CHAPTER 58 Toxic Contamination of Nutraceuticals and Food Ingredients Fernando Gil, Antonio F. Hernández and M. Concepción Martín-Domingo

INTRODUCTION AND BACKGROUND ingredients (NFI) consist of dietary supplements (e.g., vitamins, minerals, co-enzyme Q, carnitine), herbal Medicinal plants have been used therapeutically products (e.g., flavonoids, Ginseng, Ginkgo biloba, Saint throughout the world as tools of traditional medicine, John’s wort, Saw Palmetto), and bioengineered and pro- from Ayurveda to Chinese traditional medicine. In west- cessed foods (e.g., probiotics, omega-3, fish oil, trans- ern countries, despite the development and production genic plants) purported to benefit health DeFelice,( 1995; of synthetic medicines, the popularity of over the counter Pirotta et al., 2000; NNC, 2014). health foods, nutraceuticals, and medicinal products Lockwood (2007) defines nutraceutical as a term used from plants or any other natural source has recently to describe a medicinal or nutritional component that increased because of the belief that they could be more includes a food, plant, or naturally occurring material effective than conventional therapies for preventing or that may have been purified or concentrated and that treating diseases. This attitude indirectly indicates the is used for the improvement of health by preventing or lack of confidence of the general public in conventional treating a disease. Dolan et al. (2003) considered the term medical treatments. “dietary supplement” to describe a product that contains Several reasons may account for the worldwide one or more of the following ingredients: vitamins; min- growing use of nutraceuticals and dietary supplements, erals; herbs or other botanicals, amino acids; dietary sub- among them being the lower rate of adverse effects as stances used as diet supplements to increase the total compared to conventional drugs and the higher costs of daily intake; or concentrates, metabolites, constituents, many traditional pharmaceutical formulations (Meena extracts, or combinations of these ingredients. The Dietary et al., 2010; Rao et al., 2011; Martínez-Domínguez et al., Supplement Health and Education Act (DSHEA) defines 2014). nutraceuticals as a dietary supplement that may contain Consumers increasingly rely on dietary supplements an herb or other botanical, or a concentrate, metabolite, to maintain mental acuity and to overcome age-related constituent, extract, or combination of any ingredient problems, such as menopause, benign prostate hypertro- from the other categories (Frankos et al., 2010). However, phy, and elevated blood pressure and cholesterol levels, toxic contamination of NFI during any stage of produc- and also to relieve stress (Raman et al., 2004). tion can lead to changes in their quality and safety. The The term nutraceutical was coined by DeFelice to health hazard of these products largely depends on the define any substance that may be considered a food presence of unusually high concentrations of chemical or part of a food providing medical or health benefits. ingredients that may result in toxicity or even fatality if Nutraceuticals may be useful for the prevention and they are consumed (Chan, 2003). treatment of disease or as an alternative to nonconven- Furthermore, the consumption of NFI contaminated tional medicine in primary health care. They include with environmental pollutants by vulnerable subgroups herbal medicines (Ayurvedic, Chinese, Tibetan, African, of the population, such as pregnant women, children, Amazonian, Herbalism), naturopathy, vitamin and min- and elderly adults may become a serious problem if the eral therapy, and homeopathy. Nutraceuticals and food tolerable level of exposure is exceeded. For example,

Nutraceuticals. DOI: http://dx.doi.org/10.1016/B978-0-12-802147-7.00058-9 825 © 20122016 Elsevier Inc. All rights reserved. 826 58. Toxic Contamination of Nutraceuticals and Food Ingredients children and toddlers have greater susceptibility to poi- certain NFI, including herbal medicines and raw herbs, soning by heavy metals and metalloids, as opposed to are quite general and only related to microbiological adults because of their lower body weight, resulting in properties and registration of items themselves. In turn, higher dosage of these substances. there are no specific guidelines for active constituents or Risk assessment paradigms might underestimate the toxic contaminants (Cooper et al., 2007). effects of these chemicals on children and the elderly Little information is available on the toxic contamina- (Gomez et al., 2007). For instance, these subpopulations tion of nutraceuticals and dietary supplements except may be more susceptible to adverse effects associated for herbal drugs. Hence, this review is based largely with ingestion of low doses of heavy metals found in on medicinal plants, assuming that they comprise the Hypericum perforatum, commonly known as St. John’s majority of NFI. wort, widely used for the treatment of mild to mod- Herbs can be collected indiscriminately from non- erate forms of depression. Herbal remedies are also cultivated and nonenvironmentally friendly areas by frequently used by pregnant women regardless of the untrained people and placed into the market with- scarce information about their safety during pregnancy out any control. This means that consumers might be and breastfeeding. In addition to the risk of miscarriage exposed to herbal products potentially contaminated from several herbs (e.g., nettle, passionflower, and aloe), with pesticides, heavy metals and metalloids, myco- a number of epidemiologic studies suggest that cer- toxins, or radioactivity, or adulterated with drugs tain herbs (e.g., those rich in unsaturated pyrrolizidine (Figure 58.1). The presence of forbidden pesticides or alkaloids) are associated with embryotoxic or fetotoxic excessive amounts of regulated pesticides and heavy effects (de Smet, 2002; Gurib-Fakim, 2006; Rodriguez- metals depends on the source of herbal materials and Fragoso et al., 2008). whether or not they are grown in a contaminated area. Because potential interactions between pharmaceu- Moreover, chemical toxins may come from unfavorable tical formulations and herbal products (or other NFI) or wrong storage conditions or chemical treatment dur- cannot always be predicted, unexpected effects can ing their storage. In turn, the presence of drugs could be observed because of a change in the magnitude of the be related to unprofessional practices of manufactur- effect of conventional drugs (Shaw et al., 1997). These ers. Ideally, the consumption of NFI products should be types of interactions can be of either a pharmacokinetic strictly controlled and is pertinent to better knowledge or a pharmacodynamic nature (Rodriguez-Fragoso of the levels of different contaminants (specifially heavy et al., 2008). One of the most important pharmacoki- metals and pesticides) in raw materials (Chan, 2003; netic interactions occurs between herbal products and Meena et al., 2010; Harris et al., 2011). drug-metabolizing enzyme systems, particularly the Phytotherapy has a very long tradition and has been cytochrome P450 (CYP450) isoenzymes. This type of inter- popular for centuries. Medicinal plants have a long his- action has great importance in clinical practice because tory of therapeutical use throughout the world and still CYP450 isoenzymes metabolize a large number of drugs constitute an important part of traditional medicine. In and chemicals. Also, important genetic polymorphisms the last quarter of the past century, the therapeutical use of CYP450s have been reported to modify drug disposi- of herbal products has increased in developed countries tion in different populations. For instance, the interac- tion between peppermint oil and some CYP450 isoforms (CYP1A2, CYP2C19, CYP2C9, and CYP3A4) may modify levels of drugs metabolized by these isoforms (Maniacal Heavy metals and Wanwimolruk, 2001; Unger and Frank, 2004). Metalloids Oligoelements Pesticides Good manufacturing practices (GMP) and other legal requirements must be met to avoid adverse reactions Nutraceuticals or product quality deficiencies as a consequence of the and rapid growth of health-related foods and supplements Food (Genuis et al., 2012). Ingredients In the United States, the DSHEA establishes regula- Radioactive tions and limits label claims on dietary supplements. In Mycotoxins Contamination Europe, food supplements are converged by the Directive Adulteration 2002/46/EC and herbal medicinal products are converged and by Directive 2004/27/EC. However, there is no formal Undeclared Chemical legislation regulating nutraceutical products (Gulati and Substances Berry Ottaway, 2006; Martínez-Domínguez et al., 2014). Although conditions for importation of pharmaceutical FIGURE 58.1 Main toxic contaminants in nutraceuticals and food products are rigorous, the guidelines for importation of ingredients.

NUTRACEUTICALS Chemical Contamination in Nutraceuticals and Food Ingredients 827 because of their popularity among consumers and the Although raw materials are typically and supposedly widespread opinion that “natural products” implies free of substances that are currently added to patent “harmless products” and the lack of toxic effects. Their medicines, they could contain relatively low levels of easy accessibility and relatively low cost also have con- toxic compounds, particularly heavy metals and pesti- tributed to an increased use; however, their popularity cides. The majority (>95%) of the samples analyzed by and global expansion have raised concerns about the Harris et al. (2011) contained concentrations of heavy safety and quality of herbal products for public health metals or pesticide residues that were considered of (Arpadjan et al., 2008; Kosalec et al., 2009). negligible concern. Nevertheless, the elevated number Consumers are increasingly careful in choosing com- of samples showing detectable levels of contaminants ponents for their diet and intend to incorporate high makes it advisable to monitor the concentration of heavy nutrient levels into their standard diet, preferably from metals (especially cadmium and chromium) and pesti- natural sources such as plants (Bhat et al., 2010). The cide residues (especially organophosphorus and organo- increased scientific interest and consumer demand have chlorine compounds) in raw materials. promoted the development of herbal products such as However, if medicinal plants are used for infusion NFI. Medicinal plants behave as authentic medicines preparations, then the extractable component of toxic because the chemical substances of which they are compounds would only be available for consumers. formed can have a biological activity in humans. For In this case, the concentration of contaminants (e.g., example, because phenolic acid and flavonoids are natu- heavy metals) dissolved in the infusions should usually ral antioxidants and free radical scavengers, there is a be below safety levels for human consumption (Kalny growing interest in their pharmacological applications et al., 2007). (Choudhury et al., 2006). In many countries traditional herbal preparations can be sold on the market as food supplements, which do CHEMICAL CONTAMINATION not require prior safety evaluation. The safety and ben- IN NUTRACEUTICALS AND efit of plant products are directly related to the qual- FOOD INGREDIENTS ity of the raw materials from which they are derived (Salgueiro et al., 2010). Heavy Metals, Metalloids, and Oligoelements Herbal medicines are widely used in the United States, with approximately one-quarter of adults reporting their The sources of environmental pollution from heavy use for the treatment of a medical illness. Herbs are metals are quite diverse and mainly consist of indus- considered dietary supplements in the United States and trial and traffic emissions, agricultural effluents, batter- therefore are subjected to a very limited form of regula- ies containing cadmium, organic mercury fungicides, tion and oversight. Although herbs are often believed to and lead arsenate as an insecticide. The concentration of be safe, many side effects have been reported, including these contaminants in ecosystems has increased in past direct toxic effects, allergic reactions, toxicity from con- decades as a result of anthropogenic activities. Because taminants, and interactions with drugs and other herbs of the intrinsic toxicity of heavy metals, their presence (Bent and Ko, 2004). Other adverse effects include pos- in the environment may pose a potential threat to terres- sible mutagenicity and mistaken plant identities (Ernst, trial and aquatic biota. In fact, metal pollution adversely 1998). For instance, liver problems have been reported affects the density and diversity of biotic communities, following the use of Chinese herbal medicine for skin including human (Gomez et al., 2007). disorders, there have been allergic reactions to royal jelly The content of essential and trace elements in medici- and propolis, and heavy metal poisoning was found in nal plants vary depending on various factors (Haider the Indian subcontinent from Ayurvedic remedies con- et al., 2004). Among these factors are geoclimatic condi- taminated with these compounds (Shaw et al., 1997). tions, geochemical characteristics of the soil, anthropo- Overall, adverse effects of herbal drugs can be divided genic activities (e.g., chemical industries in the vicinity), into two main groups: intrinsic and extrinsic. The for- plant species (some can selectively accumulate toxic mer includes predictable toxicity, overdose, pharmaco- elements), and the part of the plant used for preparing logical interactions, idiosyncratic reactions (e.g., allergy the herbal medicine. The level to which metals accumu- and anaphylaxis), and delayed effects such as carcino- late in plants is also influenced by the physicochemical genicity and teratogenicity. The extrinsic undesirable properties of the soil where plants grow (characteristics effects largely involve the quality of the herbal medici- of soil or sediments, pH level, exposure period, disper- nal products, which can be challenged by substitution, sion range, and presence or absence of other elements). adulteration, contamination, misidentification, lack of These properties determine the nature of the associa- standardization, and inappropriate labeling (Koh and tion of trace elements with soil components and are Woo, 2000; Mazzanti et al., 2008; Rao et al., 2011). key elements for the bioavailability of metal elements

NUTRACEUTICALS 828 58. Toxic Contamination of Nutraceuticals and Food Ingredients

(Sarma et al., 2011). While plants readily assimilate trace may cause neurological disorders and kidney damage elements through the roots, these compounds can also (Goyer and Clarkson, 2001; Garcia-Rico et al., 2007). be absorbed through the leaves. Rainfall, atmospheric Aluminum (Al) can be accumulated in certain tissues dusts, plant protection agents, and fertilizers are addi- and has been associated with serious health problems, tional sources of trace elements for plants (Łozak et al., such as Alzheimer’s disease (AD), dysfunction of the 2002). blood–brain barrier (BBB), and inhibition of hydroxy- Although some trace metals present in foods play a apatite formation, which may lead to decreased skeletal physiological role as essential compounds and cofactors, mineralization (osteopenia) (Rubio et al., 2012). In addi- contamination or adulteration of NFI with heavy metals, tion, Pb and Hg may reach and cross the placental bar- metalloids, and mineral nutrients (Table 58.1) is a mat- rier and interfere with placental transport systems, thus ter of concern. Although lower concentrations of trace increasing prenatal exposure to these toxic compounds elements have health benefits, higher levels may pose (Gupta, 2009, 2011). Despite these well-known adverse health risks (Bhat et al., 2010). Because of their cumu- effects, the interpretation of total metal concentration lative properties and toxicity, heavy metal concentra- (e.g., all ionic forms) and toxicity should be based on tions could reach levels potentially leading to hazardous analytical results for the more toxic form. effects on human health. The International Agency for Research on Cancer Cadmium (Cd), mercury (Hg), lead (Pb), and arse- (IARC) has classified certain trace elements (As, Sb, Be, nic (As) are nonessential toxic elements of special con- Cd, Cr, Co, Pb, Ni, and V) as potentially carcinogenic cern because of their toxicity even at low concentrations to humans because of their potential to induce DNA (Hsu et al., 2006; Rao et al., 2011). Lead exposure has damage. Thus, it should be noted that it is important to been associated with renal tumors, reduced cognitive monitor the presence of such metal elements in medici- development, and increased blood pressure and cardio- nal plants used as NFI to prevent excessive human expo- vascular toxicity. Cd may induce kidney dysfunction, sures (Sarma et al., 2011). osteomalacia, and reproductive deficiencies. Mercury Medicinal plants are good sources of mineral ele- ments (Özcan et al., 2008) and they might be used as food supplements or for food fortification of new NFI TABLE 58.1 Toxic Contaminants Reported in Nutraceuticals and Food Ingredients with health-promoting properties. Thus, measuring mineral elements and heavy metal concentrations in NFI Trace metals Heavy metals Al, Cd, Cr, Hg, Ni, Pb is important not only from a nutritional point of view Metalloids As but also for the assessment of their quality and safety (Bhat et al., 2010). Oligoelements Cu, Zn, Mo, Se In contrast to the extensive research performed on Pesticides Organophosphorus Chlorpyrifos, methyl- nutrient elements to define their role in the human diet, pesticides chlorpyrifos, coumaphos, diazinon, dichlorvos, studies reporting the mineral content of NFI (e.g., spices dimethoate, ethion, and herbs) are limited. Malik et al. (2008) reported that fenchlorphos, malathion, infusions of some nontraditional plant species used for parathion, methyl-parathion the preparation of stimulant beverages could be a valu- propenophos able source of nutrient elements for the human diet. In Organochlorine Hexachlorocyclohexane (HCH), particular, because zinc (Zn), copper (Cu), and molyb- pesticides lindane, dichlorodiphenyl denum (Mo) are vital for many physiological functions, trichloroethane (DDT), their intake is considered good for human health. Zinc benzene hexachloride (BHC), pentachloronitrobenzene acts as a catalyst, coactive, or structural unit for some (PCNB), tecnazene (TCNB) enzymes and is a cofactor of metalloenzymes. Zinc deficiency can impair normal growth and development, Pyrethroid pesticides Cypermethrin, esfenvalerate, fenvalerate, permethrin reproduction, and immune function. Copper acts as a co- factor in cuproenzymes, which are essential enzymes for Nitrogen-containing Atrazine pesticides normal functioning of the body. Molybdenum is a com- ponent of the sulfite oxidase enzyme, a molybdoenzyme Mycotoxins Aflatoxin, ochratoxin A, fumonisins, citrinin that catalyzes the last step in the degradation pathway Radioactive 137Cs, 210Pb, 238U, 234U, 226Ra of sulfur amino acids (Kosalec et al., 2009). contamination However, these essential metals are toxic above Adulteration/ Caffeine, ephedrine, norpseudoephedrine, certain thresholds. High supplementation of oligoele- undeclared synephrine, phosphodiesterase type 5 inhibitors ments such as Cu or Zn has been related to adverse chemical effects; for example, high Cu levels induce liver damage substances and high Zn concentrations reduce immune function

NUTRACEUTICALS Chemical Contamination in Nutraceuticals and Food Ingredients 829 and high-density lipoproteins levels and also produce levels of trace metals in herbal medicines by regulatory adverse interactions with Cu. Thus, eating contami- governmental entities (Sarma et al., 2011). The World nated or adulterated foods that are not considered usual Health Organization (WHO) has regulated maximum dietary sources of essential compounds may result in an permissible limits of toxic metals like As, Hg, Cd, and inadvertent overdose, especially because consumers are Pb in herbal medicines, which amount to 10, 1.0, 0.3, often recommended to use food supplements as sources and 10 ppm, respectively (WHO, 1998). However, the of essential elements. Exposure to these compounds WHO has not set permissible limits for essential metals from various sources could lead to additive effects as because many of them are considered micronutrients. well (Tongesayi et al., 2013). Rai et al. (2007) analyzed herbal formulations for Pb and Although herbal medicines may be as efficient as syn- Cd, and results were within the aforementioned permis- thetic drugs for certain health disorders, the presence of sible limits. Markert (1994) reported the normal Cr, Mn, unacceptably high levels of heavy metals makes them and Zn compositions of plants, which were 1.5, 200, and less safe (Blicharska et al. 2010). Thus, herbal medicines 50 ppm, respectively. may be responsible for the growing number of cases of The United States has standardized recommended adverse health consequences caused by their use (WHO, daily dietary allowances (RDA) for essential dietary trace 2003). Heavy metals have been widely found in raw elements, but not for toxic metals (Sarma et al., 2011). In herbal products (Wong et al., 1993; Rai et al., 2001), and Europe, the European Commission discussed the need for some plant species are known to be heavy metal hyper- setting maximum levels of Pb, Cd, and Hg in food sup- accumulators (Pollard et al., 2002). This property has plements to amend the Commission Regulation (EC) No. made some plants an effective source for bioaccumula- 1881/2006, because monitoring studies found high levels tion of heavy metals from contaminated soils (phytore- of these metals in certain food supplements. The follow- mediation). Thus, the consumption of these plants as ing limits were then set: 3.0 mg/kg for Pb; 1.0 mg/kg for food or therapeutic agents in traditional medicine may Cd (except for seaweed products, where the limit was prove to be hazardous (Barthwal et al., 2008). set at 3.0 mg/kg); and 0.10 mg/kg for Hg (Commission Patent herbal medicines (which consist of a mix of Regulation EC No. 629/2008; Gasser et al., 2009). different substances in either pill or extract form) may In 2009, the WHO and the Food and Agriculture contain intentionally added heavy metals (Saper et al., Organization (FAO) jointly proposed acceptable levels 2008). This is the case with Ayurvedic traditional herbal for toxic substances that can be ingested on a weekly preparations, which may contain high levels of metals basis—the Provisional Tolerable Weekly Intake (PTWI). (Pb, Hg) or metalloids (As) as a result of the intentional The following PTWI levels were set: Cd (7 μg; 420 μg/ incorporation of certain metallic preparations such as person); Pb (25 μg; 1,500 μg/person); inorganic As (15 μg; lead monoxide, red mercury sulfide, mercurous chlo- 900 μg/person); and Hg (5 μg; 300 μg/person) (JECFA, ride, red mercury oxide, arsenic sulfide, disulfide or tri- 1988, 1999, 2005). sulfide, and arsenic trioxide Martena( et al., 2010). Few reports have addressed metal intakes from dietary Heavy metal contamination has been well docu- supplements. A limited study on the safety of the botani- mented and numerous studies have identified tradi- cal dietary supplements (Echinacea, garlic, G. biloba, gin- tional medicines containing high levels of toxic heavy seng, grape seed extract, kava kava, Saw palmetto, and metals (Ernst and Thompson Coon, 2001; Dwivedi and St. John’s wort) evaluated the presence of undesired Dey, 2002; Caldas and Machado, 2004; Saper et al., 2004; heavy metals and did not find unacceptable concentra- Kauffman et al., 2007; Rai et al., 2007; Meena et al., 2010; tions that might pose a health risk to consumers, includ- Rubio et al., 2012), sometimes exceeding permissible lev- ing pregnant women and children (Raman et al. (2004). els for some analyzed species (Sarma et al., 2011). Garcia-Rico et al. (2007) investigated the presence of Cu, Toxic amounts of heavy metals in herbal prepara- Zn, Cd, Pb, and Hg in dietary supplements and the esti- tions and case reports of heavy metal poisoning from mated daily intakes were below those recommended by traditional herbal products have been reported in the WHO (1989), thus suggesting that little intake of metals literature (Ernst, 2002; Tait et al., 2002; van Vonderen is associated with the consumption of dietary supple- et al., 2000; Fuh et al., 2003). The potential severe conse- ments. However, daily intake of certain metals, like Pb, quences from side effects of certain herbal products have might increase due to the number of consumed dietary also been noted (Gurib-Fakim, 2006; Bush et al., 2007). supplements, so the intake of combined supplements Trace metal monitoring in herbal products represents plus fortified foods may be a cause for health concern. an important field of research that continuously pro- Cooper et al. (2007) found that certain traditional vides relevant information for risks assessment and for Chinese medicines (TCMs) were severely contaminated setting permissible levels (Rubio et al., 2012). One con- with As, Pb, and Hg. This unacceptable finding can place tinuing problem in protecting consumers of plant-based consumers at risk for severe or even fatal heavy metal/ medicines is the lack of standardization of permissible metalloid poisoning from the consumption of these

NUTRACEUTICALS 830 58. Toxic Contamination of Nutraceuticals and Food Ingredients natural therapies. However, the calculated dose of heavy [DDT]), nitrogen-containing pesticides (e.g., atrazine), metals/metalloids ingestion from these products and pesticides of plant origin (e.g., pyrethroids and rot- is expected to be lower if their bioavailability is less enoids) (Kosalec et al., 2009). than 100%. Thus, the risk assessment of nonprescrip- Although the use of pesticides contributes to produc- tion medicines or supplements needs to consider data tivity and higher agricultural yield, there is growing on bioavailability to ensure that future guidelines are concern about their adverse effects on human health. achievable and efficient in preventing undue intake of Because of their lipophilicity, OP insecticides are read- dangerous ingredients. ily absorbed through ingestion, dermal absorption, or The importance of good quality control for medicinal inhalation. Although their primary toxicity results from herbs to protect consumers from heavy metals contami- acetylcholinesterase (AChE) inhibition in neural tissue, nation should be emphasized (Başgel and Erdemoğlu, OPs, carbamate, and OC pesticides can also induce cellu- 2006). The estimation of trace metal residues in nutra- lar oxidative stress via affecting mitochondrial function ceuticals and food formulations should be mandatory and may disrupt the neuronal and hormonal status of the for pharmaceuticals and food ingredients to assure con- body (Karami-Mohajeri and Abdollahi, 2011; Sarkhail sumers of their quality. et al., 2012; Gupta and Milatovic, 2012; Milatovic et al., 2013; Gupta et al., 2015). Pesticides Unfortunately, incidents concerning relatively high levels of pesticide residues have repeatedly been found Pesticides are among the most widely used chemicals in herbs exported from different countries (Leung et al., in the world, and are also one of the most dangerous 2005). Residues of malathion, dimethoate, chlorpyrifos, contaminants to humans. The application of pesticides and propenophos have been reported in medicinal herbs in modern cultivation becomes indispensable for a from Egypt, with malathion being the OP insecticide growing demand of quantity and quality in products, most often detected (Ahmed et al., 2001). Malathion, fol- particularly to increase crop productivity and minimize lowed by profenofos, was also the OP most frequently any possible loss due to uncontrollable pests. found in leafy vegetables and some aromatic medicinal Exposure to pesticide residues can result in many dif- plants collected from different areas of Egypt (Dogheim ferent adverse health consequences, from acute problems et al., 2004). Residues of malathion and diazinon have such as skin rashes and asthma attacks to chronic prob- also been reported in herbal drugs from Iran (Sarkhail lems including cancer, neurological, reproductive, and et al., 2012). The relatively high occurrence of malathion respiratory disorders (Zuin and Vilegas, 2000; Calvert residues may be accounted for by the fact that it is usu- et al., 2004; Leung et al., 2005; Parrón et al., 2011, 2014). ally the recommended pesticide to be used on medicinal Consumers of medicinal plants expect these herbs to plants worldwide (Ahmed et al., 2001). be produced under good agriculture practices, with no Although OC pesticides possess comparatively long residues of environmental pollutants, including pesti- residual actions, most of them are now banned because cides. However, pesticides are often used to improve of their low biodegradability and persistence in the envi- the production of Chinese medicinal plants, and thus ronment, where they can be detected for long periods the presence of pesticide residues in these materials of time. These pesticides also show long-term toxicity, become pitfalls for safety (Qing et al., 2009). Therefore, particularly cancer and endocrine disruption potential it becomes absolutely essential to ensure the quality of (Mnif et al., 2011). medicinal plants and to detect the potential presence Xue et al. (2008) analyzed different TCMs for OC pes- of pesticides (Table 58.1). However, analyses of crude ticides, and those most commonly found were benzene herbal materials have often shown the presence of pesti- hexachloride (α-BHC), pentachloronitrobenzene (PCNB), cide residues. High levels of pesticide residues in medic- hexachlorobenzene, and tecnazene. Approximately inal plants can impact the marketing of these products, three-quarters (75.8%) of the herbal medicines studied especially those prepared for exports, because regula- by these authors contained at least one OC insecticide, tions in most of the importing countries are very strict. and more than 50% of samples contained two of them. Nevertheless, in practice, the large-scale cultivation of Nevertheless, their concentration was below the MRLs medicinal and food plants is not possible without using set in the regulatory Pharmacopeia. pesticides (Ahmed et al., 2001; Sarkhail et al., 2012). Quintozene, endosulfan, and BHC were the pesticide According to chemical structure, pesticides are clas- residues most commonly detected in ginseng products sified as organophosphorus (OP) pesticides (e.g., chlor- (Sohn et al., 2004). Despite DDT series and γ-BHC being pyrifos and methylchlorpyrifos, coumaphos, dichlorvos, pesticides frequently encountered in Chinese herbal ethion, fenchlorphos, malathion, parathion), organo- medicinal products, their concentrations were, in gen- chlorine (OC) pesticides (e.g., hexachlorocyclohexanes eral, below the allowable limits set by different countries [HCH], lindane, dichlorodiphenyl trichloroethane (Wong et al., 2007).

NUTRACEUTICALS Chemical Contamination in Nutraceuticals and Food Ingredients 831

Harris et al. (2011) analyzed pesticide content in com- including genetic features, substrate, humidity, CO2:O2 monly prescribed individual raw Chinese herbal medi- ratio, and the presence of fungicides or other competi- cines and reported the following proportion of samples tive microbial species (Kosalec et al., 2009; Martins et al., with an elevated level of background pesticide expo- 2001). sure: 26% for chlorpyrifos; 1.4% for methyl-parathion; Although the presence of toxicogenic molds in an and 0.3% for esfenvalerate, fenvalerate, fipronil, lindane, herbal plant does not imply the presence of mycotoxins and quintozene. Overall, pesticides most often found in in the product, it represents a potential risk of contami- Chinese herbal products include BHC, DDT, and PCNB, nation with mycotoxins. Microbiological and mycotoxi- with pyrethroids and aminoformins rarely being found. cological quality assessment of medicinal herbs should Among the potentially polluting pyrethroids, atten- include mycotoxin contamination (Table 58.1), espe- tion has been focused on permethrin and cypermethrin cially of herbs grown in hot and humid climates, and in (Qing et al., 2009). In contrast, there are also studies in the herbal parts showing higher risk of contamination which residues of OC, OP, and pyrethroid pesticides (Kabelitz and Sievers, 2004). have not been detected in any herbal medicinal plants Raman et al. (2004) evaluated several botanical sup- (Rao et al., 2011). plements and found the presence of molds in a great Residues of hexachlorocyclohexane (HCH) isomers number of samples. The most frequently isolated molds and metabolites have been detected in some herbal in medicinal plants were Penicillium sp., Aspergillus Ayurvedic formulations (Rai et al., 2007). Although some niger, and Fusarium sp. (Abou-Arab et al., 1999). Packed DDT metabolites were also found, HCH concentrations samples of medicinal plants have a higher probability were comparatively higher than those of DDT. γ-HCH of being infected with molds than nonpacked samples, was the HCH isomer found in the greatest concentra- because of the increased humidity inside the pack and tions in all samples, as it is one of the more persistent unsuitable storage methods. isomers. Rai et al. (2008) also reported the presence of A number of studies have reported the presence of total HCH and its isomers in “Dashmoola,” a popular mycotoxins in herbal products. Bugno et al. (2006) stud- Ayurvedic herbal formulation with immunomodulatory ied medicinal herbs collected from a Brazilian market and febrifugal properties. α-HCH and γ-HCH, the main and found molds of the Aspergillus and Penicillium gen- constituents of commercial HCH formulations, were era to be the most common contaminants of raw medici- detected in 97.5% samples, with γ-HCH being more nal herbs. Santos et al. (2009) found that all the analyzed prominent in comparison to the rest of the isomers. Only medicinal herb samples collected in Spain were contami- 5% of the samples contained DDT (or metabolites) resi- nated with several mycotoxins, and nearly 87% showed dues. These findings indicate that residual HCH build- the combination of four or more mycotoxins. up is more prevalent in plant samples than DDT. However, Romagnoli et al. (2007) studied aflatoxin With regard to nutraceuticals other than herbal medi- B1 contamination in different kinds of spices, aromatic cines, residues of HCB, HCH isomers, and DDT as well herbs, and medicinal plants from Italy, and none of the as polychlorinated biphenyls (PCBs) have been found plants analyzed showed detectable levels of aflatoxins. in cod liver oil used as a dietary supplement (Storelli Aflatoxin B1 is the most common and toxic metabo- et al., 2004). HCB contributed very little to the overall lite produced by Aspergillus flavus, and its toxic effects contaminant burden of dietary supplement oils, whereas include immunosupressive, mutagenic, teratogenic, and HCH isomers were generally below instrument detec- hepatocarcinogenic activity (Prado et al., 2012). tion limits (Bengtson Nash et al., 2014). In light of these The European legislation has set maximum levels of findings, and considering that these and other persistent certain mycotoxins (aflatoxin B1 and the sum of B1, B2, organic contaminants are accumulated in the lipid stores G1, and G2) for a variety of foodstuffs and spices. The of organisms from marine ecosystems, there is a need European Pharmacopeia has also provided a limit of for strict and continuous monitoring of their content in 2 µg/kg for aflatoxin B1 and 4 µg/kg for the sum of afla- fish and krill oil products to reduce as much as possible toxins B1, B2, G1, and G2 for some medicinal herbs. For the risks for human health (Bengtson Nash et al., 2014). ochratoxin A (OTA), a limit of 20 µg/kg has been adopted in liquorice root (Glycyrrhiza glabra) (Commission Mycotoxins Regulation EC No 472/2002; EDQM Chapters 2.8.18 and 2.8.22, 2007a,b). Mycotoxins are secondary metabolites produced OTA is a mycotoxin with nephrotoxic, hepatotoxic, by fungi that are capable of causing toxicity and even embryotoxic, teratogenic, neurotoxic, immunotoxic, death in humans. They are low-molecular-weight toxic genotoxic, and carcinogenic properties. It is a second- metabolites produced by molds that can contaminate ary low-molecular-weight metabolite produced by medicinal herbs and their preparations and products. some fungal species, notably Penicillium verrucosum and Mycotoxin production depends on several factors, Aspergillus ochraceus, and to a lesser extent Aspergillus

NUTRACEUTICALS 832 58. Toxic Contamination of Nutraceuticals and Food Ingredients carbonarius and Aspergillus niger. All these mold species wild edible fungi. These compounds are a known source can contaminate medicinal herbs with OTA, particularly of chronic poisoning and can also contribute markedly in temperate and colder zones (Pitt, 2000; Ostry et al., to the contamination with radioactive chemicals such as 2013). 137Cs (Table 58.1) (Borchers et al., 2004). Higher radiocae- Fumonisins are mycotoxins mainly produced by sium activity particularly occurs in mycorrhizal species. Fusarium verticillioides (F. moniliforme) and Fusarium pro- While most samples of wild edible fungi have detectable liferatum, with the most abundant being fumonisin B1 levels of radioactive Cs, radiation doses to individuals (FB1), which is considered as possibly carcinogenic (class resulting from edible fungi consumption are dominated 210 2B). Only a few studies have investigated FB1 contami- by Pb (de Román et al., 2006). nation of medicinal herbs and herbal products. Omurtag Phosphate feed supplements may contribute to the and Yazicioğlu (2004) measured the potential levels of radiation exposure of the population because phosphates FB1 contamination in herbal teas and medicinal plants usually contain appreciable quantities of uranium (U) 238 234 226 210 210 that are consumed regularly in Turkey. FB1 was detected and its daughters U, U, Ra, and Po ( Pb). in two samples (0.160 and 1.487 μg/g). Sewram et al. The exposure of consumers depends on the degree of (2006) investigated the presence of FB1 in 19 dietary and equilibrium of the decay chain in the feed and through 30 medicinal wild plants used by residents of Eastern the metabolic process. Poultry products, particularly South Africa. Eight plants, four dietary and four medici- chicken meat, organs, and eggs, have shown to be con- nal, were positive for FB1 at levels ranging from 34 to taminated through poultry feed supplements containing 524 μg/kg and from 8 to 1,553 μg/kg, respectively. phosphorus (Izak-Biran et al., 1989). Citrinin is another mycotoxin produced by Monascus sp. (M. purpureus, M. ruber) and Penicillium sp. (P. citrinum, P. expansum, P. radicicola, and P. verrucosum). Although ADULTERATION AND UNDECLARED this mycotoxin has a powerful nephrotoxic effect, it is CHEMICAL SUBSTANCES IN devoid of mutagenic and carcinogenic potential. Citrinin NUTRACEUTICALS AND FOOD has been found in foodstuffs of vegetable origin (e.g., INGREDIENTS cereals, pomaceous fruits, black olive, roasted nuts, spices), food supplements based on rice fermented with Terms such as “natural,” “herbal,” and “dietary sup- the red microfungi Monascus purpureus, and in food- plement” are sometimes used as a means to mislead con- stuffs of animal origin (e.g., cheese) (Ostry et al., 2013). sumers, health professionals, and health authorities, and Monascus-fermented rice has recently become a popular to obscure the potential adulteration of these products dietary supplement to reduce serum cholesterol level with synthetic active compounds (Table 58.1). because many of its bioactive constituents, particularly Dietary supplements have widespread use in sports, monacolins, are inhibitors of 3-hydroxy-3-methylgluta- and most athletes competing at the highest levels use some ryl-coenzyme A reductase. However, controversy about form of dietary supplementation. A number of contami- its safety has been raised because citrinin is produced nants have been identified in some supplements, including along with the Monascus secondary metabolites by cer- a variety of anabolic androgenic steroids (e.g., testosterone, tain strains or under certain cultivation conditions (Lin nandrolone, and their pro-hormones), ephedrine, and caf- et al., 2008). feine. While in most cases this contamination comes from poor manufacturing practices, there is some evidence of a deliberate adulteration (Maughan, 2005). RADIOACTIVE CONTAMINATION A doping case associated with the use of an ephe- OF NUTRACEUTICALS AND FOOD dra-labeled dietary supplement has been reported INGREDIENTS (Ros et al., 1999), which illustrates that the potential adul- teration of herbal food supplements with undeclared Mushrooms are a good source of digestible proteins agents has to be considered. The predominance of norps- and fiber, are low in fat and calories, and provide a valu- eudoephedrine over ephedrine indicates deliberate spik- able vitamin and mineral intake. For this reason, there is ing with the former compound. While manufacturers a growing interest in the use of dried mushroom extracts usually meet their labeling claims for ephedra-free prod- as NFI for health promotion. Some mushroom species ucts (Tam et al., 2006), special attention should be given have been shown to lower cholesterol levels, have anti- to the presence of drugs like caffeine, synephrine, and oxidant activity, and may modulate mononuclear cell botanical sources of caffeine because these ingredients activation and the phenotypic expression of cytokines, have replaced ephedra in some dietary supplements. thus enhancing the immune system function. Despite Many products labeled “herbal” or “all natural” these beneficial effects, there are concerns because of the (herbal/natural) are marketed as over the counter dietary high heavy metal concentrations that may be found in supplements for the treatment of erectile dysfunction

NUTRACEUTICALS Concluding Remarks and Future Directions 833 because they claim to enhance sexual performance. Maximum However, adulteration with undeclared phosphodiester- Acceptable ase type 5 inhibitors (e.g., sildenafil and its structurally Concentration (MAC) modified analogs), which have proven efficacy in the treatment of erectile dysfunction, appears to be wide- JECF spread among the Chinese over the counter medicines Reference EP A labeled for the treatment of sexual dysfunction in men Dose (RfD) A (Campbell et al., 2013).

Minimal AT RISK ASSESSMENT OF TOXIC Risk SDR CONTAMINANTS IN Level (MRL) NUTRACEUTICALS AND FOOD FIGURE 58.2 Proposed values for risk assessment of toxic con- INGREDIENTS tamination in nutraceuticals and food ingredients.

The maximum amounts of toxic metals and nonmet- als in medicinal plant materials can be given based on the safety of this type of product (Martínez-Dominguez the provisional tolerable intake (PTI) values. The lim- et al., 2014). its for toxic metals in herbal medicines and products Harris et al. (2011) interpreted the toxicological vary throughout the world. The use of herbal medicinal significance of levels of contaminants found in NFI products is not generally expected to contribute sig- (Figure 58.2) following three different comparisons. nificantly to the exposure of the population to heavy First, levels of contaminants were expressed as percent metal contaminants. However, it should be understood of the reference dose (RfD) or population-adjusted dose that the heavy metal content of herbal medicines adds (PAD) from the Joint FAO/WHO Expert Committee on to the burden originating from food, so it is recom- Food Additives (JECFA) and EPA IRIS (Integrated Risk mended that heavy metal contamination is minimized Information System). Second, concentration of contami- (WHO, 2007). nants was also compared with the minimal risk levels Limits for pesticide residues should be established (MRLs) provided by the Agency for Toxic Substances following the recommendations of the Joint FAO/WHO and Disease Registry (ATSDR), which are based on toxi- Meeting on Pesticide Residues (JMPR), which includes cological studies in animals and on reports of human an acceptable daily intake (ADI) and the analytical occupational exposure. MRL is an estimate of the daily methodology for the assessment of pesticide residues. human exposure to a hazardous substance (based on an However, there are no standard procedures for assign- average body mass of 70 kg) that is likely to be with- ment of MRLs to medicinal plants. The methodology out appreciable risk of adverse noncancer health effects used for food commodities could be applied when a over a specified duration of exposure. Third, levels of botanically identical medicinal plant is used as food. If contaminants were also compared with the maximum so, then the established MRL for a specific pesticide in acceptable concentration limits proposed for dietary the latter could be regarded as the relevant MRL for the supplements from the NSF/ANSI Standard 173 and specific raw medicinal plant material. The toxicological the European Pharmacopoeia for dried herbal infusions evaluation of pesticide residues in herbal materials (Harris et al., 2011). Based on the assumptions of the should be based on the likely intake of the material by mode of consumption of raw Chinese herbal medicines, consumers. In the absence of a full risk assessment, it is the 99% and 95% of samples tested by Harris et al. (2011) recommended that the intake of residues from herbal for heavy metals and pesticide residues, respectively, materials should account for no more than 1% of total were likely to be of negligible concern. intake from all sources (WHO, 2007). Maximum residue levels (MRL) have been set for food and animal feed by different world organizations, like CONCLUDING REMARKS WHO, the Food and Agriculture Organization (FAO), AND FUTURE DIRECTIONS the US Environmental Protection Agency (EPA), and the European Union (EU). The European Regulation EC The general public as well as health care profession- 396/2005 defines MRL as the upper legal concentration als should be better informed regarding the basic con- limit for a pesticide residue in or on food or feed, based cept of NFI and their usefulness, and should also be on good agricultural practices and the lowest consumer warned about the potential adverse effects associated exposure. Because this regulation only concerns raw with their use because of the potential of toxic con- materials, there is a need to define MRL for NFI to assure tamination. Because of the increased use of a variety of

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