Quantitative Analysis of Ingenol in Euphorbia species via Validated Isotope Dilution Ultra-high Performance Liquid Chromatography Tandem Mass Spectrometry.

INTRODUCTION: Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol-3-angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis. OBJECTIVE: To develop a rapid and reliable method for quantification of ingenol in various plant extracts. METHODOLOGY: Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) after methanolysis and solid-phase extraction (SPE) purification. The 18 O-labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation. RESULTS: The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates. CONCLUSION: The newly established UHPLC-MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.

Essential oil yield and composition of Pistacia vera 'Kerman' fruits, peduncles and leaves grown in California.

BACKGROUND: Pistacia vera 'Kerman' is the predominant pistachio nut cultivar in the United States (California), the world's second largest producer. Despite several reports on the essential oil (EO) content in the genus Pistacia, data on 'Kerman' are limited. The EO content and volatile organic compound (VOC) emissions of tree nut orchards are of current interest to researchers investigating insect pests and the potential role of EO and VOCs as semiochemicals. To establish a basis for the VOC output of pistachios, the EO content of fruits, peduncles, and leaves was analyzed. RESULTS: Evaluated plant parts contained limonene as the primary EO component, followed by alpha-terpinolene. Peduncles were unique in containing relatively high levels of alpha-thujene. The results were reproducible between two different geographical locations. In situ solid phase microextraction (SPME) studies demonstrated the volatile emission was representative of the EO composition. CONCLUSION: This is the first report detailing the content and distribution of EO and the unique limonene-dominant profile for this Pistacia vera cultivar which may influence pistachio insect pest semiochemical research.

Effects of kava alkaloid, pipermethystine, and kavalactones on oxidative stress and cytochrome P450 in F-344 rats.

Kava-containing products remain popular in the United States and continue to be sold in health food stores and ethnic markets regardless of the fact that it was banned in Western countries such as Germany, France, Switzerland, Australia, and Canada, following reports of alleged hepatotoxicity. It is therefore critical to establish efficacy and verify adverse effects and/or herb-drug interactions for kava-kava (Piper methysticum). We have previously demonstrated that kava alkaloid, pipermethystine (PM), abundant in leaves and stem peelings, induces mitochondrial toxicity in human hepatoma cells, HepG2, as compared with the bioactive components, kavalactones (KL), abundant in the rhizome. The current study compared short-term toxic effects of PM in Fischer-344 (F-344) rats to acetone-water extracts of kava rhizome (KRE). Treatment of F-344 rats with PM (10 mg/kg) and KRE (100 mg/kg) for 2 weeks failed to elicit any significant changes in liver function tests or cause severe hepatic toxicity as measured by lipid peroxidation and apoptosis markers such as malondialdehyde, Bax, and Bcl-2. However, PM-treated rats demonstrated a significant increase in hepatic glutathione, cytosolic superoxide dismutase (Cu/ZnSOD), tumor necrosis factor alpha mRNA expression, and cytochrome P450 (CYP) 2E1 and 1A2, suggesting adaptation to oxidative stress and possible drug-drug interactions.

Traditional kava beverage consumption and liver function tests in a predominantly Tongan population in Hawaii.

PURPOSE: To determine the effects of traditionally prepared kava beverages on the liver function tests of regular kava beverage consumers in a population of Tongan and non-Tongan residents of Hawaii (Oahu). METHODS: The liver function tests of 31 healthy adult kava drinkers were compared against a control group of 31 healthy adult non-kava drinkers. Subjects were recruited from the general population, a kava bar, and Tongan kava drinking circles. The liver function profile included AST, ALT, ALP, GGT, and bilirubin (total and direct). Other tests included total protein, albumin, and screens for viral hepatitis and hemochromatosis when indicated. RESULTS: Chronic kava beverage consumption was associated with elevation of GGT in 65% of the kava drinkers versus 26% in the controls (P = .005). ALP was elevated in 23% of kava drinkers versus 3% in the controls (P = .053). CONCLUSION: Heavy kava beverage consumption was associated with significantly elevated GGT levels.

In vitro cytotoxicity of nonpolar constituents from different parts of kava plant (Piper methysticum).

Kava (Piper methysticum), a perennial shrub native to the South Pacific islands, has been used to relieve anxiety. Recently, several cases of severe hepatotoxicity have been reported from the consumption of dietary supplements containing kava. It is unclear whether the kava constituents, kavalactones, are responsible for the associated hepatotoxicity. To investigate the key components responsible for the liver toxicity, bioassay-guided fractionation was carried out in this study. Kava roots, leaves, and stem peelings were extracted with methanol, and the resulting residues were subjected to partition with a different polarity of solvents (hexane, ethyl acetate, n-butanol, and water) for evaluation of their cytotoxicity on HepG2 cells based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and lactate dehydrogenase and aspartate aminotransferase enzyme leakage assays. Organic solvent fractions displayed a much stronger cytotoxicity than water fractions for all parts of kava. The hexane fraction of the root exhibited stronger cytotoxic effects than fractions of root extracted with other solvents or extracts from the other parts of kava. Further investigations using bioassay-directed isolation and analysis of the hexane fraction indicated that the compound responsible for the cytotoxicity was flavokavain B. The identity of the compound was confirmed by (1)H and (13) C NMR and MS techniques.

In vitro toxicity of kava alkaloid, pipermethystine, in HepG2 cells compared to kavalactones.

Kava herbal supplements have been recently associated with acute hepatotoxicity, leading to the ban of kava products in approximately a dozen countries around the world. It is suspected that some alkaloids from aerial kava may have contributed to the problem. Traditionally, Pacific Islanders use primarily the underground parts of the shrub to prepare the kava beverage. However, some kava herbal supplements may contain ingredients from aerial stem peelings. The aim of this study was to test the in vitro effects of a major kava alkaloid, pipermethystine (PM), found mostly in leaves and stem peelings, and kavalactones such as 7,8-dihydromethysticin (DHM) and desmethoxyyangonin (DMY), which are abundant in the roots. Exposure of human hepatoma cells, HepG2, to 100 microM PM caused 90% loss in cell viability within 24 h, while 50 microM caused 65% cell death. Similar concentrations of kavalactones did not affect cell viability for up to 8 days of treatment. Mechanistic studies indicate that, in contrast to kavalactones, PM significantly decreased cellular ATP levels, mitochondrial membrane potential, and induced apoptosis as measured by the release of caspase-3 after 24 h of treatment. These observations suggest that PM, rather than kavalactones, is capable of causing cell death, probably in part by disrupting mitochondrial function. Thus, PM may contribute to rare but severe hepatotoxic reactions to kava.

Piperidine alkaloids from Piper methysticum.

Pipermethystine (1), 3alpha,4alpha-epoxy-5beta-pipermethystine (2) and awaine (3) were isolated from the aerial parts of kava (Piper methysticum G. Forster, Piperaceae) and identified by HRMS and NMR spectroscopic analysis. 1 was concentrated in the stem peelings and leaves. 2 and 3 are new alkaloids with 2 found only in cv. Isa among the 11 cultivars examined, and 3 occurred primarily in young leaves of all cultivars. The stem peelings have been used in recent years as a source of kavalactones in kava dietary supplement industry. Quantitative aspects of these piperidine alkaloids in P. methysticum and their potential activities on human physiology are discussed.

Isolation of natural products by ion-exchange methods.

The primary goal of many natural products chemists is to extract, isolate, and characterize specific analytes from complex plant, animal, microbial, and food matrices. To achieve this goal, they rely considerably on highly sophisticated and highly hyphenated modern instrumentation. Yet, the vast majority of modern instrumentation typically found in the laboratories of natural products chemists is founded on the simple principles of intermolecular forces to achieve separation. Ion-exchange chromatography (IEC) is, at heart, the most fundamental, and strongest, of these interactions and is considered a relatively inexpensive and effective medium in which to "clean-up" a sample. Additionally, IEC offers high recoveries of key analytes and offers the ability to modify the stationary and mobile phases in order to selectively "catch and release" compounds of interest.

Synephrine content of juice from Satsuma mandarins (Citrus unshiu Marcovitch).

Synephrine, the main protoalkaloid in Citrus species, is commonly analyzed as the active component in citrus peel-containing herbal supplements, but the edible parts of mandarins have been largely ignored. The synephrine concentration has been determined in the juices of Citrus unshiu mandarins harvested from 10 different groves located in a major growing region in California. For comparison, the physicochemical properties of the juices, including pH, conductivity, soluble solids content, and titratable acidity, were also measured. The synephrine values among 10 groves ranged from 73.3 to 158.1 mg L (-1). Repeat sampling of fruit from the 10 locations showed that the intragrove variability in synephrine concentrations ranged from 1.0 to 27.7% CV and was grove dependent. Among the physicochemical properties, titratable acidity weakly correlated with synephrine, and for one sample a low maturity index was linked to high synephrine content. The overall mean synephrine concentration of 92.8 mg L (-1) is up to 6-fold higher than values previously determined for orange juices and suggests that mandarin juice could constitute a significant dietary source of synephrine. Furthermore, the results suggest that grove location and maturity affect synephrine content.

Isolation and identification of the first C-17 limonin epimer, epilimonin.

Limonoids are a family of highly oxygenated triterpenoid secondary metabolites found in significant quantities in Citrus and reported to possess multiple health promoting properties. This is the first known report of the isolation and characterization of an epimer of limonin. The epimer, named epilimonin, was isolated by fractional crystallization from a mixture consisting mainly of limonin and epilimonin obtained as byproduct from our efforts to isolate limonin glucoside. Side-by-side comparison of the MS, IR, and (1)H and (13)C NMR data of epilimonin and limonin lead to the assignment of C-17 as the site of epimerization. An earlier study on the bioavailability of limonin glucoside in humans had indicated that limonin glucoside was metabolized to give limonin and a second limonin metabolite. Results from analyzing epilimonin by the same chromatographic conditions used for the bioavailability study suggest that the second limonin metabolite was epilimonin.