Safety assessment of plant food supplements (PFS)

Botanicals and botanical preparations, including plant food supplements (PFS), are widely used in Western diets. The growing use of PFS is accompanied by an increasing concern because the safety of these PFS is not generally assessed before they enter the market. Regulatory bodies have become more aware of this and are increasing their efforts to ensure the safety of PFS. The present review describes an overview of the general framework for the safety assessment of PFS, focusing on the different approaches currently in use to assess the safety of botanicals and/or botanical compounds, including their history of safe use, the tiered approach proposed by the European Food Safety Authority (EFSA), the Threshold of Toxicological Concern (TTC) and the Margin of Exposure (MOE) concept. Moreover, some examples of botanical compounds in PFS that may be of concern are discussed. Altogether, it is clear that "natural" does not equal "safe" and that PFS may contain compounds of concern at levels far above those found in the regular diet. In addition, the traditional use of a PFS compound as a herb or tea does not guarantee its safety when used as a supplement. This points at a need for stricter regulation and control of botanical containing products, especially given their expanding market volume.     

Multi-targeted screening of botanicals in food supplements by liquid chromatography with tandem mass spectrometry

Safety, quality and composition assessments of food supplements based on botanical ingredients are of major concern, as they have usually not been through a rigorous testing process as required for the approval of therapeutic phytopreparations. Therefore, an efficient multi-targeted method was developed to screen selected botanicals of interest in herbal food supplements. Liquid chromatography coupled with a hybrid triple quadrupole linear ion trap was used for this purpose. Botanicals were characterised by means of appropriate biomarkers, which were unambiguously identified by mass spectrometry using an information dependent acquisition experiment which combined a multiple reaction monitoring survey with dependent enhanced product ion scans. During this procedure, product ion scans of targeted analytes were generated at three collision energies and compared with an in-house library of MS/MS spectra acquired from reference standards of all biomarkers. This generic method enables detection, identification and quantification of 98 biomarkers intended to characterise 79 selected plants.     

Botanical species being used for manufacturing plant food supplements (PFS) and related products in the EU member states and selected third countries

A great wealth of plants and plant derived preparations are used in the intention to supplement the basic nutrition in order to sustain and promote health. They may be used directly or consumed as manufactured plant food supplements (PFS) in dosed form. The use of these plants may already have a long tradition as fruit, vegetable or (folk) medicinal plants. Due to globalisation, more and more plants originating from all over the world are now offered and marketed in European countries, including species from China, South Africa and the American continent. For reasons of security, EU wide lists of plants accepted or prohibited to be used in food supplements are in elaboration. A crucial point is the correct identification of the plant material. The identity can be assessed by morphological, chemical and DNA specific methods. The active substances usable in PFS are secondary plant products that are often characteristic for certain plant groups (taxa), species or plant parts. They comprise not only polyphenols, essential oils, carotenoids and phytosterols, but also glucosinolates or saponins. The quality of the plant material used for PFS depends on a variety of factors, including the natural phytochemical, intraspecific variation with the occurrence of chemotypes, the ontogenetic variation, the considered plant parts and environmental influences during plant growth. In the production of the raw materials for PFS international standards (good agricultural practice, fair trade) should be applied.     

Plant food supplement (PFS) market structure in EC Member States, methods and techniques for the assessment of individual PFS intake

The popularity of herbal products, especially plant food supplements (PFS) and herbal medicine is on the rise in Europe and other parts of the world, with increased use in the general population as well as among specific subgroups encompassing children, women or those suffering from diseases such as cancer. The aim of this paper is to examine the PFS market structures in European Community (EC) Member States as well as to examine issues addressing methodologies and consumption data relating to PFS use in Europe. A revision of recent reports on market data, trends and main distribution channels, in addition an example of the consumption of PFS in Spain, is presented. An overview of the methods and administration techniques used to assess individual food consumption as a starting point, including their uses and limitations, as well as some examples of studies that collect Food Supplement (FS) information, including herbal/botanical/plant-derived products are also discussed. Additionally, the intake estimation process of food nutrients is described and used to propose the PFS ingredients intake estimation process. Nationally representative PFS consumption data is scarce in Europe. The majority of studies have been conducted in Scandinavia and the UK. However the heterogeneity of definitions, study design and objectives make it difficult to compare results and extrapolate conclusions.     

Regulations applicable to plant food supplements and related products in the European Union

This paper deals with the current regulatory and legal settings of traditional plant food supplements and herbal medicinal products in the European Union (EU). Marketing of botanicals in foods and food supplements in the EU is subject to several provisions of food law, which cover aspects of safety, production, labelling and product composition, including the use of additives and maximum levels of contaminants and residues. However, due to limited harmonization at the EU level, specific national regulations adopted at a Member State level also apply and mutual recognition is the mechanism through which such products can be marketed in EU countries other than those of origin. Unlike food supplements, marketing of traditional herbal medicinal products is regulated by an ad hoc Directive (i.e. Directive 2004/24/EC) covering in detail all the relevant aspects of these products, including a facilitated registration procedure at national level. However, by distinguishing traditional herbal medicinal products from plant food supplements and establishing selective marketing modalities for these two product categories, the EU has been confronted with implementation difficulties for traditional herbal medicinal products and a lack of homogeneity in the regulatory approaches adopted in different EU Member States. In fact, currently the nature of the commercial botanical products made available to consumers as traditional medicinal products or food supplements, depends largely on the EU Member State under consideration as a consequence of how competent National Authorities and manufacturing companies interpret and apply current regulations rather than on the intrinsic properties of the botanical products and their constituents. When the EU approach is compared with approaches adopted in some non-European countries to regulate these product categories, major differences become evident.     

Botanical origin authentication of dietary supplements by DNA‐based approaches

Herbal products, such as dietary supplements, have become a subject of increasing global importance for their health benefits and economic considerations. However, they have also been targets of adulteration practices, being the accurate identification of botanicals in herbal products of utmost importance to protect the health and expectations of consumers. Particularly, in the case of dietary supplements, which can have different types of formulations, the identification of plant material used in their production is often a research challenge. DNA‐based techniques have played a crucial role on the development of a wide range of tools for the authentication of herbal products. Therefore, this review intends to describe their main progresses, critically discussing their advantages and drawbacks when applied to authenticate herbal products, focusing on dietary supplements. DNA barcoding is particularly emphasized because it has provided the highest number of applications, followed by the advances on high‐resolution melting analysis combined with DNA barcodes. A special emphasis is also given to the promising approaches relying on DNA metabarcoding and isothermal amplification.     

Free amino acids in botanicals and botanical preparations

ABSTRACT:  Numerous studies were carried out about aminoacidic composition of vegetable proteins, but information about the free amino acid pool and the role of these substances is very incomplete. The aim of this paper was to contribute to the scarce knowledge concerning the composition of free amino acids in botanicals and botanical preparations widely used as food, in dietary supplements, and in pharmaceutical products. This work studied the composition of free amino acids, identified the major components of 19 species of plants, and evaluated the influence of different types of extraction on the amino acid profile. Amino acids were determined using an automatic precolumn derivatization with fluorenylmethyl-chloroformate and reversed-phase liquid chromatography with fluorescence and ultraviolet detection. The amounts of total free amino acids varied widely between plants, from approximately 12 g in 100 g of Echinacea pallida extract to less than 60 mg in the same amount of Coleus forskohlii, Garcinia cambogia , and Glycine max. In 13 plants arginine, asparagine, glutamine, proline, and γ-aminobutyric acid were the free amino acids found in preponderant quantities. The levels of free amino acids above the quantification limit in 36 assayed samples of botanicals, extracts, and supplements are shown.     

Feasibility of Including Green Tea Products for an Analytically Verified Dietary Supplement Database

The Dietary Supplement Ingredient Database (DSID) is a federally funded, publicly accessible dietary supplement database that currently contains analytically derived information on micronutrients in selected adult and children's multivitamin and mineral (MVM) supplements. Other constituents in dietary supplement products such as botanicals are also of interest and thus are being considered for inclusion in the DSID. Thirty‐eight constituents, mainly botanicals were identified and prioritized by a federal interagency committee. Green tea was selected from this list as the botanical for expansion of the DSID. This article describes the process for prioritizing dietary ingredients in the DSID. It also discusses the criteria for inclusion of these ingredients, and the approach for selecting and testing products for the green tea pilot study.     

The PlantLIBRA project: how we intend to innovate the science of botanicals

The main aim of the EC-financed R&D project PlantLIBRA (PLANT food supplements: Levels of Intake, Benefit and Risk Assessment) is to foster the safe use of food supplements containing plants or botanical preparations, by enabling science-based decision making by regulators and stakeholders. To make informed decisions, competent authorities and industry need more accessible and quality-assured information, as well as better tools (e.g., databases) and procedures for safety and benefit assessments, supported by broadly accepted methodologies. Consequently, PlantLIBRA is working to develop, validate and disseminate data and methodologies for risk and benefit assessment of plant food supplements, and to implement sustainable international cooperation. International cooperation will help ensure the quality of botanicals imported in the EU. Moreover, the project will provide data on intake by conducting a harmonized consumption survey. Existing composition and safety data will be collated into a meta-database. New analytical data and methods will be investigated and validated. The consortium is working closely with competent authorities and stakeholders.     

An overview of consumer attitudes and beliefs about plant food supplements

The use of dietary supplements is increasing globally and this includes the use of plant food supplements (PFS). A variety of factors may be influencing this increased consumption including the increasing number of older people in society, mistrust in conventional medicine and the perception that natural is healthy. Consumer studies in this area are limited, with a focus on dietary supplements in general, and complicated by the use of certain plant food supplements as herbal medicines. Research indicates that higher use of dietary supplements has been associated with being female, being more educated, having a higher income, being white and being older, however the drivers for consumption of supplements are complex, being influenced by both demographic and health-related factors. The aim of this paper is to provide an overview of current knowledge about the users and the determinants of usage of plant food supplements. With growing consumption of these products, the need for effective risk-benefit assessment becomes ever more important and an insight into who uses these types of products and why is an important starting point for any future science-based decisions made by policy makers, PFS manufacturers and ultimately by consumers themselves.     

Quality control of plant food supplements

It is essential to guarantee the safety of unprocessed plants and food supplements if consumers' health is to be protected. Although botanicals and their preparations are regulated at EU level, at least in part, there is still considerable discretion at national level, and Member States may choose to classify a product either as a food supplement or as a drug. Accurate data concerning the finished products and the plant used as the starting point are of major importance if risks and safety are to be properly assessed, but in addition standardized criteria for herbal preparation must be laid down and respected by researchers and manufacturers. Physiologically active as well as potentially toxic constituents need to be identified, and suitable analytical methods for their measurement specified, particularly in view of the increasing incidence of economically motivated adulteration of herbal raw materials and extracts. It remains the duty of food operators to keep up with the scientific literature and to provide sufficient information to enable the adaptation of specifications, sampling schemes and analytical methods to a fast-changing environment.     

Liquid chromatography/mass spectrometry based fingerprinting analysis and mass profiling of Euterpe oleracea (açaí) dietary supplement raw materials

Chemical fingerprinting and mass profiling methods to identify biologically active compounds in botanical dietary supplements is gaining much attention in recent years. Euterpe oleracea (açaí) has been reported to be rich in health-beneficial chemical constituents. We have developed LC/MS based fingerprinting and mass profiling methods to identify fatty acids, anthocyanins and non-anthocyanin polyphenols in three processed raw materials; non-organic açaí powder (ADSR-1), raw-organic açaí powder (ADSR-2) and freeze-dried açaí powder (ADSR-3) that are used in the preparation of botanical dietary supplements. For LC/MS analysis of fatty acids and non-anthocyanin polyphenols, the açaí samples were extracted sequentially with dichloromethane followed by methanol. To study fingerprinting analysis of anthocyanins, açaí samples were extracted with acidic methanol–water. The LC separation of fatty acids, non-anthocyanin polyphenols and anthocyanins in açaí raw materials was achieved using a C18 column with a gradient mobile phase consisting of solvents A (0.1% formic acid in water), and B (0.1% formic acid in methanol). MS experiments were carried out with negative and positive mode electrospray ionization. LC/MS analysis of dichloromethane extracts of (ADSR-1), (ADSR-2) and (ADSR-3) açaí powders have shown to contain fatty acids, γ-linolenic acid, linoleic acid, palmitic acid, and oleic acid. Whereas, the fingerprinting analysis of methanol extracts of ADSR-1, ADSR-2 and ADSR-3 led to the identification of phenolic acids, anthocyanin and non-anthocyanin polyphenols. The results from our study may be useful for the authentication and quality assessment of açaí dietary supplement raw materials.     

New Electrochemistry-Based Approaches to Brandy Quality Evaluation Using Antioxidant Parameters

The electrochemical approaches based on coulometric titration, differential pulse voltammetry (DPV), and chronoamperometry have been tested for applicability to evaluation of brandy quality. The antioxidant properties of samples have been considered as markers of adulteration. Total antioxidant capacity (TAC), ferric reducing power (FRP), as well as ellagic acid equivalent antioxidant capacity (EAE AOC) have been evaluated for ten samples five of which have been recognized as adulteration by gas chromatography. Electrochemical data for adulterations and brandies are significantly different. TAC and FRP values for all adulterated samples are approximately the same (10?±?3 and 6?±?2 C/100 mL for TAC and FRP, respectively) while brandies show TAC in the range of 24–108 and FRP of 20–88 C/100 mL depending on denomination and origin. DPV profile of adulterations is strongly different in comparison with brandy (the required peaks are absent and irrelevant peaks are appeared), allowing sample discrimination. Addition of vanillin-containing flavoring agents has been confirmed. The corresponding EAE AOC of brandies is 8–15-fold higher (depending on denomination) than that for adulterations. Two adulterations did not show EAE AOC, reflecting the absence of aging step during beverage production. Chronoamperometric EAE AOC equals to zero for four investigated adulterations. Standard antioxidant parameters antioxidant activity and total phenolic content can be applied for the preliminary screening only. These parameters are ineffective for adulteration of the beverage age using oak extracts. Electrochemical methods developed are characterized by simplicity, cost-efficiency, and reliability of results and can be successfully applied for the brandy quality control.     

The Regulatory Framework Across International Jurisdictions for Risks Associated with Consumption of Botanical Food Supplements

Dietary supplements, including those containing botanical ingredients and botanical‐derived compounds, have been marketed to consumers globally for many decades. However, the legislative framework for such products remains inconsistent across jurisdictions internationally. This study aims to compare the regulatory framework of botanical food supplements in the EU, USA, Canada, Australia, New Zealand, India, Japan, and China. The study also aims to investigate and describe safety assessment criteria for botanical food supplements where they are present in the above said jurisdictions, and attempts to analyze whether these criteria are suitable for addressing the toxicological risks associated with the use of botanical food supplement products, based on the evaluation of reported adverse effects related to botanical food supplement use as examples. Finally, this study discusses some future issues that need further attention, such as the consideration of less than lifetime exposures, potential for misidentification, and adulteration of botanical supplements by pharmacologically active substances. It is concluded that the regulatory approaches towards botanical food supplements differ significantly across jurisdictions. In addition, national authorities are increasingly considering having more regulatory oversight for such products. Further consideration of the actual impact of adverse events arising from botanical food supplement usage will be helpful in guiding such decisions.     

ELISA as a new method to measure genistein and daidzein in food and human fluids

Widely distributed in the plant kingdom, phytoestrogens, like soy isoflavones are found in plant protein extracts at varying levels depending on culture conditions and cultivars. Their increasingly used in human, as natural estrogens and their varying levels in raw materials raise the question concerning the measurement of isoflavones both in food or food supplements as well as in human fluid. To validate our new ELISA technique, isoflavones measurements in food-supplements, in soy food and in human fluid from volunteers participating to a kinetic study or a survey, were done. Our method was also compared with other physico-chemical techniques in an inter-laboratory assay (23 laboratories). In conclusion, our new ELISA technique is reliable, sensitive, cheap, rapid and can be used either for a great number of human fluid analysis, for crude matter or food analysis as long as the appropriate extraction technique is performed prior to ELISA procedure.     

Authenticity Determination of Meat and Meat Products on the Protein and DNA Basis

Adulterated food can be defined as food incompatible with the declaration of the seller. In the case of meat and meat articles, adulterations refer not only to the replacement of ingredients but also to inappropriate information concerning the origin of raw materials. Methods aiming at investigating meat and meat product authenticity may be based either on the analysis of protein composition or on the analysis of nucleic acids. At the present time, meat and meat product authenticity investigations based on protein analysis employ electrophoretic, enzymic, and chromatographic methods, sometimes supported by the mass spectrometry technique. On the other hand, species identification is often based on polymerase chain reaction (PCR). Biochips present a promising technology.     

Review of the efficacy of green tea, isoflavones and aloe vera supplements based on randomised controlled trials

We assess the evidence for health benefits of three commonly consumed plant food supplements (PFS), green tea, isoflavone and aloe vera, based on published systematic reviews of randomised controlled trials (RCTs). Whilst the potential benefits of green tea have been reported in a wide range of health areas, it is only in the area of the metabolic syndrome that the number of RCTs is approaching sufficient to judge such efficacy. Isoflavone supplements are widely used, and RCTs indicate that they affect bone resorption at lower doses in postmenopausal women undergoing estrogen-related bone loss, but this is only translated to attenuation of bone loss at higher doses of isoflavones. A systematic review on RCTs concluded that the effects of isoflavones on hot flashes in postmenopausal women were highly variable and no conclusions could be drawn. Despite the popularity of aloe vera as a PFS, the evaluation of its efficacy as a coadjuvant therapy for certain metabolic or digestive pathologies remains scarce; it constitutes a typical example of a naturally occurring ingredient whose efficacy in topical applications presupposes its efficacy in systemic applications. Nevertheless, its possible toxic effects on oral consumption call for caution in its utility as a PFS. Since 2007, efficacy evaluation of PFS in Europe has been covered by European Union Nutrition and Health Claims legislation. The European Food Safety Authority has adopted an approach relying on RCTs, while medicinal effects are accepted based on traditional use. In general, there are insufficient RCTs for claims to be made, and conclusive results on PFS should be obtained in the future by conducting studies with more homogeneous populations, by using supplements with optimised and measured bioavailability, and by conducting larger RCTs.     

Occurrence of aflatoxins in milk thistle herbal supplements

Milk thistle (MT) dietary supplements are widely consumed due to their possible liver-health-promoting properties. As botanicals they can be contaminated with a variety of fungi and their secondary metabolites, mycotoxins. The aflatoxigenic fungus Aspergillus flavus has been previously isolated from these commodities. Currently, there is no published method for determining aflatoxins (AFs) in MT. Therefore, a liquid chromatography (LC) method validated for aflatoxin analysis in botanicals was evaluated and applied to MT. The method consisted of acetonitrile/water extraction, immunoaffinity column clean-up, LC separation, post-column photochemical reaction derivatisation and fluorescence detection. The average recoveries for AFs added to MT seeds, herb, oil-based liquid extract and alcohol-based liquid extract were 76% or higher. The mean relative standard deviation was <10%. The limit of detection (LOD) was 0.01 μg kg(-1) and the limit of quantification (LOQ) was 0.03 μg kg(-1). The method was used to conduct a small survey. A total of 83 MT samples from the US market were analysed. AFs were detected in 19% of the samples with levels ranging from 0.04 to 2.0 μg kg(-1). Additionally, an aflatoxigenic A. flavus strain from ATTC and an A. parasiticus strain isolated from MT herb powder were found to produce high amounts of aflatoxins (11,200 and 49,100 μg kg(-1), respectively) when cultured in MT seed powder. This is the first study reporting on aflatoxin contamination of MT botanical supplements and identifying methodology for AF analysis of these commodities.     

Determination of synthetic drugs used to adulterate botanical dietary supplements using QTRAP LC-MS/MS

Adulteration of botanical dietary supplements with prohibited synthetic drugs has become a serious problem. In this paper, a method for testing synthetic drugs used to adulterate botanical dietary supplements was developed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) coupled with a linearity ion-trap system in the multiple reaction monitoring (MRM) plus enhanced product ion (EPI) mode. Twenty-three drugs exhibiting various pharmacological effects, comprising blood pressure and lipid-lowering agents, sedative drugs, anti-diabetic drugs, weight-reducing agents and aphrodisiac compounds, were studied. For all drugs, a single transition was monitored using protonated molecules as precursor ions. EPI spectra were stored in a library and recognized by library searching. Several undeclared drugs were identified in herbal remedies, e.g., glibenclamide, sibutramine hydrochloride and sildenafil. Overall, 35 positive samples were found out of a total of 105 botanical dietary supplements tested. The method was selective, sensitive, rapid, high-throughput and reliable.