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The Adulteration of Commercial Bilberry Extracts

Editor’s note: This paper is part of the series being published under the aegis of the ABC-AHP-NCNPR Botanical Adulterants Program, an educational program led by the American Botanical Council, the American Herbal Pharmacopoeia, and the National Center for Natural Products Research at the University of Mississippi. The Program is financially supported and/or endorsed by a coalition of herb and dietary supplement industry members, third-party analytical laboratories, professional and trade associations, nonprofit educational groups including accredited schools of natural medicine, and others.


Bilberry fruit (Vaccinium myrtillus, Ericaceae; heath family) is a common ingredient in food, health products, and cosmetics. In European countries the berries are sold fresh, frozen, in jams and preserves, and as a juice ingredient. Finished products made with bilberry (dried fruit, dried powdered fruit, and powdered extracts) are sold in the form of dietary supplements in the United States and as phytomedicines in the European Union (EU) and elsewhere.

The genus Vaccinium includes more than 140 mostly circumpolar species, with the highest concentration of representatives in North America.1 Bilberry is an erect-to-freely-branching shrub, from 15-25 cm (up to 60 cm) in height, spreading from a creeping rhizome. Flowers are in axillary racemes with 1-2 flowers per group. The bluish-black fruit (including skin and flesh throughout) are globose and 6-10 mm in diameter. Bilberry is found throughout most of Europe, particularly in heaths, moors, and woods in northern Europe, and largely restricted to mountainous areas in southern Europe.2 It is so common in much of Europe that in some areas it represents as much as 25 percent of the vegetation in forest understory. Based on the available evidence, there is no commercial cultivation of bilberry; the world’s entire commercial supply is wildcrafted, mainly in Scandinavia and Eastern European countries.

Bilberry is a popular dietary supplement in the United States, where it ranked 15th best-selling in the mainstream market (i.e., grocery stores, drug stores, mass-market retail stores — referred to as the FDM channel), although its sales in this channel have dropped by about 10 percent per year in the past 2 years for reasons that are not clear (Table 1). It is possible that the increased price of raw material, due to the relatively poor harvest in the past 2 years, might be responsible for finished-product price increases. That, in turn, may have had a negative effect on sales. However, contrary to the sliding sales seen in the mainstream market, 2011 sales for bilberry dietary supplements in the natural foods channel increased slightly (1.5%; $17,632) compared to 2010, to a total of $1,196,845 (sales in Whole Foods Markets are not included), according to market-tracking statistics from SPINS, a Schaumburg, Illinois-based market-research firm. In the natural food store channel, bilberry is ranked 53rd in sales, significantly lower than its rank in the FDM channel.3

In the United States, only V. myrtillus is allowed to be sold as “bilberry,” according to The American Herbal Products Association’s Herbs of Commerce, 2nd ed., a book that enumerates the accepted common names of approximately 1,650 herbs and medicinal plants and their corresponding Latin binomials (scientific names).4 This book, which also lists European blueberry, huckleberry, and whortleberry as other acceptable common names for bilberry, has been accepted by the US Food and Drug Administration (FDA) as a guide to botanical nomenclature for herbal products sold in commerce in the United States.5 No other plant or plant material is acceptable for the commercial designation “bilberry” in the United States.

Health Benefits of Bilberry

Bilberry fruit extracts are among the best-selling herbal dietary supplement products in the US market, with benefits in the management of retinopathy and vascular conditions including venous insufficiency and capillary fragility.5 Since the 1960s, numerous pharmacological and clinical studies have suggested  bilberry's benefits for both vascular health and vision problems; however, many of the studies suffered from poor design, small population samples, lack of placebo controls, and other methodological deficiencies. Many early clinical reports or observational studies lacked the scientific rigor necessary for reproducibility. More recent trials suggest that bilberry fruit extract can decrease vascular permeability and increase capillary resistance.11 Bilberry extracts often are used to treat vascular insufficiency and associated symptoms such as edema, varicosities, paraesthesias (tingling or numb sensation in extremities), and cramping. By decreasing capillary fragility, an associated tendency toward bruising may be reduced. Pharmacological evidence shows that bilberry extract decreases vascular permeability, inhibits elastase and collagenase production and platelet aggregation, and is vasorelaxant and antioxidant.5,12,13

The vast majority of scientific and clinical studies have been conducted with the bilberry fruit extracts Myrtocyan® or Tegens®, both of which contain 36% anthocyanins* (equivalent to 25% by weight expressed as anthocyanadins). Myrtocyan is manufactured by Indena SpA, Milan, Italy. Tegens® is a proprietary formula from Indena’s affiliated company, Inverni della Beffa, in partnership with Sanofi-Synthelabo,12 and is the same extract as Myrtocyan. The extract is now marketed by Indena as Mirtoselect®.

Brinckmann (2011) emphasizes that reproducible results for safety and efficacy are intrinsically linked to consistent and reproducible quality. In world markets, botanicals are available in a wide range of grades and qualities from inexpensive grades of inferior quality to the highest quality grade; therefore, higher-priced ingredients tend to demonstrate reproducible efficacy and safety for a specified health benefit.14 The health benefits expected from a bilberry extract were demonstrated in various clinical studies using a bilberry preparation with a quality marker based on standardization to anthocyanin content, which is believed to be the primary contributing constituent to therapeutic activity.15

For bilberry, reproducible benefits are relative to the extract equivalence used in the majority of clinical trials involving a standardized bilberry extract containing 36% anthocyanins at a dosage of 320-480 mg/day, corresponding to 100-200 mg/day anthocyanins.16 Cassinese et al. (2007) analyzed 40 typical bilberry preparations from 24 different brands found in the American, European, and Japanese marketplaces and found that only 15 percent of the products provide the dosage of anthocyanins shown to be effective in clinical trials.17

Bilberry Supply Sources and Market Dynamics

Bilberry’s broad distribution throughout much of northern Europe and mountainous areas of southern Europe, coupled with its widespread use and market acceptance has made it one of the most successful wild-harvested, non-timber forest ingredients of the region. Nordic countries, including Norway, Sweden, Finland, and Iceland have cooperated in detailed research on market needs, quality issues, plant biology, biodiversity, production, and utilization for global markets.18,19

The cooperation of governments and private-sector companies has given Nordic countries a distinct advantage in global markets in the supply of bilberry as a raw material. A survey of companies involved in the wild-berry industry in Nordic countries resulted in the creation of a database of 1,300 Nordic companies dealing with wild berries, including approximately 750 Swedish, 350 Norwegian, and 200 Finnish companies, both small- and large-scale. The focus of research is to develop uniform wild-berry quality within Nordic countries, a uniform traceability system, and the Nordic wild-berry brand as a guarantee of quality. As much as half of the Nordic bilberry product is exported to China and Japan. To help ensure authenticity of identity, DNA testing methods have been developed to assure that bilberry exports are not contaminated with other wild berries.18,19

Estimates of potential bilberry harvests have been calculated in yield variation studies for various Scandinavian countries. For example, in Finland, inventory yield data on wild berries was collected by the Finnish Forest Research Institute from 1997 to 2008. During that time period, annual bilberry potential yields in Finland varied from 92 to 312 million kg. Of the total yield estimate, 5 to 10 percent of berries are collected every year. Picking of wild berries, as well as mushrooms, has social and cultural significance in Finland. It is viewed as a traditional household and recreational activity, with approximately 60 percent of the population participating in wild-berry picking today, compared with 69 percent in 1981, indicating that its popularity as a recreational activity has remained relatively stable. In Nordic countries, the traditional social concept of “everyman’s right” allows for open access to both private and public lands and the right to pick wild berries and mushrooms on them. The harvest also extends to commercial pickers, though commonly permission is obtained from the landowner or berry associations that negotiate exclusive rights for harvest on private lands. In Finland, where most people enjoy a high standard of living, berry picking is viewed as a leisure activity, providing healthy exercise and the opportunity to enjoy nature.20

Wild-berry picking in other Scandinavian countries is trending downward. A study conducted in the late 1970s estimated that Swedes collected 7 percent of available wild-berry volume for home consumption; 20 years later, participation in berry collection and volume of berries picked declined dramatically. In Russia, it is estimated that between 10-15 percent of available wild-berry volume is collected.20

In Russia, Balkan countries, and elsewhere in Eastern Europe, wild-berry picking provides an important additional income source in populations with high unemployment in rural areas. For example, one 12-year-old girl interviewed in August of 2011, in the Prokletije Mountains bordering the north of Montenegro and Albania, said that she expected to collect over 200 kg of bilberries in 2011. She sold fresh bilberries at a roadside stand for 3 €/kg. (It takes approximately 10-12 kg of fresh berries to produce 1 kg of dried fruit.)19

The quantity of bilberries picked during the past year has averaged 35 million kg compared to 2005, when nearly 55 million were harvested, primarily in Scandinavia and the Ukraine. In terms of anthocyanin assay content of the berries, the highest level observed was 0.37% in 2009, with the average over recent years being 0.35%. (Ris G. email to M. Blumenthal, October 2, 2012).

Timing of harvest is an important factor in quality. When bilberry buyers purchase the fruit from collectors, berry ripeness is determined with a handheld analog or digital refractometer. Values of less than 12 to 14 percent extractable solids are generally considered indicative of unripe berries. Ripeness is an important factor in the quality of bilberries. As fruit ripens, concentrations of flavonols and procyanidins decrease, while concentrations of the anthocyanins increase. Studies also suggest that bilberries must be handled with care, as damage of the skin or flesh can result in oxidization of the antioxidant anthocyanins. Bilberry is harvested traditionally by hand-picking. However, there is increased reliance on the use of berry rakes, which agronomists say damages the bushes and reduces flower buds, hence lowering berry production for the following year. Berry rakes also collect extraneous leaf and bud material which must be cleaned from the berries and capture both green and ripe berries at the same time. Depending upon the location in Europe, harvest of bilberry occurs between mid-July and the end of September, with about a 2-week harvest season of berry ripeness.19

The economics of obtaining raw materials suggest that there is adulteration in the marketplace. While pricing for labor in Asia and other parts of the world is generally lower than the cost in Europe, the relatively small region of growth for bilberries suggests that there is not much elasticity in the price of raw material. The range of pricing for the Indena bilberry standardized extract per kg is around “the high six hundreds [US dollars] in previous years up to the high eight hundreds this year” due to a poor crop last year, according to Greg Ris, vice-president of sales for Indena USA in Seattle, WA (personal communication to M. Blumenthal, October 1, 2012). His parent company is Indena SpA in Milan, Italy, universally acknowledged as the world’s leading producer of bilberry extract and pharmacological and clinical research on such extract.

Ris emphasized that it takes 100 kg of hand-picked bilberry fruit to make 1 kg of the 100:1 Indena bilberry extract, at an average range of 2.5 euros ($3.25 USD) to a “near record high” of 4.6 euros ($6.00 per kilo) in 2011. This variability is primarily due to weather conditions (either too damp to too dry). At such prices, a 100:1 extract would cost from $325 to $600 USDper kg of extract just for the raw material (Ris G., email to M. Blumenthal, October 2, 2012), plus the cost of refrigeration and/or frozen storage and transportation to keep the material fresh, as well as extraction costs and other overhead, plus profit. Therefore, says Ris, some of the bilberry extract currently being offered on the global market for as low as $200 per kg, and up to $400 per kg, is presumably or definitely adulterated. “You just can’t make an extract that meets Indena’s specifications for such a low price,” he said (Ris G., personal communication to M. Blumenthal, October 1, 2012).

This pricing information is corroborated by Don Stanek, director of sales for Linnea, a European supplier of botanical extracts with US offices in Easton, PA. According to Stanek, bilberry fruit raw material costs range from $4-7 per kg; his company, a joint venture between Germany’s W. Schwabe Pharmaceuticals and Ipsen-Beaufour in France, produces — like Indena — a bilberry extract at a 100:1 ratio of raw material to finished extract. Therefore, the cost of the bilberry fruit raw material in the Linnea extract would cost $400-700 USD per kg before shipping, storage, and extraction costs, plus a modest profit. He acknowledges that his company sells its bilberry extract for as low as $650 per kg and up, depending upon raw material costs and quantities purchased by customer, among other factors.

“With so much high cost of raw materials and such compression of profitability due to the market being virtually flooded with cheap, adulterated ‘bilberry extract,’ this item is not one of our most profitable extracts,” said Indena’s Ris, lamenting the downward pricing pressure that fraudulent extracts have had in the market.


The Confusing Morass of Adulterants

Given global demand for this relatively high-cost, wild-harvested berry, bilberry supplies are reportedly rife with economic adulteration.

Presumably, most of this adulteration is intentional, and not an accident based on poor or inadequate use of quality control techniques. In addition, anthocyanosides from unrelated plants, such as elderberry (Sambucus nigra, Caprifoliaceae), also have been identified as potential adulterants in bilberry extracts.21 A leading independent analytical laboratory in the United States, Chromadex, Inc., has reported testing samples of “bilberry extract” determined to be adulterated with extract of Chinese mulberry (Morus australis and M. spp., Moraceae) (Jaksch F., email, September 10, 2012).

Research by Indena and others affirms that the anthocyanosides are the major active ingredients in bilberry, and that the mixture of delphinidin, cyanidin, malvidin, peonidin, and petunidin in bilberry produces a unique pattern set that distinguishes bilberry from all other anthocyanoside sources of both dietary and non-dietary origin,21 although V. corybosum (North American blueberry) contains the same anthocyanins in significantly lower weight percentages; blueberry also contains significant amounts of proanthocyanins, which are almost entirely absent in bilberry extracts (Tempesta M., e-mail, September 11, 2012). And yet, the relatively high price of authentic bilberry extract has made it a target for sophisticated adulteration.

In addition, extracts of 2 circumboreal species, V. uliginosum and V. vitis-idaea, which grow in northern areas of Europe, North America, and Asia, are being wild-harvested in China and offered to world markets as “homemade Chinese bilberry” and “Chinese domestic bilberry” extracts at prices as low as $10 per kg. According to a “Research Report of Chinese Blueberry Extract Market, 2009-2010,” the Chinese market is divided into “European bilberry extract” and “Chinese bilberry extract,” “standardized from 10%, 15%, 25%, to up to 40% anthocyanidins.” “Home-made raw materials” (V. uliginosum and V. vitis-idaea) are wild-harvested in Northeast China and the Shaanxi Province. According to the report, in 2008, Chinese bilberry extract (excluding “European bilberry,” V. myrtillus) production was approximately 60 tons, 95 percent of which was exported, mostly to the United States.22

Another recently documented adulterant is amaranth dye (also known as azo dye or Red Dye No. 2).15,21,23 The HPTLC (high-performance thin-layer chromatography) analytical method for determining azo dye adulteration has been developed by CAMAG, a manufacturer of scientific laboratory instruments and methods of analysis in Muttenz, Switzerland. (Editor’s note: Amaranth dye has no relation to amaranth [Amaranthus spp., Amaranthaceae], a traditional plant food of the Aztecs in what is present-day Mexico.)

Amaranth dye also has been found as an adulterant in bilberry extract due to its color being similar to the color of bilberry extract, according to information from Indena,21 and its presence as an adulterant in bilberry extracts is documented sufficiently enough to merit its appearance as the only bilberry adulterant mentioned by AHPA in its list of “Known Adulterants.”23

The detection of aamaranth dye and/or charcoal in commercial bilberry extracts is clearly the result of intentional adulteration.21

Further, confidential reports from third-party laboratories indicate determination of profiles consistent with black soybean hull in some commercial “bilberry” samples. Soybean hull (Glycine max; Fabaceae) extracts, at 35 percent and 50 percent anthocyanidins, contain mainly cyanidin 3-O-glucoside and delphinidin-and petunidin-3-O-glucoside.

In addition, some laboratories have uncovered the adulteration of bilberry fruit extract with extract of black rice (Oryza sativa, Graminae), which is known to contain anthocyanins that can trick a total anthocyanin content by UV-detection assay.

Language issues may contribute to the adulteration problem, because various Vaccinium species are translated from one language to another as “blueberry,” “bilberry,” or variations on the theme, depending on the language into which they are translated. Most refer to various species of Vaccinium cultivated or wild-harvested in Europe, North America, South America, and temperate regions of Asia.

In a recent study, for example, an Andean Vaccinium species called “Colombian wild bilberry” or “Colombian bilberry” (V. meridionale), was shown to have high antioxidant activity and a unique anthocyanin pattern with high proportions of both delphinidin and cyanidin, which can be used to authenticate and identify this species compared with other Vaccinium species.24

This is a good example of the application of a variation on the common English name “bilberry” in order to analyze, assess, and introduce a less well-known Vaccinium species to possible commercial potential among national or international markets. Called agraz in Colombia, V. meridionale is wild-harvested and available in local markets. The size, color, morphology, and tart fruit flavor give it a superficial food experience much more akin to cranberry than to bilberry. A simple Google search for “Vaccinium meridionale also leads to websites that refer to it as “Jamaican bilberry.” The adulteration of language usage in popular and scientific literature, and in particular on the Internet, contributes to consumer confusion and also may contribute materially to the intentional or unintentional adulteration of consumer products.

“In fact,” said Frank Jaksch, founder and CEO of ChromaDex, Inc., a leading analytical laboratory, “virtually any anthocyanin-rich fruit can be a potential source of an adulterant to bilberry extract, or, in some cases, a lower-cost substitute for it, if, obviously, the fruit raw material is significantly lower in price than fresh bilberries. This would allow for the incentive for economic adulteration, that is, assuming that the adulteration with such fruits is not accidental” (personal communication to M. Blumenthal, October 8, 2012). Jaksch notes another important point about the growing list of anthocyanin-containing fruit extracts — such as acai berry (Euterpe oleracea, Arecaceae), cranberry (Vaccinium macrocarpon, Ericaceae), maqui berry (Aristotelia chilensis), etc. — is that there usually will be another anthocyanin “super fruit” popping up on the market. “It is very important to understand the different anthocyanin profiles of these different fruits as the anthocyanin profiles of adulterated bilberry extracts will inevitably vary from one fruit source to the next,” he said.

According to Roberto Pace, PhD, corporate quality control manager at Indena, the anthocyanoside profiles of other species of Vaccinium are well established by reliable analytical methods (e.g., HPLC) and can be “unequivocally” determined via appropriate analytical testing (personal communication to M. Blumenthal, October 9, 2012). Such plants could include V. angustifolium (low-bush blueberry), V. corymbosum (high-bush blueberry), and their hybrids and cultivars, as well as V. oxycoccos (European cranberry) and V. macrocarpon (cranberry), plus non-Vaccinium anthocyanin-rich fruits, e.g, black currant (Ribes nigrum, Grossulariaceae), raspberry (Rubus idaeus, Rosaceae), and wild cherry (Prunus avium, Rosaceae).

Michael Tempesta, PhD — managing partner of Phenolics LLC in Omaha, NE, and an expert in phenolic chemistry — noted that adulteration of bilberry extract with anthocyanosides from these plants, or preparations made from them (e.g., juice concentrates), would not be economically competitive, as the price of raw materials of these plants and/or their concentrations are too high to warrant their use as economic adulterants (personal communication to M. Blumenthal, October 9, 2012).

Industry-Inspired Analytical Identification and Problem-Solving

Following passage of the Dietary Supplement Health and Education Act (DSHEA) of 1994, herb product sales experienced a meteoric increase in the late 1990s and early 2000s, resulting in many new companies entering the herb market supply chain at both the wholesale and retail levels. Prior to the market boom, many standardized herb extracts available in the market were produced by well-established European firms that were not only major suppliers to world markets, but also had significant scientific expertise with the ingredient. Such is the case with the Myrtocyan product sold by Indena SpA, which essentially established the market for bilberry extract and the pharmacological and clinical research to support the chemically defined ingredient.

As international markets increased for bilberry, many new extract suppliers raced to gain market share and a highly competitive industry rapidly evolved, especially for dramatically lower-priced extracts from Asian countries, particularly China. Adulteration of bilberry supplies and extract was relatively limited prior to the market boom. The 2001 American Herbal Pharmacopeia (AHP) monograph on bilberry fruit noted that historically, bog bilberry (V. uliginosum) and lingonberry (V. vitis-idaea) appeared as adulterants, but that was considered to be rare. Microscopic and macroscopic differentiation of these species from bilberry are included in this 2001 AHP monograph. Microscopic identification of V. myrtillus is also included in an extensive microscopy text by Upton et al. (2001), but without details on microscopic identification of purported adulterants.12

(Editor’s note: A number of methods for detecting bilberry adulteration have been published and some will be discussed here in general and in more detail in a forthcoming “Laboratory Guidance Paper on Bilberry Extract Adulteration.”)

Anthocyanins are ubiquitous compounds in fruits, flowers, and vegetables, often responsible for bright colorations such as reds, blues, and violets. In the 1990s, technical interest in natural colorants grew in response to consumer demand for natural products in general. In the mid-to-late 1990s and early 2000s, a growing body of scientific evidence and subsequent reports in the popular press began to draw more attention to anthocyanins for their potential health benefits as anti-inflammatory agents and antioxidants. Various common foods and beverages — including juices, wines, grapes, berries and vegetables — morphed into functional food products or dietary supplements. Analytical papers were published on the analysis of anthocyanins in various common food and beverage items, but according to Zhang et al., (2004),25 few papers dealt with analysis of anthocyanins in botanical extracts used in the dietary supplement industry. More important, of the growing number of known anthocyanins, now estimated at more than 1,000, fewer than 100 anthocyanin reference compounds — necessary for the accurate chemical analysis in a laboratory — are commercially available.26 Zhang et al. developed an acid hydrolysis-HPLC (high-performance liquid chromatography) method for quantifying the 6 major individual anthocyanidins in bilberry extracts, including pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin.25 A direct HPLC method was deemed useful for verification of raw material origin and standardization, and Zhang’s approach completely separated 5 anthocyanidin aglycones (core compounds without a sugar residue attached), with the exception of petunidin (no reference compound was then available).

Concurrent with the continued commercial and consumer interest in anthocyanin-containing products and their potential health benefits, more refined and perhaps less expensive laboratory analytical refinements are frequently published.

Recently, a Turkish research group published an analytical method for the rapid determination of the 6 most abundant free anthocyanins in foodstuffs using HPLC-DAD (HPLC with diode-array detection).27 The 3-glucoside forms of pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin, using the aglycone cyanidin as an internal standard, could be separated using HPLC-DAD within 18 minutes. The innovation includes a fast-sample preparation method allowing for the direct injection of samples into the analytical equipment (the HPLC column), eliminating the step for chemical extraction. The concentration range of 80-420 ng/mL was demonstrated in 28 different vegetable, fruit, and commercial product samples. The accuracy of the method was stated to be 99.2 ± 0.2% with an average precision of 0.8%. The authors suggest that the method is a robust, lower-cost alternative to previous analytical methods relying on multi-step protocols of sample treatment. Developing technical innovations should help laboratories continue to make refinements in accuracy of methods and lowering costs, both of which will contribute to helping to solve adulteration problems.

Despite the advances in accurate identification and quantification of bilberry anthocyanins, by the mid-2000s, Australian researchers published a paper revealing that the method based on the single-wavelength (528 nm) spectrophotometric assay, calculating anthocyanin content based on cyanidin-3-glucoside chloride specific absorbance values as published in the 2004 British Pharmacopoeia — then in common use to determine percentage of anthocyanins in bilberry fruit extracts — yielded false-positive results in the presence of intentional adulteration.15 Therefore, the simple detection method published in the British Pharmacopoeia was not adequate to detect deliberate or accidental adulteration. The AHP monograph12 (on bilberry) fruit warned that the same spectrophotometric assay, calculating total anthocyanin content as cyanidin-3-glucoside, was useful only if appropriate methods had assured authenticity and purity of the source material prior to chemical analysis. The AHP monograph further notes the inability of the method to detect intentional adulteration with added colorants including FD&C Red, cochineal (a natural red coloring derived from a small insect residing on species of prickly pear cacti, Opuntia spp., Cactaceae), or powdered beet (Beta vulgaris, Chenopodiaceae). The herb and natural products industry had been alerted.

The study by Penman et al. (2006) also revealed that one extract obtained from China through an Australian distributor, which claimed to be a bilberry standardized dry extract powder with 25% anthocyanins, had a total measured anthocyanin content of 24% when analyzed using the simple spectrophotometric method from the 2004 British Pharmacopoeia.15 When the same extract was analyzed with a more sophisticated HPLC method, only 9% anthocyanins were found. Further testing by HPLC, mass spectroscopy (MS), and nuclear magnetic resonance (NMR) confirmed that the “bilberry powdered extract” from China was adulterated with the napththylazo sulfonic acid dye known as amaranth dye (as noted above, not to be confused with plant members of the genus Amaranthus). Amaranth dye, [3-hydroxy-4-[(4-sulfo-1-naphthalenyl)azo-]2,7-naphthalenedisulfonic acid trisodium salt], also known as the coloring agent FD&C Red No. 2, or, more commonly, as Red Dye No. 2, was banned by FDA in 1976 due to its suspected carcinogenicity.15 (The paper by Penman et al. was reported in the natural products industry trade literature in the United States.28

In 2007, scientists at Indena SpA developed and validated a new liquid chromatography method for measuring anthocyanins and anthocyanidins in dried, powdered extract of fresh bilberry fruit and in 40 commercial bilberry extract products representing 24 different brands.21 This method, which measures free anthocyanins that are often associated with poor product quality, was modified in a relatively minor fashion (e.g., removing the molecular weight correction for the content calculation, use of primary or secondary references), and has been adopted as the official analytical method for bilberry by the European Pharmacopoeia (EP).29

The EP started working on a bilberry fruit dry extract in 2005 with a proposal in Pharmeuropa, which became official in 2008 and was published in 2010. The monograph describes an authentication method by thin-layer chromatography (TLC) and an identification test by HPLC based on EP reference standards.29

USP 35/National Formulary 30 (2012) authentication method for bilberry powdered extract is a TLC identification test, based on USP Reference Standards.30

In the United States, the herb industry has formally recognized the adulteration of commercial bilberry extracts. AHPA provides guidance to its member companies on the proper identification and authentication of bilberry.31 In 2007, AHPA published a press release and update to its members regarding the adulteration of bilberry extract with Red Dye No. 2 (azo dye).32 According to the AHPA release, 2 methods of analyses were being posted to the AHPA website for members’ access and utilization: “One method is a fairly simple procedure of raising the pH of dilute bilberry extract; the resulting color change from red to blue indicates the presence of anthocyanins. The other method utilizes high-performance thin-layer chromatography (HPTLC) to provide a visual image that separates anthocyanins from amaranth dye that has been discovered as an adulterant in some powdered material labeled as bilberry extract.”33


The AHP monograph on bilberry fruit, although somewhat dated, contains nearly all of the information necessary for scientific validation of authentic bilberry supply sources.12 In addition, the analytical methods cited in this paper, including Cassinese et al., 2007;17 Pace et al., 2010 (Indena, SpA);21 Zhang et al., 2004 (Nature’s Sunshine Products);25 and Penman et al., 2006 (MediHerb);15 were all created by industry analytical labs in association with academic colleagues in an effort to solve the problem of bilberry adulteration, discovered through routine vetting of raw material suppliers. The problem could be solved with relative ease if companies offering retail consumer products comply with appropriate current Good Manufacturing Practices as required by law in the United States and many other countries.

The intentional, illegal adulteration of bilberry (V. myrtillus) extracts with synthetic, potentially dangerous, and banned dye materials, as well as ubiquitous fraudulent ingredients such as charcoal and other lower-cost anthocyanin-containing fruits creates problems for the natural products industry worldwide, in addition to eroding consumer confidence in bilberry itself and the herb and dietary supplement industry in general. The intermingling of species of Vaccinium as a “type” of bilberry because of linguistic confusion or purposeful language adulteration to enhance sales further complicates the matter. Various producers of authentic bilberry raw material and products, AHPA, non-governmental bodies producing authentication methods and monographs, and the academic community have taken the lead in helping to solve the problem of economic adulteration of bilberry.

Editor’s note: An expert reviewer of this article noted that it may be inappropriate to compare the values of any compound via UV and HPLC and suggest that one is more “accurate” than the other. As stated in the endnote on the previous page, it depends on the analytical endpoint. If the goal is to calculate total anthocyanidins, which is the case in the analysis of bilberry extract, and those include all known and possibly unknown similarly related compounds for which analytical reference compounds are unavailable, then UV is a better method. If the goal is quantitation of a few specific anthocyanidins for which analytical reference markers are available, and the analyst wants only to quantify those particular (not total) anthocyanidins, then HPLC is more accurate. The quantitation of bilberry anthocyanidins initially began with UV calculation of the compounds. Commercial interest moved the analysis to HPLC to detect adulteration, per the focus of this article. That does not make the use of a UV method inappropriate. The analytical goal has to match the nature of the method being used. The reason UV may be a superior method for quantitation in many cases is that not all compounds associated with activity in plant-based medicinal preparations are known, so general methods like UV can capture a range of compounds. UV is also a faster and less expensive method than HPLC, which can capture the presence of all compounds but takes more time, and it is much more expensive to utilize all the reference compounds. Most companies promoting the use of HPLC do it as a marketing tool because of the more distinct and accurate detection; one will rarely obtain an HPLC value to match a UV-determined value of 25% anthocyanins (the original standard applied to bilberry worldwide and what most clinical studies were based on). There is almost always a huge disparity because HPLC is calculating only a few analytes (according to the reviewer) and UV captures a broader range of compounds.

UV can be useful and applicable for these analyses if all other compendial standards are met, especially if the analytical standards for identity of the raw material are properly employed. However, in this context, the situation may be described as an effort to defeat UV analysis by adulteration with added anthocyanins/anthocyanidins from other, less-expensive sources. Differentiation of UV and HPLC is important because reliance on UV alone exposes a manufacturer (or consumer) to a greater risk of adulteration. It is possible that a less-than-scrupulous manufacturer can purchase bilberry (in this case) raw material that is authentic, then dilute it, and add anthocyanins from other, lower-cost sources. If this were done, the compendial (e.g., pharmacopeial) standards for identity of the material can be met, as well as the UV standards, but the resulting (adulterated) extract is not a true, legitimate bilberry extract.


*Definition of anthocyanin, anthocyanidin, and anthocyanoside: (From Greek anthos [flower] and kyanos [dark blue]). Chemically, anthocyanins are phenolic compounds of flavonoid structure and an attached glucose (sugar) moiety, and anthocyanidins are anthocyanin counterparts without an attached glucoside group. These plant colorants are responsible for the red, purple, and blue hues in many fruits, vegetables, cereal grains, and flowers and have been counted as having up to 600-plus molecular structures.34 Some sources claim there are over 1,000 such structures. Anthocyanoside is a synonym of anthocyanin.

Anthocyanidins are present in low quantities in fresh bilberry fruits and in Indena’s Mirtoselect (at levels less than 1%); they “are anthocyanins without the sugar moiety and should be considered anthocyanin degradation products occurring when there has been incorrect extract production and/or storage. Anthocyanidins are rare in nature and the metabolism of the anthocyanins produces only trace amounts of bioavailable anthocyanidins.”34

It is worth clarifying the limits noted for all UV methods. A standard HPTLC and HPLC analysis also can be fooled if an analyst does not know what to look for in terms of ratios of the detected compounds. It is important to note that the inclusion of a pharmacopeial method in a quality control monograph means that all the identity and quantitative tests on the investigated botanical material must conform with the monograph. Thus, the bilberries would first have to have been properly identified by one of the identity tests given in the monograph. UV methods are not listed in any pharmacopeia to confirm identity. The application of a particular method (UV versus HPLC) is dependent on the analytical endpoint.




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