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Nutrition for Optimal Wellness

Bilberry Fruit Extract: Quality Control Measures to Detect Counterfeiting

By Ted Waszkuc, Ph.D., NOW Foods Method Development Scientist
Bilberry extract is prepared from the fruit of the Bilberry shrub (Vaccinium myrtillus L.), a variant of blueberry native to unfarmed fields and woods of mainland Europe, Britain, Siberia and North America. The material of commerce is obtained by harvesting from wild regions of Europe when ripe, from July to September. In traditional European medicine the Bilberry fruit has been used for nearly one thousand years as an astringent for the digestive tract and to maintain vascular integrity. 1
Clinical trials have demonstrated benefits of Bilberry fruit preparations in vision care, including retinal conditions in eyes and the management of microcirculatory function. 2 Modern scientific studies have linked Bilberry extract’s effectiveness to a group of flavonoids called anthocyanins (from Greek anthos = flower + kyanos = blue substance).3
As one of the water-soluble plant pigments (with more than 250 identified), anthocyanins are found mostly in flowers and fruits, giving them bright red, blue, purple and violet colors. The compounds, also referred to as anthocyanosides, are composed of a highly colored flavonoid part, called anthocyanidin, with one or more sugar molecules attached. Anthocyanidin glucosides (anthocyanins) are particularly associated with dark pigmented berries like blueberry, blackberry, chokeberry, elderberry, blackcurrant, mulberry etc., and are considered their most important pharmacologically active ingredients. 4, 5 Red and black legumes, particularly black beans (Phaseolus vulgaris L.) have also been reported as potentially rich sources (~2% in seed coat) of anthocyanins.6
Modern analytical techniques such as High Performance Liquid Chromatography (HPLC) can be used to identify fruits rich in anthocyanin content. Different anthocyanin profiles obtained by an HPLC “fingerprint” have been reported for produce other than Bilberry Vaccinium species and red-colored fruits.7,8  An HPLC fingerprint can be specific for a given berry species, e.g. there have been at least 15 individual anthocyanosides identified in authentic Bilberry extract.
The anthocyanins content of fresh Bilberry fruit is typically between 0.1-0.5%, while extracts available as dietary supplements on the American market are typically standardized to 25%  anthocyanosides.10  In the past, herbs were harvested and processed traditionally by herbalists, the individuals versed in the knowledge of herbs and devoted to practice the therapeutic use of plants. Today, dietary supplement manufacturers use and rely on purchased plant extracts prepared from herbs. This may lead and has led to the possibility of deliberate adulteration. A high quality and expensive herbal extract may have a similar, but less costly, plant species or even a synthetic ingredient mixed in with it. So called “economic adulteration” may be a recurring problem with imported plant extracts. Recently, there have been suppliers offering counterfeited Bilberry extract actually containing different berries (mulberry, elderberry or black bean skin extract) and even synthetic additives.11 
Last year, Australian scientists detected the use of synthetic azo dye to mimic the dark red-blue color of the Bilberry fruit in a commercial Bilberry extract.12 The anthocyanin levels in two different extracts from local markets have been assayed and compared using the HPLC and the official, pharmacopoeial method.13   While the UV-visible spectrophotometric assay described in the British Pharmacopoeia has shown the anthocyanin value of 24% for both extracts - close to the 25% claimed by manufacturers - HPLC analysis revealed significant differences. The extract originated from Europe, Mirtoselect® (Indena, Italy), has shown to be in compliance with the claimed anthocyanin level and the HPLC fingerprint profile was typical to the authentic Bilberry. The other extract of unknown origin coming from China was found to contain only 9% anthocyanosides with the HPLC peaks pattern excluding Bilberry fruit source. Additionally, a coloring agent isolated from that extract proved to be the naphthylazo dye, not related to anthocyanins dark-red substance, called Amaranth (FD & C Red No. 2). Amaranth has UV-visible spectral characteristics similar to those of anthocyanins and that resulted in the false 24% value obtained for the counterfeited extract when using spectrophotometric assay.
Deliberate addition of azo dyes to imitate the bright color of some plant extracts has happened in the past, as it was recently reported in Europe. Specific azo dyes, called Sudans, were found to be present in imported chili and curry powders as well as tomato products.14  The use of synthetic colors in food industry started in the late 1800’s without of any knowledge of their safety and it is still in practice, especially in the developing countries.15  There is now a growing movement worldwide to restrict the use of synthetic colorants due to their potential toxicity.16 Amaranth dye was banned by the FDA in 1976 for use in  foods, drugs, and cosmetics due to suspected carcinogenic activity. The azo dyes have been also reported to react with other food components, particularly reducing agents (antioxidants) to form even more toxic aromatic amines.17 Ascorbic acid (vitamin C) - widely used as a supplemental antioxidant - may also cause the azo dyes to degrade to undesirable compounds.18 
At NOW Foods, before any herbal extract is purchased, a sample of the lot being offered to us is tested by the Quality Control Lab and results compared with specified quality criteria. We strongly recognize the necessity for more than a simple spectroscopic assay to assess the components present in herbal extracts. To ensure the safety and efficacy of NOW products, Method Development scientists have adapted and performed the published HPLC profiling for Bilberry extract authenticity.12 Additionally, our QC lab has been recently equipped with a High Performance Thin Layer Chromatography (HPTLC) sample application instrument, called Linomat 5. That allows us to perform an industry accepted quick test for the presence/absence of unwanted impurities, including artificial food colorants.19
By paying careful attention to both the scientific requirements and the marketplace realities, we can properly screen samples of raw materials and finished products in order to assure the identity, purity and quality of herbal extracts used in NOW® products. That way, no matter where in the world our materials are sourced from, we have the analytical data necessary to document that our herbal extracts have not been adulterated.
1  Morazzoni, P. and Bombardelli, E., Vaccinum myrtillus L. Fitoterapia 67(1),   329 (1996)
2  Canter, P.H.; Ernst, E. Anthocyanosides of Vaccinum myrtillus (bilberry) for     night vision – a systematic review of placebo-controlled trials. Surv. Ophthamol. 49, 38-50 (2004)
3  Bridle, P. & Timberlake, C.F.  Anthocyanins as natural food colours – selected aspects.  Food Chem. 58, 103-109 (1997)
4  Nakajima, J. et al., LC/PDA/ESI-MS profiling and radical scavenging activity of anthocyanins in various berries.   J. Biomed. Biotech. 5, 241-247 (2004)
5  Chen, P.N. et al., Mulberry anthocyanins, cyaniding 3-rutinoside and   cyaniding 3-glucoside, exhibited an inhibitory effect on the migration and invasion of a human cancer cell line.  Cancer Lett. 235, 248-259 (2006)
6  Takeoka, G.R. et al., Characterization of Black Bean (Phaseolus vulgaris L.) Anthocyanins. J. Agric. Food Chem. 45, 3395-3400 (1997)
7  Prior, R.L. et al., Identification of procyanidins and anthocyanins in   Blueberries and Cranberries (Vaccinium Spp.) using High-Performance Liquid Chromatography/Mass Spectrometry.  J. Agric. Food Chem. 49, 1270-1276 (2001)
8  Goiffon, J.P., et al., High-performance liquid chromatography of red fruit      anthocyanins. J. Chromatogr. 537, 1010-121 (1991)
9  Baj, A., et al., Qualitative and quantitative evaluation of Vaccinium myrtillus anthocyanins by high-resolution gas chromatography and high-performance liquid chromatography. J. Chromatogr. 279, 365-372 (1983)
10  Valentova, K., et al., Cytoprotective effect of a bilberry extract against oxidative damage of rat hepatocytes.  Food Chem. 101, 912-917 (2007) and references cited herein
11  www.nutraingredients.com
12  Pennman, K.G., et al. Bilberry adulteration using the food dye Amaranth. J. Agric. Food Chem. 54(19), 7378-7382 (2006)
13  British Pharmacopoiea Commission Office. Bilberry fruit; in British Pharmacopoeia; London, U.K., 2004; vol.1, p. 258
14  Daood, H.G., et al.  Simultaneous determination of Sudan dyes and carotenoids in red pepper and tomato products by HPLC. J. Chromatogr. Science 43, 461-465 (2005)
15  Tripathi, M., et al., Surveillance on use of synthetic colours in eatables vis a vis Prevention of Food Adulteration Act of India. Food Control 18, 211-219 (2007)
16  EC regulation No.178/2002. Off.J. Eur.Union 31, (2002)
17  Bitsch, A. et al., Genotoxic and chronic toxic effects in the carcinogenity of aromatic amines. Recent Results Cancer Res. 143, 209-223 (1997)
18  Marovatsanga, L., et al., The determination of added azo dyes in soft drinks via its reduction products. Food Chemistry 24, 83-98 (1987)
19  http://www.mediherb.com.au/articles/BilberryPoster.pdf