salmeterol. CONCLUSION: Salmeterol distribution is enantioselective in the central lung. This suggests duration of action is more likely associated with specific B2ADR localisation effects rather than non-specific physiochemical factors which would not be enantioselective.

Beta2-adrenergic stimulation increases energy expenditure at rest, but not during submaximal exercise in active overweight men.

PURPOSE: ß2-Agonists have been proposed as weight-loss treatment, because they elevate energy expenditure. However, it is unknown what effect ß2-agonists have on energy expenditure in overweight individuals. Furthermore, the influence of ß2-agonist R- and S-enantiomer ratio for the increased energy expenditure is insufficiently explored. METHODS: Nineteen males were included in the study of which 14 completed. Subjects were 31.6 (±3.5) years [mean (±95% CI)] and had a fat percentage of 22.7 (±2.1)%. On separate days, subjects received either placebo or inhaled racemic (rac-) formoterol (2 × 27 µg). After an overnight fast, energy expenditure and substrate oxidation were estimated by indirect calorimetry at rest and during submaximal exercise. Plasma (R,R)- and (S,S)-formoterol enantiomer levels were measured by ultra-performance liquid chromatograph-mass spectrometry. RESULTS: At rest, energy expenditure and fat oxidation were 12% (P ≤ 0.001) and 38% (P = 0.006) higher for rac-formoterol than placebo. Systemic (R,R):(S,S) formoterol ratio was correlated with change in energy expenditure at rest in response to rac-formoterol (r = 0.63, P = 0.028), whereas no association was observed between fat percentage and rac-formoterol-induced change in energy expenditure. During exercise, energy expenditure was not different between treatments, although carbohydrate oxidation was 15% higher (P = 0.021) for rac-formoterol than placebo. Rac-formoterol-induced shift in substrate choice from rest to exercise was related to plasma ln-rac-formoterol concentrations (r = 0.75, P = 0.005). CONCLUSION: Selective ß2-adrenoceptor agonism effectively increases metabolic rate and fat oxidation in overweight individuals. The potential for weight loss induced by ß2-agonists may be greater for R-enantiopure formulations.

Bronchopulmonary pharmacokinetics of (R)-salbutamol and (S)-salbutamol enantiomers in pulmonary epithelial lining fluid and lung tissue of horses.

AIMS: Salbutamol is usually administered as a racemic mixture but little is known about the enantioselectivity of salbutamol pharmacokinetics in the lung. This study was designed to investigate enantiomer concentrations in lung tissue after inhaled dosing. METHODS: Horses (n = 12) received racemic salbutamol 1000 µg via inhalation. Enantioselective ultra performance liquid chromatography-tandem mass spectrometry was used to determine salbutamol concentrations in pulmonary epithelial lining fluid (PELF) sampled 2, 5, 10 and 15 min after administration, in central lung (endoscopic bronchial biopsy) and peripheral lung (percutaneous pulmonary biopsy) tissues (at 20 and 25 min respectively), and in plasma samples. RESULTS: Mean ± 95% confidence interval (CI) yield of PELF was 57 ± 10 mg. Initial mean ± 95%CI (R)- and (S)-salbutamol PELF concentrations were 389 ± 189 ng g-1 and 378 ± 177 ng g-1 respectively, and both reduced approximately 50% by 15 min. Mean ± 95%CI central lung levels of drug were higher than peripheral lung tissue for both (R)-salbutamol (875 ± 945 vs. 49.5 ± 12 ng g-1 ) and (S)-salbutamol (877 ± 955 vs. 50.9 ± 12 ng g-1 ) respectively. There was no evidence of enantioselectivity in PELF or central lung but minor (~2%) enantioselectivity was observed in the peripheral lung. Enantioselectivity was clearly evident in plasma with (S):(R) ratio of 1.25 and 1.14 for both area under the concentration-time curve (0-25 min) and Cmax respectively. CONCLUSIONS: PELF sampling in horses offers sufficient yield allowing direct detection of drug and, combined with tissue sampling, is a valuable model to investigate bronchopulmonary pharmacokinetics. Salbutamol did not demonstrate enantioselectivity in PELF or central lung tissue uptake following acute dosing, however, enantioselective plasma concentrations were demonstrated, with minor enantioselectivity in the peripheral lung.

Enantioselective disposition of (R)-salmeterol and (S)-salmeterol in urine following inhaled dosing and application to doping control.

Salmeterol (USAN, INN, BAN) is a long-acting beta2-adrenoceptor agonist (LABA) widely used in the treatment of airways disease. Although salmeterol is permitted via inhalation by athletes and supratherapeutic dosing may enhance performance, no urine threshold has been established by the World Anti-Doping Agency (WADA). Salmeterol is a chiral compound consisting of (R)- and (S)-enantiomers, normally administered as racemic (rac-) mixture via inhalation. Levels of rac-salmeterol in urine are often below detectable levels and there is surprisingly little information regarding the enantioselectivity of salmeterol pharmacokinetics. In this study, subjects inhaled either 50 (n = 6) or 200 µg (n = 4; generally regarded as maximum therapeutic dose) of salmeterol and urine was then collected for 24 h and analyzed by enantioselective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Maximum rac-salmeterol urine concentrations were obtained at 2 h for both doses with medians of 0.084 ng/mL after the 50 µg dose and 2.1 ng/mL after the 200 µg dose, with an individual maximum of 5.7 ng/mL. Levels were detectable at 24 h for both doses. Salmeterol displayed enantioselective pharmacokinetics, with a mean ± SD log (S):(R) = 0.055 ± 0.025 (P < 0.0001) equivalent to (S):(R) of 1.13. In conclusion, rac-salmeterol by inhalation exhibits modest enantioselectivity in urine following single dose administration and can be detected following a single 50 µg dose for up to 24 h after inhalation. The present findings are of relevance if a urine threshold limit is to be introduced for salmeterol on the list of prohibited substances. The application of an enantiomer ratio analysis may offer improved discriminatory detection capability for doping control analysis applications. Copyright © 2016 John Wiley & Sons, Ltd.

Reliability of Haemophilus influenzae biofilm measurement via static method, and determinants of in vitro biofilm production.

Information is lacking regarding the precision of microtitre plate (MTP) assays used to measure biofilm. This study investigated the precision of an MTP assay to measure biofilm production by nontypeable Haemophilus influenzae (NTHi) and the effects of frozen storage and inoculation technique on biofilm production. The density of bacterial final growth was determined by absorbance after 18-20 h incubation, and biofilm production was then measured by absorbance after crystal violet staining. Biofilm formation was categorised as high and low for each strain. For the high biofilm producing strains of NTHi, interday reproducibility of NTHi biofilm formation measured using the MTP assay was excellent and met the acceptance criteria, but higher variability was observed in low biofilm producers. Method of inoculum preparation was a determinant of biofilm formation with inoculum prepared directly from solid media showing increased biofilm production for at least one of the high producing strains. In general, storage of NTHi cultures at -80 °C for up to 48 weeks did not have any major effect on their ability to produce biofilm.

Hypolipidaemic effect of crude extract from Carpobrotus rossii (pigface) in healthy rats.

Carpobrotus rossii (CR) was used by the Aboriginal population and early European settlers both as a food and therapeutic agent. Based on the presence of flavonoids in CR and results from our previous in vitro investigations, this study aimed to determine whether consumption of CR crude leaf extract: (a) affected lipoprotein profile, resting glucose, systolic blood pressure and vascular function, and (b) produced toxic effects (haematological measures, organ weight) in healthy rats. Male Hooded-Wistar rats (~230 g) were supplemented for 4 weeks with CR extract in their drinking water (35 mg/kg body weight daily). CR extract produced a significant decrease (18%, p=0.033) in atherogenic lipoproteins (but not high density lipoprotein). CR supplemented animals showed no signs of haematological toxicity and body and organ weight, daily fluid and food consumption and in vitro vascular responsiveness were similar for both groups. CR also increased (40%, p=0.049) the renal concentration of 3-hydroxy-3-methylglutaric acid (HMG), consistent with HMG-containing CR flavonoids being bioavailable, and therefore possessing the potential to interfere with cholesterol synthesis pathways. CR extract appears to be safe to ingest and may reduce cardiovascular risk.

Protoanemonin content variation between Clematis spp.: leaf, stem and root.

The content of protoanemonin, a known biologically active constituent of Clematis spp., was determined by GC-MS in the leaf, stem and root extracts of one Chinese species and four Australian Clematis taxa. The results showed that protoanemonin concentrations varied between different plants and that leaves contained higher concentrations than stems and roots. To our knowledge this is the first study to determine protoanemonin content variation in leaf, stem and root of Clematis spp.

Plasma glutathione peroxidase (GSH-Px) concentration is elevated in rheumatoid arthritis: a case-control study.

Plasma glutathione peroxidase (GSH-Px) by enzyme-linked immunosorbent assay (ELISA) offers a complimentary measurement approach to traditional GSH-Px activity methods. The aim was to investigate whether GSH-Px measured by ELISA in rheumatoid arthritis patients was elevated compared to controls. This was a case-control study with rheumatoid arthritis patients recruited from private practice and gender and age-matched controls randomly selected from the electoral role. GSH-Px concentration was measured by ELISA. Plasma malondialdehyde was used as a measure of oxidative stress, and antioxidant capacity was measured based on reduction of Cu(++) to Cu(+) by antioxidants in the sample. Disease severity was measured using the Health Assessment Questionnaire-Disability Index (HAQ-DI) and C-reactive protein was measured using an immunoturbidometric method. A total of 74 patients were recruited, consisting of 35 rheumatoid arthritis cases and 39 healthy controls. There were no differences between rheumatoid arthritis cases and controls for oxidative stress and antioxidant capacity; however, GSH-Px concentration was markedly elevated in the rheumatoid arthritis sufferers (85.9 ± 147.7 versus 17.3 ± 13.0 mg/L, respectively; mean ± SD; p < 0.01). GSH-Px levels were not associated with severity measured by the HAQ-DI or C-reactive protein. Patients with rheumatoid arthritis demonstrated increased GSH-Px consistent with an adaptive upregulation of GSH-Px to protect against oxidative stress.

Size and sulfation are critical for the effect of heparin on APP processing and Aß production.

Alzheimer's disease is associated with abnormal accumulation of Aß, which is produced from the ß-amyloid precursor protein (APP) by the ß-site APP-cleaving enzyme (BACE1) and γ-secretase. Our previous studies showed that heparin can decrease APP processing by decreasing the levels of BACE1 and ADAM10. In this study, we examined the effects of glycosaminoglycans (GAGs) on APP processing and Aß production with the aim of understanding the specificity of the effects. Various GAG analogs were incubated with primary cortical cells derived from APP (SW)Tg2576 mice and the level of APP, proteolytic products of APP and APP-cleavage enzymes were measured. The effect of GAGs on APP processing was both size- and sulfation-dependent. 6-O-Sulfation was important for the effect on APP processing as heparin lacking 6-O sulfate were less potent than native heparin. However, deletion of carboxyl groups on heparin had no significant effect on APP processing. Our studies suggest that there is structural specificity to the effect of GAGs on APP processing and that certain GAGs have a greater effect on Aß production than others. This suggests that it might be possible to alter the structure of GAGs to achieve more specific inhibitors of APP processing that can cross the blood-brain barrier.

Effects of heparin and enoxaparin on APP processing and Aß production in primary cortical neurons from Tg2576 mice.

BACKGROUND: Alzheimer's disease (AD) is caused by accumulation of Aß, which is produced through sequential cleavage of ß-amyloid precursor protein (APP) by the ß-site APP cleaving enzyme (BACE1) and γ-secretase. Enoxaparin, a low molecular weight form of the glycosaminoglycan (GAG) heparin, has been reported to lower Aß plaque deposition and improve cognitive function in AD transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS: We examined whether heparin and enoxaparin influence APP processing and inhibit Aß production in primary cortical cell cultures. Heparin and enoxaparin were incubated with primary cortical cells derived from Tg2576 mice, and the level of APP and proteolytic products of APP (sAPPα, C99, C83 and Aß) was measured by western blotting. Treatment of the cells with heparin or enoxaparin had no significant effect on the level of total APP. However, both GAGs decreased the level of C99 and C83, and inhibited sAPPα and Aß secretion. Heparin also decreased the level of ß-secretase (BACE1) and α-secretase (ADAM10). In contrast, heparin had no effect on the level of ADAM17. CONCLUSIONS/SIGNIFICANCE: The data indicate that heparin and enoxaparin decrease APP processing via both α- and ß-secretase pathways. The possibility that GAGs may be beneficial for the treatment of AD needs further study.

In vitro antioxidant, antiplatelet and anti-inflammatory activity of Carpobrotus rossii (pigface) extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Carpobrotus rossii (CR) has a history of use as a food and therapeutic agent by Australian indigenous peoples and early European settlers and is believed to contain a number of pharmacologically active polyphenolic compounds. AIMS OF THE STUDY: Oxidation of low density lipoprotein (LDL), platelet aggregation, and inflammation contribute to the development and progression of atherosclerosis. The aim of the present study was to investigate the antioxidant, antiplatelet and anti-inflammatory activity of CR extract using human blood components. MATERIALS AND METHODS: An assay employing in vitro copper-induced oxidation of serum lipids was used to assess antioxidant activity of CR extract (and tannin, flavonoid and pre- and post-flavonoid fractions). The effects of CR extract on ADP- and collagen-induced platelet aggregation, and on basal (unstimulated) and lipopolysaccharide (LPS)- and phytohaemagglutinin A (PHA)-stimulated cytokine release from peripheral blood mononuclear cells (PBMC) were also investigated. RESULTS: CR extract increased the lag time of serum oxidation (maximum of ∼4-fold at 20µg/ml) in a concentration-dependent manner. The antioxidant activity resided only in the tannin and post-flavonoid fractions. CR had no effect on ADP-induced platelet aggregation, but significantly decreased collagen-induced platelet aggregation. LPS, but not PHA, significantly increased the release of IL-1ß and TNF-α from PBMC. CR extract alone inhibited monocyte chemoattractant protein (MCP)-1 release and in the presence of LPS, inhibited IL-10, TNF-α and MCP-1 release compared to LPS alone. CONCLUSION: CR has significant in vitro antioxidant, antiplatelet and, potentially, anti-inflammatory activity.

Effective reversed-phase ion pair high-performance liquid chromatography method for the separation and characterization of intact low-molecular-weight heparins.

A simple, selective, and efficient reversed-phase ion pair high-performance liquid chromatography (RPIP-HPLC) method was developed for the separation of various commercially available intact low-molecular-weight heparins (LMWHs). The developed method uses a C(18) column (150 x 4.6 mm) with diode array detection at 230 nm, flow rate at 1.0 ml/min, and a mobile phase containing acetonitrile/water (32:68%), tetrabutylammonium hydroxide (15 mM), and ammonium acetate (50 mM) at pH 7.0. The performance of this method was assessed in terms of selectivity, linearity, intra- and interday precision, and accuracy. The novel application of RPIP-HPLC with evaporative light scattering detection (ELSD) for the analysis of intact LMWHs was demonstrated. Intact LMWHs were analyzed with superior resolution and peak shape. Different chromatographic profiles were obtained for different LMWHs showing significant structural diversity. This method clearly showed chemical changes that occurred to LMWH under the stress condition. This method can be applied for the separation, identification, characterization, and pharmaceutical stability analysis of various LMWHs.

Investigation of the effect of heating on the chemistry and antifactor Xa activity of enoxaparin.

The objective of this study was to investigate the effects of heating on the chemistry, physical properties and antifactor Xa activity of enoxaparin. Samples of enoxaparin heated at 70 degrees C lost 27% of their initial AFXa activity after 8 h, then activity increased to 94% of the initial activity over the next 4 h. Activity then decreased to 84% of control after 48 h and further to 80% of control over 22 days. The initial activity loss correlated with desulfation as demonstrated by sulfate and amine analysis. Fragmentation of oligosaccharides occurred, as demonstrated by reducing capacity and capillary electrophoresis analysis. Individual enoxaparin fractions obtained by high performance size exclusion chromatography were analysed. Early eluting fractions, containing aggregated oligosaccharides, increased in concentration following heating. Up to 65% of sulfate was lost from some fractions, containing hexa- and octa-saccharides, after 8 h, corresponding with decreased activity. Low mass oligosaccharide fractions increased in concentration and had increased activity between 8 and 12 h. Reversed-phase ion-interaction HPLC analysis supported these findings. Deca-, dodeca- and tetradeca-saccharides were resistant to thermal degradation. Desulfation, fragmentation and aggregation occur during the heating of enoxaparin and result in the initial rapid loss, recovery and subsequent gradual loss of activity.

Investigation of freezing- and thawing-induced biological, chemical, and physical changes to enoxaparin solution.

This study investigated the effect of freezing and thawing on the biological, physical, and chemical properties of enoxaparin solution. Solutions were frozen and thawed under different conditions, in the presence or absence of dimethyl sulfoxide (DMSO) or 1,2-propanediol (1,2-PD), and the antifactor Xa (AFXa) activity was determined. Enoxaparin solution lost more than 60% of its AFXa activity when thawed rapidly after freezing at -196 degrees C. The loss of AFXa activity was less with higher freezing temperatures and increased with the number of freeze/thaw cycles, but was independent of the duration of freezing. Slow freezing to -196 degrees C with rapid thawing, or rapid freezing with slow thawing, resulted in negligible loss of AFXa activity. The loss of AFXa activity did not involve the loss of N-sulfate groups, the breakdown of glycosidic bonds or the glassy state transition. Controlling the freezing or thawing conditions, dilution with water or addition of a small percentage of DMSO ameliorated the loss of enoxaparin AFXa activity. The loss in AFXa activity was found by size exclusion chromatography to be primarily due to aggregation and was reversed by sonication in the presence of DMSO. These results may provide insight into solutions for the long-term storage of concentrated or diluted enoxaparin.