Human, Animal and Plant Health Benefits of Glucosinolates and Strategies for Enhanced Bioactivity: A Systematic Review

Glucosinolates (GSs) are common anionic plant secondary metabolites in the order Brassicales. Together with glucosinolate hydrolysis products (GSHPs), they have recently gained much attention due to their biological activities and mechanisms of action. We review herein the health benefits of GSs/GSHPs, approaches to improve the plant contents, their bioavailability and bioactivity. In this review, only literature published between 2010 and March 2020 was retrieved from various scientific databases. Findings indicate that these compounds (natural, pure, synthetic, and derivatives) play an important role in human/animal health (disease therapy and prevention), plant health (defense chemicals, biofumigants/biocides), and food industries (preservatives). Overall, much interest is focused on in vitro studies as anti-cancer and antimicrobial agents. GS/GSHP levels improvement in plants utilizes mostly biotic/abiotic stresses and short periods of phytohormone application. Their availability and bioactivity are directly proportional to their contents at the source, which is affected by methods of food preparation, processing, and extraction. This review concludes that, to a greater extent, there is a need to explore and improve GS-rich sources, which should be emphasized to obtain natural bioactive compounds/active ingredients that can be included among synthetic and commercial products for use in maintaining and promoting health. Furthermore, the development of advanced research on compounds pharmacokinetics, their molecular mode of action, genetics based on biosynthesis, their uses in promoting the health of living organisms is highlighted.


Introduction
Glucosinolates (GSs) are natural, sulfur-rich anionic secondary metabolites, widely distributed in plants of the order Brassicales [1], mainly in the angiosperms families like Brassicaceae. Together with glucosinolate hydrolysis products (GSHPs), they are collectively described as mustard oil glucosides [2]. Only about 137 GSs have been characterized so far in plants [3]. Their core structure is composed of a β-D-glucosyl residue linked by a sulfur atom to a cis-N-hydroxyminosulfate ester, and a variable R group derived from a modified amino acid chain (which is the precursor used to group GSs into

Results
The search identified a total of 2198 articles that were retrieved from various databases, out of which 467 were considered potentially relevant based on their titles and abstracts in the initial screening. In a second screening, 282 articles were excluded after a thorough search through the text. Finally, 185 articles met the inclusion criteria, as shown in the PRISMA flow diagram (Figure 1). A majority of these GSs identified from natural sources, were in plants of the Brassicaceae/Cruciferae family. Specifically, a large number belonged to the aliphatic group, followed by the aromatic/benzyl and then the indole group. The analysis of GSs/GSHPs was performed using high performance liquid chromatography (HPLC) analysis of desulfo-GSs derivatives according to the ISO-9167-1 method [32]. Among other GSs compounds mentioned, we identified that some only Studies on health benefits of GSs/GSHPs 1. Bio fumigation/ biocidal activity and pesticide (n = 19) 2. Antimicrobial (n = 23) 3. Antioxidant (n = 9) 4. Anti-inflammatory (n = 12) 5. Cholinesterase (ChE) inhibitory (n = 3) 6. Cytotoxic/Anti-cancer (n = 45) 7. Controlling blood sugar levels (n = 1) 8. Antiglycation (n = 1) 9. Suppression of mutagenicity (n = 1) 10. Antifibrotic (n = 1) 11. Antispasmodic (n = 1) 12. Bone formation (n = 1) 13. Parasiticidal activity (n = 1) A majority of these GSs identified from natural sources, were in plants of the Brassicaceae/Cruciferae family. Specifically, a large number belonged to the aliphatic group, followed by the aromatic/benzyl and then the indole group. The analysis of GSs/GSHPs was performed using high performance liquid chromatography (HPLC) analysis of desulfo-GSs derivatives according to the ISO-9167-1 method [32]. Among other GSs compounds mentioned, we identified that some only occur in specific families like glucocapparin in Capparaceae and glucomoringin (GMG) (4-α-L-rhamnopyranosyloxy) benzyl GSs in Moringaceae plant species.
Herbal preparations of GSs-rich extracts mostly focused on seeds and fully mature stages (flowers, leaves and roots) of the plants however some studies considered the utility of sprouts such as cabbage, broccoli, cauliflower, kale, brussels, radish and leaf mustard, probably due to their increased use as side dishes and salads in many countries.
The seeds, sprouts and young plant extracts, predominantly contained aliphatic groups of GSs (such as broccoli (Brassica oleracea) [14,33,34]), whose contents decreased with plant maturity whereas indole GSs contents increased. Furthermore, most of the plant, roots had higher concentration of total GSs than shoots.
Root extracts of horseradish exhibited nematicidal activity against Meloidogyne incognita [65], while those from turnip (Brassica rapa L.) had a stimulatory effect in bone formation in rats [66], while from Bunias erucago showed chemopreventive activity [67] Sprouts extracts of Raphanus sativus L. induced apoptosis and cytotoxicity in human hepatocarcinoma cells [68] and human breast cancer cells [69]. The sprout extracts of Chinese kale (B. oleracea var. alboglabra) exhibited antioxidant activity [70]. Raphasatin in radish sprouts was also identified to be more potent as a chemopreventive compound than its corresponding GSHP [71]. Table 1 summarizes the health benefits of GSs including the herbal extracts mentioned above.  Anti-inflammatory synthetic [28]

Biofumigation/ Biocidal Activity and Pest Management
Natural GSs and their derivatives possess biocidal activity and show toxicity to a range of soil borne as well as plant pathogens and pests [55]. GSs rich plant sources and extracts with the ability to inhibit the growth of pathogens, offer the opportunity to explore them in controlling many plant diseases and as potential bio fumigants [72]. GSs and GSHPs volatiles, too, such as ITCs, have at low concentrations been used to control plant pathogens and/or are included as active ingredients among synthetic commercial nematicidal compounds [6]. These compounds possess bioherbicidal, larvicidal, soil fungicidal, insect feeding deterrence, nematicidal activities, and growth retardation [49,50,65,[73][74][75][76][77]126,128]. Their activity and effectiveness mainly increase with exposure time and in a dose-dependent manner [73,126].
Disease suppression in soil has been done using rotations of GSs-rich green manure. Brassica juncea L. seed meal soil supplement displays greater nematotoxicity to Meloidogyne incognita pathogens and in combination with Sinapsis alba seed meal, the manure helps deter nematodes and suppresses weeds in pepper plants [47]. A similar combination displays potential in managing Verticillium wilt and soil-borne diseases [136]. B. juncea L. leaf green manure rich in 2-propenyl GSs also exhibit high nematicidal activity both In vitro and in soil microcosms [55]. Boscia senegalensis seed wastewater, rich in methyl ITCs, has allelopathic effects which cause inhibition of germination of weeds seeds [78] while non-volatile ITCs like moringin facilitates feeding deterrence of Brassicales specialists by exhibiting antifeedant activities [43].
Besides, synthetic ITCs also display potential fungicidal structure-activity relationship and they have prospective acceptability as alternatives to traditional fungicides. They include p-nitrophenyl which inhibits Rhizoctonia solani and Erwinia carotovora pathogens [25].
The bioprotective role of some GSs is successively promoted by the action of other compounds present in the plant. For instance, indole GSs together with the phytoalexin camalexin cause Arabidopsis thaliana resistance to the oomycete pathogen Phytophthora brassicae. While indole GSs individually inhibit host penetration and play initial defensive roles, the phytoalexin camalexin enhances the defense at later stages by adding to this initial protection [137].

Antimicrobial Activity
Microbial drug resistance, pathogen resistance, food spoilage, clinical and oral microbial pathogens are becoming a global problem. Thus, there have been increasing efforts to evaluate natural products as a source of compounds against these agents. GSs/GSHPs are among the most important natural products whose antimicrobial mode of action has been characterized [10,36,[82][83][84].
Natural and synthetic volatile ITCS compounds have also been shown to inhibit the growth of drug-susceptible and multidrug-resistant (MDR) pathogens at concentrations comparable to the commonly used antimicrobial drugs [22]. Furthermore, they show even higher activity against MDR strains compared to susceptible strains, as in the case of Mycobacterium tuberculosis [22].
The effectivity of GSHPs as antimicrobial compounds can be increased by combining them with less efficient antibiotics [84,127], (which results in synergism) and/or having a mixture of several ITCs that display a broad-spectrum antimicrobial activity even to MDR pathogens [83,85]. Bioactivation of GSs with myrosinase enzyme also maximizes their effectiveness and antimicrobial activity, as demonstrated by enzyme bioactivated GMG activity against Pseudomonas aeruginosa and Staphylococcus aureus [44]. Food-borne bacteria synthesize myrosinase enzyme, which also influences the conversion of GSs to their GSHPs and increases their potency as antimicrobials [86].

Antioxidant Activity
Reactive oxygen species and free radicals produced by several metabolic processes have very high potential to harm cells through oxidation. Their continued presence speeds up aging resulting in age-related illnesses [138]. Several antioxidants studies show that GSs/GSHPs can scavenge and eliminate the damaging effects of the reactive oxygen species directly or indirectly [64].

Anti-Inflammatory Activity
GSs and GSHPs have shown great potential as anti-inflammatory agents through their ability to suppress inflammatory mediators independently or with other substances [131]. Phenethyl ITCs down-regulate the nuclear factor κB pathway, subsequently inhibiting transcription of genes involved in inflammatory effects, on a mouse ear topically applied with an inflammation-inducing substance [130]. In combination with 3-methoxyphenyl ITCs, phenyl ITCs cause approximately 99% inhibition on the human cyclooxygenase-2 enzyme [129].
The anti-inflammatory activity of GSs/GSHPs is presented in various ways for different cells. Plant extracts of Wasabi koreana, containing allyl ITCs, protect neuronal cells from activated microglia induced toxicity by inhibiting activation of nuclear factors [58] while sulforaphane isolated from broccoli sprouts improve cognitive function among schizophrenic patients [95]. Myrosinase-activated GMG [45] and glucoraphanin [97] act as potent anti-inflammatory agents by inducing inflammatory pathways.
Anti-inflammatory activity may be established in a concentration-dependent manner, as shown by sinigrin which suppress the production of inflammatory mediators in activated macrophages by inhibiting lipopolysaccharide-induced nitric oxide pro-inflammatory mediators [93]. To achieve maximal activity it is worth activating compounds as in the case of bioactive glucoraphanin that preserves tight junctions' integrity preventing dysfunctional blood-brain barriers in autoimmune encephalomyelitis, causing multiple sclerosis [96]. Furthermore, synthetically produced GSHPs are worth evaluating for their activity like indole GSs [28], and aromatic GSs [27] which have an inhibitory effect in the secretion of tumor necrosis factor-α in human monocytic leukaemia T peripheral helper-1 cells stimulated with lipopolysaccharides.
In addition to natural GSs, synthetic GSHPs and their derivatives also show good cholinesterase inhibitory activity. This has been illustrated in phenyl ITCs and its derivatives that display the most promising inhibitory activity with potential applications in treating Alzheimer's disease [129].

Cytotoxic and Anti-Cancer Activity
The mechanisms contributing to the anti-tumor and/or chemopreventive properties of GSs/ GSHPs are varied, as shown in various studies included in this review. Compounds may act by inducing/modulating systems of carcinogen detoxifying/metabolizing enzymes [103,117] which include increased detoxification, they may also act by upregulating phase II enzymes such as glutathione-S-transferase [104,123,125] and quinone reductase [11,13,18,71,106,107]. These mechanisms may be organ specific [123], may depend on the compound enantiomer involved [103], on the amounts of compounds [142], and on the availability of various compound which produce synergistic action [13].
GSHPs-ITCs may inhibit enzyme activity, as evidenced in 2-propenyl and 3-butenyl ITC that inhibit cytochrome P450 1A enzyme activity [99]. The compounds may also display selective anticancer activity that results in the inhibition of deoxyribonucleic acid replication in cancer cells as shown by sulforaphane and phenethyl ITCs, causing more double strand breaks in prostate cancer cells [110].
Furthermore, GS/GSHP compounds exhibit radical scavenging ability and cytotoxic effects in various cancer cells. Compounds like butenyl thiocyanates, allyl, phenethyl ITCs [100], and GMG GSs [133] reveal such effects. The compounds' effectivity lies in their ability to inhibit cell proliferation, [37,39,61] arrest the cell cycle [38], causing morphological changes, cytotoxicity, and cell apoptosis [35,36,40,41,54,56,59,69,101,113,115,119]. These activities may be dose dependent [37] and may also be defined by the chemical nature of the compound [143]. Sometimes, the dynamism of these activities occurs through synergic effects [105] of various compounds present in an extract and the particular extract's vigor is affected by the method of preparation [102].
Some GSs/GSHPs display their potential by possibly inhibiting compounds or activities related to cancers. Among them include, phenyl ITCs that inhibit transcriptional activity regulated by androgen receptor modulating growth of prostate cancer cells through down-regulation of an androgen receptor-regulated gene [134]. ITCs may influence changes and activity of compounds that are normally overexpressed during cancer such as histone deacetylase [112,114] or compounds which may cause intoxication such as doxorubicin [124].
GSs/GSHPs exhibit the ability to suppress healthy cell invasion and abolish the growth of tumors by affecting pathways that adjust carcinogen metabolizing compounds. These include sulforaphane ITCs, which down regulates the expression of matrix metalloproteins [108]. Such ITCs may activate the antioxidant response element electrophile response element/antioxidant responsive element, modulating estrogen signaling in different tissues that leads to induction of the inhibitory effects of estrogen in breast cancer cells [109], while in cortical neurons, they activate the extracellular signal regulated kinase pathway, transcriptional factors, and upregulate detoxification enzymes [111]. Benzyl and phenethyl ITCs show significant inhibition of the proteasome activity and also suppress the growth of multiple myeloma cells [135].
The photoprotective properties of 3-methoxybenzyl ITCs and 3-methoxyphenyl acetonitrile, in Limnanthes alba, display their anti-photocarcinogenic and anti-photoaging properties. These ITCs induce human skin cell proliferation activity against ultraviolet B rays and prevent hyperplasia induced by these rays in reconstructed skin epidermal tissue [48].

Other Activities
Sulforaphene and raphasatin ITCs reduce the risks of sugar-related diseases. In addition, these compounds cause spargel expression, which modulates energy metabolism, inhibiting α-amylase, and α-glucosidase in vitro [120]. Elsewhere, sulforaphane and phenethyl ITCs suppress urinary mutagenicity activity as illustrated on rats treated with oral doses of heterocyclic amine 2-amino-3-methylimidazo[4 ,5-f]quinoline, a food carcinogen. Long term intake of the ITCs decreases urinary mutagenic activity, implying enhanced/modulation of the metabolism of the quinoline [121].
Sinigrin, a common GS in most cruciferous vegetables, exhibits the great potential of inhibiting non-enzymatic aglycation [119], while its GSHP allyl ITCs has hepatoprotective roles (reduction of hepatic fibrosis). In rats exposed to carbon tetrachloride (CCl 4 ), the ITCs reduce alanine aminotransferase and aspartate aminotransferase activity, activate macrophages and regulate Kupffer cells [118].
The potential stimulatory effect of GSs in bone formation, and as natural sources of promising neutraceutical agents has been shown in B. rapa L. roots GSs, that show stimulatory effects on human osteoblast-like MG-63 cell differentiation. Administration to young rats increases serum osteocalcin as well as some bone microstructural parameters [66]. In addition, 5-phenylpentyl ITCs isolated from A. rusticana exert significant spasmolytic activity on rats' distal colon [59].
Functional foods based on GSs display parasiticidal potential against Lepeophtheirus salmonis lice, ectoparasites that infect Atlantic salmon. In an in vivo study that involved various feeding trials on fish, beneficial effects on expression of genes functioning in detoxification and regulation of iron metabolism in several tissues are shown. Such properties affect the availability of heme to lice, deterring them from infesting the fish [144].

Effects of GSs/GSHPs Used in Preclinical and Clinical Studies
Clinical and preclinical studies evaluating GSs/GSHPs components and diets provide the basis for further explaining the mechanism of action of these components. Broccoli sprouts, rich in sulforaphane administered in a pilot study on melanoma patients display chemoprevention ability [145] and shows favorable effects in reduction of serum insulin while at the same time enhances insulin resistance in type 2 diabetes patients, therefore improving their glycaemic control and decreasing risk factors of cardiovascular diseases [146]. Broccoli soup consumption also reduces the risk of prostate cancer progression [147] and daily administration of free sulforaphane shows promising evidence in managing biochemical recurrence after radical prostatectomy in patients with prostate cancer [148]. Similarly, in asthmatics, sulforaphane enhanced bronchoconstrictor effect, improving major defects and even mild asthma [149] and in schizophrenic patients, sulforaphane from broccoli sprouts helped in improving their cognitive function [95].
Clinical studies, however, require a good design that follows a standardized protocol which includes selecting a representative sample size and various doses [148] of the phytochemicals to minimize bias and allow generalization.
Likewise, masking and the duration of treatments [146,150]/interventions/exposure/supplementation [151] should be well considered to confirm specific effects of the components. Variabilities in concentrations of GSs/GSHPs consumed need to be minimized in the diet within these studies. Furthermore, research on the mechanism of action, probably in the biomarker-based approach of GSs/GSHPs is highly recommended to understand various relationships between exposure and risks of the phytochemicals as reported in some studies [152,153]. Table 2 summarizes some preclinical and clinical studies evaluating the health benefits of GSs and GSHPs.

Improving Plant GSs, GSHPs Content, Their Bioavailability, and Bioactivity
There are several factors that affect GSs contents/composition as well as their biological activities. These include (a) post-harvest treatment of GSs rich plant [154][155][156]  Evaluation of Symptoms using positive and negative syndrome scale and cognitive function Sulforaphane potentially improved cognitive function [95] Salt stress, in a concentration-dependent manner, is a potential inducer of producing these health-promoting compounds. Sodium chloride treatment increases glucoraphasatin on radish sprouts [161], and sulforaphane in broccoli sprouts [162]. Salt stress in combination with elevated carbon dioxide increasing sulphur cellular partitioning and metabolism which affects the GSs-myrosinase system [163]. Similarly, sulphur salts supplementation during the cultivation of vegetables increases GSs content and their biological activities [68].
Overexpression of genes involved in the breakdown of GSs defends plant against pathogens. In A. thaliana, overexpression of myrosinase gene TGG1, affects the metabolism of GSs compounds enhancing plant stomatal defense against Pseudomoas syringae bacteria [172].
The treatment of plants with trace/essential elements affects the GSs-myrosinase system. Selenium treatment for instance, increases sulforaphane in broccoli cultivars sprouts, enhancing their myrosinase activity [34] while in R. sativus an increment in glucoraphanin is observed [173]. In A. thaliana and B. oleracea Var. italica, ammonium nutrition stimulates the accumulation of GSs and induces myrosinase activity as well [174]. Managing plants using chemicals and light may increase GSs content, for instance 6-benzylaminopurine [175] alone and in combination with 1-methyl-cyclopropene [176] increases GSs in broccoli and green light emitting diode lights increase broccoli's florets' total GSs content and sulforaphane [177]. Elsewhere, GSs in plants may accumulate through natural occurrences, in response various stresses like yearly seasonal variations that cause increased temperatures, water stress [168], larval/herbivore infestation [166,178,179], ozone fumigation [180], and biotechnological transformations [172,181].
Dietary intake of GSs rich products is positively correlated to their health benefits and biological activity. Their bioavailability can be enhanced by increasing the hydrolysis enzyme activity and/or treating extracts in mild heat [182]. Continuous consumption of GSs rich foods is exogenously treated with myrosinase [183][184][185][186] or intake of diverse GSs rich foods which increase the activity of the GSs degradation enzyme in the gut results into the bioavailability of GSs/GSHPs [187]. The content of these compounds in prepared and processed food is essential and therefore, post-harvest processing techniques, food processing, and treatment methods that affect the contents of GSs and the biological activities should be carefully chosen. Pasteurization [92], high pressure [188,189] treatments and freezing [190,191], thermal boiling [133,157,192], steaming [193], microwaving [46,194,195], storage time [139], storage temperatures [196] affect to GSs contents. On the other hand, blanching is considered to prevent loss of phytochemicals [102] in vegetables. Tables 3 and 4 summarize the factors and treatments which affect the availability and activity of GSs/GSHPs in plant or diet and the hydrolysis activity of myrosinase enzyme. Several substances that have been used in the elicitation of GSs/GSHPs have shown not only to induce physiological changes but also stimulate various biological activities, as illustrated.

Search Strategy and Selection Criteria
Peer-reviewed articles were searched using keywords in various scientific databases, including PubMed, ResearchGate.net, Wiley Online Library, and Google Scholar, between March 2010 and March 2020. The search terms used to obtain articles for this review were "glucosinolate" OR "glucosinolate extract" OR "plant glucosinolate" OR "glucosinolate isolate" OR "pure glucosinolate" OR "synthetic glucosinolate" OR "glucosinolate hydrolysis product" OR "Glucosinolate degradation product" OR "mustard oil glucoside" OR "myrosinase end product" OR "isothiocyanate" OR "benzyl glucosinolate" OR "indole glucosinolate " combined with "profile" OR "availability" OR "variability" AND "activity" OR "Bioactivity" OR "health benefits" OR "specific biological activities" AND "improvement" OR "elicitor". Data extraction included the specific glucosinolate/ glucosinolate hydrolysis products, their sources, biological activities, mode of action, availability, strategies used to increase their content availability and activity. Articles published in a non-English language, before and/or after the set period, retracted from the databases, having self-reported outcomes, and reviews and editorials, were excluded. The review focused on the peer-reviewed original studies with a particular outcome of interest, depending on the study objective.

Conclusions
GSs/GSHPs offer a wide variety of health benefits, including disease prophylactic and therapeutic effects. This review shows the usefulness of these compounds in preventing and reducing disease progression in humans and animals, their biocidal, biofumigation capabilities in plants, and their antimicrobial use in the food industry as food preservatives.
Although their composition and concentration vary in various crop species, organs, cultivars, and at different stages of development, sometimes in response to both abiotic and biotic factors, strategies to improve specific compounds have been successful, leading to improved crop varieties with both nutritional and pharmacological benefits. Biological activity assays on GSs-rich extracts exhibit a positive correlation between concentrations of GSs/GSHPs-related benefits in organisms. Taking this into consideration, the bioavailability of these compounds should be well maintained in their sources through choosing and having handling, preparative techniques, extraction methods that maintain them.
This review also reveals the necessity to maintain GSs content as bioactive compounds, to explore and improve GSs rich plant as a source of these natural compounds, which have potential as active ingredients among synthetic and commercial products to maintain and promote health. Considering that most research evaluating these natural compounds mainly focuses on the plant of Brassicaceae family, there is furthermore a need to probe the compounds even more in vivo studies, to understand their primary mechanism of actions and their molecular targets should also be emphasized.