Identification and Comparison of Constituents of Aurantii Fructus and Aurantii Fructus Immaturus by UFLC-DAD-Triple TOF-MS/MS.

Although Aurantii Fructus (AF) and Aurantii Fructus Immaturus (AFI) are both the fruits of the same rutaceae plant at different stages of growth, they exert similar yet distinct clinical effects. The chemical composition is crucial for quality control as well as therapeutic application. To address this concern, it is significant to evaluate the similarities and differences of the constituents in both AF and AFI. The extract of AF and AFI were comprehensively analyzed by ultra fast liquid chromatography-photodiode array detector-triple-time of flight-tandem mass spectrometry (UFLC-DAD-Triple TOF-MS/MS). Among the 40 compounds detected, 19 metabolites were detected in both the AF and AFI; whereas 13 compounds were only detected in AF and five constituents were exclusively detected in AFI. In particular, even in AFI, three compounds were only identified in AFI (Citrus aurantium' L. and its cultivar). Among the 18 compounds confirmed by standard database, 13 compounds were reported in AF and AFI for the first time. Furthermore, the distinction was also revealed by the content of naringin, hesperidin, neohesperidin, and synephrine. The study directly contributed to the similarities and differences of AF and AFI. Herein, similarities and the differences in chemical profiles of AF and AFI could explain the current clinical applications.

Fast hepatic biotransformation of p-synephrine and p-octopamine and implications for their oral intake.

Citrus aurantium (bitter orange) extracts have been used in products for weight management and sports performance. These extracts contain large amounts of p-synephrine and much smaller amounts of p-octopamine. Both protoalkaloids exert lipolytic and glycogenolytic activities at similar concentrations. The biotransformation of p-synephrine and p-octopamine is not as well-known as those of other adrenergic amines. For this reason transformation of these amines was investigated in the isolated perfused liver. Special attention was devoted to the single pass extraction of each compound as well as to the kinetics of uptake. The assay of the amines in the outflowing perfusate was done by means of high performance liquid chromatography (HPLC). The single pass extraction of p-synephrine was higher than 90% at a portal concentration of 10 µM. It declined with the concentration, but was still around 30% at the concentration of 500 µM. At low concentrations (10-50 µM) the decreasing sequence of single pass extractions was p-synephrine > p-octopamine ≈ epinephrine > norepinephrine. Rates of uptake versus p-synephrine concentration resulted in a Michaelis-Menten type of relationship, with a KM value of 290.7 ± 32.1 µM and a Vmax of 0.762 ± 0.042 µmol min(-1) g(-1). The rates of uptake of p-octopamine did not present clear saturation and could be approximated by a linear relationship with a first order rate constant of 1.5 min(-1). The rapid hepatic transformation of p-synephrine and p-octopamine means that their concentration in the portal vein exceeds that in the systemic circulation during absorption. Their metabolic effects will, thus, be exerted predominantly in the liver.

The Small Molecule R-(-)-ß-O-Methylsynephrine Binds to Nucleoporin 153 kDa and Inhibits Angiogenesis.

R-(-)-ß-O-methylsynephrine (OMe-Syn) is a naturally occurring small molecule that was identified in a previous screen as an inhibitor of angiogenesis. In this study, we conducted two animal model experiments to investigate the in vivo antiangiogenic activity of OMe-Syn. OMe-Syn significantly inhibited angiogenesis in a transgenic zebrafish model as well as in a mouse retinopathy model. To elucidate the underlying mechanisms responsible for the antiangiogenic activity of OMe-Syn, we used phage display cloning to isolate potential OMe-Syn binding proteins from human cDNA libraries and identified nucleoporin 153 kDa (NUP153) as a primary binding partner of OMe-Syn. OMe-Syn competitively inhibited mRNA binding to the RNA-binding domain of NUP153. Furthermore, depletion of NUP153 in human cells or zebrafish embryos led to an inhibition of angiogenesis, in a manner similar to that seen in response to OMe-Syn treatment. These data suggest that OMe-Syn is a promising candidate for the development of a novel antiangiogenic agent and that inhibition of NUP153 is possibly responsible for the antiangiogenic activity of OMe-Syn.

A review of the receptor-binding properties of p-synephrine as related to its pharmacological effects.

Bitter orange (Citrus aurantium) extract and its primary protoalkaloid p-synephrine are used widely in weight loss/weight management and sports performance products. Because of structural similarities, the pharmacological effects of p-synephrine are widely assumed to be similar to those of ephedrine, m-synephrine (phenylephrine), and endogenous amine neurotransmitters as norepinephrine and epinephrine. However, small structural changes result in the receptor binding characteristics of these amines that are markedly different, providing a plausible explanation for the paucity of adverse effects associated with the wide-spread consumption of p-synephrine in the form of dietary supplements as well as in various Citrus foods and juices. This paper summarizes the adrenoreceptor binding characteristics of p-synephrine relative to m-synephrine, norepinephrine, and other amines as related to the observed pharmacological effects.

Biochemistry of neuromodulation in primary headaches: focus on anomalies of tyrosine metabolism.

Recent studies have suggested that abnormalities of dopamine and trace amines (tyramine, octopamine, and synephrine), products of tyrosine metabolism, may constitute the metabolic events that predispose to the occurrence of cluster headache (CH) and migraine attacks. This hypothesis is supported by the following evidences: the discovery of trace amine associated receptors (TAARs), expressed on the olfactory epithelium, amigdala, hypothalamus, periacqueductal gray, and the biochemical anomalies of dopamine and trace amines. The possible effects of these biochemical abnormalities on TAARs and dopamine receptors, located in different areas of CNS, may explain the behaviour (restlessness, anxiety and, at times, hypersexuality) and the autonomic signs during the painful attacks of CH, and the premonitory symptoms of migraine crisis (thirst, craving, yawning, alteration of smell, depression etc.).

Applications of LC-MS in the study of the uptake, distribution, metabolism and excretion of bioactive polyphenols from dietary supplements.

Specific and quantitative analyses of the bioactive components and their metabolites in body fluids are essential to assess the interaction between groups of compounds in dietary supplements and preparations of psychoactives. Reverse-phase LC separations combined with tandem mass spectrometry provide the necessary specificity and sensitivity. In this paper, applications of these methods are described for the analysis of isoflavones, salvinorin A, synephrine isomers and their metabolites in serum, urine and aqueous humor.

Elusive amines and primary headaches: historical background and prospectives.

Although the role of trace amines such as tyramine, octopamine and synephrine in the pathogenesis of migraine has been debated for decades, this issue remains still unresolved. In spite of a relevant body of work, the inability to demonstrate specific receptors for these compounds and the lack of sensitive non-radioactive methods for the detection of trace amines in biological samples have limited their investigation in humans. However, the recent identification of a new, large family of G protein-coupled receptors, some of which bind and are activated by trace amines, has focused renewed attention on these compounds. This discovery, together with the possibility of providing novel insights for evaluation of the pathophysiological role of trace amines in primary headaches, may offer new opportunities for pharmacological strategies acting on these receptors. In light of the new scientific background, this review outlines a historical perspective and summarizes evidence supporting a role of trace amines in the pathogenesis of migraine and cluster headache.

The unique nature of the serine interactions for alpha 1-adrenergic receptor agonist binding and activation.

Activation of the beta2- and alpha2-adrenergic receptors (AR) involves hydrogen bonding of serine residues in the fifth transmembrane segment (TMV) to the catechol hydroxyls of the endogenous agonists, epinephrine and norepinephrine. With the beta2-AR both Ser204 and Ser207 but not a third TMV serine (Ser203) are required for binding and full agonist activity. However, with the alpha2a-AR only one of two TMV serines (Ser204, equivalent to Ser207 in the beta-AR) appears to contribute partially to agonist-binding and activation. Because the alpha1a-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in alpha1-AR activation. Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine- substitution mutants that lacked either Ser188 (Ser188--> Ala) or Ser192 (Ser192-->Ala). With the substitution of both serines in the double mutant, Ser188/192-->Ala, binding of all three ligands was significantly reduced (10- 100-fold) consistent with a single hydrogen bond interaction. However, receptor-mediated inositol phosphate production was markedly attenuated only with the Ser188-->Ala mutation and not with Ser192-->Ala. In support of the importance of Ser188, binding of phenylephrine (meta-hydroxyl only) by Ser192-->Ala increased 7-fold over that observed with either the wild type receptor or the Ser188-->Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser192-->Ala mutation. In addition, when combined with a recently described constitutively active alpha1a-AR mutation (Met292-->Leu), only the Ser188-->Ala mutation and not Ser192-->Ala relieved the high affinity binding and increased agonist potency observed with the Met292-->Leu mutation. A simple interpretation of these findings is that the meta-hydroxyl of the endogenous agonists preferentially binds to Ser188, and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser192, that allows receptor activation. Furthermore, since Ser188 and Ser192 are separated by three residues on the TMV alpha-helix, whereas Ser204 and Ser207 of the beta2-AR are separated by only two residues, the orientation of the catechol ring in the alpha1-AR binding pocket appears to be unique and rotated approximately 120 degrees to that in the beta2-AR.

Activities of octopamine and synephrine stereoisomers on alpha-adrenoceptors.

1. The activities of the (-)- and (+)-forms of m- and p-octopamine and m- and p-synephrine on alpha 1-adrenoceptors from rat aorta and anococcygeus and alpha 2-adrenoceptors from rabbit saphenous vein were compared with those of noradrenaline (NA). 2. The rank order of potency of the (-)-forms on alpha 1-adrenoceptors from rat aorta and alpha 2-adrenoceptors was NA greater than m-octopamine = m-synephrine greater than p-octopamine = p-synephrine. The two m-compounds were 6 fold less active than NA on alpha 1-adrenoceptors from rat aorta and 150 fold less active on alpha 2-adrenoceptors. The two p- compounds were 1,000 fold less active than NA on both alpha 1-adrenoceptors from rat aorta and alpha 2-adrenoceptors. The rank order of potency of the (-)- forms on alpha 1-adrenoceptors from rat anococcygeus was NA = m-synephrine greater than m-octopamine greater than p-octopamine = p-synephrine. m-Octopamine was 4 fold less active than NA and (-)-m-synephrine. The two p- compounds were 30 fold less active than NA. 3. The rank order of potency of the (+)- forms was NA greater than m-octopamine greater than m-synephrine greater than p-octopamine greater than p-synephrine on both alpha 1- and alpha 2-adrenoceptors. The potency of each (+)- form was 1-2 orders of magnitude less than that of the (-) counterpart, the differences being greater for the stereoisomers of synephrine than for those of octopamine on both alpha 1- and alpha 2-adrenoceptors. 4. The yohimbine diastereoisomer antagonists, rauwolscine and corynanthine, were tested against (-)-NA and (-)-m-octopamine-induced contractions in both preparations. Based upon the known selectivities of these isomers for alpha-adrenoceptor subtypes, it is concluded that the rat aorta contains only alpha 1-adrenoceptors while the rabbit saphenous vein possesses predominantly alpha 2-adrenoceptors. 5. Ligand binding data for the octopamine and synephrine stereoisomers at alpha 1- and alpha 2-binding sites from rat cerebral cortex was also obtained. (-)-Forms were more active than (+)-forms. The rank order of affinity of the (-)-forms for both alpha 1- and alpha 2-binding sites was NA greater than m-octopamine = m-synephrine greater than p-synephrine greater than p-octopamine. The relative affinities of the members of the series against alpha 1-binding sites were very similar to their relative functional activities on rat aorta. However, the affinities of both m- and p-compounds relative to that of ( -)-NA were much greater at the x2-binding sites than were the relative activities in rabbit saphenous vein, possibly suggesting low intrinsic efficacy. Functional antagonist responses to NA by the (-)-octopamine and synephrines could not, however, be demonstrated on rat aorta or rabbit saphenous vein. 6. The activities of m-octopamine and m-synephrine were not significantly different from each other on either a,-adrenoceptors from rat aorta or x2-adrenoceptors; however, m-synephrine is more active than m-octopamine on a,-adrenoceptors from rat anococcygeus. Both m-octopamine and msynephrine can be considered to be naturally occurring x,-selective amines. However, if m- and poctopamine are co-released with NA in amounts proportional to their concentration, it is concluded that their activities on m,- and x2-adrenoceptors are too low to be physiologically significant.

Kinetic study of sinephrine oxidation by mushroom tyrosinase.

Mushroom tyrosinase catalyzes the oxidation of sinephrine showing a marked lag period during appearance of adrenochrome and simultaneously adrenaline accumulation in the reaction medium can be detected. The adrenaline accumulation follows a sigmoidal curve until a constant level of adrenaline is reached when the system is in the steady-state. These experimental results agree with a model of enzymatic catalysis that includes the chemical evolution of adrenoquinone and permit us to explain these phenomenon as well as the influence that enzyme and sinephrine concentration present on the lag period and the level of adrenaline accumulated in the steady-state.

Purification and properties of synephrinase from Arthrobacter synephrinum.

Synephrinase, an enzyme catalyzing the conversion of (-)-synephrine into p-hydroxyphenylacetaldehyde and methylamine, was purified to apparent homogeneity from the cell-free extracts of Arthrobacter synephrinum grown on (+/-)-synephrine as the sole source of carbon and nitrogen. A 40-fold purification was sufficient to produce synephrinase that is apparently homogeneous as judged by native polyacrylamide gel electrophoresis and has a specific activity of 1.8 mumol product formed/min/mg protein. Thus, the enzyme is a relatively abundant enzyme, perhaps comprising as much as 2.5% of the total protein. The enzyme essentially required a sulfhydryl compound for its activity. Metal ions like Mg2+, Ca2+, and Mn2+ stimulated the enzyme activity. Metal chelating agents, thiol reagents, denaturing agents, and metal ions like Zn2+, Hg2+, Ag1+, and Cu2+ inhibited synephrinase activity. Apart from (-)-synephrine, the enzyme acted upon (+/-)-octopamine and beta-methoxysynephrine. Molecular oxygen was not utilized during the course of the reaction. The molecular mass of the enzyme as determined by Sephadex G-200 chromatography, was around 156,000. The enzyme was made up of four identical subunits with a molecular mass of 42,000.

The metabolism and biosynthesis of (+/-)-o-octopamine and (+/-)-o-synephrine in the rat.

The metabolism of (+/-)-o-octopamine and (+/-)-o-synephrine by rats was studied quantitatively by a gas chromatography-mass spectrometry-selected ion monitoring (g.c.-m.s.-s.i.m.) method using deuterated internal standards. When o-octopamine was injected intraperitoneally into rats four metabolites were excreted in the urine: (i) unconjugated o-hydroxymandelic acid (OHMA) (16%), (ii) unconjugated o-hydroxyphenylglycol (OHPG) (4.5%), (iii) an acid-hydrolysable conjugate of OHPG (28%) and (iv) unconjugated o-octopamine (10%). When o-synephrine benzoate was similarly administered six metabolites were excreted in urine: (i) unconjugated OHMA (13.5%), (ii) unconjugated OHPG (3.3%), (iii) an acid-hydrolysable conjugate of OHPG (15.6%), (iv) unconjugated o-synephrine (10%), (v) an acid-hydrolysable conjugate of o-synephrine (8.5%) and (vi) unconjugated o-octopamine (0.3%). Adult rats normally excreted OHMA (1.0 micrograms day-1) but OHPG, o-octopamine and o-synephrine could not be detected in urine. After the administration of a monoamine oxidase inhibitor, unconjugated o-octopamine (0.3 micrograms day-1) was excreted in urine but OHPG and o-synephrine could not be detected. o-Tyramine given to rats afforded urinary o-octopamine (75 ng day-1) and this was increased 10-fold upon co-administration of a monoamine oxidase inhibitor and o-tyramine.

Identification and quantitative determination of m-hydroxyphenylglycol in mammalian urine.

m-Hydroxyphenylglycol (MHPG) has been identified in mammalian urine by gas chromatography mass spectrometry selected ion monitoring. (2H0) MHPG and (2H5) MHPG were synthesized for use as authentic sample and internal standard, respectively. After acid hydrolysis or treatment with sulfatase, the glycol was extracted from urine with ethyl acetate, converted to its tris-pentafluoropropionyl (PFP) derivative and identified by comparison of the retention times and relative intensities of the characteristic ions, m/z 592, m/z 428 and m/z 415, with those from the authentic sample. Using the internal standard the following quantitative results were obtained: 10 normal human adults excreted 7 ng MHPG mg-1 creatinine (range 2-18) as a sulfate conjugate. Ten rats excreted 0.8 micrograms day-1 (range 0.5-1.1) of MHPG also in the conjugated form. Urinary m-hydroxymandelic acid (MHMA) was also measured quantitatively. The ratio MHMA:MHPG was 1:1 in the rat and 6:1 in the human. This indicates that the overall reductive pathway of m-octopamine and m-synephrine metabolism is more important in the rat than in the human. o-Hydroxyphenylglycol (OHPG) and the three isomeric monomethoxy phenylglycols could not be detected in human or rat urine by these techniques.

A radioenzymatic assay for free and conjugated normetanephrine and octopamine excretion in man.

A radioenzymatic method for the measurement of free and conjugated normetanephrine (NMN) and octopamine (OCT) is described for human urine. The assay is based on the conversion of NMN or OCT to radiolabeled metanephrine (MN) or synephrine (SYN) by phenylethanolamine-N-methyl transferase (PNMT), using tritium-labeled S-adenosyl-methionine [3H]SAM as methyl donor. Thin-layer chromatographic separation yields an assay of high specificity. The sensitivity of the assay is 5 and 2.5 pg of NMN or OCT, respectively. The normal value of free and conjugated NMN was found to be 23 +/- 13 and 102 +/- 49 ng/mg creatinine. Four patients with pheochromocytoma had highly increased levels of free and conjugated NMN. The urinary excretions of free and total octopamine were 5.7 +/- 2.8 and 34.8 +/- 16.6 ng/mg of creatinine, respectively, in normal patients.

[Pharmacokinetics and metabolism of 3H-synephrine (author's transl)]. / Pharmakokinetik und Metabolismus von 3H-Synephrin.

The physiological disposition of 1-(4-hydroxyphenyl)-2-methylamino-ethanol-tartrate (synephrine, Sympatol) in man had not been investigated to date. Therefore, we studied pharmacokinetics and metabolism of tritiated synephrine in man. Following short i.v. infusion in six patients about 80% of the administered radioactivity was recovered in urine. Two-thirds of the urinary tritium activity consisted of the deaminated p-hydroxymandelic acid. 10% were excreted as unchanged synephrine only. Following oral ingestion in ten patients the total urinary radioactivity was quite comparable to the i.v. experiments. Therefore, complete enteric absorption has to be stated. The amount of unchanged synephrine amounted, however, only to 2.5% of the dose. The resulting bioavailability has to be calculated to 22% only. The pharmacokinetic parameters are quite comparable to the sympathomimetics similar in structure. The biological half-life was about 2 h. After oral ingestion absorption was fast, the peak concentrations were observed between 1 and 2 h after administration.

Degradation of (+/-)-synephrine by Arthrobacter synephrinum. Oxidation of 3,4-dihydroxyphenylacetate to 2-hydroxy-5-carboxymethyl-muconate semialdehyde.

1. Cell-free extracts of Arthrobacter synephrinum catalyse the oxidation of 3,4-dihydroxy-phenylacetate. 2. The product of oxidation was characterized as 2-hydroxy-5-carboxymethylmuconate semialdehyde from its chemical behaviour as well as from nuclear-magnetic-resonance spectra. 3. A 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15) was partially purified from A. synephrinum. 4. The enzyme had a Km of 25 micrometer towards its substrate and exhibited typical Michaelis-Menten kinetics. 5. The enzyme also catalysed the oxidation of 3,4-dihydroxymandelate and 3,4-dihydroxyphenylpropionate, at reaction rates of 0.5 and 0.04 respectively of that for 3,4-dihydroxyphenylacetate. 6. The enzyme was sensitive to treatment with thiol-specific reagents. 7. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography was approx. 282000.

Conversion of (+/-)-synephrine into p-hydroxyphenylacetaldehyde by Arthrobacter synephrinum. A novel enzymic reaction.

A partically purified enzyme from Arthrobacter synephrinum was found to catalyse the conversion of (+/-)-synphrine into p-hydroxyphrenylacetaldehyde and methylamine. The enzyme is highly specific for synephrine and is distinctly different from monoamine oxidase.

Microbial metabolism of phenolic amines: degradation of dl-synephrine by an unidentified arthrobacter.

Microorganisms capable of degrading dl-synephrine were isolated from soil of Citrus gardens by enrichment culture, with dl-synephrine as the sole source of carbon and nitrogen. An organism which appears to be an arthrobacter, but which cannot be identified with any of the presently recognized species was predominant in these isolates. It was found to metabolize synephrine by a pathway involving p-hydroxyphenylacetaldehyde, p-hydroxyphenylacetic acid, and 3,4-dihydroxyphenylacetic acid as intermediates. Some of the enzymes of this pathway were demonstrated in cell-free extracts. An aromatic oxygenase, which could also be readily obtained in a cell-free system, was found to degrade 3,4-dihydroxyphenylacetic acid by meta cleavage.