Review
Chromatographic and electrophoretic methods for the analysis of phenetylamine alkaloids in Citrus aurantium

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Abstract

Citrus aurantium (bitter orange) is a plant belonging to the family Rutaceae, whose fruit extracts have been used recently for the treatment of obesity. The most important biologically active constituents of the C. aurantium fruits are phenethylamine alkaloids (i.e. octopamine, synephrine, tyramine, N-methyltyramine and hordenine). Synephrine is a primary synthesis compound with pharmacological activities such as vasoconstriction, elevation of blood pressure and relaxation of bronchial muscle. Synephrine is present in the peel and the edible part of Citrus fruit. Of the adrenergic amines of natural origin, synephrine has been found to be the main constituent of C. aurantium fruits and extracts; the other alkaloids are either absent or present in only low concentrations. It is known that synephrine and the other amines found in C. aurantium have adverse effects on the cardiovascular system, owing to adrenergic stimulation. In light of the great commercial proliferation of C. aurantium herbal medicines in recent years, this review provides an overview of various extraction, separation and detection techniques employed for the qualitative and quantitative determination of the alkaloids in C. aurantium and related species. The application of chromatographic and electrophoretic methods for the separation and determination of these active components in C. aurantium plant material and derivatives are described. Since synephrine is a chiral compound, enantioselective chromatographic and electrophoretic techniques for the analysis of synephrine enantiomers in natural products are presented. Furthermore, examples of identification of these active compounds in complex matrices by hyphenated methods, such as gas chromatography–mass spectrometry and high-performance liquid chromatography–mass spectrometry, are described. The advantages and limitations of these separation and identification methods are assessed and discussed.

Introduction

Citrus aurantium (bitter orange) is a plant belonging to the Rutaceae family, whose fruit extracts have been used recently for the treatment of obesity in humans. The compounds responsible for this activity are the phenethylamine alkaloids octopamine, synephrine, tyramine, N-methyltyramine and hordenine [1], [2].

Synephrine is a primary synthesis compound with pharmacological activities such as vasoconstriction, elevation of blood pressure and relaxation of bronchial muscle. Synephrine is a known constituent of the peel and the edible part of Citrus fruits [2], [3]. Weathon and Stewart [1] have speculated that the biosynthesis of synephrine in plants could follow the biosynthetic pathway tyramine  N-methyltyramine  synephrine (Fig. 1) rather than the tyramine  octopamine  synephrine pathway believed to predominate in animals. Tyramine is derived from the amino acid tyrosine by decarboxylation.

Synephrine is an alkaloid that is similar in structure to ephedrine (Fig. 2), the main active component of plants of the genus Ephedra. Only two substitutions are needed to obtain synephrine from ephedrine: one of the ring carbon atoms is hydroxylated (OH replaces H), and a side-chain methyl group (CH3) is replaced by hydrogen.

Synephrine has effects on human metabolism that could help to reduce fat mass in obese people, since it stimulates lipolysis, raises the metabolic rate and promotes the oxidation of fat through increased thermogenesis [4], [5]. However, it is known that synephrine and the other alkaloids found in C. aurantium have effects on the cardiovascular system by means of adrenergic stimulation. The effects of repeated oral administration of two commercially available C. aurantium fruit hydroalcoholic synephrine-standardized extracts on food intake and cardiovascular functions in rats have been studied [6]. C. aurantium extracts reduced food intake and body weight gain. Mortality was observed in all C. aurantium-treated groups. ECG alterations were observed in animals treated with both extracts. Haller et al. [7] evaluated the hemodynamic effects of dietary supplements containing C. aurantium in 10 healthy human adults, and demonstrated that some of the dietary supplements raised blood pressure in healthy, normotensive subjects. These findings indicated that weight loss supplements have a cardiovascular stimulant action and could cause adverse effects in some individuals. More recently, several case reports indicated that products containing C. aurantium are suspected of being associated with cardiovascular arrhythmias, including tachycardia, cardiac arrest, ventricular fibrillation, transient collapse, etc. [8]. All cases were considered serious. Evaluation of these reports is challenging, because of many factors, such as the lack of information on the ingested dose of synephrine, the contributory effects of other (multiple) ingredients and the ambiguity of the reported information.

Many industries have responded to the negative publicity on Ephedra by introducing new “Ephedra-free” dietary supplements. However, Ephedra-free is not necessarily danger-free [9], [10]. C. aurantium is an ingredient in many of these Ephedra-free dietary supplements, which often contain concentrates of other herbs that are rich in caffeine. The combination of synephrine and caffeine has the same potential to induce arrhythmia, hypertension, heart attacks and strokes as the combination of ephedrine and caffeine. Many people mistakenly believe that Ephedra-free supplements are safe, and consumers may be injured or even die before the problem is recognized. Haller et al. [7] have observed that the cardiovascular stimulant action of Ephedra-free products are not likely caused by C. aurantium alone, but may be attributable to a synergistic action with caffeine and other stimulants present in the multi-component formulations.

Furthermore, C. aurantium is used experimentally to inhibit intestinal cytochrome P450 (CYP) 3A4, an enzyme responsible for the metabolism of a large number of drugs. Thus, it can cause an alarming increase in the blood levels of many drugs [11], [12].

Haaz et al. [8] have concluded that larger and more rigorous clinical trials are necessary to reach adequate conclusions regarding the safety and efficacy of C. aurantium dietary supplements for promoting weight loss.

Section snippets

Chromatographic and electrophoretic methods

Most dietary supplements are crude plant extracts, i.e. a complex mixture of different phytochemicals (plant secondary metabolites). The chemical nature of these compounds differs significantly between species. Even extracts of the same species may vary, depending on plant origin, growth conditions, climate, soil quality, harvest season, drying process, powdering methods, extraction and stabilization procedures. There are various “standardized herbal extracts” on the market that provide a

Hyphenation procedures

The use of chromatographic separation coupled with mass spectrometry (MS) for the chemical characterization and composition analysis of botanicals has been growing rapidly in recent years. The use of hyphenated techniques, such as gas chromatography–mass spectrometry (GC–MS), high-performance liquid chromatography–mass spectrometry (HPLC–MS), high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) to perform on-line composition and structural analyses provides a wealth of

Other methods

A sensitive determination of synephrine by flow-injection chemiluminescence (CL) was described recently [61]. It was found that the light emission produced by the oxidation of luminol by potassium ferricyanide in a basic medium was enhanced by synephrine. The proposed method was applied to the determination of synephrine in Citrus fruits and in spiked biological fluids (plasma and urine). The method was validated, and showed good sensitivity and level of recovery. The application of the method

Conclusions

C. aurantium fruit extracts have been widely explored for the treatment of obesity. Several extraction, separation and detection methods have been developed and applied for the qualitative and quantitative analysis of phenethylamine alkaloids (octopamine, synephrine, tyramine, N-methyltyramine, hordenine), which are the main active components, in Citrus plant material, extracts and dietary supplements. Considering the possible optical isomerization of synephrine during the extraction procedure,

References (63)

  • I. Stewart et al.

    J. Biol. Chem.

    (1964)
  • C.O. Moro et al.

    Fitoterapia

    (2000)
  • G. Calapai et al.

    Fitoterapia

    (1999)
  • C.A. Haller et al.

    Am. J. Med.

    (2005)
  • M. Hamburger et al.

    J. Pharm. Biomed. Anal.

    (1989)
  • X.J. He

    J. Chromatogr. A

    (2000)
  • K. Hashimoto et al.

    J. Chromatogr.

    (1992)
  • S.M. Vieira et al.

    Food. Chem.

    (2007)
  • F. Pellati et al.

    J. Pharm. Biomed. Anal.

    (2002)
  • F. Tang et al.

    J. Chromatogr. A

    (2006)
  • M. Ganzera et al.

    Talanta

    (2005)
  • D.S. Bell et al.

    J. Chromatogr. A

    (2005)
  • P.F. Cancalon et al.

    J. Chromatogr. A

    (1993)
  • G. Chen et al.

    Anal. Bioanal. Chem.

    (2002)
  • J. Haginaka

    J. Pharm. Biomed. Anal.

    (2002)
  • J. Gal et al.

    J. Pharmacol. Methods

    (1986)
  • F. Kusu et al.

    Anal. Biochem.

    (1996)
  • K. Arai et al.

    J. Pharm. Biomed. Anal.

    (1997)
  • F. Pellati et al.

    J. Pharm. Biomed. Anal.

    (2005)
  • J. Haginaka

    J. Chromatogr. A

    (2001)
  • H. Henriksson et al.

    FEBS Lett.

    (1996)
  • A.G. Peterson et al.

    J. Chromatogr. B

    (1996)
  • E. Marchei et al.

    J. Pharm. Biomed. Anal.

    (2006)
  • X.-G. He et al.

    J. Chromatogr. A

    (1997)
  • Q. Li et al.

    Luminescence

    (2006)
  • R. Wang et al.

    Luminescence

    (2006)
  • T.A. Weathon et al.

    Phytochemistry

    (1969)
  • T.A. Weathon et al.

    Lloydia

    (1970)
  • C. Carpéné et al.

    Naunyn-Schmiedebergs Arch. Pharmacol.

    (1999)
  • S. Haaz et al.

    Obes. Rev.

    (2006)
  • D.M. Marcus et al.

    Science

    (2003)
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