Marketplace Analysis Demonstrates Quality Control Standards Needed for Black Raspberry Dietary Supplements

There is currently no standard for the minimum anthocyanin concentration a black raspberry dietary supplement must contain for legal sale in the US. All consumer available black raspberry products (n = 19), packaged as dietary supplements or otherwise prepared (freeze-dried whole and pre-ground powders), were purchased and analyzed for their anthocyanin composition and concentration. Seven of the 19 samples contained no anthocyanins from black raspberry fruit, while three of those seven (without black raspberry fruit) had no anthocyanins of any kind. There was a wide range of anthocyanin concentration within the remaining products (18.1–2,904.8 mg/100 g; n = 12). When expressed as per capsule or per ∼1 teaspoon, concentration ranged from 0.1 to 145.2 mg (average 28 mg; n = 12). Until US dietary supplement labeling comes under regulatory oversight similar to food guidelines, foods are a more dependable source for dietary phenolics than supplements.


Introduction
The USDA (United States Department of Agriculture) has put together several campaigns advocating a healthy eating diet by having one that is color diverse (USDA 5 A Day campaign, USDA ChooseMyPlate.gov, HealthierUS School Challenge-HUSSC, etc.). As consumers have become increasingly aware of the benefits from eating healthier, sales of black raspberry supplements have also increased, and were further raised after a popular US media program promoted black raspberry consumption for its cancer fighting potential. This popular television show made the assertion that an adult should consume 600 mg of anthocyanins from black raspberry dietary supplements daily (300 mg twice a day), followed by the unreasonable claim that each 300 mg capsule (featured product) contained the equivalent content of four cups of fresh black raspberries. Two samples linked with the group making these statements were examined in this study, and are discussed later. Although the potential health benefits of black raspberry fruit and its specific mechanisms are still under investigation [1][2][3], unwitting consumer demand has increased the availability of products described as black raspberry supplements in the dietary supplement marketplace.
Black raspberry (Rubus occidentalis L.; native to Eastern North America) fruit has been traditionally used as a food and a natural colorant, but renewed US consumer interest has brought an upsurge in the number of commercial black raspberry products available (from desserts to dietary supplements; [4]). This can partially be explained by increased awareness of the potential health benefits high-pigmented fruit might provide [1-3, 5, 6], but their distinct flavor, unlike blackberries or red raspberries, may also help. Unfortunate side effects of intensified consumer demand have been occurrences of product adulteration, though some likely arose from the limited production of black raspberries, which due to their unique growing requirements make Oregon the only US state with notable acreage [4].
Black raspberry (Rubus occidentalis L.) has fruit, plant phenology, plant morphology, and anthocyanin profile distinctly different from red raspberry, blackberry, or any other genus Rubus berries [5,[7][8][9]. Since most people are unable to distinguish black raspberries, a simple fact sheet has been generated (www.black-raspberry.com) to help educate researchers, health professionals, industry, and consumers the differences among black raspberry, red raspberry, and blackberry. It is apparent that black raspberry dietary supplement producers and sellers might not be able to differentiate between black raspberry and blackberry, as the images on the supplements' packaging were incorrect in four of the products examined in this study (summarized in Table 1). The inaccurate images used were that of either blackberries or an altered image of blackberries (white core was colored in black). The confusion about black raspberry fruit's correct morphology [8,9], and the sudden boost in available black raspberry products (although the fruit production is limited; [10]), caused us to examine the anthocyanin concentration of marketplace black raspberry dietary supplements, and other forms that can be used as supplements (i.e., freeze-dried black raspberries, extracts).

Materials and Methods
Samples, Reagents, Chemical, and Standards An effort to purchase all commercially available black raspberry supplements and dried fruit (powder and whole fruit forms) were made (n=19) from May to July of 2013 (Amazon.com, Inc., Seattle, WA, USA). No purchased products were past their expiration or best use by date. These samples represented products from 17 companies. Products A09, B01, and B02 were from one company. The rest of the samples were from different companies. Sample information is summarized in Table 1. Dietary supplements in capsules or extract were coded A01 to A15, with one sample (A09) in liquid form. The four available dried fruit products were purchased and coded B01 to B04. One sample was freeze-dried whole fruit (B01); while the remaining dried samples were in powder form. Five capsules and their contents were weighed in triplicate to determine the weight of the powder within the capsules, and to convert our findings into per capsule. Capsule contents of A11 were suspicious (see Table 1) and a second example was purchased to double check that the original product was not random error.
All chemicals, reagents, and standards used in this study were analytical or HPLC grade from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Cyanidin-3-glucoside was purchased from Polyphenols Laboratories AS (Sandnes, Norway).
Extraction and Sample Preparation All powder contents of the capsules were removed and stored at −75°C until extraction. The one example of freeze-dried whole fruit (B01) was ground (using a coffee bean grinder, model K2M2; Braun GmbH, Kronberg, Germany) prior to storage and subsequent extraction. Each sample group's collected powders were pooled and kept frozen until the start of chemical extraction.
Samples were extracted and expressed as-is, since that represented the form of intended consumption. Powders (initially 1.5 g) were extracted with high purity water (initially 15 mL; Millipore Simplicity UV, Millipore Corp., Billerica, MA, USA) by sonication for 15 min, centrifuged 10 min at 4,000 rpm, then filtered (Millipore 0.45 μm Millex-FH syringe filter, Bedford, MA, USA) prior to injection onto the HPLC system [12,13]. Solid to liquid extraction ratio had to be altered for samples that indicated very low to zero levels of anthocyanins (3.0 g powder extracted in 10 mL of water). A09 was diluted (1.5 g:15 mL high purity water; by weight due to its high viscosity) and put through the same process as the other samples (sonication, centrifugation, and filtration) prior to HPLC analysis. 'Munger' fruit extract was obtained from previous study [10,11,14,15].
HPLC (High Performance Liquid Chromatography) Condition for Individual Anthocyanin Separation HPLC/DAD (diode array detector)/MS (mass spectrometry) was used for anthocyanin elution as described in detail in our previous work [13], except for the use of a longer analytical column [8]. Briefly, an Agilent HPLC 1100 (Agilent Technologies Inc., Palo Alto, CA, USA) was used for this investigation. Individual peaks were monitored at 520, 280, and 255 nm. Anthocyanins were expressed as cyanidin-3-glucoside (Polyphenols Laboratories AS, Sandnes, Norway). Anthocyanin peaks were identified by retention time, UV-VIS spectra, external standards (when available), verified fruit with known anthocyanin profiles, and prior published research [8,10,11,14,[16][17][18]. Analyses were conducted in duplicate. Results were expressed as mg of cyanidin-3-glucoside/100 g of powder, mg of cyanidin-3-glucoside/capsule for samples coded A01-A08, or 5 g (∼1 teaspoon) for samples coded A09 and B01-B03. Peaks 2 and 3 peak areas were split at each apex, where it was not co-eluting, and multiplied by two to obtain total peak area prior to calculations.

Results and Discussion
Relevant information from packaging, prices, and observations are summarized in Table 1; including the labeled contents from the products that were found to contain no black raspberry fruit. Photo examples of six dietary supplements can be found in Fig. 1. Supplements ranged in visual color from light pink to dark red, except for A11 capsules (see Fig. 1.e) that contained no red hue. An example of no filler added in the capsule contents ( Fig. 1.a) compared to high amounts of filler used in Fig. 1.b through Fig. 1.d. Red appearance cannot be used to indicate the presence of black raspberry fruit since some of the manufacturers used pink colored fillers (A10 and A12) and other fruit powder (A12, A14, A15, and B04). Contained compounds that absorb at 520 nm, but not black raspberry anthocyanins (see Fig. 2.). One side of the bag was transparent. This powder had a different hue than samples B02 and B03 (appeared to be not as intense red-purple).
Seven (A10-A15 and B04) of the 19 samples had no black raspberry anthocyanins. Out of those seven, A10, A11, and A13 contained no 520 nm absorbing compounds (chromatogram of A10 as an example in Fig. 2). Samples A12, A14, A15, and B04 contained 520 nm absorbing peaks, but their profiles did not match that of black raspberry (examples in Fig. 2.a and b). One anthocyanin profile, B04 from four freeze-dried samples (which were the B coded samples), did Fig. 1 Photos of six black raspberry dietary supplements purchased. In photo example b through d, contained high amounts of filler. In photo example e (coded A11 in Table 1), contained no anthocyanin (no black raspberry fruit) Fig. 2 Anthocyanin profiles of black raspberries (a-'Munger' and b-A09) and selected supplement samples (c-A10, d-A12, e-A14, and f-B04) that contained questionable materials other than black raspberry fruit. The chromatograms were monitored at 520 nm (280 and 255 nm traces not shown). Clearly, black raspberry anthocyanin profile is suitable for authenticity work (comparing 'Munger' to sample A09, leaves no doubt that A09 contains black raspberry). Corresponding peak identification for 'Munger' and A09 listed in Table 2. Additional black raspberry anthocyanin profiles can be found in our past work [7,8,10,11] not match black raspberry either (see Fig. 2). We suspect B04 was blackberry (Rubus spp.; [5,19]) freeze-dried powder and sold as black raspberry. Hydrochloric acid was used to confirm that A10, A11, and A13 contained no anthocyanins, as a color shift (redness due to oxonium formation) after acidification would also indicate the presence of anthocyanin [20][21][22], but this visual change did not occur in these three extracts. Some example chromatograms of these questionable materials are shown in Fig. 2 (c through f). Our second purchased A11 sample had an identical appearance (olivebrown-black powder; see Fig. 1.e) and the same HPLC profile (data not shown) as the first A11 sample, again with no detectable 520 nm absorbing peaks. Sample A03's label claimed 550 mg of black raspberry in each capsule, though capsule entire content's weight was measured at 480 mg (Table 1).
A07 and A08 appeared to be pure fruit powders as indicated by their uniform dark red-purple powders (whole black raspberry seeds present) and high anthocyanin levels (A07-1052.4 and A08-1,138.8 mg/100 g). Based on observing A07 and A08, it would be possible to fill capsules without bulking Table 2 Individual anthocyanin concentrations are listed in the order of HPLC elution (peak 1: cyanidin-3-sambubioside, peak 2: cyanidin-3xylosylrutinoside, peak 3: cyanidin-3-glucoside, peak 4: cyanidin-3-rutinoside, peak 5: pelargonidin-3-glucoside, peak 6: pelargonidin-3rutinoside, and peak 7: peonidin-3-rutinoside) Total expressed as mg of cyanidin-3-glucoside/100 g and mg/capsule or 5 g (∼1 teaspoon). Values within parenthesis indicate standard errors. Seven samples (A10, A11, A12, A13, A14, A15, and B04) either contain no detectable anthocyanins or no black raspberry anthocyanins (see Table 1) a A01-A08 were expressed as per capsule, A09 and B01-B03 as per ∼1 teaspoon (5 g) b nd, not detected agents and fillers as seen in the lower quality capsule samples. The highest anthocyanin concentration was from A09 (the liquid example) at 2,904.8 mg/100 g. If one consumed two capsules from sample A08 or A09, they ingested only ∼7.0 mg of anthocyanins. Samples B01, B02, and B03 if packaged as capsules, would provide amounts similar to A07 and A08, since those two capsule products contained 100 % freeze-dried berries. Samples A04 and A11 are linked to the unnamed group mentioned in the introduction. A04 contained 0.2 mg/capsule, while A11 contained the mysterious olive-brown-black powder with a strange medicinal odor. A04 contained ingredients other than fruit powder that were not listed on the label (Table 1). In fact, three samples (A04, A06, and A11) did not indicate additional ingredients in capsules, but based on the visual appearance and HPLC anthocyanin results it is clear they did contain substances other than black raspberry fruit powder.
Using anthocyanin profile for identifying food and dietary supplement adulteration is not a new concept and has been demonstrated before [7,8,24,25]. Misidentification of plant source material is a known issue in the US dietary/herbal supplement industry [26][27][28][29] and it is an obvious problem that needs to be corrected. It is possible that due to supply demand that the dietary supplement companies were fraudulent, or made honest mistakes from not testing constituents prior to production. Some issues surrounding dietary supplements can be resolved by improved dietary supplement regulations, and by endorsing proposed rules [30][31][32][33][34] for the safety of US consumers before another "ephedrine" scale incident occur [29].

Conclusion
While there are companies (four companies herein) that provide consumers with high black raspberry anthocyanin containing products (A07, A08, A09, B01, B02, and B03), the majority (>70 %) of companies are selling low quality products, some containing unknown/unreported ingredients and very little black raspberry fruit. Until US dietary supplement products are better regulated and quality control standards for safety, purity, and dosage are defined and endorsed, the safer source for dietary phenolics as a consumer is from food intake [6]. From past research [24,35,36] and findings herein, there is a real need to create standards for dietary supplements made from plant sources. At the moment, a consumer who assumes the US dietary supplement marketplace is free from risk is unfortunately naive. Forty percent (seven out of 19) of the black raspberry supplements and products purchased and evaluated here contained no black raspberry fruit anthocyanin.