Transformation of ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834. Southern blot analysis of the established roots revealed that only the TL-DNA was integrated into the plant genome without incorporation of the TR-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degrees C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.     

Single dose pharmacokinetics of syrup of ipecac

Syrup of ipecac (SI) has been used medicinally since the 1500s; however, little is known about the pharmacokinetics in humans of SI's active ingredients, emetine and cephaeline. The objective of this study was to evaluate the rate of absorption and the rate of elimination of emetine and cephaeline. Ten healthy, adult, human volunteers between 18 and 45 years of age who were of ideal body weight (body mass index 20-25) completed this study. After an overnight fast, 30 mL SI were ingested. Blood samples were collected 30, 45, 60, 90, 120, 150, and 180 minutes post-ingestion and urine was collected throughout the study period. Plasma and urine concentrations of emetine and cephaeline were measured by reverse-phase HPLC with fluorescence detection. In virtually all subjects, emetine and cephaeline were detected within 5-10 minutes of dosing with the time to maximum concentration being approximately 20 minutes. The mean areas under the concentration-time curve (AUC) for both emetine and cephaeline were similar; however, the ratio of mean cephaeline maximum concentration (Cmax) to emetine Cmax was approximately 1.5. Four of the ten subjects exhibited a type of concentration-time profile in which the levels of cephaeline were substantially higher than those of emetine and the levels of cephaeline were substantially higher than noted for the other six subjects. In these remaining six subjects, the levels of emetine and cephaeline were lower than 10 ng/mL at all time-points. An initial elimination phase was noted in some subjects but not in others. Individuals in whom an initial elimination phase was not observed also exhibited low levels of both alkaloids as compared with the other subjects suggestive of a slower distribution phase. Less than 0. 15% of the administered emetine and cephaeline was recovered in the urine at 3 hours. No relationship between vomiting episodes and peak concentrations of emetine or cephaeline was found. Administration of SI results in rapid appearance and disappearance of emetine and cephaeline in plasma becoming almost undetectable at 3 hours. Very little of either alkaloid is eliminated in the urine within this time period, suggesting extensive distribution. The length of time that an administered dose of SI can result in the detection of emetine and/or cephaeline in the urine has not been determined; future studies in humans are required.     

Studies for the emetic mechanisms of ipecac syrup (TJN-119) and its active components in ferrets: involvement of 5-hydroxytryptamine receptors

Ipecac syrup, prepared from a galentical ipecac, contains the nauseant alkaloids cephaeline and emetine. The involvement of receptors and serotonin- and dopamine-metabolizing enzymes in the emesis induced by ipecac syrup and these components was investigated. 1) In ferrets, the selective 5-HT3-receptor antagonist ondansetron (0.5 mg/kg, p.o.) prevented each emesis induced by TJN-119 (0.5 mL/kg, p.o.), cephaeline (0.5 mg/kg, p.o.) and emetine (5.0 mg/kg, p.o.), but the intraperitoneal administration of the selective dopamine D2-receptor antagonist sulpiride failed to significantly suppress the TJN-119, cephaeline and emetine-induced emesis at a dose of 0.1 mg/kg that blocked apomorphine-induced emesis. 2) In the receptor binding assays, cephaeline and emetine had a distinct affinity to 5-HT4 receptor, but no or weak affinity to 5-HT1A, 5-HT3, nicotine, M3, beta1, NK1, and D2 receptors. 3) Cephaeline and emetine did not affect activities of metabolic enzymes of 5-HT and dopamine (MAO-A, MAO-B, tryptophan 5-hydroxylase and tyrosine hydroxylase) in vitro. These results suggest that 5-HT3 receptor plays an important role in the emetic action of TJN-119, cephaeline and emetine, and the 5-HT4 receptor may be involved in their mechanisms.     

Metabolism of ipecac alkaloids cephaeline and emetine by human hepatic microsomal cytochrome P450s, and their inhibitory effects on P450 enzyme activities.

In this study, we identified the metabolites and the CYP forms that are specifically involved in emetine O-demethylation in human liver microsomes, and cleared the inhibitory potential of cephaeline and emetine on the activity of the major drug-metabolizing CYP enzymes. Incubation of emetine with human liver microsomes yielded three metabolites identified by using HPLC by comparison of the retention time with the authentic sample of cephaeline, 9-O-demethylemetine and 10-O-demethylemetine. CYP3A4 and CYP2D6 were able to metabolize emetine to cephaeline and 9-O-demethylemetine, and CYP3A4 also participated in metabolizing emetine to 10-O-demethylemetine. Cephaeline and emetine inhibited probe substrates metabolism. IC50 for cephaeline against CYP2D6 and CYP3A4 were 121 and 1000 microM, respectively. For the emetine, CYP2D6 and CYP3A4 were 80 and 480 microM, respectively. Inhibition constants (Ki) for both compounds on the CYP2D6 and CYP3A4 activities were determined by graphic analysis of Dixon plots at various concentrations. The obtained Ki values of cephaeline for CYP2D6 and CYP3A4 were 54 and 355 microM, respectively, and the values of emetine were 43 and 232 microM, respectively. We concluded that these in vitro inhibitions of cephaeline and emetine would hardly increase plasma concentrations of co-administered drugs in clinical therapy.     

Eine neue Methode zur Bestimmung der Gesamtalkaloide und des Cephaelins in Ipecacuanha

A new Determination Method for Total Alkaloids and Cephaeline in Ipecacuanha Graf's procedure¹) to determine the total alkaloids of Ipecacuanha root using adsorption chromatography is revised and improved. Both the total alkaloid content and the cephaeline content can be determined directly in the chloroform eluate, total alkaloids by spectrophotometry at 286 nm, cephaeline by reaction with 2,6-dichloroquinone-chloroimide in presence of methanolic potassium hydroxide, followed by spectrophotometry at 595 nm.     

Biotransformation of the ipecac alkaloids cephaeline and emetine from ipecac syrup in rats.

The metabolism of cephaeline and emetine, which are the primary active components of ipecac syrup, were investigated in rats. Cephaeline-6'-O-glucuronide was found to be a biliary metabolite of cephaeline. Cephaeline (6'-O-demethylemetine) and 9-O-demethylemetine were observed to be enzyme-hydrolyzed biliary metabolites of emetine. Cephaeline was conjugated to glucuronide, while emetine was demethylated to cephaeline and 9-0-demethylemetine, and may be conjugated to glucuronides afterwards. Urine, feces and bile were collected from rats within 48 hours following the administration of ipecac syrup containing tritium (3H)--labeled cephaeline or emetine. Metabolites were separated and quantified by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Biliary and urinary excretion rates of 3H-cephaeline were 57.5% and 16.5% of the dose, respectively. Cephaeline-6'-O-glucuronide was comprised 79.5% of biliary radioactivity and 84.3% of urinary radioactivity. Unchanged cephaeline was detected in 42.4% of the dose in feces. Biliary excretion rate of 3H-emetine was 6.9% of the dose. Emetine, cephaeline and 9-0-demethylemetine comprised 5.8%, 43.2% and 13.6% in hydrolyzed bile, respectively. There were no emetine-derived metabolites in urine or feces. The occurrence of unchanged emetine was 6.8% and 19.7% of the dose in urine and feces, respectively.     

Determination of a new reversible proton pump inhibitor,DBM-819, in human plasma and urine,and rat tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new proton pump inhibitor, DBM-819, in human plasma and urine and rat tissue homogenates using KR-60461 as an internal standard. A 100-microl aliquot of acetonitrile (containing 0.5 microg/ml of the internal standard) and a 200-microl aliquot of 0.1 M Na(2)HPO(4) (adjusted pH 11 with 1 N NaOH) were added to a 100-microl aliquot of biological sample. After vortex-mixing, the mixture was extracted with 1 ml of ethylacetate. After centrifugation at 12000 x g for 3 min, the organic layer was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100-microl aliquot of mobile phase, and a 40-microl aliquot was injected onto the HPLC column. The mobile phase, 0.02 M phosphate buffer (pH 5): acetonitrile: methanol (46:44:10, v/v/v), was run at a flow rate of 0.5 ml/min and the column effluent was monitored by the fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 470 nm. The retention times for DBM-819 and the internal standard were approximately 10.5 and 12 min, respectively. The detection limits of DBM-819 in human plasma and urine, and rat tissue homogenates were 0.01, 0.02 and 0.02 (or 0.05) microg/ml. respectively. The coefficients of variation (CV) of the assay were below 11% for human plasma and urine, and rat tissue homogenates. No interferences from endogenous substances were found.     

Characteristics of apoptosis induction by the alkaloid emetine in human tumour cell lines

Cytotoxic and apoptosis-inducing effects of the alkaloid emetine from Psychotria ipecacuanha (Rubiaceae) were studied in human cell lines. In Jurkat T-cells emetine leads to phosphatidylserine exposure, mitochondrial depolarisation, and DNA fragmentation. Furthermore, activation of several caspases (caspase-3, -9/6, and -8) was demonstrated in a fluorescent caspase assay. Bcl-2 over-expressing cells are less sensitive to emetine while caspase-8-deficient Jurkat T-cells react similarly to wild-type cells. This indicates that apoptosis induction is mediated via the mitochondrial pathway. By using hepatoma cell lines with differing p53 expression, it was concluded that p53 does not seem to play a role in apoptosis induction by emetine. Alterations of protein profiles during emetine-induced apoptosis were analysed by 2D-PAGE and MALDI-TOF-MS. A new protein spot was apparent after treatment with emetine: It could be identified as the N-terminal fragment lamin B1, which is released after cleavage by caspase-6.     

RPLC Determination of Tinidazole and Diloxanide Furoate in Tablets

A simple reverse phase liquid chromatographic method has been developed and subsequently validated for simultaneous determination of tinidazole and diloxanide furoate. The separation was carried out using a mobile phase consisting of acetonitrile, methanol and 0.2 M potassium dihydrogen phosphate (pH 5) in the ratio 2:3:2.The column used was SS Wakosil-II C-18 with a flow rate of 1 ml/min and UV detection at 282 nm. The described method was linear over a concentration range of 10-70 μg/ml and 10-90 μg/ml for the assay of diloxanide furoate and tinidazole, respectively. The mean recovery was found to be 100-101% for tinidazole and 97-103% for diloxanide furoate when determined at three different levels.     

Urinary excretion of ipecac alkaloids in human volunteers

Limited data are available regarding urinary excretion of ipecac alkaloids in humans. In this study, ipecac syrup was administered po to 12 healthy human volunteers at a dose of either 20 mL or 30 mL, and urinary excretions of cephaeline and emetine as well as blood and vomit concentrations were detected by HPLC. All participants showed vomiting after the 30 mL dose within 1 h, whereas 2/6 did not show vomiting within 4 h after the 20 mL dose. Percentage recovery of alkaloids in vomit were 39 +/- 38 or 76 +/- 14% after the 20 mL or 30 mL doses, respectively. In most participants, plasma alkaloids reached their maximum levels within I h and became undetectable after 6 h. Total excretions of ipecac alkaloids into the urine within the first 48 h were less than 2%, but both alkaloids were detectable in the urine at 2w in all participants and could be detected up to 12w in 1/2 participants who did not vomit. These results show that ipecac alkaloids may be detectable in urine several weeks after ingestion and suggest that their detection in urine may be helpful to identify the Munchausen syndrome by proxy using ipecac syrup.     

A high performance liquid chromatography method for the detection of brodifacoum in serum

A procedure for the detection of brodifacoum (BDF) in serum was developed. Extraction of BDF was achieved by acidification of 2 ml of serum with 1 ml of 1.5% acetic acid followed by dual extractions with 10 ml diethyl ether and ether: acetonitrile [1:1]. In spiking experiments, 68 +/- 3, 61 +/- 4 and 65 +/- 5% of added BDF was recovered from serum containing 1000, 100 and 25 ng BDF/ml, respectively. Two high performance liquid chromatography solvent systems were used for chromatographic separation (A: 1.5% acetic acid, pH 4.5: acetonitrile [1:2] with 1% dibutylamine; and B:O.2 M tris(hydroxymethyl)aminomethane, pH 7.5:acetonitrile [1:3]). Detection limits were 75 and 3 ng BDF/ml of serum using ultraviolet absorption (254 nm) and fluorescence measurement (313 nm excitation, 375 nm emission), respectively. This method has been used successfully to monitor serum concentrations of BDF in experimental and field cases of exposure.     

A reverse-phase HPLC assay for the simultaneous determination of enrofloxacin and ciprofloxacin in pig faeces

A reverse-phase HPLC assay is described for the simultaneous assay of enrofloxacin (ENR) and ciprofloxacin (CPX) in pig faeces. Extraction used dichloromethane, 2-propanol and 0.3M ortho-phosphoric acid (1:5:4 v/v/v). Separation was achieved using a Spherisorb S5 C8 column, heated to 50 degrees C and a mobile phase of 0.16% ortho-phosphoric acid (adjusted to pH 3.0 with tetrabutylammonium hydroxide solution) with 20 ml acetonitrile per litre solution. The method used fluorescence detection (Ex 310 nm; Em 445 nm), a flow rate of 1 ml/min and a 20 microl injection volume. Retention times were approximately 6 min for ciprofloxacin and 10 min for enrofloxacin. The linearity range for both compounds was 0-20 mg/kg, lowest limit of quantification 0.3 mg/kg and recoveries were >92%.     

Simultaneous RPHPLC Determination of Nitazoxanide and Ofloxacin in Combined Tablet Dosage Form

A simple, precise, accurate, rapid and reproducible reverse phase high performance liquid chromatographic procedure was developed for simultaneous determination of nitazoxanide and ofloxacin in tablet dosage form at a single wavelength. The mobile phase used was a combination of acetonitrile:0.25M potassium dihydrogen phosphate buffer (80:20) with 0.5%v/v of triethylamine and the pH was adjusted to 2.5 by adding orthophosphoric acid. The detection of the combined dosage form was carried out at 320 nm and flow rate was set to 1ml/min. Linearity was obtained in the concentration range of 5 to 25 μg/ml of nitazoxanide and ofloxacin with correlation coefficients of 0.9987 and 0.9995, respectively. The results of the analysis were validated statistically and recovery studies confirmed the accuracy of the proposed method.     

Development an ion-pair liquid chromatographic method for determination of sotalol in plasma using a monolithic column

A rapid and sensitive ion-pair HPLC method using a monolithic column and fluorescence detection has been developed for quantification of sotalol in plasma. The assay enables the measurement of sotalol for therapeutic drug monitoring with a minimum quantification limit of 10 ng ml(-1). The analytical method involves simple, one-step protein precipitation and no extraction procedure is needed. Sample preparation is fast and the analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column at ambient temperature. The mobile phase was 10% acetonitrile, 0.001 M heptane sulfonic acid, 0.02 M sodium dihydrogen phosphate, and distilled water to 100%, adjusted to pH 5.5 at a flow rate of 1.8 ml/min. The excitation wavelength was set at 235 nm, emission at 300 nm. The calibration curve was linear over the concentration range 20-1500 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 7%. The method has been applied to the determination of sotalol in plasma from 12 subjects dosed with racemic sotalol.     

Quantification of carvedilol in human plasma by liquid chromatography using fluorescence detection: application in pharmacokinetic studies

A simple, rapid and sensitive isocratic reversed-phase HPLC method with fluorescence detection using a monolithic column has been developed and validated for the determination of carvedilol in human plasma. The separation was performed on a Chromolith Performance (RP-18e, 100mm x 4.6mm) column with an isocratic mobile phase consisting of 0.01 M disodium hydrogen phosphate buffer-acetonitrile (40:60, v/v) adjusted to pH 3.5. The sample preparation involves protein precipitation procedure and analytical recovery was complete. Letrozole was used as internal standard. The assay enables the measurement of carvedilol for therapeutic drug monitoring with a minimum quantification limit (LOQ) of 1 ng ml(-1). The excitation and emission wavelengths were set at 240 and 340 nm, respectively. The calibration curve was linear over the concentration range 1-80 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8.0%.     

Determination of scopolia extract in commercial gastrointestinal drugs by high-performance liquid chromatography]

Tropane alkaloids, contained in gastrointestinal drugs including Scopolia Extract, were determined using high-performance liquid chromatographic method. Tropane alkaloids in commercial drugs were extracted with 0.5 N hydrochloric acid test solution. This solution was adjusted to pH 10.0 with a strong ammonia solution, extracted with ethyl ether, and separated on TSKgel ODS-120T (4.6 mm i.d. x 15 cm; temperature, 30 degrees C; flow rate, 1.5 ml/min; detector, UV 210 nm) with a mixture of 1/15 M sodium phosphate buffer (pH 2.5) and acetonitrile (68:32) containing 12 mM sodium dodecyl sulfate. The contents of atropine in the gastrointestinal drugs were 0.25-0.34 mg (coefficient of variation = 0.20-2.61%) a day.     

Improved high-performance liquid chromatographic determination of amphotericin B in human serum and plasma

An improved reversed-phase high-performance liquid chromatographic procedure is described for the determination of Amphotericin B (AMB) in human serum and plasma. The procedure involves the addition of the internal standard, p-nitroaniline, to the sample (0.1 ml) followed by protein precipitation with acetonitrile. The supernatant is injected directly onto a C8 chromatographic column and eluted with an acetonitrile-aqueous 0.01 M sodium acetate buffer, pH 7.4, mobile phase. A spectrophotometric detector operated at 405 nm is used. Retention times for internal standard and AMB are 5.2 and 6.6 min, respectively. The assay standard curve is linear between 0.05-2.0 micrograms cm-3. Within- and between-run relative standard deviations (RSD) for high and low concentrations of the drug are less than 5.30%. Analytical recovery of added AMB in serum is 98.4-101.4%. Data obtained by microbiological assay correlated well (r = 0.936) with LC results. Some commonly co-administered drugs and high concentrations of bilirubin are shown not to interfere.     

Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin in commercial St. John's wort preparations

A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's Wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol–water (80:20, v/v) containing 5% HP-β-cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 °C, were injected into a C₁₈ column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection. Quantification of the marker compounds (hypericin, pseudohypericin, hyperforin) was achieved by use of standard curves generated by plotting peak heights versus concentrations. Validation studies demonstrated that this HPLC method is simple, rapid, reliable, and reproducible. The standard curves were linear over the concentration ranges, 0.5–2.5 μg/ml (hypericin), 0.35–1.6 μg/ml (pseudohypericin) and 5–50 μg/ml (hyperforin). The intra-day coefficients of variation obtained for hypericin, pseudohypericin and hyperforin were 4.4%, 5.4%, and 2.8%, respectively; inter-day CVs were 5.8%, 4.9%, and 2.5%, respectively. This method may be applied for the routine standardization of St. John's Wort products against hyperforin and the hypericins, the putative antidepressant principles in the herbal.     

Simultaneous determination and pharmacokinetic analysis of seven alkaloids and two flavonoids from rat plasma by HPLC-DAD after oral administration of Wuzhuyu decoction

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic analysis of seven alkaloids dehydroevodiamine (DHED), 10-hydroxyrutaecarpine (HDR), evodiamine (EDM), rutaecarpine (RCP), 1-methyl-2-n-nonyl-4(1H)quinolone (MNQ), evocarpine (ECP), and dihydroevocarpine (DHE), and two flavonoids isorhamnetin-7-O-rutinoside (RIM) and diosmetin-7-O-β-d-glucopyranoside (GRD) in rat plasma after oral administration of Wuzhuyu decoction. The flow rate was kept at 1.0?ml/min and the detection wavelength was set at 300?nm. The calibration curves were linear in the range of 0.5013-30.076?μg/ml for DHED, 0.2161-21.608?μg/ml for RIM, 0.161-12.876?μg/ml for HDR, 0.2146-21.457?μg/ml for GRD, 2.0464-40.928?μg/ml for EDM, 1.0398-31.194?μg/ml for RCP, 0.5970-35.818?μg/ml for MNQ, 0.8371-20.928?μg/ml for ECP, and 0.5167-31.003?μg/ml for DHE. The precision (relative standard deviation (RSD), %) for all was less than 10% and the accuracy (relative error (RE), %) was within ±?10%. The results demonstrated that the assay had remarkable reproducibility with acceptable accuracy and precision. The lower limit of quantifications for the compounds in plasma ranged from 0.12 to 0.23?μg/ml and the lower limit of detections ranged from 0.024 to 0.076?μg/ml. This validated method has been successfully applied in the pharmacokinetics study of seven alkaloids and two flavonoids after orally administrating the Wuzhuyu decoction to rats.     

High-performance liquid chromatographic determination of metoprolol and alpha-hydroxymetoprolol concentrations in human serum, urine, and cerebrospinal fluid

A sensitive and simplified high-performance liquid chromatographic procedure was developed for the simultaneous quantification of metoprolol and alpha-hydroxymetoprolol in human serum, as well as cerebrospinal fluid and urine. Following protein precipitation with trichloroacetic acid, the sample was alkalinized with 1 M NaOH and extracted with dichloromethane. The mobile phase consisted of acetonitrile-water (50:50) containing 0.005 M 1-heptanesulfonic acid in 0.001% acetic acid. Using pronetalol as an internal standard, compounds were quantitated using fluorescence detection at 230 nm with a 300-nm emission filter and 0.02 AUFS. Extraction recovery is approximately 80% for both compounds. The lower limits of detection are 5 ng/mL and 4 ng/mL for metoprolol and alpha-hydroxymetoprolol, respectively.