Research Journal of Pharmacognosy (RJP) 5(2), 2018: 71-89 Received: 22 Mar 2017 Accepted: 23 Sep 2017 Published online: 13 Mar 2018 DOI: 10.22127/RJP.2018.58511 Review article

Isothiocyanates: a Review

Chandra Kala1*, Syed Salman Ali 2, Nabeel Ahmad3, Sadaf Jamal Gilani1, Najam Ali Khan2

1Glocal School of Pharmacy, The Glocal University, Uttar Pradesh, India. 2School of Pharmaceutical Sciences, IFTM University, Uttar Pradesh, India. 3Department of Biotechnology, IFTM University, Uttar Pradesh, India.

Abstract (ITCs) are naturally occurring molecules belonging to highly reactive organosulphur synthons, with the general structure R–N=C=S. The precursor molecule anions are hydrolyzed enzymatically (under the effect of enzymes) or unenzymatically to produce nitriles or isothiocyanates depending upon conditions such as pH and temperature. Family is known to contain abundant ITCs. A significant number of isothiocyanates has been isolated from different plant sources and some of them have been synthesized. Several isothiocyanates have demonstrated significant pharmacological activities including anti-cancer, anti-inflammatory, anti- microbial activities, etc. Pharmacokinetic profiles of these sulphur containing compounds are well established. However, safety profiles of ITCs need consideration and a well-designed study with appropriate control, for their production as lead compounds. This review summarises the chemistry, sources, pharmacokinetics, pharmacological activity and toxicity profiles of the isothiocyanates.

Keywords: Brassicaceae; ; isothiocyanates; pharmacokinetics; pharmacological activity Citation: Kala C, Ali SS, Ahmad N, Gilani SJ, Ali Khan N. Isothiocyanates: a review. Res J Pharmacogn. 2018; 5(2): 71-89.

Introduction Isothiocyanates (ITCs) are naturally occurring watercress [5]. Sixteen families of dicotyledonous molecules belonging to highly reactive angiosperms contain , which are organosulphur synthons [1], with the general precursor of ITCs. In several reviews, it has been structure R–N=C=S [2]. Isothiocyanates present mentioned that Brassicaceae family primarily in a vegetable are responsible for the plant sharp contains ITCs [6]. taste and active in its defence system [3]. They Previously, these compounds were considered for are derived from glucosinolates. Glucosinolates having a negative impact on health as some anti- are thioesters, which upon loss of cellular nutritional or goitrogenic glucosinolates were integrity activate the enzyme glucosinolates found in widely grown oilseed crops and in myrosinase leading to generation of several domesticated crops. However, more recent unstable intermediates which rearrange into studies have reported that therapeutic and degradation products. These degradation products prophylactic properties of these compounds were are transformed into , oxozolidine- revealed by numerous researches [6]. They 2-thiones, nitriles, etc. depending upon different possess strong anti-oxidant, anti-inflammatory factors like pH, presence of myrosinase- activity, anti-microbial, neuroprotective and interacting protein, and availability of ferrous ion cardioprotective activity. Isothiocyanates are [4]. considered to be safe and no serious adverse Isothiocyanates are found in cruciferous effects have been reported in humans. They vegetables such as broccoli, rocket, cauliflower, possess potent anti-carcinogenic activity and are Brussels sprouts, cabbage, radish, turnip and *Corresponding author: [email protected] © 2018. Open access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/) Kala C. et al. active against many forms of cancers and cardiovascular diseases CVDs and diabetes. The tumours, but when given after chemical pharmacological benefits of the ITCs are carcinogens in high concentration, i.e. 25-250 attributed to the electrophilic nature of carbon times more than normal dietary intake, they residue which reacts with biological nucleophiles promote bladder cancer in rats [7]. However, [8] and with nutrients such as amines, amino there is some information showing that acids, proteins, thiols and alcohols to give variety isothiocyantes act against bladder and prostate of compounds such as thioureas and cancer [7]. Some ITCs are known to form adducts dithicarbamates [10]. ITCs react with N-terminal with DNA and induce gene mutation and residues of alpha-amino group of lysine through aberration. Thus, there is need of considering the alkylation. [11] genotoxicity of ITC [8]. Physicochemical properties such as solubility and The present review summarizes the sources, acid-base property also exert effects on biological chemistry, pharmacokinetics, pharmacological properties of isothiocyanates. The activity of activity and toxicity profile of ITCs. ITCs against 4-(methylnitrosamino)-1-(3- pyridyl)-1- butanone (NNK)-induced lung tumorigenesis is favoured at their high Methods Available scientific databases including Science lipophilicity and low reactivity against GSH [9]. direct, Pub med, Elsevier, Scopus, Research Exchange of substituents does not result in scholar, Google scholar were explored for remarkable changes in reactivity of NCS group. chemistry, pharmacokinetics and pharmacology In ITCs of aryl alkyl type or substituted of ITCs. US patents were also referred for details phenylsulphide and phenylsulphone type, of Lepidium meyenii roots. Reports of lengthening of ITC does not affect NCS group reactivity [12]. toxicological profile of ITCs were obtained from Oriental seed (OMS) US EPA office of pesticide program and Bureau of pest Pharmacokinetics of ITCs management, New York. Absorption ITCs are rapidly absorbed across intestinal cell Results and Discussion membrane via passive diffusion [13]. Allyl Chemistry of isothiocyanates isothiocyanate (AITC) is known to be absorbed ITCs are basically esters of isothiocyanic acid from small bowel and colon and its metabolite (H-NCS). They may also be considered as sulfur can be easily detected using urine determination analogues of isocyanates (R-NCO). They are of [14C] AITC (25 or 250 μmol/kg body weight) isomeric with (R-SCN) and [14]. About 90% of orally administered AITC is isoelectric with thioketones (R2C=C=S) [1]. The absorbed, confirming its high bioavailability [15]. hydrogen atom on the adjacent carbon to the In a research, human intestinal effective isothiocyanate moiety is also responsible for the permeability and intestinal metabolism of tautomeric structure of methylene-isothiocyanate was studied in human jejunum (loc- moiety to beta-unsaturated thioketones [10]. It has 1-gut). The percentage absorption of been suggested that ITCs possess high sulforaphane through the jejunum was found to electrophilicity due to the presence of the carbon be 74±29% [16]. atom and high nucleophilicity associated with In another study, it was found that broccoli sulfur atom. The dipole moments of ITCs as sprouts ITCs were largely absorbed with a peak determined by several workers indicate that – concentration of 0.943-2.27 µm/L in plasma, NCS group possess electron accepting character serum and erythrocytes 1 h after feeding [17]. and negative end of dipole and hence may serve One of the interesting facts is that, absorption of as a versatile building block for preparation of ITC is lower from cooked food compared to raw wide class of nitrogen, sulphur and oxygen vegetables because the colon microflora is able to heterocycles. They possess extended pi-electron catalyse the hydrolysis of glucosinolate in a system contributing to its ability to variety of cooked form [7]. target molecules. [1] There are various reports mentioning the health Distribution benefits of ITCs in ailments such as in cancer, In a study conducted in mice fed with purified 72 RJP 5(2), 2018: 71-89

Isothiocyanates: a review sulforaphane and erucin, both metabolites were with urinary ITC metabolite following ingestion found in skin, liver, kidney, bladder, lungs and of broccoli. The HPLC cyclocondensation assay plasma by HPLC-MS/MS analysis. Erucin was was used for measuring urine ITC metabolites the favoured form in the liver, kidney and and matrix assisted laser desorption/ionization bladder, suggesting systemic benefits of both time of flight was used for GSTM1, GSTT1 [18]. In another report regarding tissue deletion GSTP1 11e105Val and GSTA1*A/*B distribution and metabolism of sulforaphane in genotyping. The result of the study revealed that wild type and Nrf2 knockout mice, sulforaphane there were no substantial differences in ITC metabolites were detected in all tissues at 2 and 6 levels among genotypes, supporting the idea of h following administration, and the highest alternative routes of ITC metabolism [24]. concentration was observed in the small intestine, Comparative metabolism of different ITCs was prostate, kidney and lungs [19]. studied on F344 rat liver microsomes [25]. The enzyme responsible for ITC metabolism was Metabolism induced by treating the rats with Aroclor 1254, Organic ITCs are conjugated with glutathione beta-napthoflavone, isosafrole or phenobarbital. (GSH) to form dithiocarbamates. These The result of the study revealed that rate of dithiocarbamates are metabolized by γ-glutamyl metabolic conversion followed the order of transpeptidase (GGT) to form cysteinyl glycine 1-naphthyl>>phenyl>benzyl & phenethyl ITC conjugates. These are further metabolized to >>propyl, ethyl and methyl [25]. form cysteine-ITC conjugates and by N-acetyl ITCs are potent inducers of phase-II enzymes transferase to form NAC-ITC conjugate [20,21]. such as UDP-glucoronosyltransferase (UGTs), GSTM1 and GSTT1 catalyse the conjugation and sulphonyltransferase (SULTs) and quinone excretion of ITCs, and these conjugates are reductase (QRs) and hence detoxify activated substrates of ATP-binding cassette (ABC) carcinogens. For example, consumption of transporter [22]. P-glycoprotein (P-gp, ABCB1), broccoli and watercress inhibit CYP450 enzymes multi drug resistance protein (MRP1, ABCC1), and reduces the risk of lung cancer [25]. (MRP2, ABCC2) and breast cancer resistance In a research, it was revealed that heme protein (BCRP ABCG2) are known to interact destruction and protein modification was with ITCs [22]. To support the above fact the responsible for the inactivation of CYP2E1 by kinetic property of the glutathione S-transferases phenyl ethyl isothiocyanate (PEITC). GSH (GSTs) GSTs A1-1, M1-1, M4-4 and P1-1 with metabolize PEITC to form PIC (phenethyl 14 ITCs (propyl-, butyl-, pentyl-, hexyl-, (±)-2- isocyanates). PIC bounds to CYP2E1 but does hexyl, cyclohexyl-, cyclopropyl-, cyclooctyl-, not inactivate them. Instead, the reactive sulphur benzyl-, phenethyl-, allyl-, erucin, sulforaphane, atom generated during desulfurization of PEITC erysolin) was investigated. The result of that is involved in inactivation of CYP2E1, as study showed that GST M1-1 and GST P1-1 revealed by LCMS of inactivated CYP2E1 apo- were the main contributor of metabolism of ITC. protein [26]. Liver tissue was detected with 0.1 mM concentration of GST A1-1 and GSTM1-1 Excretion revealing their major role in conjugation of ITCs The mercapturic acid pathway was found to be [22]. the major route of elimination of ITCs. Four genotypes of GST (GSTP1, GSTM1-1, Defecation, exhalation and perspiration were GST-A1-1 and GSTM2-2) were tested for found to be the minor routes. Several studies conjugation with GSH for allyl-benzyl-PEITC support his fact e.g. pure benzyl isothiocyanate and sulphoraphane. Among these four genotypes, (BITC administered in a human was 54% GST-P1-1 and GSTM-1 were the most efficient recovered as mercapturic acid in the urine. catalysts and GSTM2-2 was least efficient. Similarly PEITC from watercress was 47%

Among the four ITCs, benzyl ITC was the most recovered as mercapturic acid [27]. A study on rapidly conjugated while, sulforaphane was only metabolism and excretion of BITC revealed that slowly conjugated [23]. 53.7% of BITC was excreted as metabolites by Steck et al. conducted a study with 114 the renal route. The metabolite was completely individuals to assess the association of the GST excreted 10-12 h after administration [28]. 73

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A similar study was conducted to find out the The anticancer activity of phenethyl disposition of AITC in rat and mouse. The urine isothiocyanate (PEITC) was studied by several was found to be the major route of excretion of researchers [33]. The anti-proliferative and pro- 14C AITC about 50-80%, with smaller amounts apoptotic effect of PEITC was studied on human about 6-12% in faces and 3-7% in expired air cervical HeLa cell line in cancer stem like cells [29]. Moreover, inorganic , using a cell proliferation assay, immunoblotting allylthiocarbomoylmercapturic acid and and flow cytometry [33]. It was found to possess allylthiocarbomoylcysteine were found in mice anti-cancer activity. but no cysteine conjugate was found in rat urine. The anticancer potency of SFN, along with other Also, approximately 80% of 14C were present in anti-proliferative agents was evaluated against mercapturic in rat, suggesting that hydrolysis is Barrett oesophageal adenocarcinoma (BEAC) the major metabolic pathway of AITC in mouse [34]. Various parameters including the effects on whereas glutathione conjugation is the major drug resistance by Rhodamine efflux assay and metabolic pathway of AITC rats [29]. the induction of apoptosis using annexin V Borghoff et al. evaluated age related changes in labelling and Western blot analysis of poly excretion of AITC [30]. The study was conducted (ADP-Ribose) polymerase cleavage were in male Fischer rats of age 3, 16 and 27 months. evaluated. The study showed that SFN induced a Difference in elimination of AITC in urine decline in cell survival, cell cycle arrest and (which is a major route of excretion) was not apoptosis. It also reduced tumour volume and significant. However, faecal excretion of AITC increased the activity of other anti-proliferative decreased with age and bile excretion increased agents [34]. Sulphoraphane fractions (standard at 16 month and decreased afterward as SFN, extract and purified SFN) of Brassica compared to 3 month age group. Age related oleraceae var rubra were also evaluated for anti- 14 decrease in CO2 was also observed [30]. cancer activity against human epithelial The relationship between dietary total ITC and carcinoma HEp-2 and Vero cells [35]. GST M1/T1/P1 genotype was reported in one Sulphoraphane was found to be potent against observational study by measuring total ITC in pancreatic cancer cell Mia Paca-2, Panc-1, AsPc- urine sample of 246 Singapore Chinese 1, and BxPc-3 and inhibited pancreatic cancer individuals [31]. The results revealed that there cell growth and induced apoptosis [36]. It was was no difference in ITC level of GSTM1-null also found to be potent against ovarian cancer and GSTM1-positive subjects. However, both in human ovarian cancer cell line SKOV3 significant difference was observed in urine ITC and mouse ovarian cancer cell lines C3 and T3 level of GSTT1 null and positive individual. The [37]. Similarly, SFN was found to be a potent null genotypes were associated with lower inhibitor of A-549 lung cancer cell growth [38]. excretion levels [31]. Similarly, AITC which commonly occurs in cruciferous vegetables is a Pharmacological activity of ITCs potent inhibitor of many types of cancer [39]. It Cytotoxic activity of isothiocyanates has inhibited both human bladder cancer UM- Anti-carcinogenic activity of isothiocyanates has UC-3 cells and rat bladder cancer AY-27 cells been known for decades. Recently anticancerous proliferation as evaluated using orthotropic rat activity of synthetic ITC, CM9, and its fullerene bladder cancer model [40]. It has also exhibited derivative was studied in human jurkat T- potent anti-proliferative activity against MDA- leukemic cell [32]. CM9 is an MB-468 human breast cancer cells [41] and naphthalenetetracarboxylic diimide (NDI) against human A-549 and H-1299 non-small cell consisting of two side chains: a N,N'-bis [3,3'- lung cancer (NSCLC) cells in vitro [42]. (dimethylamino) propylamine]-naphthalene- 1,4,5,8-tetracarboxylic diimide (N-BDMPrNDI) Mechanism of action derived protonated dimethylaminoethyl side 1. Modulation of B-cell lymphoma-2 (Bcl-2), chain and an ITC group as a second side chain. Bcl associated X protein (Bax), Tumor The result of the study showed that CM9 reduced protein p53 ()p53, vascular endothelial Jurkat leukemic cell viability in a concentration growth factor (VEGF), cyclin B1 and dependent manner [32]. capsases-3 expression leading to activation of

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intrinsic pathway of apoptosis as determined 5. γH2AX (phophorylated form of H2A histone western blotting and reverse family, member X) and Fanconi anemia (FA) transcription polymerase chain reaction (RT complementation group proteins D2 PCR) in human epithelial carcinoma HEp-2 (FANCD2) foci, ataxia telangiectasia and verocells, and orthotrophic rat bladder mutated protein kinase (ATM) / ataxia cancer model [32,35,40]. telangiectasia and Rad3 relatde protein 2. The mechanism of anti-proliferating effect of kinase (ATR) - and S and G2/M cell cycle phenylethyl isothiocyanate (PEITC) was arrest are identified as potential targets [42]. found to be mediated by up regulation of death receptor 4 (DR4) and death receptor 5 Antimicrobial activity (DR5) of TNF-related apoptosis-inducing Antimicrobial activity of ITCs has been reported ligand (TRAIL)-mediated apoptotic by several researchers. ITCs possess inhibitory pathways [33]. activity against food spoilage bacteria species 3. The anti-cancer activity of SFN was such as Escherichia. coli, Klebsiella spp., attributed to induction of caspase 8 and p21 Salmonella spp., Bacillus spp., Serratia spp., and down regulation of heat shock protein 90 Staphylococcus spp. and Listeria spp. Gram (hsp90) as determined by western blot and negative bacteria were found to be more sensitive RT-PCR in human epithelial carcinoma HEp- to ITCs compared to Gram positive bacteria [44]. 2 and Vero cells [35]. It also targets Kelch- Isothiocyanate isolated from horseradish was like ECH-associated protein 1 (Keap1)- evaluated for its anti-bacterial property against 4 Nuclear factor (erythroid-derived 2)-like 2 strains of antibiotic-resistant bacteria: (Nrf2) complex and induced phase 2 genes. It methicillin-resistant Staphylococcus aureus targets Bcl-2 protein, death receptor, (MRSA), vancomycin-resistant S. aureus Mitogen-activated protein kinases (MAPK), (VRSA), multidrug-resistant Acinetobacter mitochondria-associated apoptotic protein baumanii (MRAB), and multidrug-resistant and induce programmed cell death, it targets Pseudomonas aeruginosa (MRPA), and 3 clinical cyclins, cyclin-dependent kinases (CDKs), strains of pathogenic bacteria: S. aureus, A. Cdc25C (gene), histone deacetylases baumanii, and Pseudomonas aeruginosa by (HDAC), p21, microtubules and cause cell measuring minimum bactericidal concentrations cycle arrest mainly G2/M phase. It targets (MBC) [45]. ITCs showed maximum activity matrix metalloproteinases (MMP)-2 and against A. baumanii and P. aeroginosa, with MMP-9 and inhibits invasion and metastasis MBC values substantially less than the standard [43]. Degradation of heat shock protein antibiotics vancomycin, levofloxacin, and were (HSP) 90 and blockade of interaction comparable with that of norfloxacin [46]. between HSP90 and its cochaperone p50 in 4-(α-L-Rhamnosyloxy) benzyl isothiocyanate pancreatic cancer cells are also targets of and 4-(4’-O-acetyl-α-L -rhamnosyloxy)-benzyl sulforaphane as determined by western blotting [36]. isothiocyanate isolated from Moringa oleifera 4. Inhibition of Akt (protein kinase - B) - signal was evaluated for antibacterial activity against transduction pathway as determined by effect Staphylococcus aureus, S. epidermidis, of sulforaphane in human ovarian cancer cell Bacillus subtilis, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and line SKOV3 (IC50 40 μmol/L) and mouse Pseudomonas aeruginosa, and for their ovarian cancer cell lines C3 and T3 (IC50 25 μmol/L each) using 2,3-bis(2-methoxy-4- antifungal activities against Candida albicans, nitro-5-sulfophenyl)-2H-tetrazolium-5- Trichophyton rubrum, and Epidermophyton carboxanilide inner salt (XTT) assay [37]. floccosum using the disk diffusion method. Both Increased ubiquitination and degradation of of these compounds exhibited moderate to good α- and β-tubulin leading to inhibition of antimicrobial activity [47]. mitosis and induction of mitochondrial Phenethyl, benzyl, benzoyl ITCs are known to mediated apoptosis in bladder cancer cell as inhibit harmful intestinal bacteria Clostridium determined in orthotopic rat bladder cancer difficile, Clostridium perfringes and E. coli. SFN, model [40]. BITC are known to be active against Gram

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Kala C. et al. positive and negative bacteria of human faecal neurons [52]. PD also involves autooxidation of isolates. β-Phenylethyl ITCs are known to dopamine, leading to the formation of neurotoxic possess strong activity against Vibrio species including electrophilic DA quinone, ROS parahaemolyticus and S. aureus; considerable and hydrogen peroxide, which mediate α- activity against Bacillus cereus and Vibrio synuclein-associated neurotoxicity [52]. Loss of parahaemolyticus. Allyl, benzyl, 4-methylthio-3- function and mutation in genes of phase-II butenyl are also known to possess strong anti- detoxification pathway (especially glutathione bacterial activity against E. coli ATCC 11775, metabolism) also contribute to neuronal death Enterobacter cloacae ATCC 13047, Salmonella accompanying enhancement of α-synuclein typhimurium ATCC 13311, Proteus vulgaris expression. Sulforaphane is potent against both of ATCC 6380, S. aureus S-6, Staphylococcus these neurodegenerative conditions [52]. SFN epidermidis ATCC 14990, Bacillus subtilis has been able to overcome neuronal loss in ATCC 6633, B. cereus var. mycoides ATCC various models of Parkinsonism like Drosophila 11778 and Campylobacter jejuni [48]. model of alpha-synucleinopathy and Drosophila parkin mutants. [52] SFN is also protective against Mechanism of action spinal cord injury [53]. Erucin, an isothiocyanate, 1. The action is attributed to interaction of these possesses neuroprotective effects on compounds with sulfhydryl of glutathione dopaminergic like neuroblastoma SH-SY5Y cell containing enzymes followed by inhibition of line. [54] Neuroprotective effects of synthetic sulfahydryl enzymes activities [49]. The ITC 4-iodophenyl isothiocyanate (4-IPITC) was linkage between ITC and enzyme limits evaluated in several in vitro and in vivo models of enzymatic activity and leads to formation of neurodegenration including the exposure of oxygen and other free radicals thus reducing primary cortical neurons to glutamate, oxidative the content of important thiol group and stress, oxygen, glucose deprivation, 1- reducing the viability of bacterial cell [50]. methylphenylpyridinium (MPP+) and The rate of spontaneous degradation of ITC- experimental autoimmune encephalomyelitis thiol conjugates and bacterial detoxification (EAE). 1-methyl-4-phenyl-1,2,3,6- mechanism determine the efficacy of tetrahydropyridine (MPTP) induced Parkinson’s particular ITC [51]. disease [55]. That study revealed that 4-IPITC 2. The effect on cell membrane and leakage of significantly reduced neuronal cell death and cellular metabolite and increase in 3- possessed neuroprotective and neurotrophic galactosidase activity [46]. properties [55]. In light of these researches, it 3. ITCs interact with cytochrome-450 and get could be concluded that isothiocyanates possess oxidized to produce more reactive potent neuroprotective activity. isocyanates which are more toxic than the parent compound as determined by Mechanism of action cytochrome-450 dependant oxidative 1. Sulforaphane has induced proteasome desulfuration of 2-naphthylisothiocyanate to expression in murine neuroblastoma yield 2-naphthylisocyanate [50]. Neuro2A cells via carbonyl formation and hydrogen peroxide induced cytotoxicity and Activity against neurodegenerative diseases ultimately protected neuronal cells from Sulforaphane, one of the potent isothiocyanates amyloid beta peptide mediated cytotoxicity which possesses various pharmacological in Aleizhmer’s disease [54]. activities, is also active in neurodegenerative 2. SFN has been able to enhance glutathione disorders [52]. Alzheimer’s disease is one of the synthesis or glutathione conjugation activity. most common neurodegenerative diseases. It is SFN also caused reduction of DA quinone characterized by accumulation of amyloid beta level as revealed in various in vitro models peptides resulting in oxidative damage and [52]. inflammation [52]. Another neurodegenerative 3. SFN has prevented nigral dopaminergic disease, Parkinson’s disease (PD), is neurons cell death and causes astrogliosis, characterized by loss of dopaminergic (DA) microgliosis inhibition in basal ganglia as

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revealed in 1-methyl-4’-phenyl-1,2,3,6- The extract reduced RNA and acid phosphatase tetrahydropyridine mouse model of PD [55]. levels significantly, revealing its radioprotective 4. L-SFN has activated Nrf2-antioxidant activity. response (ARE) transcriptional pathway which protects neuron degeneration in rat Cardioprotective activity nigrostraital tissue slice model. [56] Cardiovascular disease (CVD) is the largest 5. SFN has also modulated extracellular signal– cause of disability and morbidity in developing regulated kinases (ERK)1/2 neuronal survival countries. Despite advancement in understanding pathway in brain of mice [57]. the pathobiology and treatment of CVD, we are 6. SFN is known to inhibit caspase-3 enzyme facing the global problem in alleviating the activation and has reduced the burden of CVD. At present, much research phosphorylation of mitogen activated protein focuses on validation of nutraceuticals. Many kinase (MAPK) signaling pathway, oxidative studies have indicated that consumption of stress. It has enhanced intracellular cruciferous vegetables is associated with glutathione level, methylglyxalase decreased risk of CVD [63]. This category is found detoxifying system and activity of glyoxalase to be rich in isothiocyanates [63]. 1 [58,59]. Consumption of broccoli (which is rich in 7. In spinal cord injury, treatment with sulforaphane) is associated with reduced risk of sulforaphane has up regulated phase 2 coronary artery disease in postmenopausal antioxidant response, decreased mRNA level women. It has also reduced LDL and total of Matrix MetalloProteinase (MMP)-9, an cholesterol and increases HDL cholesterol. inflammatory cytokine and increased the Furthermore, broccoli consumption has reduced serotonergic axon caudal counting to the ischemic reperfusion injury mediated lesion site [52]. cardiomyocyte death [64]. 8. Treatment with erucin has resulted in 4-Carboxyphenyl isothiocyanate (4-CPI) was significant elevation of GSH levels and evaluated for its cardioprotective effect in reduction of reactive oxygen species and O•− Lagendroff perfused heart of wistar rat and formation resulting in resistant to neuronal C57BL/6J mice, an ex vivo model of ischemia cell death [53]. reperfusion injury [65]. 4-CPI significantly improved functional parameters and reduced the Radioprotective activity diameter of ischemic areas. It showed Radioprotective activity of AITC and PITC was vasorelaxation effects on the coronary artery, and evaluated in Swiss albino mice. The whole body membrane hyperpolarisation effects on vascular of the mice were irradiated by gamma radiation smooth muscle cells [65]. by a Co60 source (dose rate = 0.98 Gy/min). Parameters such as serum alkaline phosphatase Mechanism of action (ALP), liver glutamate pyruvate transaminase 1. SFN activates the AKT / Protein kinase B (GPT), haemoglobin, total erythrocyte count and and extracellular signal-regulated kinas 1 and total leucocyte count were monitored. Both ITCs 2 (ERK1/2) signaling pathway, these reduced the level of these enzymes significantly, pathways are implicated in cardiac cell which had been elevated due to irradiation. ITCs survival. It also modulates expression and also enhance the GSH content of intestinal activity of glutathione reductase, glutathione- mucosa and liver. [60] Radioprotective activity of S-transferases, thioredoxin reductase and seed extract of Brassica campteris which is rich NAD(P)H: quinone oxidoreductase via in glucosinolates and isothiocyanates was also activation of phosphatidyl 3-kinase/AKT evaluated and the extract was found to be pathway [66]. effective against radiation induced heamtological 2. Nuclear factor-E2-related factor 2 (Nrf2), a and biochemical changes [61]. basic leucine zipper transcription factor, Radioprotective activity of seed extract of signaling pathway plays a significant role in Moringa oleifera, which is rich in regulation of gene expression of phase II isothiocyanates, was studied against gamma cytoprotective enzymes. In absence of radiation (8 gy) induced irradiation in mice. [62] oxidative stress (Nrf2) pathway remains

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inactive as Nrf2 is sequestrated and degraded reported to possess antidiabetic activity, in the cytoplasm. As soon as the cell confirmation that the activity is due to the experiences oxidative stress, Nrf2 is presence of isothiocyanates has not been translocated to the nucleus where it induces established. There are few reports e.g. SFN transcription of antioxidant genes. It also affords protection against diabetes. It is reported promotes the transcription of many to block the development of type 1 diabetes in cytoprotective genes. Another factor named streptozotocin treated mice by inhibiting cytokine Kelch-like chicken erythroid-derived cap “n” and oxidative stress induced beta cell damage and collar homology (ECH) factor-associated protects against diabetes induced complications protein 1- (Keap1) is a negative regulator of [63]. SFN is also known to protect aortic damage Nrf2. Keap1 forms complex with Nrf2 and in streptozotocin (STZ) induced diabetes in T1D promotes its rapid proteasomal degradation. mice [66].

But during oxidative stress, disruption of Keap 1-Nrf2 complex takes place (through Mechanism of action transduction of phosphorylation and redox Activation of Nrf2 leading to suppression of NF- modification of the complex) which κB mediated pathway. These pathways make ultimately promotes translocation of Nrf2 to antioxidant defense system. Oxidative stress and the nucleus. SFN, an isothiocyanate exerts inflammation plays pivotal role in diabetes and cytoprotective effect by activation Nrf2 its complication [67]. pathway in three ways: 1) Direct effect on Nrf2 related signaling pathway , as it is an Anti-platelet activity electrophile that interact with Keap cysteine 1-Isothiocyanato-4-methylsulfinylbutane has residue and block degradation and been evaluated for its anti-platelet activity using polyubiquitination of Nrf2 and induce in vitro techniques. In these studies, collagen, significantly higher level of Nrf2 expression adenine diphosphate and thrombin were used for in cardiac cell nuclei. 2) The indirect effect platelet aggregation. 1-Isothiocyanato-4- on Nrf2 related signaling pathway, as it methylsulfinylbutane has inhibited collagen induced platelet aggregation significantly. It also activates protein kinase B and extracellular inhibited collagen and epinephrine induced signal regulated kinase 1 and 2 signaling pulmonary embolism [68]. 1-Isothiocyanato-4- pathway, which play role in cardiac cell methylsulfinylbutane anti-platelet activity was survival. 3) SFN also mediate long term confirmed invivo using ADP-induced acute protection against free radical induced cell pulmonary thromboembolism in mice [69]. Allyl damage. Therefore, SFN is found to be isothiocyanate was evaluated for its anti-platelet potent against many cardiovascular diseases activity via in vitro techniques [68]. The study like hypertension, atherosclerosis, and suggested that it possessed remarkable anti- cardiac ischemia reperfusion injury [63]. platelet activity. 3. The cardioprotective effect of broccoli is mediated through inhibition of phase I Mechanism of action enzyme and DNA adduct, angiogenesis, 1. Anti-platelet activity of 1-Isothiocyanato-4- inflammation and induction of phase II methylsulfinylbutane may be attributed to enzymes and cell cycle arrest [64]. activation of intrinsic pathway and inhibition 4. The mechanism of action of 4-CPI in of cycloxygenase and glycoprotein IIb/IIIa cardioprotection was attributed to H2S donor [69]. capacity (H2S play pivotal role in regulation 2. Activation of adenyl cyclase/cAMP pathway of blood pressure and cardiac function) and followed by inhibition of intracellular activation of mitochondrial K+-ATP channel cascades such as PI3-kinase/Akt and activation [65]. collagen-induced phosphorylation of phospholipase C (PLC) γ2- protein kinase C Anti-diabetic activity (PKC) -p47 pathway and eventually Studies on antidiabetic activity of isolated inhibition of platelet activation and isothiocyanates are scarce. Although crude aggregation [68]. extracts of plants rich in isothiocyanates are 3. Anti-platelet activity of AITC was attributed 78 RJP 5(2), 2018: 71-89

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to the inhibition of TXA2 production, ATP (mainly lymphocytes and monocytes) and release, cellular calcium increase and increased neutrophils count. At a dose of 100-150 phosphorylation of PKCδ, p38, ERK, Akt mg/kg, AITC increased AST levels. However, [70]. the toxicity was suggested to be dose dependant [73]. AITC was moderately and acutely toxic and Toxicity profile caused skin and eye irritation, although it has The risk benefit ratio of isothiocyanates is been systemically safe in subchronic feeding controversial and hence the toxicity profile of study in laboratory animals [75]. A dose of 25 ITCs needs to be elaborated. Various data from mg/kg/day for 90 days was reported to be safe in vitro studies suggest that ITCs exhibit and no clinical effects were observed. According genotoxic potential as they form adducts with to one report, fourteen individual tests were DNA. However, the dietary concentration of conducted to determine the mutagenic effects of ITCs was found to be several orders of AITC. Among these, eight were considered to be magnitude lower compared to the doses used in negative as they did not develope any genotoxicity studies. A precise evaluation of mutagenecity, three were positive (developed toxicological profile is required to predict mutagenicity) and two were equivocal potential genotoxicity of ITC and for any (ambigous) [74]. recommended clinical use [71]. Listed here are (containing 93-97% AITC) was used the toxicity profiles of some individual ITCs on to access the developmental toxicity and the basis of which some conclusion can be mutagenecity. Low observed adverse effect level drawn. (LOAEL) of AITC was found to be 2.8 and 23.8 mg/kg/day (in rabbits and hamsters) for Benzyl isothiocyanate (BITC) incidences of extra sternebrae and incomplete Daily dose of BITC (12 µm) was found to be safe ossification of sternebrae [74]. and did not possess any toxicity. However, in rats treated with a substantially higher dose (200 Phenylethyl isothiocyanate (PEITC) mg/kg), alteration in various parameters were PEITC has been reported to be safe and does not observed including reduction in haemoglobin, possess any apparent toxicity when administered lymphocyte count and elevation in neutrophils, at high doses (as revealed by safety studies in rats eosinophils and platelets and increased and dogs) [33]. At doses of 80 and 160 mg/kg cholesterol and renal dysfunction. Adverse PEITC i.p., increased weight of liver and spleen. effects were not observed at a dose of 50 mg/kg Effective concentration of PEITC were 0.12 to 14 and hence that dose was considered to be safe at µM [75]. therapeutic dose. BITC induced mutagenesis in PEITC is currently in phase I trials and found to the Ames assay and also caused chromosomal be active against lymphoproliferative disorders. aberration and DNA strands breaks. It is also The trial is conducted by University of Texas – known to induce bladder carcinogenesis [72]. M.D Anderson Cancer Centre. [75] In a study examining the sub-acute toxicity of BITC in male rats at oral doses of 0, 50, 100 and Sulforaphane 200 mg/kg B.W/day for 4 weeks, BITC induced There is lack of confirmatory evidences whether haematological alteration of toxicological SFN is safe or possesses some toxicity. SFN at a relevance and decreased TG level at 200 mg/kg dose of 64 mg/kg (in mouse) has been reported to and increased serum cholesterol at all doses [73], induce hepatotoxicity and there are studies which indicating renal dysfunction. contradicts such toxicity [76].

Allyl isothiocyanate (AITC) Various isothiocyanates from plant sources As per the report of agency vegetable oil RED, and their potential benefits Dec 1993, AITC is not likely to cause adverse Glucosinolate and their breakdown product, human health effects and the oral lethal dose in isothiocyanates, exhibit diverse therapeutic rats was found to be 339 mg/kg [74]. AITC at a potentials. For decades, studies have been dose of 20 mg/kg s.c. has reduced WBC counts conducted to explore more and more potential

79

Kala C. et al. benefits of isothiocyanates and their toxicity. has summarised those plants which have Numerous plants have been exploited by workers undergone studies for their isothiocyanate using different extraction methods to extract out contents, method of extraction and their ITCs of significant therapeutic potential. Table 1 pharmacological activities.

Table 1.Various isothiocyanates from plant sources with their pharmacological and biological activity Extraction Identification Pharmacological/biological Scientific name Part ITCs Ref technique technique activity Allyl ITC Brassica oleracea var. Solid Phase micro Phenyl ITC Active against Alteria rot in Leaves GC/MS 77 capitata extraction Benzyl ITC Bell Pepper Phenylethyl ITC Skin anti-inflammatory Seeds Steam distillation GC, NMR, IR methylthiobutylITC 78 effect Allyl ITC Eruca sativa Steam distillation 3-butenyl ITC Activity against harmful Seed oil GC/MS 79 HS/SPME 2-phenylethyl ITC effect of Alfatoxin Sulphoraphane Brassica oleracea var. R-sulforaphane Inhibit quoram sensing in Sprout GC/MS 80 italica Erucin pseudomonas aeroginosa Phenylethyl ITC Solvent 7-methylsulfinylheptyl Rorippa nasturtium- Inducer of Phase-II enzymes Tissue extraction with LC-MS/ UV ITC 81 aquaticum (L.) Hayek (quinone reductase) 70% ethanol 8-methylsulfinyloctyl ITC Preparative Sulfadex G-15 HPLC, Wasabia japonica 6-(Methylsulfinyl) Root Gel filtration and FAB-MS, Anti-inflammatory activity 82 Matsum hexyl ITC reverse HPLC EI-MS, IR & NMR

4-[(4’-O-acetyl-α-L- rhamnosyloxy)benzyl] ------Hypotensive effect 83 Moringa oleifera Pods ITC 4(α-L-rhamnosyloxy) benzyl ITC 4-(methylthio)-3- Raphanus sativus ------Antimutagenic activity 84 butenyl ITC Brassica alba Seeds ------Allyl ITC Acaricidal activity 85 Seed RP-HPLC GC/MS Sulforaphane --- 86 Brassica oleraceae var Stem Hydrodistillation GC/MS Allyl ITC Anti-microbial activity 87 botrytis c.v. Calabrese Stem Hydrodistillation GC/MS Butane-1-ITC Anti-microbial activity 87 Stem Hydrodistillation GC/MS 2-phenylethyl ITC Anti-microbial activity 87 4- Aerial GC-FID / Cardariadraba (1) desv Hydrodistillation (methylsulfanyl)butyl Anti-microbial activity 88 pats GC/MS ITC 2-phenylethyl ITC Brassica junica Seeds SPME --- Benzyl ITC Fungicidal activity 89 2-butenyl ITC Brassica nigra Seeds SPME 2-phenylethyl ITC Fungicidal activity 89 Hydrodistillation- Degeniavelebitica Leaves GC/MS Pentyl-4-enyl ITC Antimicrobial activity 90 adsorption Activity against gram Salvadoraperseca Roots GC/MS Benzyl ITC 91 negative bacteria E-2-hexenal methyl Flowers, Hydrodistillation- ITC Eruca sativa leaves, adsorption, GC, GC-MS 3-butenyl ITC --- 92 bean hydroditillation Hexyl ITC Erucin Wasabia japonica Whole Supercritical fluid Gas Allyl ITC --- 93 Matsum plant extraction chromatography Extraction from pretreated Brassica juncea Seed --- Allyl ITC --- 94 mustard meal by distillation

80 RJP 5(2), 2018: 71-89

Isothiocyanates: a review Table 1. Continued Extraction Identification Pharmacological/biological Scientific name Part ITCs Ref technique technique activity Solvent Allyl ITC Seed extraction with n- GC/MS Benzyl ITC --- 95 hexane Phenethyl ITC Stems, leaves Alliaria petiolata Hydrodistillation GC & GC/MS Allyl ITC --- 96 and flower Allyl ITC 3-butenyl ITC 2-pentyl ITC β-phenylethyl ITC Isopropyl ITC Armoracia ruticana Root Steam distillation GC/MS Butyl ITC --- 98 2-pentyl ITC Phenyl ITC 3-methylthiopropyl ITC Benzyl ITC GC/MS & Leaves --- 3-butenyl ITC --- 99 Brassica rapa subsp. LC/MS pekinensis GC/MS & Midrib --- 3-butenyl ITC --- 99 LC/MS Solvent 4-pentenyl ITC Flower extraction with GC/MS 2-phenylethyl ITC --- 100 Brassica campestrisspp. Dichloromethane 4-methylpentyl ITC pekinensis 3-butenyl ITC Table 1. Continued Seeds --- GC/MS 4-pentenyl ITC --- 101 2-phenylethyl ITC Brassica Rapa ------Benzyl ethyl ITC --- 102 Isopropyl ITC Allyl ITC 3-butenyl ITC 3-methylbutyl ITC Pentyl ITC 4-methylpentyl ITC Seeds/ Hexyl ITC Brassica Rapa HS/SPME GC-IT-MS --- 102 sprout 3-methylbutyl ITC Pentyl ITC 4-methylpentylhexyl ITC 3-methylthiopropyl ITC Phenylethyl ITC Seeds ------Benzyl ITC --- 103 Roots Leptidium sativa and non- flowering ------Benzyl ITC --- 103 aerial parts Solvent Tropaeolum majus Seeds extraction using HPLC Benzyl ITC --- 104 90% methanol 4-[(4’-O-acetyl-α-L- rhamnosyloxy)benzyl] ITC 4(α-L-rhamnosyloxy) benzyl ITC Seeds & 1H-NMR & (4-[(2’-O-acetyl-α-L- Moringa oleifera Lam --- 105 leaves LC-MS rhamnosyloxyl)benzyl ITC (4-[(2’-O-acetyl-α-L- rhamnosyloxyl)benzyl ITC

Hydrodistillation HPLC Benzyl ITC --- 106 Raphanus sativus L. 4-methylthio-3- Roots --- GC/MS --- 107 butenyl ITC

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Kala C. et al. Table 1. Continued Extraction Identification Pharmacological/biological Scientific name Part ITCs Ref technique technique activity Drypetes roxburghii ------108 Carica papaya Seeds --- GC/MS Benzyl ITC --- 109 Isopropyl ITC Sisymbrium officinale ------GC/MS --- 110 Sec-butyl ITC Benzyl ITC Lepidium meyenii ------111 p-metoxybenzyl ITC Leaves Isopropyl ITC --- 13 C-NMR --- 112 oil Butyl ITC Methyl ITC Leaves --- GC/MS --- 113 Capparis spinosa Benzyl ITC Methyl ITC Ripe fruit ------Isopropyl ITC --- 113 and roots Sec-butyl ITC Capparis Ovata Oil --- GC/MS Methyl ITC --- 113 GC/MS & GC- Brassica hirta Seed --- 4-hydroxybenzyl ITC --- 92 FID Capsella bursa pastoris Seed --- GC/MS 3-Butenyl ITC --- 92 Erucin Sulforaphane Erysimum allionnii Seed --- GC/MS --- 92 Erucin nitrile Sulforaphane nitrile Lesquerella fendleri ------3-butenyl ITC --- 92 Lobularia maritima ------3-butenyl ITC --- 92 Matthiola longipelata ------Sulforaphene nitrile --- 92 Leaves & Brassica fruticulosa SPME GC/MS 3-butenyl ITC --- 114 root 4-isothiocyanato-1- Aerial Butene Morettia phillaeana Hydrodistillation GC/MS --- 115 parts Isothiocyanatomethyl Benzene 3-benzyl ITC 4-methylpentyl ITC Broccolini (Brassica 1-isothiocyanato oleraceaItalica X Seeds --- GC/MS --- 116 butane Alboglabra) Phenylethyl ITC Sulforaphane Allyl ITC Butyl ITC 3-butenyl ITC Whole 3-butenyl ITC Iberis sempervirens Hydrodistillation GC & GC/MS --- 117 plant 3-methylthippropyl ITC 4-methylthiobutyl ITC 2-phenylethyl ITC Isothiocyanates (ITCs), Gas chromatography-Mass spectroscopy (GC/MS), Nuclear magnetic resonance (NMR), Infrared (IR), Liquid chromatography- mass spectroscopy (LC-MS), Ultra violet (UV), High performance- liquid chromatography (HPLC), Reverse phase-HPLC (RP-HPLC), Solid phase micro extraction (SPME), Gas chromatography – flame ionization detector (GC- FID). Fast atom bombardment mass spectroscopy (FAB-MS), Electron ionization mass spectroscopy (EI-MS)

Conclusion anti-tumour and anti-inflammatory effects have Isothiocyanates which could be found abundantly been known but accurate doses of ITC have not in cruciferous vegetables have dragged lots of been established by researchers. Different attention during the past years for their ability to researchers used different doses of ITC. Some exert beneficial effects on human health. displayed their therapeutic activity in micrograms Numerous epidemiological studies have proved while others much more concentrations. Also, the their beneficial effects. An attempt has been therapeutic window and toxicity profile of ITCs made by authors to distinguish minor to major have not been defined. aspects of isothiocyanates because some aspects Therapeutic activity of ITC is also influenced by have not been discussed previously and thus still the presence of aromatic and aliphatic side chain need a profound consideration and study. in their structure; thus, the structure activity Pharmacological activities e.g. anti-carcinogenic, relationship should be further studied thoroughly.

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4-iodophenyl isothiocyanate; ABC: ATP-binding minimum bactericidal concentration; MMP: cassette; AITC: allyl isothiocyanate; ALP: matrix MetalloProteinas; MMP: matrix alkaline phosphatase; ARE: activated Nrf2- metalloproteinases; MPP+: antioxidant response; ATM: ataxia telangiectasia methylphenylpyridinium; MPTP: 1-methyl-4- mutated protein kinase; ATR: ataxia phenyl-1,2,3,6-tetrahydropyridin; MRAB: telangiectasia and Rad3 related protein kinase; multidrug-resistant Acinetobacter baumanii; Bax: Bcl associated X protein; Bcl-2: B-cell MRP: multi drug resistance protein; MRPA: lymphoma-; BCRP: breast cancer resistance multidrug-resistant Pseudomonas aeruginosa; protein; BEAC: barrett oesophageal MRSA: methicillin-resistant Staphylococcus adenocarcinoma; BITC: benzyl isothiocyanate; aureus; NDI: naphthalenetetracarboxylic diimide; BITC: benzyl isothiocyanate; CDK: cyclin- NMR: nuclear magnetic resonanc; Nrf2: nuclear dependent kinases; CVD: cardiovascular diseases; factor (erythroid- derived2)-like 2; Nrf2: DA: dopaminergic; DR4: death receptor 4 ; DR5: nuclear factor-E2-related factor 2; NSCLC: non- death receptor 5; EAE: experimental autoimmune small cell lung cancer; OMS: oriental mustard encephalomyelitis; EI-MS: electron ionization see; PD: Parkinson’s disease; PEITC: phenyl mass spectroscopy; ERK: extracellular signal– ethyl isothiocyanate; P-gp: P-glycoprotein; PIC: regulated kinase; FAB-MS: fast atom phenethyl isocyanates; PKC: protein kinase C; bombardment mass spectroscopy; FANCD2: PLC: phospholipase ; QR: quinone reductase ; Fanconi anemia complementation group proteins RP-HPLC: reverse phase-HPLC ; RT-PCR: D; GC/MS: gas chromatography-Mass reverse transcription polymerase chain reaction; spectroscopy; GC-FID gas chromatography - SFN: Sulphoraphane; SPME: solid phase micro flame ionization detector; GGT: γ-glutamyl extraction ; STZ: streptozotocin; SULT: transpeptidase; GPT: glutamate pyruvate sulphonyltransferase; TRAIL: TNF-related transaminase; GSH: glutathione; GST: apoptosis-inducing ligand; UGT: UDP- glutathione S-transferas; HDAC: histone glucoronosyltransferase; UV: ultra violet; VEGF: deacetylases; HPLC: high performance- liquid vascular endothelial growth factor; VRSA: chromatograph; HSP: heat shock protein; IR: vancomycin-resistant S. aureus; XTT: 2,3-bis(2- infrared; ITC: isothiocyanate; Keap 1: Kelch-like methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium- ECH-associated protein 1; LC-MS: liquid 5 carboxanilide inner salt chromatography- mass spectroscopy; MAPK: mitogen-activated protein kinases; MBC:

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