Ralph L. Reed

Pharmacokinetics of xanthohumol and metabolites in rats after oral and intravenous administration

SCOPE Xanthohumol (XN), a dietary flavonoid found in hops, may have health-protective actions against cardiovascular disease and type 2 diabetes. Yet, there are limited data on the pharmacokinetics (PK) of XN. This study provides PK parameters for XN and its major metabolites in rats. METHODS AND RESULTS A PK study was conducted in male jugular vein-cannulated Sprague-Dawley rats. Rats (n = 12/group) received an intravenous (IV) injection (1.86 mg/kg BW) or an oral gavage of a low (1.86 mg/kg BW), medium (5.64 mg/kg BW), or high (16.9 mg/kg BW) dose of XN. Plasma samples were analyzed for XN and its metabolites using LC-MS/MS. The maximum concentration (C(max) ) and area under the curve (AUC(0-96 h) ) of total XN (free and conjugated) were 2.9±0.1 mg/L and 2.5±0.3 h*  mg/L in IV group, 0.019±0.002 mg/L and 0.84±0.17 h*  mg/L in the oral low group, 0.043±0.002 mg/L and 1.03±0.12 h*  mg/L in the oral medium group, and 0.15±0.01 mg/L and 2.49±0.10 h*  mg/L in the oral high group. CONCLUSION The bioavailability of XN is dose-dependent and approximately 0.33, 0.13, and 0.11 in rats, for the low-, medium-, and high-dose groups, respectively.

Human pharmacokinetics of xanthohumol, an antihyperglycemic flavonoid from hops

SCOPE Xanthohumol (XN) is a bioactive prenylflavonoid from hops. A single-dose pharmacokinetic (PK) study was conducted in men (n = 24) and women (n = 24) to determine dose-concentration relationships. METHODS AND RESULTS Subjects received a single oral dose of 20, 60, or 180 mg XN. Blood was collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h. Plasma levels of XN and its metabolites, isoxanthohumol (IX), 8-prenylnaringenin (8PN), and 6-prenylnaringenin (6PN) were measured by LC-MS/MS. Xanthohumol (XN) and IX conjugates were dominant circulating flavonoids among all subjects. Levels of 8PN and 6PN were undetectable in most subjects. The XN PK profile showed peak concentrations around 1 h and between 4-5 h after ingestion. The maximum XN concentrations (C(max)) were 33 ± 7 mg/L, 48 ± 11 mg/L, and 120 ± 24 mg/L for the 20, 60, and 180 mg dose, respectively. Using noncompartmental modeling, the area under the curves (AUC(0→∞)) for XN were 92 ± 68 h × μg/L, 323 ± 160 h × μg/L, and 863 ± 388 h × μg/L for the 20, 60, and 180 mg dose, respectively. The mean half-life of XN was 20 h for the 60 and 18 h for the 180 mg dose. CONCLUSION XN has a distinct biphasic absorption pattern with XN and IX conjugates being the major circulating metabolites.

Xanthohumol lowers body weight and fasting plasma glucose in obese male Zucker fa/fa rats

Obesity contributes to increased risk for several chronic diseases including cardiovascular disease and type 2 diabetes. Xanthohumol, a prenylated flavonoid from hops (Humulus lupulus), was tested for efficacy on biomarkers of metabolic syndrome in 4 week old Zucker fa/fa rats, a rodent model of obesity. Rats received daily oral doses of xanthohumol at 0, 1.86, 5.64, and 16.9 mg/kg BW for 6 weeks. All rats were maintained on a high fat (60% kcal) AIN-93G diet for 3 weeks to induce severe obesity followed by a normal AIN-93G (15% kcal fat) diet for the last 3 weeks of the study. Weekly food intake and body weight were recorded. Plasma cholesterol, glucose, insulin, triglyceride, and monocyte chemoattractant protein-1 (MCP-1) levels were assessed using commercial assay kits. Plasma and liver tissue levels of XN and its metabolites were determined by liquid-chromatography tandem mass spectrometry. Plasma and liver tissue levels of xanthohumol were similar between low and medium dose groups and significantly (p<0.05) elevated in the highest dose group. There was a dose-dependent effect on body weight and plasma glucose levels. The highest dose group (n=6) had significantly lower plasma glucose levels compared to the control group (n=6) in male but not female rats. There was also a significant decrease in body weight for male rats in the highest dose group (16.9 mg/kg BW) compared to rats that received no xanthohumol, which was also not seen for female rats. Plasma cholesterol, insulin, triglycerides, and MCP-1 as well as food intake were not affected by treatment. The findings suggest that xanthohumol has beneficial effects on markers of metabolic syndrome.

Protective effects of butylated hydroxyanisole against the acute toxicity of monocrotaline in mice

Abstract Dietary butylated hydroxyanisole (BHA) at levels of 0.25 and 0.75% was found to protect young female mice against the acute toxicity of monocrotaline. The protective effect was associated with reduced levels of pyrrole metabolites in liver, decreased activity of hepatic aminopyrine demethylase, and a reduced rate of in vitro microsomal conversion of monocrotaline to pyrrole metabolites. BHA also increased liver sulfhydryl levels and the activity of cytosolic glutathione S -transferase. Dietary cysteine (1%) was less protective than BHA against monocrotaline toxicity. The LD50 values of monocrotaline in control and cysteine fed mice were 259 and 335 mg/kg, respectively, but for BHA-fed mice, the LD50 value was 497 (0.25% dietary BHA) or 542 mg/kg (for 0.75% dietary BHA with or without 1% cysteine). Dietary cysteine did not produce an increase in liver sulfhydryls but it increased the glutathione S -transferase activity and the in vitro production of pyrroles from monocrotaline. The ip administration of monocrotaline (280 mg/kg) did not affect liver sulfhydryl levels or aminopyrine demethylase activity but produced a significant decrease in the in vitro formation of pyrroles from monocrotaline. Although alterations in microsomal monooxygenase activities occurred after BHA, cysteine or monocrotaline treatment, the concentration of cytochrome P -450 in liver was unchanged.

Coupling Supercritical CO2 and Subcritical (Hot) Water for the Determination of Dacthal and Its Acid Metabolites in Soil

Dacthal and its mono- and diacid metabolites were sequentially extracted from soils by first performing a supercritical carbon dioxide extraction to recover Dacthal, followed by a subcritical (hot) water extraction step to recover metabolites. Dacthal was recovered from soil in 15 min by supercritical carbon dioxide at 150 °C and 400 bar. The mono- and diacid metabolites were extracted from soil in 10 min under the subcritical water conditions of 50 °C and 200 bar. The metabolites were trapped in situ on a strong anion-exchange disk placed over the exit frit of the extraction cell. Metabolites are combined with Dacthal by placing the disk into the GC autosampler vial containing the SFE extract. The metabolites then are simultaneously eluted from the disk and derivatized to their ethyl esters by adding 100 μL of ethyl iodide and heating the vial at 100 °C for 1 h. Using this approach, only a single sample is analyzed, and because the disk-catalyzed alkylation reaction does not transesterify Dacthal, the speciation of Dacthal is maintained. In addition, no sample cleanup steps are required, the use of diazomethane for derivatization is avoided, and the method consumes a total of 5 mL of nonchlorinated organic solvent.

Characterization of Phytoecdysteroid Glycosides in Meadowfoam (Limnanthes alba) Seed Meal by Positive and Negative Ion LC-MS/MS

Meadowfoam ( Limnanthes alba) is an oilseed crop grown in western Oregon. The seed meal has potential value as a biopesticide due to glucosinolate degradation products and phytoecdysteroids, a group of polyhydroxylated triterpenoids with potent activities as arthropod molting hormones. Liquid chromatography in combination with tandem mass spectrometry operated in the precursor ion mode revealed the presence of four ecdysteroid glycosides in meadowfoam seed meal. The carbohydrate sequence and the identity of the ecdysteroid aglycones, ponasterone A and 20-hydroxyecdysone, were determined by product ion scanning. Ecdysteroids were detected in the negative ion mode as [M + formate] (-) ions, which yielded [M - H] (-) and alpha-cleavage fragments with retention of hydroxyl groups in MS/MS experiments (not seen in the positive ion mode), allowing the determination of the number of hydroxyl groups in the side chain and in the steroid ring system. MS/MS of glycoside ions ([MH] (+) or [M + formate] (-)) provided carbohydrate sequence information.

Microsomal formation of a pyrrolic alcohol glutathione conjugate of the pyrrolizidine alkaloid senecionine.

1. Pyrrolizidine alkaloids (PAs) are metabolized primarily to putative dehydroalkaloid (PA pyrrole) metabolites and to PA N-oxide by rat liver microsomal monooxygenases. 2. The dehydroalkaloids are highly reactive and either bind covalentely to tissue nucleophiles or are hydrolysed to the more stable pyrrole, (R,S)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), and the corresponding necic acid. 3. Addition of glutathione (GSH 1 mM) to incubation mixtures containing rat liver microsomes and the PA senecionine (SN), resulted in the formation of a conjugate of DHP with GSH. 5. The mass spectrum of this DHP-GSH conjugate was identical to that of the chemically-synthesized dehydroretronecine (the R enantiomer of the racemic DHP) and GSH. 6. Only negligible amounts of DHP-GSH conjugate were formed when DHP itself was incubated with GSH at physiological pH. 7. These findings provide strong evidence for the microsomal conversion of SN to a highly reactive metabolite, presumably dehydrosenecionine, which then reacts with GSH to form the DHP-GSH conjugate. 8. It is likely that a similar mechanism is responsible in vivo for the formation of GSH conjugates of DHP from SN and other PAs.

Herbicidal Activity of Glucosinolate Degradation Products in Fermented Meadowfoam (Limnanthes alba) Seed Meal

Meadowfoam ( Limnanthes alba ) is an oilseed crop grown in western Oregon. After extraction of the oil from the seeds, the remaining seed meal contains 2-4% of the glucosinolate glucolimnanthin. This study investigated the effect of fermentation of seed meal on its chemical composition and the effect of the altered composition on downy brome ( Bromus tectorum ) coleoptile emergence. Incubation of enzyme-inactive seed meal with enzyme-active seeds (1% by weight) resulted in complete degradation of glucolimnanthin and formation of 3-methoxybenzyl isothiocyanate in 28% yield. Fermentation in the presence of an aqueous solution of FeSO(4) (10 mM) resulted in the formation of 3-methoxyphenylacetonitrile and 2-(3-methoxyphenyl)ethanethioamide, a novel natural product. The formation of the isothiocyanate, the nitrile, and the thioamide, as a total, correlated with an increase of herbicidal potency of the seed meal (r(2) = 0.96). The results of this study open new possibilities for the refinement of glucosinolate-containing seed meals for use as bioherbicides.

Protective action of zinc against pyrrolizidine alkaloid-induced hepatotoxicity in rats.

The influence of Zn on the acute hepatotoxicity of pyrrolizidine alkaloids (PAs) was determined in male rats. Zinc, 72 mumol/kg as ZnCl2, was administered ip for 3 consecutive days, followed 16 h after the last dose by a single ip injection of purified mixed PAs (80, 120, or 160 mg/kg) obtained from tansy ragwort (Senecio jacobaea). Hepatotoxicity of the PAs was assessed by measuring the activities of plasma glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) and by histological examination of the liver. There was a dose-dependent increase in plasma GOT and GTP 24 h after PA administration, whereas no significant increase of these enzymes was seen after administering Zn alone. The 7-fold increase in plasma GOT and 12-fold increase in GPT after PA (120 mg/kg) were reduced to 2.4- and 2.1-fold, respectively, by Zn pretreatment. The PA-induced liver necrosis was either reduced in severity or abolished by Zn when the PA dose was 80 or 120 mg/kg. These results suggest a protective effect of Zn against PA hepatotoxicity. The protective effect was associated with a marked increase in liver metallothionein and a significant decrease in hepatic cytochrome P-450 content, aminopyrine N-demethylase activity, and in vitro microsomal conversion of the PAs to pyrroles. Liver nonprotein sulfhydryls were unchanged. The possible role of metallothionein in the sequestration of pyrrole metabolites merits further investigation.

Mechanisms for Pyrrolizidine Alkaloid Activation and Detoxification

The pyrrolizidine alkaloids (PAs) constitute a large group of hepatotoxic and carcinogenic plant constituents of wide geographic and botanical distribution. These alkaloids are responsible for the death of livestock throughout the world and for occasional human poisonings following the consumption of contaminated foods or the injudicious use of herbal medicines (Bull et al., 1968; Mattocks, 1986; Hirano, 1981; Huxtable, 1980; Peterson and Culvenor, 1983). PAs are relatively nontoxic but are bioactivated in vivo primarily via the liver, through enzymatic dehydrogenation to form highly reactive pyrrole- type metabolites. It is thought that PAs are initially converted to the corresponding dehydropyrrolizidine alkaloids (PA pyrroles) which then can either alkylate protein, DNA or other cellular nucleophiles (Hsu et al. 1975; Reed et al. 1988; Wickramanayake et al., 1985) or be hydrolyzed to the more stable pyrrolic alcohol, such as (R)-6,7-dihydro-7-hydroxy-l-hydroxymethy1-5H-pyrrolizidine (DHP) in the case of retronecine or heliotridine based PAs (Jago et al., 1979; Kedzierski and Buhler, 1985, 1986; Mattocks, 1986; Mattocks and White, 1971). PAs also can be oxidized in vivo to relatively nontoxic PA N-oxides and hydrolyzed to the corresponding amino alcohol (Kedzierski and Buhler, 1986; Mattocks, 1986; Ramsdell et al., 1987).