Posted in HPLC on May 23, 2007
How many of you out there picked or even ate fresh bilberries? Probably not too many, unless you are from Northern Europe, Russia, or the Rocky Mountains of the US. The rest of us find bilberries in the form of purple capsules in dietary supplements.
Bilberry is small shrub to a one foot in height with sweet, plump blue-black berries with deep purple flesh that taste somewhat similar to blueberries but slightly tarter and much more aromatic. Bilberry is one of my favorite berry, and if I happen to be in Europe in July, I can never resist bilberry vareniki (East European stuffed dumplings) in dark-purple bilberry sauce.
Bilberry fruits have been used as herbal medicine since the Middle Ages as treatment for various intestinal conditions, thyroid fever, and a number of infections. In the modern European herbal medicine, bilberry fruit extracts are used to enhance poor micro-circulation, including eye conditions such as night-blindness and diabetic retinopathy. In the 90s, several laboratory studies in Europe have confirmed a number of activities associated with bilberry extracts such as antioxidant effects, an ability to inhibit aggregation of blood platelets (reduce stickiness), produce a slight relaxation effect on vascular smooth muscles, and a possible role in reducing factors associated with chronic inflammatory diseases.
The potency of the fruit extracts was attributed to a group of compounds called “anthocyanosides.” These compounds are derivatives of anthocyanins – the pigments responsible for the red, blue or violet colors in flowers and fruits. Most bilberry extracts available as dietary supplements in the US and European markets are standardized to contain 25% anthocyanins. The usual method of determining the anthocyanin content is a single-wavelength UV-Visible spectroscopy assay.
Australian research team headed by Reginald P. Lehmann (doi:10.1021/jf061387d) analyzed commercially available bilberry extracts using UV/Vis method and anthocyanin levels of two extracts were found to be 25% as claimed by the manufacturers. However, when high-performance liquid chromatography (HPLC) was used, one extract was found to contain 9% anthocyanins probably not derived from bilberry. This adulterant was subsequently identified, using HPLC, mass spectrometry (MS), and nuclear magnetic resonance (NMR), as amaranth – 3-hydroxy-4-[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt-a synthetic dark red sulfonic acid based naphthylazo dye.
These research findings were published in 2006; however, now there are reports that mulberry or black bean skins are being used to increase the anthocyanin content of the extracts. Marketing director of Italian botanical derivatives supplier Indena S.p.A. commented: “Based on our routine analysis, at least 15 to 20 per cent of the bilberry samples we collect on the market and analyze are adulterated.The adulteration with azo-dyes, that we also found in the past, is much less present then the sophistication with other fruits.”
I could not find the method that Idena uses to characterize the source of anthocyanins, but my guess is that there is an HPLC fingerprint profile of polyphenols for each kind of fruit so one I can tell the origin of anthocyanins.
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any details of the HPLC method? column? mobile phase?
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