Issue:
96
Page: 64-73
The Adulteration of Commercial Bilberry Extracts
by Steven Foster, Mark Blumenthal
HerbalGram.
2012; American Botanical Council
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.
Background
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 USD
per 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
Conclusions
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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