Enhanced cellular delivery of idarubicin by surface modification of propyl starch nanoparticles employing pteroic acid conjugated polyvinyl alcohol

Enhanced intracellular internalization of the anti-cancer active idarubicin (IDA) was achieved through appropriate surface modification of IDA loaded propyl starch nanoparticles. This was conducted by synthesizing pteroic acid modified polyvinyl alcohol (ptPVA) and employing this stabilizer for formulating the said nanoparticles. Pteroic acid attached at the nanoparticles improved the surface protein adsorption of the nanoparticle, a condition which the nanoparticles would largely experience in vitro and in vivo and hence improve their cellular internalization.Spherical, homogenous IDA nanoparticles (214 ± 5 nm) with surface modified by ptPVA were formulated using the solvent emulsification-diffusion technique. The encapsulation efficiency and drug loading amounted around 85%. In vitro release studies indicated a controlled release of IDA. Safety and efficacy of the nanoparticles was confirmed by suitable cellular cytotoxicity assays. Protein binding studies indicated a higher adsorption of the model protein on nanoparticles formulated with ptPVA as compared to PVA. Cellular uptake studies by confocal laser scanning microscopy revealed a higher cellular uptake of ptPVA stabilized nanoparticles thus confirming the proposed hypothesis of higher protein adsorption being responsible for higher cellular internalization.     

Quantification of the major metabolites of bromhexine in human plasma using RRLC–MS/MS and its application to pharmacokinetics

(E)-4-hydroxydemethylbromhexine (E-4-HDMB) and (E)-3-hydroxydemethylbromhexine (E-3-HDMB) were found as major metabolites, while (Z)-4-hydroxydemethylbromhexine and (Z)-3-hydroxydemethylbromhexine as minor metabolites of bromhexine in human plasma. These compounds were identified in comparison with synthetic authentic samples. A sensitive and selective rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS) method was developed to quantify the concentration of bromhexine and its two major metabolites (E-4-HDMB and E-3-HDMB) in human plasma. Following solid phase extraction, the analytes were separated on a Zorbax 1.8 μm particle size reversed-phase C₁₈ column, using a gradient elution program with solvents consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in 5 mM ammonium acetate at a flow rate of 0.7 mL/min. Detection was carried out with an Agilent 6460 triple-quadrupole mass spectrometer operated with an electrospray ionization source mode operated in the positive ion mode. The recovery of bromhexine, E-4-HDMB, E-3-HDMB, and internal standard (IS) was 63.1–70.9%, 60.5–68.4%, 57.0–63.5%, and 87.8%, respectively. The matrix factors of bromhexine, E-4-HDMB, E-3-HDMB, and IS were 89.9–96.7%, 89.6–94.8%, 90.4–91.4%, and 103%, respectively. After an oral administration of 8.0 mg bromhexine to five healthy male subjects, AUC_0–24 h values of bromhexine, E-4-HDMB, and E-3-HDMB were found to be 93.5 ± 31.9, 34.0 ± 14.5, and 15.8 ± 6.89 ng h/mL, respectively; while C_max values were 24.6 ± 5.16, 3.11 ± 1.13, and 5.36 ± 2.55 ng/mL, respectively. Plasma concentration of bromhexine, E-4-HDMB, and E-3-HDMB declined with t_1/2 which gave 3.6 ± 0.5, 8.4 ± 2.7, and 6.4 ± 2.5 h, respectively.     

Development and validation of a rapid reversed-phase HPLC method for the determination of the non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles

The objective of this work was to develop and validate a rapid reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the in vitro pharmaceutical characterization of dapivirine-loaded polymeric nanoparticles. Chromatographic runs were performed on a RP C18 column with a mobile phase comprising acetonitrile–0.5% (w/v) triethanolamine solution in isocratic mode (80:20, v/v) at a flow rate of 1 ml/min. Dapivirine was detected at a wavelength of 290 nm. The method was shown to be specific, linear in the range of 1–50 μg/ml (R² = 0.9998), precise at the intra-day and inter-day levels as reflected by the relative standard deviation values (less than 0.85%), accurate (recovery rate of 100.17 ± 0.35%), and robust to changes in the mobile phase and column brand. The detection and quantitation limits were 0.08 and 0.24 μg/ml, respectively. The method was successfully used to determine the loading capacity and association efficiency of dapivirine in poly(lactic-co-glycolic acid)-based nanoparticles and its in vitro release.     

Antioxidant and hepatoprotective activities of five eggplant varieties

Eggplant is consumed throughout the world and varies in fruit color, shape, and size. In this study, five varieties of eggplant (purple colored moderate size, white-green colored moderate size, long green, green striped moderate size and pale-green colored small size, respectively, called SM1–SM5) were evaluated for total phenolic and flavonoid content, antioxidant activity and hepatoprotection against cytotoxicity of tert-butyl hydroperoxide (t-BuOOH) in human hepatoma cell lines, HepG2. Total phenolic content found in methanol extracts of SM1–SM5 ranged from 739.36 ± 1.59 to 1116.13 ± 7.30 gallic acid equivalents mg/100 g extract and total flavonoid content from 1991.29 ± 6.32 to 3954.20 ± 6.06 catechin equivalents mg/100 g extract. SM1 and SM2 which contained high total phenolic and flavonoid had better antioxidant activities than the other varieties. Pretreatment of HepG2 cells with 50 and 100 μg/mL of SM1–SM5 significantly increased the viability (p < 0.05) of t-BuOOH-exposed HepG2 cells by 14.49 ± 1.14% to 44.95 ± 2.72%. The antioxidant activities of the eggplant were correlated with the total amounts of phenolic and flavonoid (r = 0.5310−0.7961). Significant correlation was found between hepatoprotective activities and total phenolic/flavonoid content (r = 0.6371–0.8842) and antioxidant activities (r = 0.5846–0.9588), indicating the contribution of the phenolic antioxidant present in eggplant to its hepatoprotective effect on t-BuOOH-induced toxicity.     

In vivo CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene

Assessing CYP2E1 phenotype in vivo may be important to predict individual susceptibility to those chemicals, including benzene, which are metabolically activated by this isoenzyme. Chlorzoxazone (CHZ), a specific CYP2E1 substrate, is readily hydroxylated to 6-OH-chlorzoxazone (6-OH-CHZ) by liver CYP2E1 and the metabolic ratio 6-OH-CHZ/CHZ in serum (MR) is a specific and sensitive biomarker of CYP2E1 activity in vivo in humans. We used this MR as a potential biomarker of effect in benzene-treated rats and, also, in humans occupationally exposed to low levels of benzene. Male Sprague-Dawley rats (375–400 g b.w.) were treated i.p. for 3 days with either a 0.5 ml solution of benzene (5 mmol/kg b.w.) in corn oil, or 0.5 ml corn oil alone. Twenty-four hours after the last injection, a polyethylene glycol (PEG) solution of CHZ (20 mg/kg b.w.) was injected i.p. in both treated and control animals. After 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240 min from injection, 0.2 ml blood was taken from the tip tail and stored at −20 °C until analysis. A modified reverse phase HPLC method using a 5 μm Ultrasphere C18 column equipped with a direct-connection ODS guard column, was used to measure CHZ and its metabolite 6-OH-CHZ in serum. No statistically significant difference in the MR was observed, at any sampling time, between benzene-treated and control rats. The concentration-versus-time area under the curve (AUC), however, was lower (p < 0.05, Mann–Whitney test), whereas the systemic clearance was higher (p < 0.05) in treated than in control rats. Eleven petrochemical workers occupationally exposed to low levels of airborne benzene (mean ± SD, 25.0 ± 24.4 μg/m³) and 13 non-exposed controls from the same factory (mean ± SD, 6.7 ± 4.0 μg/m³) signed an informed consent form and were administered 500 mg CHZ p.o. Two hours later a venous blood sample was taken for CHZ and 6-OH-CHZ measurements. Despite exposed subjects showed significantly higher levels of t,t-MA and S-PMA, two biomarkers of exposure to benzene, than non-exposed workers, no difference in the MR mean values ± SD was found between exposed (0.59 ± 0.29) and non-exposed (0.57 ± 0.23) subjects. So, benzene was found to modify CHZ disposition, but not CYP2E1 phenotype in benzene-treated rats, nor in workers exposed to benzene, probably due to the levels of exposure being too low.     

Validation of a liquid chromatography–tandem mass spectrometric assay for the quantitative determination of hydrastine and berberine in human serum

A high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of berberine and hydrastine in human serum, after oral administration of goldenseal (Hydrastis canadensis L.), was developed using simple acetonitrile treatment of serum samples. Noscapine served as the internal standard. Lower limit of quantification for both analytes was 0.1 ng mL⁻¹ using positive ion electrospray tandem mass spectrometry (MS/MS). The intra-day (n = 5) accuracy and precision of the method for hydrastine was 82 ± 8.8%, 97.9 ± 2.4% and 96.2 ± 3.3%, respectively. The inter-day (n = 4) accuracy and precision for hydrastine was 90.0 ± 15.17%, 99.9 ± 7.1% and 98 ± 6.54%, respectively. For berberine quantitation intra-day accuracy and precision was 96.0 ± 8.4%, 92.5 ± 4.7% and 94.4 ± 3.7%, respectively. The respective values for inter-day quantitation were 91.0 ± 8.4%, 94.3 ± 4.7% and 94.4 ± 3.7%. The analytical recovery for hydrastine was 82.4–96.2% and for berberine it was 94.4–96.0%. The analytes and noscapine were stable for 24 h at room temperature (CV 5–10%). Matrix ion effects were studied by post-column infusion of hydrastine and berberine, calculation of calibration curve slope precision was obtained using serum from five different subjects, and by comparison of the response of methanol standards and extracted serum samples. The method was further validated by determination of serum pharmacokinetics of hydrastine and berberine after administration of a single oral dose of goldenseal extract containing 77 mg of hydrastine and 132 mg of berberine.     

Determination of nucleosides and nucleobases in different species of Cordyceps by capillary electrophoresis–mass spectrometry

In present study, a capillary electrophoresis–mass spectrometry (CE–MS) method was developed for the simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine, guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2′-deoxyuridine, uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside as an internal standard (IS). The CE separation conditions and MS parameters were optimized systematically for achieving good CE resolution and MS response of the investigated compounds. The optimum CE electrolyte was 100 mM formic acid containing 10% (v/v) methanol. The optimum MS parameters were as follows: 75% (v/v) methanol containing 0.3% formic acid with a flow rate of 3 μL/min was selected as the sheath liquid; the flow rate and temperature of drying gas were 6 L/min and 350 °C, respectively. The optimized CE–MS method was successfully applied for the simultaneous determination of 12 nucleosides and nucleobases in natural and cultured Cordyceps. On the basis of quantitative results, the total content of nucleosides is much higher in cultured Cordyceps (9138 ± 4823 μg/g for cultured C. sinensis; 3722 ± 1446 μg/g for C. militaris) than in natural ones (2167 ± 412 μg/g). However, the hypoxanthine (131 ± 47 μg/g) and inosine (335 ± 90 μg/g) are much higher in natural C. sinensis. Cordycepin, which is abundant in cultured C. militaris (2276.5 ± 842.6 μg/g), is only found in natural C. sinensis with very low content and cannot be detected in the cultured ones.     

A new approach for pharmacokinetics of single-dose cetuximab in rhesus monkeys by surface plasmon resonance biosensor

A novel assay method has been developed and validated, using surface plasmon resonance (SPR), for quantitation of cetuximab (C225) in monkey serum. By injecting non-labeled antibody samples onto a biosensor surface on which epidermal growth factor receptor (EGFR) was immobilized, the concentration of C225 can be accurately measured. This assay has a range of reliable response from 0.05 to 50 μg/ml C225 in monkey serum, which was well fitted with a sigmoidal model. The immobilized EGFR was found to be stable for at least 100 regeneration cycles at room temperature. Intra- and inter-assay CVs ranged from 3.20% to 8.89% and from 5.93% to 11.11%, accuracy from 92% to 107.52% and from 90% to 106.88%, respectively. Matrices such as 50% human serum, 50% Sprague Dawley rat serum, chimeric recombinant anti-CD20 monoclonal antibody, human γ-globulin and chimeric recombinant her2 antibody did not interfere with C225 analysis on the sensor surface. This is the first report on the quantitation of C225 in monkey serum by an optical biosensor technology. This method was used to characterize the pharmacokinetics of C225 in rhesus monkeys. After a single-dose of intravenous infusion administration of 7.5, 24 and 75 μg/kg, average C_max ranged from 168 ± 28 to 1624 ± 113 μg/ml, and AUC_0–∞ ranged from 15,739 ± 1059 to 295,017 ± 44,533 μg h/ml. C225 elimination followed a bi-exponential profile with t_1/2 ranging from 2.7 ± 0.7 to 6.7 ± 0.1 h. It was non-linear serum pharmacokinetics of C225 across the investigated dosage range in monkeys (7.5–75 mg/kg).     

Pharmacokinetic study of melamine in rhesus monkey after a single oral administration of a tolerable daily intake dose

To perform pharmacokinetic study of melamine in rhesus monkey, melamine was orally administered to three experimental monkeys at a single dose of 1.4 mg/kg body weight. Plasma and urine were collected for the determination of melamine and cyanuric acid with a liquid chromatography tandem mass spectrometry method. The mean ± SD area under the concentration–time curve from time zero to 48 h (AUC0–t) was 14,145 ± 2002 μg/L h. The maximum concentration of melamine in plasma (C_max) was 1767 ± 252 μg/L. The time to maximum concentration (T_max) was 2.67 ± 1.16 h and the half-life of melamine in plasma (t_1/2) was 4.41 ± 0.43 h. Following oral administration, melamine was rapidly excreted, mainly through urinary clearance. No significant correlation was found between melamine and cyanuric acid, suggesting that cyanuric acid may not be derived from melamine.     

Contents of major bioactive flavones in proprietary traditional Chinese medicine products and reference herb of Radix Scutellariae

A simple and efficient HPLC/UV method for the simultaneous determination of six bioactive flavones, namely baicalein, baicalin, wogonin, wogonoside, oroxylin A and oroxylin A-7-O-glucuronide, has been developed and applied for their content determination in reference herb and proprietary traditional Chinese medicine (PTCM) products of Radix Scutellariae. The chromatographic separation was carried out on a Thermo C₁₈ column and linear gradient elution was employed with a mobile phase containing acetonitrile and 20 mM sodium dihydrogen phosphate buffer (pH 4.6). All the analytes were detected by PDA detector at a wavelength of 270 nm. Contents of the analytes in Radix Scutellariae containing PTCM products in forms of capsule, soft capsule, tablet and dripping pill and the reference herb of Radix Scutellariae were analyzed by sonicator extraction with methanol and water mixture (80:20) containing 1 mM HCl for 30 min followed by HPLC analysis. Separation of the six analytes was achieved within 25 min with good linearity (R² > 0.99). The R.S.D. of both the intra-day and inter-day precision for all the six analytes was below 10.14%. The accuracy at different concentrations was within the range of −7.83 to 4.06%. The extraction recovery was within the range of 89.22–107.33% for all the analytes. Contents of the six flavones were found to vary significantly among different products with glycosides, such as baicalin, wogonoside and oroxylin A-7-O-glucuronide, in much greater quantity than their corresponding aglycones. In addition to baicalin (18.54 ± 0.71%, w/w), the commonly used marker compound for Radix Scutellariae, wogonoside (3.54 ± 0.18%, w/w) and oroxylin A-7-O-glucuronide (2.84 ± 0.14%, w/w) also existed in abundant amount in the reference herb. Our findings suggested that wogonoside and oroxylin A-7-O-glucuronide should also be served as the chemical markers together with baicalin for the quality control of herbs and PTCM products of Radix Scutellariae.     

Organic cation transporter 1 mediates the uptake of monocrotaline and plays an important role in its hepatotoxicity

Monocrotaline (MCT) is a kind of toxic retronecine-type pyrrolizidine alkaloids (PAs) from plants of Crotalaria, which can be bio-activated by cytochrome P450 (CYP) enzymes in liver and then induce hepatotoxicity. Since CYPs are localized in the endoplasmic reticulum, the influx of MCT to the liver is the key step for its hepatotoxicity. The objective of the present study was to investigate the role of organic cation transporter 1 (OCT1), a transporter mainly expressed in liver, in the uptake of MCT and in hepatotoxicity induced by MCT. The results revealed that MCT markedly inhibited the uptake of 1-methyl-4-phenylpyridinium (MPP⁺), an OCT1 substrate, in Madin–Darby canine kidney (MDCK) cells stably expressing human OCT1 (MDCK-hOCT1) with the IC₅₀ of 5.52 ± 0.56 μM. The uptake of MCT was significantly higher in MDCK-hOCT1 cells than in MDCK-mock cells, and MCT uptake in MDCK-hOCT1 cells followed Michaelis–Menten kinetics with the K_m and V_max values of 25.0 ± 6.7 μM and 266 ± 64 pmol/mg protein/min, respectively. Moreover, the OCT1 inhibitors, such as quinidine, d-tetrahydropalmatine (d-THP), obviously inhibited the uptake of MCT in MDCK-hOCT1 cells and isolated rat primary hepatocytes, and attenuated the viability reduction and LDH release of the primary cultured rat hepatocytes caused by MCT. In conclusion, OCT1 mediates the hepatic uptake of MCT and may play an important role in MCT induced-hepatotoxicity.     

Quantitation of the tacrine analogue octahydroaminoacridine in human plasma by liquid chromatography–tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of octahydroaminoacridine in human plasma using tramadol as internal standard (I.S.). Sample preparation involved pH adjustment with sodium carbonate followed by solvent extraction with dichloromethane:ethyl ether (40:60, v/v). Chromatographic separation was achieved on a Venusil MP-C18 column (5 μm, 100 mm × 4.6 mm) using acetonitrile:10 mM ammonium acetate:formic acid (30:70:1, v/v/v) as mobile phase. Detection utilized an API 4000 system operated in the positive ion mode with multiple reaction monitoring of the analyte at m/z 203.1 → 175.1 and of the I.S. at m/z 264.1 → 58.0. The method was linear in the range 0.01–10 ng/ml with a lower limit of quantitation of 0.01 ng/ml. Intra- and inter-day precisions measured as relative standard deviation were <3.15% and <5.01%, respectively. The method was successfully applied to a pharmacokinetic study involving oral administration of a tablet containing 4 mg octahydroaminoacridine succinate to healthy volunteers. Pharmacokinetic parameters for octahydroaminoacridine include C_max 1.19 ± 0.53 ng/ml, T_max 0.77 ± 0.17 h, AUC_0−t 3.42 ± 1.01 ng h/ml and t_1/2 2.89 ± 0.56 h.     

Simultaneous determination of methotrexate and its polyglutamate metabolites in Caco-2 cells by liquid chromatography–tandem mass spectrometry

In normal and malignant human cells, the folate antagonist methotrexate (MTX) is converted to a series of polyglutamates (MTXGlu_n, n = 2–5) which play a role in its therapeutic efficacy. Here we report an assay to determine MTX and MTXGlu_n in Caco-2 cells exposed to MTX. After a simple protein precipitation step, cell homogenates (2 × 10⁶ cells) were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using aminopterin as internal standard. Separation was by reversed phase HPLC on a C8 column using gradient elution with 0.1% formic acid and acetonitrile. Detection was by electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the transitions of the [M+H]⁺ ions of MTX and MTXGlu_n to their common product ion at m/z 308.2 and of aminopterin at m/z 441.3 → 294.2. Calibration curves for all analytes were linear in the range 2–250 nM (r² > 0.996). Intra- and inter-day precisions (as coefficient of variation) were 3.4–15.1% and 4.3–18.4%, respectively with corresponding accuracies (as relative error) of −3.6 to +6.6% and −5.5 to +7.5%, respectively. Recoveries were in the range 60 ± 4 to 108 ± 13%. It was found that MTX undergoes only limited polyglutamation in Caco-2 cells exposed to MTX over 24 h.     

A novel approach to oral iron delivery using ferrous sulphate loaded solid lipid nanoparticles

Iron (Fe) loaded solid lipid nanoparticles (SLN's) were formulated using stearic acid and iron absorption was evaluated in vitro using the cell line Caco-2 with intracellular ferritin formation as a marker of iron absorption. Iron loading was optimised at 1% Fe (w/w) lipid since an inverse relation was observed between initial iron concentration and SLN iron incorporation efficiency. Chitosan (Chi) was included to prepare chitosan coated SLN's. Particle size analysis revealed a sub-micron size range (300.3 ± 31.75 nm to 495.1 ± 80.42 nm), with chitosan containing particles having the largest dimensions. As expected, chitosan (0.1%, 0.2% and 0.4% w/v) conferred a net positive charge on the particle surface in a concentration dependent manner. For iron absorption experiments equal doses of Fe (20 μM) from selected formulations (SLN-FeA and SLN-Fe-ChiB) were added to Caco-2 cells and intracellular ferritin protein concentrations determined. Caco-2 iron absorption from SLN-FeA (583.98 ± 40.83 ng/mg cell protein) and chitosan containing SLN-Fe-ChiB (642.77 ± 29.37 ng/mg cell protein) were 13.42% and 24.9% greater than that from ferrous sulphate (FeSO4) reference (514.66 ± 20.43 ng/mg cell protein) (p ≤ 0.05). We demonstrate for the first time preparation, characterisation and superior iron absorption in vitro from SLN's, suggesting the potential of these formulations as a novel system for oral iron delivery.     

Enhanced distribution and extended elimination of glycyrrhetinic acid in mice liver by mPEG-PLA modified (mPEGylated) liposome

A rapid and simple method of high-performance liquid chromatography with UV detector for the quantification of glycyrrhetinic acid (GA) in mice plasma and tissues has been developed and validated. With the established assay method, the pharmacokinetic profiles and tissue distribution of GA in different formulations are compared in mice after intravenous administration of the drug (25 mg/kg). The results showed that mPEG-PLA modified (mPEGylated) GA liposome (PL-GA) significantly prolonged the mean residence time (MRT) of GA in mice plasma and liver (MRT: 0.43 ± 0.13 and 1.72 ± 0.11 h, respectively) than the normal GA liposome (L-GA) (MRT: 0.23 ± 0.01 and 1.07 ± 0.31 h, respectively) and GA sodium injection (S-GA) (MRT: 0.13 ± 0.01 and 0.95 ± 0.08 h, respectively). Moreover, PL-GA specifically increased GA uptake in liver (AUC_{0–∞,liver} value of 1.6-fold and 1.3-fold higher than that for S-GA and L-GA, respectively) and reduced its distribution into other tissues after dosing. Due to these pharmacokinetic properties, it may be promising to develop PL-GA further as a new pharmaceutical preparation for GA on the treatment of various chronic hepatic diseases.     

Effects of Thai Musa species on prevention of UVB-induced skin damage in mice

The effects of oral administration of Musa sapientum and Musa suerier on prevention of UVB induced skin damages were investigated in male ICR mice. Animals were orally administered 50 mg/day ascorbic acid, or M. sapientum or M. suerier’s fruit pulps at dose of 0.5, 1 or 1.5 mg/g body weight/day for 12 weeks. Concurrently, the shaved backs of animals were irradiated with UVB for 12 weeks. The intensity of irradiation was progressively increased, from 54 mJ/cm² per exposure at week 1–126 mJ/cm² at week 11. A significant decrease (p < 0.05) in skin elasticity (from 0.82 ± 0.02 to 0.42 ± 0.09) and total glutathione (from (193.6 ± 18.7 to 152.7 ± 7.8 ng/mg protein) as compared with the control group (water-administered UVB-irradiated mice) was observed after 12 weeks of UVB exposure. When l-ascorbic acid (0.72 ± 0.01) or 1 mg/g body weight/day M. suerier (0.84 ± 0.06) were administered to UVB-irradiated mice, the reduction in skin elasticity was significantly inhibited (p < 0.05). Moreover, the significant increase (p < 0.05) in level of total glutathione was found in these groups (220.8 ± 13.3 ng/mg protein for l-ascorbic acid and 224.9 ± 20.1 ng/mg protein for M. suerier). These findings suggest the potential effect of daily consumption of M. suerier on prevention of skin damage from repeated UVB exposure.     

Chiral HPLC analysis of formoterol stereoisomers and thermodynamic study of their interconversion in aqueous pharmaceutical formulations

A chiral HPLC method was validated and successfully applied for the determination of formoterol stereoisomers and their inversion products in an aqueous matrix stored at 5–70 °C up to 3 weeks. Analysis was performed on a Chiral-AGP column (100 × 4-mm, 5-μm) using a variable mixture of mobile phase A (50-mM sodium phosphate buffer, pH 7.0) and B (10% v/v IPA) at a flow rate of 1.3 ml min⁻¹, and UV detection at 242 nm. All four formoterol stereoisomers were adequately resolved with acceptable detection and quantitation limits varying from 0.01–0.04 μg/ml and 0.04–0.1 μg/ml, respectively. The method showed acceptable accuracy (≥88%), precision (RSD ≤ 8.5%) and good linearity (r² ≥ 0.9999) over the concentration range investigated. While interconversion at 5 ± 3 °C and 25 ± 2 °C/60% RH ±5% RH was too low to be determined accurately within the study period, chiral inversion of formoterol stereoisomers measured at high temperatures followed the first order rate kinetics and occurred at a single chiral center, resulting in the reversible formation of diastereoisomers, (R,R) ↔ (S,R) and (S,S) ↔ (R,S). No enantiomerization or diastereomerization occurred. There was no significant difference in inversion of the active components in racemic (R,R/S,S)-formoterol fumarate and the single isomer (R,R)-arformoterol tartrate drug formulations, and both drugs are expected to maintain their stereochemical integrity throughout the proposed shelf-life at the recommended storage condition (5 ± 3 °C).     

Noninvasive measurement of smoking-associated N-ethyladenine and N-ethylguanine in human salivary DNA by stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry

Evidence showed that ethylating agents are contained in cigarette smoke, which damage DNA producing ethylated DNA adducts, including N³-ethyladenine (3-EtAde) and N⁷-ethylguanine (7-EtGua). These two ethylpurines can be depurinated spontaneously and be repaired by enzymes and they have been detected in human urine. In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human salivary DNA. These ethylpurines were released from DNA by neutral thermal hydrolysis and then enriched by a solid-phase extraction column before nanoLC–NSI/MS/MS analysis. The detection limits (S/N ≥ 3) of 3-EtA and 7-EtG were 15 fg (92 amol) and 10 fg (56 amol), respectively, injected on-column. The lower quantification limits of 3-EtAde and 7-EtGua were both 100 fg, i.e. 620 and 560 amol, respectively, corresponding to 9.4 and 8.6 adducts in 10⁹ normal nucleotides, respectively, starting with as little as 20 μg of DNA isolated from an average of 3 mL of saliva. The mean (±SD) levels of 3-EtAde in 15 smokers and 15 nonsmokers were 12.6 ± 7.0 and 9.7 ± 5.3 in 10⁸ normal nucleotides, respectively, while those of 7-EtGua were 14.1 ± 8.2 and 3.8 ± 2.8 in 10⁸ normal nucleotides in smokers and nonsmokers, respectively. Levels of 7-EtGua, but not 3-EtAde, were statistically significantly higher in smokers than in nonsmokers (p < 0.0001). Furthermore, salivary 7-EtGua levels are significantly correlated with the number of cigarettes smoked per day as well as with the smoking index. This highly specific and sensitive stable isotope dilution nanoLC–NSI/MS/MS assay might be feasible in measuring 7-EtGua in human salivary DNA as a noninvasive biomarker for DNA damage induced by cigarette smoking.     

Chemical composition,in vitro anti-microbial,antifungal and antioxidant activities of the essential oil and methanolic extract of Hymenocrater longiflorus Benth., of Iran

In this study we identified the chemical composition, anti-microbial and antioxidant effects of essential oil and methanolic extract of Hymenocrater longiflorus Benth. Totally 87 volatile compounds from the essential oil in H. longiflorus, were identified by gas chromatography–mass spectrometry (GC–MS). These compounds are mainly monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygenated monoterpenes and oxygenated sesquiterpenoids compounds. The anti-microbial and antifungal activity of plants extracts against several pathogenic microorganisms was studied by disc diffusion and minimum inhibitory concentration procedures. The results revealed that the essential oil and polar sub-fraction are effective mostly against Staphylococcus aureus, Aspergillus niger and Candida albicans. The antioxidant activity was also determined by 1,1′-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, β-carotene linoleic acid assay and reducing power. In addition the total phenol of essential oil (54.6 ± 1.2), polar sub-fraction (50.0 ± 1.4) and non-polar sub-fraction (64.7 ± 2.0) were determined.     

Is there any association between imidapril hydrochloride stability profile under dry air conditions and cancer initiation?

Stability study for imidapril hydrochloride (IMD) was performed under stress conditions of increased temperature (T = 373 K) and decreased relative air humidity (RH = 0%) in order to obtain and identify its degradation product. The degradation sample stored for 15 days under the above environmental conditions was analyzed by LC–MS technique and it was found that the only degradation impurity formed in the course of the investigated drug degradation was IMD diketopiperazine derivative (DKP) which was produced by dehydration and intramolecular cyclization. The kinetics of its formation was analyzed by a revalidated RP-HPLC method and the kinetic model of this reaction was established. It was concluded that the DKP formation follows Prout–Tompkins kinetics with the rate constant k ± Δk = 2.034 ± 0.157 × 10⁻⁶ [s⁻¹]. The obtained degradation impurity was further assessed with respect to its mutagenic potential using commercial Ames MPF 98/100 microplate format mutagenicity assay kit equipped with Salmonella typhimurium strains TA 98 and TA 100. Both strains were exposed to six concentrations (in a range of 0.16–5.0 mg/mL) of DKP in the presence and absence of metabolic activation system. No mutagenic effect was observed confirming that the presence of DKP in IMD final dosage form has no impact on cancer initiation.