Isolation and identification of tuliposides D and F from tulip cultivars.

6-Tuliposides A (6-PosA) and B (6-PosB) are major defensive secondary metabolites in tulip cultivars (Tulipa gesneriana), having an acyl group at the C-6 position of d-glucose. Although some wild tulip species produce 1,6-diacyl-glucose type of Pos (PosD and PosF), as well as 6-PosA/B, they have not yet been isolated from tulip cultivars. Here, aiming at verifying the presence of PosD and PosF in tulip cultivars, tissue extracts of 25 cultivars were analyzed by high-performance liquid chromatography (HPLC). Although no HPLC peaks for PosD nor PosF were detected in most cultivars, we found two cultivars giving a minute HPLC peak for PosD and the other two cultivars giving that for PosF. PosD and PosF were then purified from petals of cultivar 'Orca' and from pistils of cultivar 'Murasakizuisho', respectively, and their identities were verified by spectroscopic analyses. This is the first report that substantiates the presence of 1,6-diacyl-glucose type of Pos in tulip cultivars.

Substrate specificity of tuliposide-converting enzyme, a unique non-ester-hydrolyzing carboxylesterase in tulip: Effects of the alcohol moiety of substrate on the enzyme activity.

6-Tuliposides A (PosA) and B (PosB) are glucose esters accumulated in tulip (Tulipa gesneriana) as major defensive secondary metabolites. Pos-converting enzymes (TgTCEs), which we discovered previously from tulip, catalyze the conversion reactions of PosA and PosB to antimicrobial tulipalins A (PaA) and B (PaB), respectively. The TgTCEs, belonging to the carboxylesterase family, specifically catalyze intramolecular transesterification, but not hydrolysis. In this report, we synthesized analogues of Pos with various alcohol moieties, and measured the TgTCE activity together with a determination of the kinetic parameters for these analogues with a view to probe the substrate recognition mechanism of the unique non-ester-hydrolyzing TgTCEs. It was found that d-glucose-like structure and number of the hydroxyl group in alcohol moiety are important for substrate recognition by TgTCEs. Among the analogues examined, 1,2-dideoxy analogues of PosA and PosB were found to be recognized by the TgTCEs more specifically than the authentic substrates by lowering Km values. The present results will provide a basis for designing simple, stable synthetic substrate analogues for crystallographic analysis of TgTCEs.

One-Step Enzymatic Synthesis of 1-Tuliposide A Using Tuliposide-Converting Enzyme.

6-Tuliposides A (6-PosA) and B (6-PosB) are major secondary metabolites in tulip (Tulipa gesneriana), having an acyl group at the C-6 position of D-glucose. They serve as precursors of the antimicrobial α-methylene-γ-butyrolactones tulipalins A (PaA) and B (PaB). The conversions of 6-PosA/6-PosB to PaA/PaB are catalyzed by tuliposide-converting enzymes A and B (TCEA and TCEB), respectively. A minor Pos, 1-PosA, which has the acyl group at the C-1 position of D-glucose, has been identified in some wild tulip species, but availability of this compound is limited. Here, by using the TCEs, we established a facile enzymatic process for 1-PosA synthesis from the naturally occurring 1,6-diacyl-glucose type of Pos (PosD and PosF). We first discovered that TCEA and TCEB react preferentially with PosD and PosF, respectively, to form 1-PosA and the corresponding Pa derived from the 6-acyl group, demonstrating that the TCEs specifically acted on the 6-acyl group, but not the 1-acyl group, of the substrates. Using TCEB, 300 mg of PosF was completely converted to 1-PosA and PaB in 10 min at room temperature. Then, 160 mg of 1-PosA (75% molar yield) was purified by column chromatography. This one-step enzymatic process dramatically improves accessibility to 1-PosA.

Intoxication by gamma hydroxybutyrate and related analogues: Clinical characteristics and comparison between pure intoxication and that combined with other substances of abuse.

OBJECTIVE: To study the profile of European gamma-hydroxybutyrate (GHB) and gammabutyrolactone (GBL) intoxication and analyse the differences in the clinical manifestations produced by intoxication by GHB/GBL alone and in combination with other substances of abuse. METHOD: We prospectively collected data on all the patients attended in the Emergency Departments (ED) of the centres participating in the Euro-DEN network over 12 months (October 2013 to September 2014) with a primary presenting complaint of drug intoxication (excluding ethanol alone) and registered the epidemiological and clinical data and outcomes. RESULTS: We included 710 cases (83% males, mean age 31 years), representing 12.6% of the total cases attended for drug intoxication. Of these, 73.5% arrived at the ED by ambulance, predominantly during weekend, and 71.7% consumed GHB/GBL in combination with other substances of abuse, the most frequent additional agents being ethanol (50%), amphetamine derivatives (36%), cocaine (12%) and cannabis (8%). Among 15 clinical features pre-defined in the project database, the 3 most frequently identified were altered behaviour (39%), reduced consciousness (34%) and anxiety (14%). The severity ranged from mild cases requiring no treatment (308 cases, 43.4%) to severe cases requiring admission to intensive care (103 cases, 14.6%) and mechanical ventilation (49 cases, 6.9%). No deaths were reported. In comparison with only GHB/GBL consumption, patients consuming GHB/GBL with co-intoxicants presented more vomiting (15% vs. 3%, p<0.001) and cardiovascular symptoms (5.3% vs. 1.5%, p<0.05), a greater need for treatment (59.8% vs. 48.3%, p<0.01) and a longer ED stay (11.3% vs. 3.6% patients with ED stay >12h, p<0.01). CONCLUSIONS: The profile of the typical GHB/GBL-intoxicated European is a young male, requiring care for altered behaviour and reduced level of consciousness, mainly during the weekend. The clinical features are more severe when GHB is consumed in combination with other substances of abuse.

The main constituents of Tulipa systola Stapf. roots and flowers; their antioxidant activities.

People living on the mountains of the Kurdistan Region, Iraq make a large use of herbs in the local traditional medicine. Among them, Tulipa systola, which grows under and between rocks, is very popular as an anti-inflammatory remedy and pain-relief. The phytochemical study of an ethanolic extract obtained from flowers and roots of Tulip (T systola Stapf.) afforded three compounds, identified as (+)-1-O-E-feruloyl-3-O-E-p-coumaroylglycerol (1), (+)-6-tuliposide A (2), and (-)-kaempferol-3-O-rutinoside (3). The significant radical scavenging and antioxidant activity of the isolated compounds were evaluated on three tests, by determining the DPPH free radical scavenging activity, the total antioxidant activity and the hydrogen peroxide scavenging activity. Tuliposide A shows potent allergenic activity.

In Vitro and In Vivo Evidence for Active Brain Uptake of the GHB Analog HOCPCA by the Monocarboxylate Transporter Subtype 1.

γ-Hydroxybutyric acid (GHB) is a recreational drug, a clinically prescribed drug in narcolepsy and alcohol dependence, and an endogenous substance that binds to both high- and low-affinity sites in the brain. For studying the molecular mechanisms and the biologic role of the GHB high-affinity binding sites, ligands with high and specific affinity are essential. The conformationally restricted GHB analog HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid) is one such compound. The objective of this study was to investigate the transport of HOCPCA across the blood-brain barrier in vitro and in vivo and to investigate the hypothesis that HOCPCA, like GHB, is a substrate for the monocarboxylate transporters (MCTs). For in vitro uptake studies, MCT1, -2, and -4 were recombinantly expressed in Xenopus laevis oocytes, and the previously reported radioligand [(3)H]HOCPCA was used as substrate. HOCPCA inhibited the uptake of the endogenous MCT substrate l-[(14)C]lactate, and [(3)H]HOCPCA was shown to act as substrate for MCT1 and 2 (Km values in the low- to mid-millimolar range). Introducing single-point amino acid mutations into positions essential for MCT function supported that HOCPCA binds to the endogenous substrate pocket of MCTs. MCT1-mediated brain entry of HOCPCA (10 mg/kg s.c.) was further confirmed in vivo in mice by coadministration of increasing doses of the MCT inhibitor AR-C141990 [(R)-5-(3-hydroxypyrrolidine-1-carbonyl)-1-isobutyl-3-methyl-6-(quinolin-4-ylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione], which inhibited brain penetration of HOCPCA in a dose-dependent manner (ID50 = 4.6 mg/kg). Overall, our study provides evidence that MCT1 is an important brain entry site for HOCPCA and qualifies for future in vivo studies with HOCPCA.

Molecular diversity of tuliposide A-converting enzyme in the tulip.

Tuliposide A-converting enzyme (TCEA) catalyzes the conversion of 6-tuliposide A to its lactonized aglycon, tulipalin A, in the tulip (Tulipa gesneriana). The TgTCEA gene, isolated previously from petals, was transcribed in all tulip tissues but not in the bulbs despite the presence of TCEA activity, which allowed prediction of the presence of a TgTCEA isozyme gene preferentially expressed in the bulbs. Here, the TgTCEA-b gene, the TgTCEA homolog, was identified in bulbs. TgTCEA-b polypeptides showed approximately 77% identity to the petal TgTCEA. Functional characterization of the recombinant enzyme verified that TgTCEA-b encoded the TCEA. Moreover, the TgTCEA-b was found to be localized to plastids, as found for the petal TgTCEA. Transcript analysis revealed that TgTCEA-b was functionally transcribed in the bulb scales, unlike the TgTCEA gene, whose transcripts were absent there. In contrast, TgTCEA-b transcripts were in the minority in other tissues where TgTCEA transcripts were dominant, indicating a tissue preference for the transcription of those isozyme genes.

Psychiatric aspects of acute withdrawal from gamma-hydroxybutyrate (GHB) and its analogue gamma-butyrolactone (GBL): implications for psychiatry services in the general hospital.

OBJECTIVE: The objective of this study was to describe the psychiatric symptoms, management and outcomes in a consecutive series of patients being managed medically for symptoms of withdrawal from gamma-hydroxybutyrate (GHB) and its analogue gamma-butyrolactone (GBL) in a general hospital setting. METHODS: A toxicology database was used to identify patients presenting with a history suggestive of withdrawal from GHB and analogues. Electronic and paper medical records were searched for demographic features, neuropsychiatric symptoms, psychiatric management while in hospital and overall outcome. RESULTS: There were 31 presentations with withdrawal from the drugs involving 20 patients. Of these 17 (54%) were referred to and seen by the liaison psychiatry team. Anxiety (61.3%) and agitation (48.4%) were the most common symptoms. Of the 17 cases seen by the liaison psychiatry team, 52.9% required close constant observation by a mental health nurse and 29.4% required to be detained in hospital under mental health legislation. CONCLUSIONS: The significant proportion of patients presenting with neuropsychiatric symptoms and requiring intensive input from the liaison psychiatry team during withdrawal from GHB and its analogues points to the importance of close liaison between medical and psychiatric teams in managing these patients in the general hospital.

A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip.

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

Gamma butyrolactone (GBL) and gamma valerolactone (GVL): similarities and differences in their effects on the acoustic startle reflex and the conditioned enhancement of startle in the rat.

Gamma butyrolactone (GBL) is metabolized to gamma hydroxybutyrate (GHB) in the body. GHB is a DEA Schedule 1 compound; GBL is a DEA List 1 chemical. Gamma valerolactone (GVL) is the 4-methyl analog of GBL; GVL is metabolized to 4-methyl-GHB; GVL is NOT metabolized to GBL or GHB. The effects of GBL (18.75-150 mg/kg), GVL (200-1600 mg/kg) or vehicle on the acoustic startle reflex (ASR), and the classically-conditioned enhancement of startle, the Startle Anticipated Potentiation of Startle (SAPS) response were studied in male rats. Both compounds produced a dose-dependent reduction of ASR, with GBL 5-7 times more potent than GVL. In contrast, GBL treatment significantly reduced SAPS at doses that exerted only moderate effects on ASR, whereas GVL exerted little or no effect on the SAPS, except at doses that produced pronounced reductions in Noise Alone ASR. In a second experiment, rats were tested for Noise Alone ASR behavior following treatment with a single mid-range dose of GBL (75 mg/kg), GVL (400mg/kg) or vehicle; immediately following startle testing the animals were sacrificed and their brains and blood were collected for determination of GHB, 4-methyl-GHB, GBL and GVL. GHB was found in measurable concentrations in all of the blood specimens and 6 (of 8) of the brain specimens from the GBL-treated subjects. 4-Methyl-GHB was found in measurable concentrations in all of the blood and brain specimens of the GVL-treated subjects; the change in startle amplitude was inversely correlated to the brain concentrations of these compounds. These findings confirm the differences in the metabolic fate of GBL and GVL as pro-drugs for the formation of GHB and 4-methyl-GHB, respectively. Moreover, the dissimilarity in effect profile for GBL and GVL on ASR versus SAPS behaviors suggests that different receptor(s) may be involved in mediating these behavioral effects.

Simultaneous determination of γ-Hydroxybutyrate (GHB) and its analogues (GBL, 1.4-BD, GVL) in whole blood and urine by liquid chromatography coupled to tandem mass spectrometry.

A simple liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous identification and quantification of γ-hydroxybutyrate (GHB), γ-butyrolactone (GBL), 1.4-butanediol (1.4-BD), and γ-valerolactone (GVL) in whole blood from forensic cases. The sample preparation of whole blood involved protein precipitation by acidic methanol. Urine samples were diluted and evaluated in relation to a control at the cutoff concentration. Hexadeutero GHB (GHB-d(6)) was used as the internal standard. Separation was achieved by reversed-phase chromatography, and detection was by MS-MS in MRM mode. The linear range for all compounds was from 1.0 to 100 mg/kg in whole blood with a limit of quantification of about 1 mg/kg. The method was validated with regards to selectivity, recovery, accuracy and precision, and stability. The method is currently applied to investigations on suspected drug-facilitated sexual assaults, driving under the influence of drugs, and general intoxication with these substances.

A web-based study of gamma hydroxybutyrate (GHB): patterns, experiences, and functions of use.

GHB (gamma hydroxybutyrate) was developed as a general anesthetic. Due to dosing difficulty and side effects, regular use was discontinued. Medical uses include treating sleep and alcohol disorders. In the 1990s, it was promoted as a supplement and taken to improve mood and sex. GHB and its analogs (gamma butyrolactone and butanediol) were widely available until federal regulations were put into effect with mounting evidence of adverse events. This survey (N = 61) study was conducted to assess patterns, experiences, and functions of use. Much of what is understood regarding GHB treatment is based on hospital case studies for overdose and withdrawal. Not enough is known about prevention, reducing use and associated problems, or relapse. We know little about specific drug effect expectancies, triggers, coping skills, and consequences of use (positive/negative). While the drug treatment literature has a wealth of information to draw upon, GHB-specific information may greatly assist relapse prevention.

Structure-activity relationships of tulipalines, tuliposides, and related compounds as inhibitors of MurA.

The enzyme MurA performs an essential step in peptidoglycan biosynthesis and is therefore a target for the discovery of novel antibacterial compounds. We report here the inhibition of MurA by natural products from tulips (tulipalines and tuliposides), and the structure-activity relationships of various derivatives. The inhibition of MurA can be related to antibacterial activity, and MurA is probably one of the relevant molecular targets of the tulipaline derivatives. MurA inhibition by this class of compounds depends on the presence of the substrate UNAG, which indicates non-covalent suicide inhibition as observed previously for cnicin. With respect to selectivity, however, the reactivity against arbitrary sulfhydryl groups, such as in glutathione, could not yet be sufficiently separated from MurA inhibition in the present dataset.

Calcium and cAMP signaling induced by gamma-hydroxybutyrate receptor(s) stimulation in NCB-20 neurons.

The NCB-20 neurohybridoma cells differentiated with dibutyryl-cyclic-AMP represent an interesting model to study several components of the gamma-hydroxybutyrate (GHB) system in brain. In particular, an active Na(+)-dependent uptake and a depolarization-evoked release of GHB is expressed by these cells, together with high affinity specific binding sites for this substance. However, only little is known about cellular mechanisms following GHB receptor(s) stimulation in these neurons. Electrophysiological data indicate that GHB can differently affect Ca(2+) currents. L-type calcium channels were typically inhibited by GHB when NCB-20 cells were depolarized. In contrast, when NCB-20 cells were at resting potential, GHB induced a specific Ca(2+) entry through T-type calcium channels. In this study, we investigated the effect induced on cytosolic free Ca(2+) level and cAMP production by GHB receptor(s) stimulated with micromolar concentrations of GHB or structural analogues of GHB. Ca(2+) movements studied by cellular imaging were dose-dependently increased but disappeared for GHB concentrations >25 microM. In addition, nanomolar doses of GHB inhibited forskolin-stimulated adenylate cyclase. This effect was also rapidly desensitized at higher GHB concentrations. Acting as an antagonist, NCS-382 decreased GHB receptor(s) mediated cAMP and calcium signals. The agonist NCS-356 mimicked GHB effects which were not affected by the GABA(B) receptor antagonist CGP-55-845. Our results reveal the occurrence of Ca(2+)-dependent adenylate cyclase inhibition in NCB-20 neurons after GHB receptor(s) stimulation by GHB concentrations <50 microM. Above this dose, GHB effects were inactivated. In addition, at GHB concentrations exceeding 50 microM, GTP-gammaS binding was also reduced, confirming the desensitization of GHB receptor(s). Taken together, these results support the existence in NCB-20 neurons of GHB receptors belonging to GPCR family that may recruit various G protein subtypes.

Purification and characterization of a tuliposide-converting enzyme from bulbs of Tulipa gesneriana.

An enzyme that catalyzes the stoichiometric conversion of 6-tuliposide into tulipalin was purified and characterized from bulbs of Tulipa gesneriana. The enzyme appeared to be a dimer, the relative molecular mass (Mr) of each subunit being 34,900; it had maximum activity and stability at neutral pH and moderate temperature. The enzyme preferentially acted on such glucose esters as 6-tuliposides, and to a lesser extent on p-nitrophenylacetate.

Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain.

Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have synthesized and assayed three conformationally restricted GHB analogs for binding against the GHB-specific ligand [3H]NCS-382 [(E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-)acetic acid] in rat brain homogenate. The cyclohexene and cyclopentene analogs, 3-hydroxycyclohex-1-enecarboxylic acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported to date. Neither of the cycloalkenes showed any affinity (IC50 > 1 mM) for GABA(A) or GABA(B) receptors. These compounds show excellent potential as lead structures and novel tools for studying specific GHB receptor-mediated pharmacology.

Gamma-butyrolactone and 1,4-butanediol: abused analogues of gamma-hydroxybutyrate.

gamma-Hydroxybutyrate (GHB) is a GABA-active CNS depressant, commonly used as a drug of abuse. In the early 1990s, the US Drug Enforcement Administration (DEA) warned against the use of GHB and restricted its sale. This diminished availability of GHB caused a shift toward GHB analogues such as gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) as precursors and surrogates. Both GBL and 1,4-BD are metabolically converted to GHB. Furthermore, GBL is commonly used as a starting material for chemical conversion to GHB. As such, the clinical presentation and management of GBL and 1,4-BD intoxication shares a great deal of common ground with that for GHB. This similarity exists not only for acute intoxication but also for withdrawal in those patients with a history of extended high-dose abuse. This review examines the history of GHB analogue abuse as well as the clinical presentation and management of acute intoxication and withdrawal associated with abuse of these compounds.

Gamma-hydroxybutyrate: bridging the clinical-analytical gap.

Laboratory detection of gamma-hydroxybutyrate (GHB) has been published as early as the 1960s. However, wide-scale use of GHB during the 1990s has led to the development of current analytic methods to test for GHB and related compounds. Detection of GHB and related compounds can be clinically useful in confirming the cause of coma in an overdose patient, determining its potential role in a postmortem victim, as well as evaluating its use in a drug-facilitated sexual assault victim. Analytical method sensitivity must be known in order to determine the usefulness and clinical application. Most laboratory cut-off levels are based on instrument sensitivity and will not establish endogenous versus exogenous GHB levels. Interpretation of GHB levels must include a knowledge base of endogenous GHB, metabolism of GHB and related compounds, as well as postmortem generation. Due to potential analytical limitations in various GHB methods, it is clinically relevant to specifically request for GHB as well as related GHB compounds if they are also in question. Various storage conditions (collection time, types of containers, use of preservatives, storage temperature) can also affect the analysis and interpretation of GHB and related compounds.