Citrus fruits are promoted as one of the healthy eating options. In the UK, they are options within the five-a-day programme of the Department of Health in which people are encouraged to eat at least five portions of fruit and vegetables a day to "help reduce the risk of some cancers, heart disease and many other chronic conditions."
Like other types of healthy food, the active components in citrus are flavonoids which have been recognised to impart many health benefits. Bioflavonoids are also referred to collectively as vitamin P, although they are not actually vitamins, probably due to their health-promoting properties.
The predominant flavonoid in lemons and oranges is a compound called hesperidin. It is concentrated in the white, membranous parts and the peel and has been credited with antioxidant, antibacterial, antifungal, antiviral, anticancer and analgesic properties, among others.
Chemically, hesperidin consists of a flavanone component joined to a sugar group through the oxygen atom at the 7-position to give the rhamnoglucoside. After ingestion, the sugar group is removed in the gut to leave the parent flavanone, 3',5,7-trihydroxy-4'-methoxyflavanone, also known as hesperetin.
In order to study the actions and effects of hesperidin in the human body, scientists need to study hesperetin, the active metabolite. There are a number of reported studies on its bioactivity but they fail to take into account the chiral nature of the compound. Hesperetin exists as two enantiomers, the (R)- and (S)-forms, which will have different properties, so should be considered independently.
Scientists from Italy and Algeria recently succeeded in separating the enantiomers of several flavanones and their glycosides, including hesperetin and hesperidin, using nano-LC. They used a specially prepared chiral column, based on phenylcarbamate-modified propyl-beta-cyclodextrin, which provided baseline resolution of most of the enantiomeric pairs.
By switching from the conventional HPLC column with an internal diameter of 4.6 mm, to a nano column with an internal diameter of 100 µm, the sensitivity was increased by about 2000-fold. In addition, the switch rendered the procedure more environmentally friendly, due to the reduced volumes of the stationary phase and phases.
Now, the team has applied the column to determine the enantiomers of hesperetin in human urine after the consumption of orange juice. Salvatore Fanali and colleagues from the Institute of Chemical Methodologies of the Italian National Council of Research in Rome and the University of Science and Technology Houari Boumediene in Algiers transferred the optimised conditions from their previous work.
The mobile phase consisted of triethylamine acetate buffer (pH 4.5) in 70% aqueous methanol at room temperature. The retention times of the (R)- and (S)-isomers were 9 and 12 minutes, respectively, and no interfering peaks were observed from orange juice or urine. The LC instrument was fitted with a UV detector operating at 205 nm.
7-Ethoxycoumarin was added as an internal standard, eluting after 7 minutes. The detection limits were 0.1 µg/L for each enantiomer and linear calibration curves were obtained over 0.25-25 µg/mL. Recoveries were higher than 82%.
Under these conditions, the (R)- and (S)-enantiomers of hesperetin and hesperidin were first measured in a high quality commercial blood orange juice. Hesperetin was not detected, confirming that the flavanone exits in orange juice in its conjugated form only. The concentrations of (R)- and (S)-hesperidin were found to be high, at 112.81 and 599.55 mg/L, respectively.
A volunteer drank 1 L of the orange juice and urine was collected at intervals over 24 hours for analysis after hydrolysis. Both enantiomers appeared in urine after about 2 hours, maximised after 7 hours then fell back sharply, returning to zero after 24 hours.
The total amounts of (R)- and (S)-hesperetin excreted corresponded to 6.44 and 3.55% of the original orange juice content and most of this, 87 and 91% respectively, was excreted in the busy period from 4-8 hours.
Based on the speed, simplicity, accuracy and relatively low cost of the method, the researchers proposed the use of hesperetin as a biomarker for citrus juice uptake. It could be used in clinical studies of the bioactivity of hesperetin and could be extended to other flavanones, once their separation on the chiral column has been confirmed.
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* Journal of Pharmaceutical and Biomedical Analysis 2010, 51, 225-229: "Analysis of hesperetin enantiomers in human urine after ingestion of blood orange juice by using nano-liquid chromatography"
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