Mannino G, Di Stefano V, Lauria A, Pitonzo R, Gentile C. Vaccinium macrocarpon (cranberry)-based dietary supplements: variation in mass uniformity, proanthocyanidin dosage and anthocyanin profile demonstrates quality control standard needed. Nutrients. April 3, 2020;12(4). pii: E992. doi: 10.3390/nu12040992.
Sales of dietary supplements (DS) in Europe are ~€7 billion annually, with Italy leading the market in plant-based DS. European regulatory and monitoring systems for DS have been criticized as weak and ineffective, in part because the quality control requirements in food and drug manufacturing are not mandatory for DS. The European Commission regulates the market of food, including fortified and functional foods. According to the legislation, information on food derivation and origin must be "unblemished and unquestionable” for the consumer. DS, however, often resemble pharmaceutical drugs, with similar unit-doses and dosages. Sold in pharmacies alongside drugs, misuse of DS may result in overdosing. Some DS fail to declare all active substances, plant parts, and/or amounts of active substances on their label. Further, misidentification or adulteration of plant materials has been disclosed. Furthermore, contamination with environmental chemicals, mycotoxins, and harmful microorganisms has been reported. Consumers perceive DS as “natural” and consequently presume they are safe. Therefore, validating the safety and quality of DS is important.
Cranberry (Vaccinium macrocarpon, Ericaceae) DS share a health claim to support the well-being of the urinary tract. It is reported that 36 mg/d cranberry proanthocyanidins (PAs), as per Brunswick Laboratories 4-dimethylaminocinnamaldehyde (BL-DMAC) assay, is sufficient for significant benefits due to PAs' antiadhesion activity against P-fimbriated Escherichia coli. The authors analyzed 24 best-selling European cranberry DS for mass uniformity, total PA content (tPAC), total anthocyanin (AC) content (tAC), and actual cranberry content.
Mass uniformity was measured by individually weighing > 20 dose units from each of the 24 DS. Average mass and standard deviation were calculated. Under European Pharmacopeia (EP) criteria, DS met the standard if no more than one unit differed by > 5% (for tablets) or > 7.5% (for capsules and sachets) from the average. They failed to comply if more than one unit was outside the limits of 90-110% (for tablets) or 85-115% (for capsules and sachets) of average mass. Of the 24 DS tested, seven had more than one unit with a mass that differed > 5 or 7.5% from the average, and four had at least one unit outside the limits of 90-110% or 85-115% of average weight. Only 17 DS met EP criteria for mass uniformity; of these, two listed unit weights on their labels that differed "largely" from those measured.
Since efficacy of cranberry DS depends on a determined dose, most of them, including 16 used in this study, claim a specific dose and BL-DMAC verification (eight of the studied DS). BL-DMAC is the most accurate and rapid spectrographic method for determining tPAC, considering all grades of polymerization and excluding the interference of ACs. Extracts were prepared for assay according to the pharmaceutical form of each (tablet, capsule, sachet, or liquid). To rate the completeness of extraction, residues were also assayed. All DS analyzed, regardless of label claims, contained PAs. Of the eight claiming a specific tPAC and BL-DMAC verification, four contained between 95-105% of declared value. One had a content 85% less than the declared amount; three, 15% less. For DS that declared tPAC but no verification and those making no tPAC claim, BL-DMAC was used to determine if the suggested intake provided 36 mg/d PAs. Eight DS met this standard. For others, the suggested posology in the label provided between 0.56 and 12.96 mg/d PAs.
Powdered DS cannot be identified taxonomically; however, chemical markers can identify their original plant material. Cranberry fruit is characterized by a readily measurable level of characteristic ACs. Extracts of the DS were prepared for tAC assay according to the pharmaceutical form of each, as with samples used for tPAC verification. The pH differential method used makes it possible to determine the amount of ACs in a sample while excluding chemical colorants and other red pigments. Five of the 24 DS tested did not contain any ACs. Values for the others ranged from 0.389 ± 0.032 to 21.977 ± 1.713 mg/unit dose. Although not required, an accurate label statement of tAC would be useful for consumers.
Unlike PAs and polyphenols in general, ACs occur in each plant species in a characteristic ratio and chemical pattern of distribution, making them a good choice for detecting product adulteration. A species' qualitative AC profile is unique and remains so even after processing and storage. Chemical fingerprints were obtained via high-performance liquid chromatography (HP-LC) coupled with an ultraviolet visible (UV/Vis) detector. DS with AC profiles not attributable to cranberry were also analyzed via ultra HP-LC (UHP-LC) coupled to a quadrupole Orbitrap-mass spectrometer (MS/MS). All chromatographs were compared to those of authentic cranberry fruits. Cranberries must contain five ACs, including cyanidin-3-O-galactoside (C3Ga), cyanidin-3-O-glucoside (C3Gl), cyanidin-3-O-arabinoside (C3Ar), peonidin-3-O-galactoside (P3Ga), and peonidin-3-O-arabinoside (P3Ar). Moreover, they cannot contain significant amounts of delphinidin, malvidin, or related conjugates as they likely arise from adulteration from non-cranberry sources. The 24 DS analyzed fell into three groups. Under HP-LC/UV/Vis, five produced flat chromatographs; that is, they contained no ACs. Four had AC profiles matching those of cranberry. The remaining 15 samples had AC profiles different from cranberry. UHP-LC/MS/MS confirmed these results except for one in the third group under HP-LC/UV/Vis; UHP-LC/MS/MS detected a low amount of cranberry ACs besides atypical peaks. Non-cranberry AC profiles had delphinidin-glucoside (n = 13), delphinidin-rutinoside (12), petunidin-glucoside (two), and cyanidine-3-O-rutinoside (one) peaks.
Of the 24 "cranberry" DS tested, only one complied with EP criteria for mass uniformity; had an effective, verified tPAC; and contained cranberry ACs. Results argue for a much more vigorous regulation of DS in Europe in the interests of consumer protection for cranberry containing DS. This may also be relevant in blueberry, elderberry and other colored fruit-based DS available in the EU.