Measurement of pharmacokinetics and tissue distribution of three bioactive constituents from Zanthoxylum armatum DC in rat plasma and tissues through UFLC-MS/MS.

The compounds of N-Methylanhydrotetrahydroberberrubine A, dictamnine and eudesmin were the primary bioactive components in the roots of Zanthoxylum armatum DC (Z. armatum). To clarify the pharmacokinetics and distribution of these three compounds, an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was employed to determine the contents of these three compounds in rat plasma and seven tissues. The separation was achieved on a Kinetex XB-C18 100A column (2.1 × 50 mm, 2.6 µm, Phenomenex). The optimized mobile phase system was set with 0.1‰ formic acid aqueous solution (A) and acetonitrile (containing 0.1‰ formic acid) (B) with a programmed elution of 0.00 to 0.50 min, 2% B; 0.51-4.00 min, 30%-60% B; and 4.01-5.00 min, 2% B. All analytes were measured with optimized multiple reaction monitoring (MRM) in the positive ion ESI mode. Berberine hydrochloride was selected as the internal standard (IS). The MS/MS transitions of N-Methylanhydrotetrahydroberberrubine A, dictamnine, eudesmin and IS were 339.9135.1, 200.1 → 129.1, 387.4 → 369.0 and 337.1 → 321.1, respectively. The lower limits quantification (LLOQ) of the three analytes was 0.5-20 ng/ml. The linear ranges were 0.5-400 ng/ml for N-Methylanhydrotetrahydroberberrubine A and dictamnine and 20-4000 ng/ml for eudesmin. The present analysis showed that the two alkaloids were quickly absorbed, with Tmax in 0.167-0.292 h, and eudesmin was absorbed in 2.5 h. Moreover, all compounds were found at high concentrations in the gastrointestinal track. These results are helpful for further investigation of the clinical application of Z. armatum.

Developing an activity and absorption-based quality control platform for Chinese traditional medicine: Application to Zeng-Sheng-Ping(Antitumor B).

ETHNOPHARMACOLOGICAL RELEVANCE: Zeng-Sheng-Ping (ZSP), also called antitumor B, is a marketed Chinese traditional medicine used for cancer prevention. AIM OF THE STUDY: Currently, for the quality control of Chinese traditional medicines, marker compounds are not selected based on bioactivities and pharmaceutical behaviors in most of the cases. Therefore, even if the "quality" of the medicine is controlled, the pharmacological effect could still be inconsistent. The aim of this study is to establish an activity and absorption-based platform to select marker compound(s) for the quality control of Chinese traditional medicines. MATERIALS AND METHODS: We used ZSP as a reference Chinese traditional medicine to establish the platform. Activity guided fractionation approach was used to purify the major components from ZSP. NMR and MS spectra were used to elucidate the structure of the isolated compounds. MTT assay against oral carcinoma cell line (SCC2095) was performed to evaluate the activities. UPLC-MS/MS was used to quantify the pure compounds in ZSP and the active fraction. The permeabilities of the identified compounds were evaluated in the Caco-2 cell culture model. The intracellular accumulation of the isolated compounds was evaluated in the SCC2095 cells. RESULTS: The major compounds were identified from ZSP. The contents, anti-proliferation activities, permeabilities, and intracellular accumulations of these compounds were also evaluated. The structure of these purified compounds were identified by comparing the NMR and MS data with those of references as rutaevine (1), limonin (2), evodol (3), obacunone (4), fraxinellone (5), dictamnine (6), maackiain (7), trifolirhizin (8), and matrine (9). The IC50 of compounds 5, 6, and 7 against SCC2095 cells were significantly lower than that of ZSP. The uptake permeability of compounds 5, 6, and 7 were 2.58 ± 0.3 × 10(-5), 4.33 ± 0.5 × 10(-5), and 4.27 ± 0.8 × 10(-5) respectively in the Caco-2 cell culture model. The intracellular concentrations of these compounds showed that compounds 5, 6, and 7 were significantly accumulated inside the cells. CONCLUSION: Based on the activity against oral carcinoma cell line as well as the absorption permeability, compound 5, 6, and 7 are selected as quality control markers for ZSP. An activity and absorption-based platform was established and successfully used for the quality control of ZSP.

A validated HPLC-PDA method for identification and quantification of two bioactive alkaloids, ephedrine and cryptolepine, in different Sida species.

A simple, rapid, accurate and reproducible reverse-phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP-18 X-terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10-250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra-day and inter-day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species.

Determination of moxifloxacin and its oxidation products with kinetic evaluation under potassium permanganate treatment in acidic solution by ultra-performance liquid chromatography/tandem mass spectrometry.

A simple, sensitive, and reproducible ultra-performance LC method for the determination of moxifloxacin (MOXI) oxidation stability under permanganate treatment in acidic conditions (pH 3.0-6.0) was developed. Besides the MOXI peak [retention time (RT) = 2.58], four additional products (RT = 0.86, 0.91, 1.42, and 1.89) were observed in all conditions tested. The oxidation process followed second-order reaction kinetics and depended upon solution acidity. The highest reaction rate constant was observed at pH 3.0, and this value decreased as the pH was increased to 6.0. The oxidation products were characterized, and their fragmentation pathways, derived from MS/MS data, were proposed. Two of these products were identified as hydroxyl derivatives of MOXI and two others as their oxidation product analogs with molecular ions of 418.4 and 416.4 m/z, respectively.

Rapid and green analytical method for the determination of quinoline alkaloids from Cinchona succirubra based on Microwave-Integrated Extraction and Leaching (MIEL) prior to high performance liquid chromatography.

Quinas contains several compounds, such as quinoline alkaloids, principally quinine, quinidine, cinchonine and cichonidine. Identified from barks of Cinchona, quinine is still commonly used to treat human malaria. Microwave-Integrated Extraction and Leaching (MIEL) is proposed for the extraction of quinoline alkaloids from bark of Cinchona succirubra. The process is performed in four steps, which ensures complete, rapid and accurate extraction of the samples. Optimal conditions for extraction were obtained using a response surface methodology reached from a central composite design. The MIEL extraction has been compared with a conventional technique soxhlet extraction. The extracts of quinoline alkaloids from C. succirubra obtained by these two different methods were compared by HPLC. The extracts obtained by MIEL in 32 min were quantitatively (yield) and qualitatively (quinine, quinidine, cinchonine, cinchonidine) similar to those obtained by conventional Soxhlet extraction in 3 hours. MIEL is a green technology that serves as a good alternative for the extraction of Cinchona alkaloids.

The effect of green tea and olive oil on the mutagenic activity of heterocyclic amines extracted from common food consumed in Saudi Arabia.

The effect of green tea (GT) and green tea with olive oil (GT+OL) as antioxidants on the formation and mutagenic activity of heterocyclic aromatic amines (HCAs) extracted from beef shawerma, grilled chicken and fried beef liver was examined. HCAs were extracted by blue rayon, analyzed as spiked and unspiked samples with high-performance liquid chromatography and its mutagenic response was assessed by Sallmonela typhimurium 100 in the Ames test. Surprisingly, GT and GT+OL augmented HCAs measured in beef shawerma and grilled chicken but total HCAs measured in GT+OL were less than GT treatment. Both treatments altered the HCA profile as imidazoquinoline type became the most abundant. In control and GT+OL fried beef liver no HCAs were detected, but Trp-P1 was detected in GT treatment. Generally, the mutagenic response of HCAs measured in GT+OL was less than GT in beef shawerma and grilled chicken. However, the mutagenic response of control and 2% GT+OL fried liver was negative. These data suggest that GT concentrations used in this study may induce free radical formation during the Millared reaction due to its pro-oxidative effect, which augmented the HCAs formed and its mutagenic response. In order to optimize both safety and quality of our diets, more need to be done to fully understand the risk of HCAs in food.

FIT probes: peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery.

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.

Effect of dietary constituents with chemopreventive potential on adduct formation of a low dose of the heterocyclic amines PhIP and IQ and phase II hepatic enzymes.

We conducted a study to evaluate dietary chemopreventive strategies to reduce genotoxic effects of the carcinogens 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). PhIP and IQ are heterocyclic amines (HCAs) that are found in cooked meat and may be risk factors for cancer. Typical chemoprevention studies have used carcinogen doses many thousand-fold higher than usual human daily intake. Therefore, we administered a low dose of [14C]PhIP and [3H]IQ and utilized accelerator mass spectrometry to quantify PhIP adducts in the liver, colon, prostate, and blood plasma and IQ adducts in the liver and blood plasma with high sensitivity. Diets supplemented with phenethylisothiocyanate (PEITC), genistein, chlorophyllin, or lycopene were evaluated for their ability to decrease adduct formation of [14C]PhIP and [3H]IQ in rats. We also examined the effect of treatments on the activity of the phase II detoxification enzymes glutathione S-transferase (GST), UDP-glucuronyltransferase (UGT), phenol sulfotransferase (SULT) and quinone reductase (QR). PEITC and chlorophyllin significantly decreased PhIP-DNA adduct levels in all tissues examined, which was reflected by similar changes in PhIP binding to albumin in the blood. In contrast, genistein and lycopene tended to increase PhIP adduct levels. The treatments did not significantly alter the level of IQ-DNA or -protein adducts in the liver. With the exception of lycopene, the treatments had some effect on the activity of one or more hepatic phase II detoxification enzymes. We conclude that PEITC and chlorophyllin are protective of PhIP-induced genotoxicity after a low exposure dose of carcinogen, possibly through modification of HCA metabolism.

HPLC-NMR/HPLC-MS analysis of the bark extract of Stauranthus perforatus.

A combination of HPLC-MS and HPLC-NMR techniques has been used to analyse the cytotoxic fractions of the dichloromethane extract of bark of Stauranthus perforatus. Six furanocoumarins (byakangelicol, heraclenin, heraclenol, imperatorin, isopimpinellin and xanthotoxin) and nine quinoline alkaloids (two known compounds, veprisine and 5-hydroxy-1-methyl-2-phenyl-4-quinolone, along with seven novel compounds, stauranthine, 3',4'-dihydroxy-3',4'-dihydroveprisine, 3',4'-dihydroxy-3',4'-dihydrostauranthine, 3',6'-dihydroxy-3',6'-dihydroveprisine, 3',6'-dihydroxy-3',6'-dihydrostauranthine, 6'-hydroxy-3'-ketoveprisine and 6'-hydroxy-3'-ketostauranthine) have been identified in the fractions.

Green tea and the metabolism of 2-amino-3-methylimidazo.

The effects of green tea intake on the metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the rat was studied. IQ belongs to a new class of mutagens and carcinogens, heterocyclic arylamines, formed during cooking through browning meats and fish, thus, in the food chain of most non-vegetarians. Ten adult male and female Fischer 344 rats were placed on a 2% solution of green tea and 10 control rats were on water for 6 weeks. Then, animals were administered a single dose of 40 mg/kg body weight of [2-14C]IQ by oral gavage. Twenty-four hour urine samples were collected and metabolites were separated by HPLC and quantitated by scintillation counting. Two minor and three major metabolites were isolated, including, small quantities of IQ itself. The rats on tea showed significant differences (P < 0.05) in the recovery of the three major metabolites, namely, IQ-sulfamate, IQ-5-O-sulfate, and IQ-5-O-glucuronide, respectively. Green tea, therefore, influences the manner in which the food carcinogen IQ is metabolized and excreted in urine. Formation of glucuronides, increased by green tea, represent a key means of detoxification of the heterocyclic amine, IQ.

Exposure to agricultural treatment residues: some simple statistical considerations based on monitoring data for ethoxyquin on apples.

The data examined here concern ethoxyquin levels, mainly measured in apple samples from the retail market. In this study about three hundred measurements, carried out on apples in Northern-Central Italy and recently published, are examined, with the purpose of defining the main characteristics of value statistical distribution and variability and to optimize sampling criteria. The analysis did not indicate the presence of significantly high levels or consequently significantly high risks; nevertheless, it pointed out some important aspects in data collection and evaluation. First, it appeared that the statistical distribution of data is typically nonsymmetrical, non-Gaussian, and characterized by a tail extending toward the high values. The logarithmic transformation of data appeared useful in order to obtain a Gaussian distribution. As a rule, the data variability appeared to be high. The frequency and distribution of values above the analytical threshold resulted in significant change, both in time and in space. The percentage of positive values (above the analytical threshold) appeared to vary up to a factor of 2-3, as analogously did their geometric mean.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral ciprofloxacin therapy of infections due to Pseudomonas aeruginosa.

The efficacy and safety of oral ciprofloxacin, a fluoroquinolone, were evaluated in the treatment of infection due to Pseudomonas aeruginosa. 96 infections in 71 patients were treated. Substantial underlying disease was present in most of the patients, and 25 (35%) were seriously ill. 52% of pseudomonas isolates were carbenicillin-resistant, and 31% gentamicin-resistant. The overall clinical response rate was 77%-28 of 35 exacerbations of cystic fibrosis respiratory disease, 17 of 19 urinary infections, 4 of 6 osteomyelitis, and 11 of 15 soft tissue infections. The bacteriological cure rate was 34%-0 of 35 cystic fibrosis, 4 of 17 respiratory infections, 17 of 19 urinary infections, 4 of 6 osteomyelitis, and 8 of 15 soft tissue infections. Ps aeruginosa developed resistance to ciprofloxacin in 25 of 96 infections. Side-effects were generally slight with nausea in 14 (15%) the most common, and there were only two substantial superinfections.

Ciprofloxacin as therapy for experimental osteomyelitis caused by Pseudomonas aeruginosa.

Ciprofloxacin, a new carboxyquinoline antimicrobial agent, was compared with tobramycin in the treatment of chronic osteomyelitis due to Pseudomonas aeruginosa in rabbits. Treatment with tobramycin for four weeks was ineffective (94% had positive bone cultures). In contrast, ciprofloxacin administered for four weeks sterilized the bones of all but one (94%) of 18 treated rabbits. Treatment with ciprofloxacin for two weeks was less effective than treatment for four weeks but was more effective than either treatment with tobramycin or no therapy. Two of 10 isolates from rabbits treated with ciprofloxacin for two weeks were susceptible to MICs of ciprofloxacin that were 16- and fourfold greater than the MIC for the parent strain; the other eight isolates remained sensitive to ciprofloxacin with MICs equivalent to that of the parent strain.

High pressure liquid chromatographic determination of ethoxyquin in paprika and chili powder.

A method is described for the determination of ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) in paprika and chili powder. Ethoxyquin is extracted from the spice with hexane and partitioned into 0.3N HCl. After adjusting the solution to pH 13-14, ethoxyquin is extracted into hexane, and the hexane layer is evaporated to dryness. An acetonitrile solution of the residue is then analyzed by reverse phase high pressure liquid chromatography with detection at 254 nm. The mobile phase is water-acetonitrile with ammonium acetate buffer. Recoveries from samples fortified at 50, 100, and 200 ppm averaged 92% with a coefficient of variation of 2.3%. The method was applied to a number of commercial samples of paprika and chili powder. Ethoxyquin was found in paprika samples at levels up to 63 ppm and in chili powder samples at levels up to 20 ppm.

Antimicrobial agents from higher plants. The antimicrobially inactive components of Ptelea trifoliata L.

From the weakly antibacterial non-quaternary alkaloidal fractions from Ptelea trifoliata L. (Rutaceae), ten tertiary quinol-2-one and quinol-4-one alkaloids were isolated and identified. In addition, beta-sitosterol, beta-sitosteryl-beta-D-glucoside and bergapten were isolated. None of these compounds possessed perceptible antimicrobial activity. The weak antimicrobial activity of the neutral and alkaloidal fractions was traced to small amounts of pteleatinium chloride which had not been completely separated by bulk processes. Alkaloids previously known to be present in P. trifoliata which were found in this study ptelefoline methyl ether, pteleine and skimmianine. Alkaloids previously known but new to this plant were lunidoine and isomaculasidine. Alkaloids newly found in nature were neohydroxylunine, hydroxylunidonine, 6-methoxylunidoine, 6-methoxylunineand 6-methoxy-hydroxylunidine. The structure of the latter three bases is proposed tentatively.

Antimicrobial agents from higher plants. The quaternary alkaloids of Ptelea trifoliata.

Quaternary alkaloid extracts of Ptelea trifoliata showed potentially interesting antimicrobial activity. Chromatographic examination showed the presence of six components. Of these, choline and O-4-methyl ptelefolinium were known compounds. Rational structures are proposed for the new alkaloids, O-4-methylhydroxyluninium, neohydroxylunine and pteleatinium salts. Pteleatinium salt is responsible for the antimicrobial activity of the extracts of the plant.