Nowadays, the incidence of non-communicable diseases (NCDs), including obesity, diabetes, cancer, and other chronic conditions, is increasing and showing high mortality indexes worldwide (https://www.who.int/nmh/topics/es/
). These diseases are multifactorial, but it has been described that lifestyle, such as smoking or sedentary life, contributes to the prevalence of the NCDs. As a result, different prevention strategies have been developed, mainly the promotion of healthy habits, recommending avoiding a sedentary life, quitting smoking and the consumption of alcoholic drinks, and keeping a healthy diet [1
]. To emphasize the importance of these strategies, it has been proven that these habits have an important role in the prevention of cardiovascular diseases and type-II diabetes [2
Since one of the main points of intervention for a healthy lifestyle is diet, global health recommendations are always encouraging the message of eating a minimum of five portions of fruits and vegetables daily [3
]. Vegetables are not only a natural source of amino acids and minerals, but also of phytochemicals [5
]. In particular, cruciferous vegetables, like broccoli, cabbage, red radish, or Brussels sprouts, are consumed worldwide not only because of their culinary value being popular in many countries with plates being part of the culture legacy, but also because of their diverse content in phytochemicals with high health-promoting benefits [6
]. Between them vitamin C, polyphenols, and minerals can be found. However, one of the most relevant biomolecules are glucosinolates (GSLs), mainly found in plant species and varieties of the order Brassicales, mainly Brassicaceae as the most well-known representatives of this horticultural, botanical, and socioeconomically relevant botanical family [8
]. They consist of a basic structure of a thiohydroximate-O
-sulfate group with a glycosylation and, depending on which amino acid they are derived from, a different side chain [8
]. GSLs are stable secondary metabolites, but after tissue disruption, they are hydrolyzed by the enzyme myrosinase (EC 22.214.171.124), generating different GSL hydrolysis products (Figure 1
) including bioactive isothiocyanates (ITCs) if the pH is 5–8, in the range of neutrality. In some plants, epithiospecifier proteins exist and can modify the outcome of the hydrolysis process, typically to promote other products than ITCs, as it is the case of nitrile specifier protein originating simple nitriles and elemental sulfur [8
]. On the other hand, the epithiospecifier proteins (ESP) with similar function may also carry out a different reaction for the few aliphatic GSLs with a terminal double bond by adding the sulfur to this bond, forming an epitionitrile. The production of thiocyanates is produced at pH > 8, and oxazolidine-2-thiones can be formed if a hydroxyl group is present on carbon 2 (beta position) of the glucosinolate [10
The effect of cooking practices on the content of GSLs and degree of conversion to ITC/nitrile are also important when considering the use of cruciferous foods for clinical studies, and a relevant source for clarification is the review of Nugrahedi et al. [12
Sulforaphane was discovered by Prochazka [13
], but it became widely known after the study by Fahey et al. [14
] describing its involvement in cancer prevention. That pioneering study was in some ways misleading, as the protocol used (addition of purified myrosinase enzyme to chemical extracts of broccoli) could have masked the normal tendency of broccoli to form nitriles. Importantly, natural glucoraphanin and sulforaphane both have a chiral sulfinyl group in the side chain [15
]; the natural molecule is pure R-isomer. In contrast, some commercial glucoraphanin and sulforaphane has a sulfinyl group that is a mixture of the R and S isomers, because of semisynthetic preparation by chemical oxidation of a more easily available glucosinolate. Importantly, the artificial racemic mixture has different biological properties than the natural single stereoisomer [16
], which is an important fact to know in the evaluation of published studies.
Diverse bioactivities have been reported for ITCs, being strong inducers of phase II detoxification enzymes [18
], their anti-carcinogenic effect [19
], or their anti-inflammatory properties [20
]. Between aliphatic GSLs, glucoraphanin (GRA) is one of the most studied, since its resultant ITC, sulforaphane (SFN), has shown diverse properties on human health For example, it has been described that SFN exerts its main function acting over the nuclear factor erythroid 2-related factor 2 (Nrf2) [13
]. In normal conditions, Keap1 represses the interaction between the transcription factor Nrf2 and the sequence motif present in nuclear DNA known as antioxidative response element (ARE) through sequestration and the subsequent ubiquitination and proteasomal degradation of Nrf2 [21
]. However, the interaction between SFN and Keap1 interferes with the suppression of Nrf2, allowing the transcription of phase II cytoprotective proteins [22
]. Furthermore, also the subject of study has been the intervention of SFN in the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), decreasing its capability of binding target sequences related to inflammatory processes, such as tumor necrosis factor (TNF-α) [23
Another abundant GSL present in cruciferous vegetables is glucobrassicin (GB) [24
], whose main degradation product is ascorbigen [26
]. However, its corresponding indole-3-carbinol (I3C) and its condensation product 3,3-diindolylmethane (DIM) [27
] have been the main focus of medical research because they were readily commercially available. Glucobrassicin was discovered as the precursors of the already known ascorbigen [28
] I3C and DIM were found to be formed in vitro, but not in vivo. From the very fast formation of ascorbigen at plant pH, it is well established that I3C is neither an intermediate in the in vivo formation of ascorbigen, which must be due to reaction with ascorbic acid with an earlier intermediate in the glucobrassicin breakdown. Early authors (e.g., Bjeldanes, in the 1980s) suggested indoles to be cancer protecting; later authors noted Janus properties, being either protectors or carcinogens depending on the timing of the carcinogen and the indole, as reviewed by, e.g., Holst and Williamson [29
] and Agerbirk et al. [26
Although several studies showed that DIM interferes with diverse signal transduction pathways implied in tumorigenic and inflammatory processes, such as AKT kinase, phosphoinositide 3 kinase (PI3K), NFκB pathway, or EGFR/ERK, the exact interaction has not been yet elucidated [30
]. Furthermore, it has been studied that the growth and expansion of certain cancer types, as colorectal, are promoted by the presence of pro-inflammatory interleukins, like TNFα and IL-6. These molecules activate pathways regulated by NFκB and transcription factor STAT3, inducing and maintaining in time a pro-inflammatory microenvironment [32
]. In this way, carcinogenic cell proliferation, tumor invasion, angiogenesis, and immunosuppression are promoted [33
]. The work of Zou et al. [34
] showed that when human ovarian cancer cells (SKOV3 and A27809) were treated with DIM, a downregulation in STAT3 and a subsequent inhibition in cell adhesion and invasion was observed.
All these studies performed in vitro suggest that cruciferous vegetables mainly from Brassica
spp. (either as foods or ingredients rich in bioactive substances) are good candidates as dietary coadjutants to improve human health in NCDs. As a consequence, some clinical and pre-clinical studies have been developed during the last six years. Epidemiological studies have linked the intake of GSLs with the risk of coronary heart disease or type 2 diabetes [35
]. Nevertheless, this information from large cohorts is usually based in food frequency questionnaires, making it difficult to know the specific effects of the GSLs in the intake. On the other hand, promising anti-cancer results have been obtained from interventional studies but, due to the reduced sample size, cannot be interpreted as a generalization [37
]. In general, the studies analyzed in our review did not present an established and standardized protocol, differing in the concentrations of the component, time of the intervention, and sample size. Moreover, not all of them presented a double-blind design. For all that, this review aims to analyze the critical points of the studies performed with Brassica
-related biomolecules with the aim to establish a basis for future trials and avoid biases.
In the present work we pursued the objective of critically reviewing the clinical studies in which ingredients and bioactive compounds from cruciferous vegetables (Brassicaceae family) had been used with different pathologies. Based on the anti-cancer properties attributed to GSLs and isothiocyanates (ITCs), we opted to search according to these pathologies since they were the most studied in the past decades. Additionally, one of the main target pathways of the SFN in the organism is the Nrf2–Keap1–ARE system [13
], with a wide role in the development and progression of chronic diseases (e.g., cancers, respiratory problems), and, finally, based on their anti-inflammatory characteristics we opted also to review the more recent research data on the effects of the consumption of GSLs in the development and management of metabolic diseases, mediated by systemic inflammatory conditions. Diverse bibliographical searches were carried out in databases including PubMed, Scopus, and SpringerLink using the following terms and keywords: cancer, metabolic disease, respiratory tract disease, sulforaphane, and glucosinolates. In order to make a more concise selection, the results were limited to clinical trials. From the obtained records, articles including cruciferous foods and derived products were the selected items, and we reduced the timeframe to the last years, from 2012 to the present as well. This selection gave us 53 articles of studies developed with human subjects, with granted access to the full text of the given publication, and contributed with clinical data of the compounds and the pathologies present in the studies. The articles based on in vitro or in vivo (animal models) experiments or trials were not considered because we focused our work on the data of studies carried out with human adults with different pathologies, to back the decision-making when looking for nutritional recommendations in the daily clinical practice with such patients. Nor were articles using low-caloric diets, low-fat diets, or Mediterranean-type diets considered, because these are healthy options for the human subjects in any intervention study, and they did not provide us with specific information about consumption of cruciferous foods or ingredients derived from Brassicaceae products. Additionally, articles where the isothiocyanates (ITCs) were used as biomarkers in intra-hospitalar tumor resections or documents based on healthy volunteers were also not included in the analysis, because they were patients with pathologies unrelated to our objective of study, and therefore, 36 articles were discarded.
According to all these selected criteria, 15 clinical studies were selected to carry out this critical study of clinical evidence using bioactives from Brassica
and also one cohort study because of its ample sample size and long period of follow-up of 22 years [35
]. We also carried out the analysis of a “letter to editor” [38
] that was submitted as a response to this cohort study.
4. Future Perspectives and Challenges
The assessment of the health effects of bioactive compounds from plant-derived foods requires several aspects to be taken into consideration and a different perspective from that when evaluating single compounds of any chemical entity. In the case of Brassica
spp. and other cruciferous foods and bioactive compounds obtained from them, it is crucial to control the concentration of glucosinolates ingested or delivered in the administration, and therefore to control the factors that affect this composition in the natural matrix, namely the production practices, handling, cooking procedures, time of chewing, age of sprout/mature plant, amount ingested, and concomitant ingestion with other foods, etc. In this context, well-researched studies are being carried out that include and analyze all these factors and standardize the Brassica
-derived products by their SFN content [53
], and efforts must continue in this line. The exact calculation of the amount of raw broccoli needed to produce a supplement that delivers a specific amount of SFN and its metabolites within the body remains to be established [55
]. It is important to point out that a food product should be tested in its pattern of consumption on a daily basis or with a frequency of consumption clearly reported.
One point that deserves attention is the unreasonable but common focus in the literature on glucoraphanin and glucobrassicin that merits mention, and contrasted to the high number of other GSLs occurring in vegetables. There are evidences presented for exciting protective effects of many other GSLs, but they are not commercially available [56
]. The same could be said for the indoles, with the literature focusing to an absurd degree on compounds that happen to be commercially available, although they are not necessarily the most widespread in vegetables [58
], or when comparing a non-substituted and a substituted indole [59
Similarly, efforts on health-oriented breeding (e.g., manipulation of the activity of nitrile specifier protein in broccoli by Roman et al. [60
]) as well as the very promising bioactivities of exotic GSLs like glucomoringin and its ITC moringin from a tropical tree not from the Brassicaceae family but within the order Brassicales [57
]. This area of work together with additional efforts on the discovery of brassicaceous phytochemicals is another perspective: the widespread occurrence of epithionitriles in cabbage and new GSL products in these plants [10
]; the poorly known chemistry of some vegetables, e.g., commercial chemotype of watercress in the USA [61
]; and other derivatives including seleno-glucosinolates [62
Additionally, the potential for positive synergistic effects of combinations of GSLs have been little studied, even though some pioneering work can be found in this subject [64
]. These are all examples of the many open lines of work for the future of understanding the potential benefits of the consumption of the bioactive compounds present in cruciferous foods from the farm to the foods for health.
The CONSORT (Consolidated Standards of Reporting Trials) statement includes a check list as a guidance for reporting randomized controlled trials and hence, it serves indirectly as a guidance for the design of human intervention studies to evaluate the beneficial effects of foods [65
]. The report of the trial design must include the eligibility criteria and a participant flow diagram, complete data of the intervention administered, and well-defined primary and secondary outcome measures, among others. It is highly advisable to refer to this check list before performing an intervention to ensure that we take into account all the factors that may later influence the final result.
In human studies, sample size is an important factor to reduce inter-individual variability, especially in markers subjected to a great variability, as the expression of particular genes, but for logistical reasons it is not always possible to have a large and appropriate number of participants. However, to help to reduce the variability in clinical trials performed in parallel groups, it is essential to control variables (the so-called covariables) that can influence in the behavior of other variables. Sex, age, and BMI are covariables that greatly influence responses linked to inflammatory processes or gene expression. It is important to randomize intervention groups stratified by these covariables, in order to reduce inter-individual variability, so that significant differences are more likely to be detected, and some clinical trials do not take these aspects into account. Absorption, distribution, metabolism, and excretion (ADME) processes are also influenced by age, sex, dietary habits, or the microbiome. The COST Action POSITIVe working group has recently published the determinants involved in the ADME process of different bioactive compounds [66
], and they include the concept of metabolic phenotype (metabotype) or the similar pharmacokinetics profile common in some individuals, which may result in similar response patterns. This concept should be applied to the human host as well as to the gut microbiota metabolisms and is dependent on gene polymorphisms as well as microbiome composition. This perspective can help to understand the differences in responses observed between individuals. Stratification of individuals by metabotype is an attractive challenge for future research.
It is crucial that the outcome measure is of biological relevance; biomarkers or risk factors measured must be associated to the latter development of a disease. Validation of appropriate markers is a pending issue as very few are recognized as valid in terms of specificity and applicability to a range of population [66
Biomarkers of effect are early stage end-points, for instance the modulation of phase 2 enzymes by glucosinolates. They need more time than biomarkers of exposure to be influenced by the dietary treatment. Hence, length or duration of the study must be defined according to the biomarker measured to be modified, that is, to define perfectly the time of exposure to observe changes in relevant parameters. Gene expression is one important target for glucosinolates, and it requires a sufficient period of exposure to (de)activate signaling pathways involved. It is crucial to find appropriate biomarkers of effect that are linked to later disease outcomes, and more investigation is needed in this sense. Post-study follow-up can be of great value in assessing the persistence of certain effects, or in discovering those that appear more long-term [67
In case of clinical trials performed with patients and not healthy subjects, the appropriate timing of treatment with the nutraceutical or food product is also a pending issue. The degree of progression of the disease greatly influences the response observed. In some cases, a bioactive compound is no longer sufficient, and in other cases it can have the opposite effect. It has been suggested that the effect of SFN on Nrf2 activation is desirable in the early stages of tumorigenesis, whilst it may have a deleterious effect in later stages. It can behave as an oncogenic transcription factor and increase the cytoprotective activity in the cancer cell and cause resistance to chemotherapy [67
]. More studies are needed on the potential effects of this molecule on patients with diagnosed cancer before making any recommendations of supplements with these phytochemicals [55
]. The need for studies with larger populations is clear, as well as longer follow-ups, in order to find significant results with enough strength to support recommendations for public health in favor of increasing the consumption of cruciferous foods on a daily basis.