aAccording
to SPINS (SPINS
does not track Whole Foods Market sales, which is a major natural products
retailer in the US)
bAccording to SPINS/IRI
(the Mainstream Multi-Outlet channel was formerly known as food, drug and mass
market channel [FDM], exclusive of possible sales at Walmart from 2013-2015)
cn/a: not available
Source:
Smith T [American
Botanical Council] email communications, September 2-3 2015; K. Kawa [SPINS]
email communication, July 11, 2016.
2.2
Supply sources: Grapefruit seeds are abundantly
available as byproducts of the citrus juice industry.
2.3
Raw material forms: Whole or ground dried seeds; in some
instances, ground peel and pulp are also included.3,4
3
Adulteration
3.1
Known adulterants: The secondary metabolites of
grapefruit seeds are predominately limonoids and flavonoids. Naringin, a
diglycoside of the common flavanol naringenin, is the dominant flavonoid in
grapefruit seeds.
Limonoids are
a unique subset of triterpenes in which the conventional triterpene skeleton is
significantly oxidized and cleaved in one or more places. Seven limonoids,
along with seven limonoid glycosides (the glycosides have one or several sugars
attached to the triterpene core), have been reported from grapefruit seeds.9-14
Limonin, the most abundant grapefruit seed limonoid, comprises ~0.5% of the dry
weight of the seeds,15 and the total limonoid content could approach
1%.16
It is noteworthy that none of the
published analyses of commercial grapefruit seed extracts has indicated the
presence of either limonoids or flavonoids in those products, except for the
most recent paper in the series,4 even though limonoid glycosides
have been isolated from citrus seeds extracted with aqueous acid in the
presence of pectinase.17-19 Instead, a series of analyses of
commercial GFSE products over a span of two-and-a-half decades has revealed the
presence of a number of synthetic microbicides and preservatives, including
triclosan, methyl and propyl parabens, benzalkonium chloride, benzethonium
chloride, cetrimonium bromide, and decyl trimethylammonium chloride.
Any commercially available quaternary
ammonium salt with at least one lipophilic (hydrophobic) ring or chain could
conceivably be used as a microbicide in purported GFSE products. This class of
compounds exerts its considerable microbicidal effect by lysing (breaking down
or disintegrating) cell membranes.
Manufacturers have claimed that a
proprietary extraction process, involving treatment/reactions with ascorbic and
hydrochloric acids and ammonium chloride, produces compounds similar to the
known antimicrobial quaternary amine compound benzalkonium chloride,3
although no rational organic chemistry mechanism has been provided for this
non-credible purported transformation. Further, the compounds in question are
not just similar to benzalkonium chloride or benzethonium chloride; researchers
have isolated the compounds in question from commercial products and, using
authentic reference standards, have shown them by high-performance liquid
chromatography (HPLC) retention times and ultraviolet, nuclear magnetic
resonance (NMR), and mass spectra to be identical to those authentic reference
materials.20-23
3.2
Sources of information supporting
confirmation of adulteration: A
series of 13 publications over 25 years comprises the evidence for adulteration
of GFSE. A variety of analytical methods were utilized in studies emanating
from Japan, European countries, and the United States. The results show a consistent
pattern of adulteration; moreover, the results also show that after the first
two of the aforementioned publications appeared, the synthetic microbicides
present began to shift. Table 1 summarizes the chronological record of the
appearance of the various GFSE adulterant microbicides.
Table 2. Time sequence of the detection of adulterants
in GFSE products
Year, First Author Adulterants
|
|
1a
|
2a
|
3a
|
4a
|
5a
|
6a
|
7a
|
|
1991 Nishina et al.24
|
√
|
√
|
|
|
|
|
|
|
1996 Sakamoto et al.25
|
√
|
√
|
|
|
|
|
|
|
1999 von Woedtke et al.26
|
√
|
√
|
|
√
|
|
|
|
|
2001 Takeoka et al.20
|
|
|
|
√
|
|
|
|
|
2001 Terreaux et al.27
|
|
|
|
√
|
√
|
|
|
|
2004 Spitaler et al.28
|
√
|
√
|
√
|
√
|
√
|
|
|
|
2005 Takeoka et al.21
|
|
|
|
|
√
|
|
|
|
2006 Ganzera et al.22
|
|
√
|
√
|
√
|
√
|
|
|
|
2007 Avula et al.29
|
√
|
|
|
√
|
|
|
|
|
2007 Spinosi et al.30
|
|
|
|
√
|
|
√
|
√
|
|
2008 Sugimoto et al.31
|
|
|
|
√
|
√
|
|
|
|
2008 Bekiroglu et al.23
|
|
|
|
√
|
|
|
|
|
2016 Avula et al.4
|
|
|
|
√
|
|
|
|
a1 – triclosan; 2 – methyl paraben; 3 –
propyl paraben; 4 – benzethonium
chloride; 5 – benzalkonium chloride;
6 – cetrimonium bromide; 7 – decyltrimethyl ammonium chloride
3.3 Frequency of adulteration: A sense of the frequency of
adulteration can be gained by looking at the results reported in the 13 articles
listed in Table 2. Triclosan was reported in nine out of 21 samples analyzed in
five different studies. Parabens were found in six of 15 samples analyzed in four
different studies. Benzethonium chloride was detected in 27 of 46 samples
analyzed in six of the reports, while benzalkonium chloride was found in nine
of 15 samples analyzed in four reports. These are very high frequencies of
adulteration, even if one considers the relatively modest number of ingredient
suppliers of GFSE and the history of changing adulterants. An important additional
consideration is that, in cases where researchers compared antimicrobial or
microbicidal activity to the presence or absence of these synthetic
adulterants, such activity was observed only in those products containing one
or more of the listed adulterants.26
3.4
Possible safety issues: Triclosan is a long-used
disinfectant, labeled for external use only in the United States, found in hand
soaps and cleaning products such as dish detergents. Recent studies indicating
disruption of hormone regulation in animal models and possible impact on the
immune system have prompted the US Food and Drug Administration (FDA) to
initiate a new review of the safety and regulatory status of this compound.32,33
In September 2016, the FDA issued a rule effectively banning the use of
triclosan in hand soaps due to a lack of perceived benefits over regular hand
soap, and the above-mentioned safety concerns.34 However, it is
still approved by the FDA as an OTC drug ingredient in combination with a copolymer
in toothpaste to reduce gingivitis.
Benzethonium chloride is a broad-spectrum
microbicide, effective against many species of bacteria, fungi, and yeasts. The
FDA specifies that a safe and effective concentration in first aid treatments
is 0.1-0.2%. Benzethonium chloride is not approved for use as a food additive
in the United States or Europe.
Benzalkonium chloride is a skin, eye,
and mucosal membrane irritant, with a 0.1% solution regarded as the highest
concentration not eliciting an irritant response in skin, while eye drops use
concentrations between 0.004 and 0.01%. Benzalkonium chloride is classified
as a Category III antiseptic active non-prescription (over-the-counter) drug ingredient
by the FDA. Ingredients are placed in Category III when "available data
are insufficient to classify as safe and effective, and further testing is
required.” Benzalkonium chloride and benzethonium chloride were not included in
the recently published (2013) FDA rule on the safety and effectiveness of
consumer antiseptics and topical antimicrobial over-the-counter drug products;
thus, for now they will remain Category III ingredients.33
3.5 Analytical methods to detect adulteration: A variety of analytical methods have
been used in the analysis of GFSE products for the presence of adulterants: thin-layer
chromatography (TLC);26 high performance liquid
chromatography-ultraviolet spectroscopy (HPLC-UV);25-27 high performance
liquid chromatography-mass spectrometry (HPLC-MS);22,25,28,31 gas chromatography-mass
spectrometry (GC-MS);30 and proton nuclear magnetic resonance spectroscopy
(1H-NMR).21,23 Due to the continuing change in the
composition and content of microbicidal compounds in commercial products labeled
as GFSE over time (see Table 2), none of the published methods has been
evaluated for the detection and quantification of all the known adulterants of
GFSE. The HPLC-UV method of Avula et al.29 and the HPLC-UV-MS method
of Ganzera et al.22 have been validated, making them attractive
methods to develop further for all the potential adulterants. The very recent
paper by Avula et al.4 improves on those two methods, in that the
UHPLC-UV-MS method has been modified to resolve and identify not only the
suspected adulterant microbicides, but also the limonoids and flavonoids
expected in a true extract of grapefruit seed. Thus, one can now look at a
product labeled as GFSE with one analytical method to examine whether it is
made from grapefruit seeds or other citrus (lemon and orange seeds are
abundantly available as byproducts of the juice industry) and/or whether it contains
any adulterating synthetic microbicides.
It should be noted that cetrimonium
bromide and decyl trimethylammonium chloride (detected by Spinosi et al.30)
do not contain a UV chromophore. Therefore, a mass spectrometry-based method
might be the most appropriate approach for analyzing GFSE products for any of
the known or suspected adulterants.4,22,30 Fortunately, samples of
all the potential adulterants are commercially available, facilitating
development and validation of analytical methods.
For additional details on the
adulteration of GFSE, readers can refer to the Botanical Adulterant Program’s original
review on this topic in HerbalGram in
2012.3
4 Conclusions
The 13
published analyses of so-called “grapefruit seed extract” (GFSE) products,
conducted and reported over the course of 25 years, revealed that a
consistently high percentage of the tested materials were adulterated with
synthetic microbicidal compounds. Further, during that time period, the
specific microbicides that were detected changed, as illustrated in Table 1. A
variety of analytical methods have been developed for the purpose of
identifying and/or quantifying adulterant microbicides in GFSE and can be
relatively easily adapted for use by commercial and in-house industry laboratories.
5 References
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Tucker AO. American Herbal Products
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3, Cardellina JH. Adulteration of
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disinfectant compounds. HerbalGram.
2012;94:62-66.
4. Avula B, Sagi S, Wang Y-H, Wang M, Gafner S, Manthey
JA, Khan IA. Liquid chromatography-electrospray ionization mass spectrometry
analysis of limonoids and flavonoids in seeds of grapefruits, other citrus
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7. Harich J, Biocidal Compositions and Their Method of
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8. Caldecott T.
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RD, Hasegawa S. 7α-Oxygenated limonoids from the Rutaceae. Phytochemistry. 1982;21:2349-2354.
11. Hasegawa S, Bennett RD,
Herman Z, Fong CH, Ou P. Limonoid glycosides in citrus. Phytochemistry. 1989;28:1717-1720.
12. Bennett RD, Hasegawa S, Herman Z. Glucosides of
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13. Jayaprakasha GK, Brodbelt JS, Bhat NG, Patil BS.
Rapid methods for the separation of bioactive compounds from citrus. Presented at:
228th ACS National Meeting; August 2004; Philadelphia, PA.
14. Jayaprakasha G, Patil B, Bhat N. Process for the isolation of limonoid glucosides from citrus. US Patent App. 11/696,845,200, 2007.
15. Yu J, Dandekar DV, Toledo RT, Singh RK, Patil BS.
Supercritical fluid extraction of limonoids and naringin from grapefruit (Citrus paradisi Macf.) seeds. Food Chem. 2007;105:1026–1031.
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grapefruit seed extracts. J Agric Food
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21. Takeoka GR, Dao LT,
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grapefruit seed extracts. J Agric Food
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22. Ganzera M, Aberham
A, Stuppner H. Development and validation of an HPLC/UV/MS method for
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23. Bekiroglu S, Myrberg
O, Ostman K, Ek M, Arvidsson T, Rundlöf, T, Hakkarainen B. Validation of a
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“grapefruit seed extract” by HPLC and LC/MS. Eisei Shikenjo Hokoku (Bull Natl Inst Health Sci.). 1996;114:38-42.
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Schlüter B, Pflegel P, Lindequist U, Jülich W-D. Aspects of the antimicrobial
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27. Terreaux C,
Chevalley I, Hostettmann K, Grapefruit seed extract: a natural antibiotic? Schweizer Apotheker Zeitung. 2001;24:823-825.
28. Spitaler R, Marschall K,
Zidorn C, Markus K, Zelger R, Stuppner H. Apple scab control with grapefruit seed extract: no alternative to
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29. Avula B, Dentali S,
Khan IA. Simultaneous identification and quantification by liquid
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commercial products labeled as grapefruit seed extract. Pharmazie. 2007;62:593-596.
30. Spinosi V, Semprini
P, Langella V. Presence of chemical additives and microbial inhibition capacity
in grapefruit seed extracts used in apiculture. Veterinaria Italiana. 2007;43:109-113.
31. Sugimoto N, Tada A,
Kuroyanagi M, Yoneda Y, Yun YS, Kunugi A, Sato K, Yamazaki T, Tanamoto K.
Survey of synthetic disinfectants in grapefruit seed extract and its compounded
products. Shokuhin Eiseigaku Zasshi.
2008;49:56-62.
32. Food and Drug
Administration. Triclosan: What Consumers Should Know. Available at: http://www.fda.gov/ForConsumers/ConsumerUpdates/ucm205999.htm, accessed December 28, 2015.
33. US Food and Drug
Administration. Safety and Effectiveness of Consumer Antiseptics. The Federal Register.
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Accessed November 14, 2016.
Revision summary
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