5 SFN, TRAIL, and Cancer Stem Cells

364 S. Banerjee and S.B.
Paruthy
due to multiple mechanisms which include the induction of death receptors and
proapoptotic members of Bcl-2 family and inhibition of antiapoptotic Bcl-2 pro-
teins and activation of caspase(s). Also, by sulforaphane pretreatment, enhance-
ment of the inhibitory effect of TRAIL on PC-3 colony formation and sensitization
of TRAIL-resistant LNCaP colonies convey the message that sulforaphane aug-
ments the ability of TRAIL to inhibit colony growth. Additional data suggest that
upregulation of death receptors DR4 and DR5 by sulforaphane may be one of the
mechanisms that reinforce the proapoptotic effects of TRAIL in PC-3 cells and
sensitize TRAIL-resistant LNCaP cells. In vivo, sulforaphane inhibits the growth
of PC-3 cells orthotopically implanted in nude mice by inducing apoptosis and
inhibiting tumor cell proliferation. It has been recorded to inhibit the expression of
proteins related to invasion, metastasis, and angiogenesis by inhibiting phosphor-
ylation of AKT, ERK1/2, and FOXO3a and activation of NF-κB [130]. These
provide strong and compelling preclinical evidence that sulforaphane either alone
or in combination with TRAIL can be used to prevent and/or treat prostate cancer.
5 SFN, TRAIL, and Cancer Stem Cells
5.1 Prostate Cancer
An extension to the above report in support of SFN-sensitizing prostate cancer cells

to TRAIL-induced apoptosis, apoptosis-resistant cancer stem cells (CSCs) have been
identified in advanced androgen-independent prostate cancer (AIPC) that are not
eliminated by currently available therapeutics. Thus, the disease relapses due to the
ability of CSCs to self-renew and differentiate into multiple cell types populating
with all the cellular subtypes of the original tumor. In particular, these cells over-
express CSC markers including ALDH1, CD44, CD133, and c-Met [131]. Therefore,
numerous investigators pursued investigating innovative approach combining
TRAIL and bioactive natural compounds aimed to eliminate CSCs. In line with
such objective and utilizing advanced androgen-independent prostate cancer (AIPC)
cell lines – DU145 and PC3 – with highly enriched CSC characteristics and primary
patient-derived prostate CSCs, Labsch et al. revealed a synergistic action between
SFN and recombinant soluble TRAIL when used in combination [132]. The authors
built their conclusion from results comparing the effects of the drugs either as
monotherapy or in combination using multitude of in vitro assays including
NF-κB activity, self-renewal and differentiation potential, stem cell signaling via
spheroid, colony-forming assays, and antibody protein array. Mechanistically, the
dominance of a therapeutically relevant effect associated with the combination
regimen is a result of the inhibition of TRAIL-induced NF-κB activity by SFN
and reduction in the levels of Oct-3/4, HNF-3β, PDX-1, Otx2, TP63, GSC, Snail,
VEGFR-2, and HCG relative to monotherapy. These proteins relate to requirement
for self-renewal, differentiation, cell migration, and epithelial-mesenchymal transi-
tion (EMT) and tumorigenesis. Additionally, it has been noted that the expression of
CXCR4 receptor which is involved in migration and metastasis becomes inhibited
12 Therapeutic Paradigm Underscoring Glucosinolate Sulforaphane in Chemo- and. . . 365
following sulforaphane-only treatment and the combination with TRAIL further

reduces the expression of this receptor. Similar results were noticed for the Notch-
1 receptor and its ligand, Jagged-1, which are regulators of asymmetric and sym-
metric division, progression, and metastasis in prostate cancer. Correspondingly, the
inhibition of SOX 2 and Nanog expression which are important regulators of self-
renewal potential was much stronger in the combination group. ALDH1 activity
which represents differentiation and self-renewal potential was also significantly
inhibited by the combination regimen attesting strong inhibition of stem cell signal-
ing potential. Furthermore, in an in vivo tumor engraftment model, TRAIL and SFN
together inhibited tumor growth, reduced CSC marker expression, and eliminated
primary prostate CSCs synergistically via the induction of apoptosis and inhibited
the proliferation and CSC marker expression. Conclusively, these findings suggest
that sulforaphane shifts the balance from (TRAIL-induced) survival signaling to
apoptosis and explains the observed synergistic effect.
5.2 Pancreatic Cancer
To address the need to eliminate oncogenically transformed CSC in pancreatic

cancer (PaCa), Kallifatidis et al. pointed that pancreatic tumors harbor tumor-initi-
ating cells (TICs; aka CSC), wherein TRAIL-activated NF-κB signaling acts as a
contributor to resistance to apoptosis induction by TRAIL [109]. The reported
anticancer activity of SFN in pancreatic cancer stem cells has been detailed in the
preceding section [109]. Of interest, sulforaphane allegedly prevents NF-κB binding
and thus is able to arrogate resistance to chemotherapy without cytotoxicity to
normal cells. In a xenograft model, sulforaphane strongly blocked tumor growth
and angiogenesis, while combination with TRAIL levied an additive effect without
any obvious cytotoxicity to normal cells [109]. Further, primary tumor cells isolated
from tumors freshly resected from patients with pancreatic cancer expressing
markers for TICs could also be sensitized by sulforaphane for TRAIL-induced
cytotoxicity; together these findings provide new insights into resistance mecha-
nisms of TICs and suggest the combination of sulforaphane with TRAIL as a
promising strategy for eliminating pancreatic TICs.
6 Sulforaphane Combination with Other Natural Agents

in Cancer Therapy
Against the backdrop of foregoing thought-provoking reports from laboratory inves-

tigations confirming that SFN intercedes in sensitizing different treatment modalities
including TRAIL therapy, anticipations from combination of SFN and other well-
characterized chemopreventive bioactive natural compounds aiming to increase the
antitumor response became the focus of several reports. Based on scientific rationale
that health benefits of fruits and vegetables are from additive and synergistic
combination of phytochemicals, SFN when combined with other bioactive
366 S. Banerjee and S.B. Paruthy
phyto-compounds also displays promising results against various cancers. Such effect
may conceivably be due mainly because of the induction of a broad spectrum of anti-
cancer pathways. This concept is well illustrated in study reporting chemoprevention
of familial adenomatous polyposis (FAP) by combination of sulforaphane and
dibenzoylmethane in ApcMin/+ mouse colorectal cancer model that mimics the
human familial adenomatous polyposis (FAP) that progresses sporadically to colo-
rectal cancer with early Apc mutation [133]. In a prior study on human colon
carcinoma HT-29 cells, SFN shows growth inhibition by inducing G1 cell cycle
arrest and apoptosis, probably through the regulation of mitogen-activated protein
kinase (MAPK) without affecting the Akt pathway [134]. On the other hand,
dibenzoylmethane (DBM) – a minor constituent of licorice, inhibits cancer cell
growth by inhibiting Akt pathway activity. Thus, a study that included a combination
treatment regimen of SFN and DBM at half of the single dose was designed to
investigate their potential synergistic effect on colon cancer chemoprevention in
ApcMin/+ mouse model [133]. The results showed that dietary administrations of
SFN and DBM combination significantly inhibits the development of intestinal
adenomas and block the colon tumor development by inhibition of cell survival
and growth-related cell signaling pathways (such as Akt and extracellular signal-
regulated kinase) and induction of apoptosis and other related biomarkers (including
arachidonic acid metabolism, proliferating cell nuclear antigen, cleaved caspases,
cyclin D1, and p21) conceptually supporting the hypothesis that both SFN and DBM
in combination are potent natural dietary compounds in chemoprevention of gastro-
intestinal cancers. Moreover, the protective ability of glucosinolates due to syner-
gistic effect with other constituents in the diet cannot be ignored. In this direction,
SFN in combination with tea polyphenol – epigallocatechin (EGCG) – has been
testified in ovarian, prostate, and colon cancers [135–137]. Previous published
epidemiological studies indicate that consumption of green tea and cruciferous
vegetables is inversely associated with occurrence of ovarian cancer
[138–141]. Therefore, the combinatorial effect and underlying mechanism of
major components of green tea (epigallocatechin gallate – EGCG) and cruciferous
vegetables (sulforaphane) on ovarian and colon cancer cells as basis for innovative
therapeutic approach befitted as subject for further investigation [135]. Encouraging
data interestingly depicts that EGCG potentiates the inhibiting effect of SFN on
paclitaxel-sensitive and paclitaxel-resistant ovarian cancer cell lines in time- and
dose-dependent manner. Further examination points to SFN action in arresting
ovarian cancer cells in G2/M phase, while EGCG and SFN co-treatment causes
cell cycle arrest in both G2/M and S phases resulting in a significant escalation in
apoptosis. The combination also strongly inhibits hTERT (the main regulatory
subunit of telomerase) and Bcl-2 and alters the expression of phosphorylated
H2AX that promotes DNA damage response specifically in paclitaxel-resistant
ovarian cancer cell lines proposing the use of these compounds for overcoming
paclitaxel resistance in ovarian cancer treatment [135]. Furthermore, a synergistic
combination effect between sulforaphane and (-) epigallocatechin-3-gallate in
human colon carcinoma cells (HT-29 AP-1) has also been reported [136]. The
same group of investigators examined in prostate of Nrf2-knockout mice and
human prostate cancer cells the combination of EGCG and SFN with evidence of
regulatory cross talks between the transcription factors Nrf2 and AP-1 in prostate
tumors as several key Nrf2-dependent genes were found downregulated (three- to
35-fold) after in vivo administration of the combination of EGCG (100 mg/kg) and
SFN (45 mg/kg) [137]. In silico bioinformatics data revealed conserved transcription
factor binding site (TFBS) signatures in the promoter regions of Nrf2 and AP-1
suggesting the combination effect of SFN+EGCG is mediated via a concerted
modulation of Nrf2 and AP-1 pathways in the prostate [137].
Interestingly, SFN as a supportive partner with resveratrol and eugenol in induc-
ing apoptosis of tumor cells has also been reported [142, 143]. In glioma cells,
combination treatment with resveratrol inhibits cell proliferation and migration,
reduces cell viability, induces lactate dehydrogenase release, decreases
pro-survival Akt phosphorylation and increases caspase-3 activation [142]. This
points the worth of combining resveratrol and sulforaphane for the treatment of
glioma. However, more critical studies before translational implication of this study
are warranted. In human cervical cancer cells, concurrent sublethal doses of SFN and
eugenol act in a synergistic manner with gemcitabine culminating in loss of viable
cells by stimulation of apoptosis elicited by downregulation in the expression of
Bcl-2, COX-2, and IL-β [143].
Based on the notion that additional distinctive isothiocyanate constituents of
cruciferous vegetables such as indole-3-carbinol (I3C) or its condensation product
3,30 -diindolylmethane and SFN underscore any prognostic interactive influence,
their role in prostate and colon cancer models has been examined. In human colon
cancer cell lines, ITCs like sulforaphane (SFN) are cytotoxic, whereas indoles
including indole-3-carbinol and its condensation product 3,30 -diindolylmethane act
by cytostatic mechanisms. As a result, using defined combinations of SFN and
3,30 -diindolylmethane, the investigators interestingly found that at a total drug
concentration of 2.5 μM, all combinations of SFN and 3,30 -diindolylmethane were
antagonistic, but consecutively with increasing concentrations, the antagonistic
effect gradually turned into a synergistic interaction at the highest combined cyto-
toxic concentration of 40 μM [144]. Furthermore, SFN and DIM combination
(at 10 μM concentrations of each) results in strong G2/M cell cycle arrest, which
was not observed with either compound alone. Overall, the results suggest that at
low total concentrations (below 20 μM), which is physiologically more relevant, the
combined broccoli compounds display antagonistic interactions in terms of cell
growth inhibition. Building on their findings, further elucidation of mechanistic
interactions between bioactive food components is warranted for much better pre-
diction of beneficial health effects. With epidemiological findings weighing in favor
of high intake of cruciferous vegetables reducing the risk of prostate cancer
[145–147], the effect of the combination of glucosinolate hydrolysis products such
as I3C and SFN which are constituents of cruciferous vegetables seems logical and
therefore was examined mainly with reference to cell proliferation in a prostate
cancer cell line (PC-3) model. Cell proliferation in PC-3 prostate cancer cells was
significantly inhibited by I3C and sulforaphane at media concentrations of 0.2 mmol/
L and 0.02 mmol/L, respectively, which explicitly explains the observed protective
effect of cruciferous vegetables in relation to the risk of developing prostate

cancer [148].
At present time, there is no strong evidence for an inverse association between
consumption of cruciferous vegetables and pancreatic cancer risk, except a prospec-
tive study reporting cabbage consumption and statistically significant lower risk of
pancreatic cancer (one or more servings/week versus never consumption)
[149]. However, in support of the notion that combination of chemopreventive
bioactive compounds may afford protection against pancreatic cancer, few studies
have been conducted as presented herein. In related context, Hutzen et al. appraised
and reported bioactive isothiocyanates from cruciferous vegetables such as benzyl
isothiocyanate (BITC) and sulforaphane, both being capable of inhibiting cell
viability and inducing apoptosis in PANC-1 by mechanistically targeting inhibition
of tyrosine phosphorylation of signal transducer and activator of transcription factor-
3 (STAT-3) [150]. The STAT proteins comprise a family of latent transcription factors
with well-established roles in cell proliferation, growth, and survival and are consti-
tutively active in many human cancers; thus, inactivation of STAT-3 signaling has
emerged as an attractive target in cancer therapeutics. Interestingly, SFN has minimal
effect on the direct inhibition of STAT-3 tyrosine phosphorylation; however, combi-
nations of BITC and SFN inhibited cell viability and STAT-3 phosphorylation more
radically than either agent alone signifying that the combination of these bioactive
agents, BITC and sulforaphane, carries immense translational potential as a thera-
peutic and interventional agent. Moreover, overlapping with this report, in a 16-week
medium-term pancreatic carcinogenesis model in hamsters, the protective effects of
benzyl isothiocyanate and sulforaphane against initiation of pancreatic carcinogen-
esis have been acknowledged [151]. The multiplicity of combined pancreatic lesions
including atypical hyperplasias and adenocarcinomas was significantly decreased by
feeding a diet supplemented with 80 ppm BITC and 80 ppm SFN in the initiation but
not the post-initiation stage underpinning the fact that isothiocyanates BITC and SFN
together can block benzo(a)pyrene initiation of hamster pancreatic carcinogenesis.
Relating to pancreatic cancer therapy, another published study in literature docu-
ments that low doses of aspirin (ASP, 1 mM), curcumin (CUR,10 μM), and sulfo-
raphane (SFN, 5 μM) (ACS) combination statistically reduce cell viability and
concurrently induce apoptosis facilitated by activation of caspase-3 and poly
(ADP-ribose) polymerase (PARP) cleavage, considered as hallmark of apoptosis
[152]. Further mechanistic probing reveals that ACS inhibits NF-κB DNA binding
activity and promotes sustained activation of phosphor-extracellular signal-regulated
kinase 1/2 (p-ERK1/2), c-Jun, p38 MAPK signaling, and p53 proteins as probable
mechanism [152]. In addition, the feasibility of using solid lipid nanoparticulate
system to deliver this combination chemopreventive regimen has been investigated
for pancreatic cancer chemoprevention [153]. Referencing previously described
concept of cancer stem cells backed by in vitro data that SFN can eliminate
pancreatic CSC by inhibiting NF-κB activity alongside self-renewal capability
sensitizes the cells to undergo apoptosis [72, 109, 110]. An extension to this
observation, SFN, quercetin, and a natural mixture of a complete set of green tea
catechins (combination of green tea-derived catechins – epicatechin-2-gallate (ECG)
and catechin gallate (CG)) have been found to complement each other in the
elimination of advanced pancreatic cancer by miR-let-7 induction and K-ras inhibi-
tion [154]. Additionally, this novel combination of quercetin, sulforaphane, and GTC
also inhibits the self-renewal potential, apoptosis resistance, and migratory potential
of tumor cells; of significant interest is the fact that the combination of bioactive
agents is more effective than each single agent despite current therapeutics do not
adequately target the CSCs and novel therapeutic options are critically warranted.
Additional citation in the literature opinions sulforaphane synergizing with quercetin
to inhibit self-renewal capacity of pancreatic cancer stem cells by inhibiting stem cell
pluripotent transcription factor Nanog which could be a novel strategy to eliminate
cancer stem cell characteristics and destroy CSCs [155].
7 Sulforaphane and Clinical Trials
Given the exceptional inciting laboratory research conclusions projecting SFN as a

multifaceted bioactive natural compound that targets multiple processes associated
with tumor growth and survival and further outcome of experimental laboratory
studies in context of combined novel therapies, clinical trials are expected to be
initiated in the future to confirm therapeutic benefit of its combination with standard
chemotherapeutics for chemo- and radiosensitization of cancer. The toxicity and
effects of SFN in healthy men and patients with cancer have been recorded. In a
phase I clinical trial, SFN from broccoli sprouts has been evaluated for safety,
tolerance, and metabolism and conveyed a good safety profile [156]. In this protocol,
following a 5-day acclimatization period on a crucifer-free diet, broccoli sprout
extracts were administered orally at 8-h intervals for 7 days (21 doses). Most clinical
trials utilize broccoli sprouts or their hot water infusion as a rich and consistent
source of glucoraphanin because the young florets reportedly contain glucoraphanin
levels 20–50 times more than mature market-stage broccoli [13]. Current search for
sulforaphane and cancer at http://www.clinicaltrials.gov indicates a total of 13 trials
either ongoing, complete, or currently recruiting (Table 1). Phase II clinical trial for
treating patients with recurrent prostate cancer is currently being performed
(ClinicalTrials.gov, NCT01228084). Unfortunately, contextually relating to focus
of this chapter, none of the projected trials is currently inclined to sensitization
aspect of cancer therapy. However, the POUDER study objective which is currently
recruiting participants will provide data on the clinical feasibility and acceptability
of an supportive treatment option accompanying palliative chemotherapy
[NCT01879878]. This pilot study, POUDER trial, determines the feasibility of a
randomized controlled trial regarding the application of freeze-dried broccoli sprouts
rich in sulforaphane and quercetin in patients with advanced surgically
non-resectable pancreatic ductal adenocarcinoma (PDA) under palliative chemother-
apy. The patients in the experimental arm will receive 15 capsules containing a total
of 90 mg of active sulforaphane during the chemotherapy treatment course as a
nutrition supplement, whereas patients assigned to the placebo group will receive
inactive substance (methylcellulose) with identical capsule and potion distribution.
Table 1 Clinical trials with sulforaphane in cancer (http://www.clinicaltrials.gov)

Identifier
Official title # Study design Status
Pilot study evaluating broccoli sprouts in NCT Intervention Recruiting
advanced pancreatic cancer [POUDER trial] 01879878 participants
The effects of sulforaphane in patients with NCT Interventional Study completed
biochemical recurrence of prostate cancer 01228084
Chemoprevention of prostate cancer, HDAC NCT Interventional Not known
inhibition, and DNA methylation 01265953
Evaluating the effect of broccoli sprouts NCT Interventional Study completed
(sulforaphane) on cellular proliferation, an 00982319
intermediate marker of breast cancer risk
A human dietary intervention study to NCT Interventional Ongoing, but not
investigate the effect of sulforaphane on 01950143 recruiting
prostate cancer interception
Effect of topical application of sulforaphane NCT Observational Ongoing, but not
containing broccoli sprout extracts on 00894712 recruiting
radiation dermatitis during external beam participants
radiation therapy for breast cancer
Sulforaphane: a dietary histone deacetylase NCT Interventional Study completed
(HDAC) inhibitor in ductal carcinoma in situ 00843167
(DCIS)
Broccoli sprout extracts in healthy NCT Interventional Study completed
volunteers: a pilot study of Nrf2 pathway 02023931
modulation in oral mucosa
An intervention study to assess NCT Interventional Completed
bioavailability of sulforaphane delivered by 02300324
glucoraphanin-enriched broccoli soups in
healthy subjects
A pilot study evaluation of sulforaphane in NCT Interventional Ongoing, but not
atypical nevi-precursor lesions: assessment 01568996 recruiting
of STAT1 and STAT3 risk markers of participants
melanoma
Broccoli sprouts intervention in Qidong, PR, NCT Interventional Completed
China 01437501
Cruciferous vegetable intake and histone NCT Observational Recruiting
status in screening colonoscopy patients 01344330 participants
A randomized, double-blind, placebo- NCT Interventional Study completed
controlled, multiple ascending dose study to 02055716
evaluate the safety, tolerance,
pharmacokinetics, and pharmacodynamics
®
of Sulforadex in healthy male subjects
following daily dosing for 7 days
The outcome of this study will provide data on the clinical feasibility and accept-
ability of supportive treatment option accompanying palliative chemotherapy.
Importantly, from the outcome of the results, future clinical studies to create further
awareness for therapeutic benefit of sulforaphane in combination with chemothera-
peutic agents and radiotherapy in the field of oncology are anticipated.
8 Conclusions and Perspective
Based on the above overwhelming evidence supporting preclinical cognizance of

SFN as an adjuvant combination partner in a spectrum of organ specific antitumor
treatment modalities (including targeted therapy) currently in use, emerging per-
spective justifies practical clinical application of SFN on cancer patients in future.
However, more robust preclinical data using SFN or its analogs using available
genetically modified animal models are warranted. This will add value to combined
novel therapies and additionally prove an indisputable multipronged broad research-
based strategy toward stronger and efficacious therapeutic outcome in the field of
cancer treatment. Secondly, it is critical to explore and synthesize novel synthetic
analogs with improved bioavailability and extending their retention time in tumor
tissue with added benefit of capitalizing as innovative research strategy-based
prescription drug for prognostic anticancer treatment benefits.
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isothiocyanates: a clinical phase I study. Nutr Cancer 55:53–62
Part III
Analytical and Processing Methods
Changing Trends in the Methodologies
of Extraction and Analysis of Hydrolytic 13
Products of Glucosinolates: A Review
Rohit Arora, Sakshi Bhushan, and Saroj Arora
Abstract
The increasing resistance among various pests and pathogens to the available
synthetic medicines had lead to the compulsion for exploring new and improved
alternatives. The phytochemicals have arisen as effective and safer alternate.
Among the many plant secondary metabolites, glucosinolates surpass in biolog-
ical activity and hence have been exhaustively extracted and explored for their
activity against these dreaded diseases. This augmented demand for exploring the
biological properties of all the available glucosinolates, especially their hydrolytic
products, has lead to the development of new methods for extraction and analysis
of these metabolites. The extraction methods are designed to match the volatility
of the compound without degrading its quality. These methods have been
improved to choose the best extraction conditions including the extracting sol-
vents. The extracted glucosinolate hydrolytic products are further analyzed using
an array of analytical methods ranging from as simple as paper chromatography
to as complex as microchip analysis. These methods are designed and developed
to match the needs of accuracy, reliability, and repeatability in addition to the cost
effectiveness. This review thus is a highlight and a milestone for the scientists and
budding researchers working on the crucial task of extracting, analyzing, and
exploring the biological properties of glucosinolate hydrolytic products.
Keywords
Glucosinolate hydrolytic products • Biosynthesis • Hydrodistillation • GC-MS •
HPLC • Microchip analysis
R. Arora (*) • S. Bhushan (*) • S. Arora (*)

Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar,
Punjab, India
e-mail: rtarora23@gmail.com; bhushan.sakshi8@gmail.com; sarojarora.gndu@gmail.com
# Springer International Publishing Switzerland 2017 383

J.-M. Mérillon, K.G. Ramawat (eds.), Glucosinolates, Reference Series in
Phytochemistry, DOI 10.1007/978-3-319-25462-3_13
384 R. Arora et al.
Abbreviations
AITC Allyl isothiocyanate
DCM Methylene chloride
ELISA Enzyme linked immunosorbent assay
FID Flame ionization detector
GC Gas chromatography
GHPs Glucosinolate hydrolytic products
GSLs Glucosinolates
HILIC Hydrophilic interaction liquid chromatography
HPLC High performance liquid chromatography
HS Head space
HSCCC High speed counter-current chromatography
ITCs Isothiocyanates
MS Mass spectrometry
NIRS Near infrared reflectance spectroscopy
NMR Nuclear magnetic resonance
PAPS 3-phosphoadenosine-50 -phosphosufate
PC Paper chromatography
S-GT: UDPG Thiohydroximateglucosyl transferase
SIXCPC Strong ion-exchange centrifugal partition chromatography
SIXCPE Strong ion-exchange centrifugal partition extraction
TLC Thin layer chromatography
UHPLC Ultra high performance liquid chromatography
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
2 Glucosinolates Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
3 Bioactivities of Glucosinolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
3.1 Bactericidal/Antibacterial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
3.2 Antifungal Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
3.3 Insecticidal Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
3.4 Antimutagenic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
3.5 Antiproliferative Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
4 Glucosinolate Hydrolytic Products Extraction Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
4.1 Cold Extraction Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
4.2 Hot Extraction Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
4.3 Other Extraction Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
5 Glucosinolate Hydrolytic Products Analysis Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
5.1 Chromatography Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
5.2 Non-Chromatographic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
7 Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
13 Changing Trends in the Methodologies of Extraction and Analysis of. . . 385
1 Introduction
Glucosinolates (GSLs) are class of plant secondary metabolites that contain

“sulfur” and are present in a number of plant families [1]. These plant
metabolites are associated with the presence of enzyme myrosinase also
known as thioglucosidase. Myrosinase belong to class of ß-thioglucosidases
present in myrosin cells also known as idioblasts that are scattered throughout
the tissue of GSL containing plant families. Both GSLs and myrosinase are
present together in the different parts of the plant, viz. root, shoot, stem,
seeds, etc. [2]. The plant injury results in the activation of
“GSL –MYROSINASE” system and results in the formation of “mustard oil
bomb.” Such response of plant tissue leads to the generation of unstable
thiohydroximate-O-sulfate moieties. The nonenzymatic removal of the sulfate
group followed by rearrangement of the left out core structure results in
variety of bioactive products also known as hydrolytic products. GSLs show
abundant structural diversity due to the presence of a vast variety of side chain
structures [3]. These structures are usually associated with protein amino acids
such as alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, trypto-
phan, and methionine (Fig. 1). GSLs are mainly known due to the immense
biological properties of their hydrolytic products.
Glucosinolates are broken down into different hydrolytic products by the
action of myrosinase (Scheme 1). The hydrolytic products include
isothiocyanates (ITCs), nitriles, epithionitriles that depends upon the plant
species, side chain substitutions, pH, and ion concentration [4]. These hydro-
lytic products are responsible for various biological properties of this metabo-
lite. The breakdown products of GSL are not only responsible for the flavor and
aroma of plant families but also possess biological activities/properties includ-
ing action against different organisms (insect, fungi, and bacteria) along with
cancer chemoprevention [5]. Among the different hydrolytic products, ITCs are
known to possess anticarcinogenic properties in vitro as well as in vivo
systems.
Fig. 1 Structure of
glucosinolate
386 R. Arora et al.
Scheme 1 Formation of
glucosinolate hydrolytic
products
2 Glucosinolates Biosynthesis
Biosynthesis of glucosinolates has been widely studied for exploring the

anticarcinogenic properties of this compound. The biosynthesis of GSLs is classified
on the basis of precursor amino acid. GSL synthesis is three step process, firstly
amino acid chain elongation, secondly synthesis of glucon from the amino acid, and
lastly chain modification (glucon addition) [6].
1. Side chain elongation

GSLs are synthesized from chains elongated forms of phenylalanine, valine, and
methionine. The synthesis of GSLs is similar to the synthesis of leucine from
amino acid valine. The transamination of amino acid results in the production of
α-keto acid, followed by addition/condensation of acetyl CoA, and finally by
process of isomerization involved in the shifting of the hydroxyl group and

oxidation to ultimately attain elongated keto acid which is further used to obtain
amino acid.
2. Formation of glucone moiety
The first step in the formation of glucone moiety is the conversion of amino acid
oxime. Several studies have supported that various enzyme are involved in the
conversion of different amino acids that are responsible for biosynthesis of GSLs.
The above studies are basically carried out on the microsomal systems. The
enzymes involved in conversion of tyrosine and phenylalanine to their
corresponding oxime are the best example of such conversion involving GSL
synthesis. These enzymes are known to be responsible for the formation of
oximes that are further required for glycoside biosynthesis. The enzymatic system
involved in the chain elongation of methionine and phenylalanine is flavin
monooxygenase and that of tryptophan to indole acetaldoxime is peroxidase.
Above facts support the involvement of homologous enzyme system in such
conversions. The intermediate moieties between the oxime and thiohydroximate
have not been observed. It has been speculated that oxime is oxidized to nitro
compound which conjugates with cysteine and functions as a thiol source. The
above reaction is catalyzed by glutathione-S-transferase resulting in the formation
of S-alkylthiohydroximate. In the final step of glucone formation, the
thiohydroximate is S-glucosylated by S-GT (UDPG: thiohydroximateglucosyl
transferase) to form a desulfoglucosinolate. The above formed product is further
sulfated by 3-phosphoadenosine-50 -phosphosulfate (PAPS). Several studies have
shown the role of S-GT in sulfonation.
3. Side chain modifications
The side chain modifications of the elongated chains occur following glucone
formation. The aliphatic side chains that are attached to methionine are especially
modified. The pathway basically includes oxidation to methylsulfinylalkyl
followed by the removal of methyl sulfinyl group and desaturation. The above
step results in the formation of alkyl glucosinolates which on hydroxylation
results in hydroxyl alkenyl glucosinolates. The phenomenon responsible for
these modifications has been studied in case of 3-butenyl GSL that is catalyzed
by cytochrome P450 hydroxylase. The benzoyloxyalkyl are formed due to the
conjugation of hydroxyl alkyl GSL with benzoic acid. Interestingly, the side
chains of GSLs from branched amino acid and phenylalanine can be hydroxyl-
ated. In addition to this, tryptophan amino acid chains usually adds methoxy
group to result in its modification (Scheme 2).
3 Bioactivities of Glucosinolates
The formation of biologically important hydrolytic products such as isothiocyanates,

nitriles, epithionitriles, oxazolidine-2-thione, and thiocyanates along with other
products from glucosinolates makes this secondary metabolite an important choice
for preventing and curing diseases in plants and animals.
388 R. Arora et al.
Scheme 2 Process of biosynthesis of glucosinolates
3.1 Bactericidal/Antibacterial Activity
Glucosinolate hydrolytic products (GHPs) are known for their bactericidal activ-
ities against a wide range of bacterial strains. Among these, ITCs are effective
against Helicobactor pylori and its related strains [7]. Allyl ITC (AITC) is often
used as a preservative in industries. The aryl and alkyl GHPs have shown cytotoxic
effects against Salmonella typhimurium [8]. Benzyl ITC has been tested for
treatment against respiratory and urinary tracts [9]. The level of toxicity and
activity against pathogens varies with the type of strain. It has been shown that
GHPs present in Brassica napus have the potential to inhibit the growth of
Aphomyces euteiches along with propionibacterium [10]. Glucoraphanine contains
sulforaphane that has effective bactericidal action against H. pylori and related
strains along with strains resistant to antibiotics [11]. Hydrolytic products like
AITC, 4-hydroxybenzyl ITC, methyl ITC, 4-(Methylsulfinyl) butyl ITC, phenyl
ITC, and oxazolidienethiones showed bactericidal effects against different strains
of bacteria such as Bacillus cereus IFO-13494, Bacillus subtilis IFO-13722,
Salmonella enteritidis JCM-1891, Staphylococcus aureus IFO-12732, Escherichia
coli 0157:H7, Helicobacter pylori, and nitrifying bacteria [12–14]. Literature
survey reveals antibacterial activities of most of the GHPs, but their exact mech-
anism of action is not known. However, the action can be attributed to the tendency
of GHPs to inactivate various intracellular enzymes, thus resulting in oxidative

breakdown of sulfur bridges [15].
3.2 Antifungal Activities
The members of family Brassicaceae have the tendency to act against many fungi
that are phytopathogenic in nature [16]. Presence of ITCs such as AITC and
phenethyl ITC had resulted in antifungal activities of plant extracts belonging to
mustard family [17]. Many fungi such as Pernospora parasitica, Sclerotium rolfsii,
and Pythium ultimum were controlled using cabbage tissues due to the presence of
GHPs in them [18]. GHPs in addition to this were known to possess antifungal action
against Fusarium culmorum, Sclerotinia sclerotiorum, Pythium irregular, Rhizocto-
nia solani, and Diaporthe phaseolorum [19]. The fungi acting on wheat were also
shown to be inhibited by various GHPs [20]. It has also been observed that Brassica
napus c.v. inhibited fungal pathogens such as S. sclerotium [21]. GHPs have also
shown strong fungicidal activity against many fungal species such as Aspergillus
niger, Colletotrichum circinans, Peronospora parasitica, Fusarium graminearum,
Rhioctonia solani, Alternaria alternata, Penicillium glaucum, etc. [22]. The toxicity
and antifungal activity of GHPs depends on the chemical structure of ITCs [23]. The
mechanism of their action depends on their tendency to enhance and trigger the
defence mechanism of plants either by killing the pathogen or by providing protec-
tion by creating a plant barrier against respective fungi (pathogen) [24].
3.3 Insecticidal Activity
GHPs are bioactive agents responsible for their insecticidal properties against
different pests including nematodes [25]. The ability of these compounds to biode-
grade naturally makes it an excellent biofumigant [26]. Aromatic ITCs were found to
be effective insecticidal agents against Otiorhynchus sulcatus. GHPs have also been
reported to act against Naupactus leucoloma (weevil larvae) [27]. Inhibitory activity
of AITC, benzyl ITCs, methyl ITCs, phenyl ITCs, phenylethyl ITCs, and propenyl
ITC have been reported in a number of insects such as Cyclocephala, Musa
domestica, Rhyzopertha dominicia, Otiorhynchus sulcatus, Limonius influscatus,
Naupactus leucoloma, and Drosophila melanogaster [28–30].
Several reports have suggested nematode inhibitory effect of GHPs. It has been
observed that rotation of crops/plants belonging to Brassicaceae family resulted in
nematode control [31]. Various studies have demonstrated nematicidal activity
against Tylenchulus semipenetrans, Heterodera glycines, Meloidogyne chitwoodi,
M. incognita, M. javanica, and Globodera rostochiensis [32–35]. Plants belonging
to Brassicaceae family have also been known to possess herbicidal properties due to
the presence of various allelochemicals. Brown and Morra [36] reported the inhibi-
tion of growth of Lactuca sativa when grown along with B. napus. Such results have
suggested the ability of GHPs to play important role as a biofumigant [37].
390 R. Arora et al.
In addition to the above phenomenon, Brassica members have shown immense

potential to act as a green manure, thus limiting the use of artificial fertilizers
[38]. Vigna unguiculata when grown in the presence of Brassica showed reduction
in weeds [39]. Petersen et al. [40] have demonstrated the potential of various ITCs to
act against weeds. The Brassica species were able to suppress weeds such as
Echinochloa crus-galli (L.), Amaranthus hybridus L. (smooth pigweed), Sonchus
asper (L.) Hill (spiny sowthistle), Triticum aestivum L. (wheat), Echinochloa
crus-galli (L.) Beauv. (barnyard grass), Alopecurus myosuroides Huds. (black
grass), and Matricaria inodora L. (scentless mayweed). It has been proposed that
the mechanism of GHPs as a herbicide may be due to their interference and
inhibition in protein synthesis and other processes involved in plant growth [40].
3.4 Antimutagenic Activity
Literature survey has revealed that GHPs have strong antimutagenic effect. It was
observed that cauliflower extract acted as an effective antimutagenic agent against
quninoline induced mutagenicity [41]. It has been seen that broccoli extract admin-
istered to male BALB/C strain resulted in the reduction of mutagenicity thus
supporting antimutagenic effect of GHPs [42]. Similar results have also been
reported during studies involving metabolic activation involving S. typhimurium
TA 98. A study conducted by Murugun et al. [43] reported that broccoli extracts
prepared in ethanol resulted in suppression of mutagenicity caused by respective
mutagens on all the strains including TA 98, TA 102, and TA 1535.
3.5 Antiproliferative Activity
GHPs have also been studied for their potential to act against carcinogens and
mutagens. These phytochemicals have shown antitumor ability in tissues such as
liver, colon, pancreas, bladder, etc. [44]. Hydrolytic products of methyl sulfinyl GSL
have been observed to play important role in phase II enzymes induction [45]. Several
studies using animal models have supported anticarcinogenic effects of cruciferous
vegetables [46, 47]. Toxicity induced by administration of carcinogens in animals
has also been observed to decrease following the administration of GHPs [48–51].
4 Glucosinolate Hydrolytic Products Extraction Methods
The GHPs as discussed earlier have an array of biological properties ranging from
antioxidant, herbicidal, to anticancer activities. Their immense importance in phar-
maceutical industries plays a key role in identifying new and improved extraction
methods, which will not only increase their yield but their composition as well. The
two major types of extraction methods used for this purpose are cold extraction and
hot extraction.
4.1 Cold Extraction Methods
The cold extraction of GHPs is based on the solvent extraction of these important
metabolites from different plant sources, viz. root, shoot, leaves, seeds, etc. [52,
53]. In this method, the plant material is washed to remove any dust particle if
present. The material is then crushed and immediately added into solvent to prevent
any loss of metabolite due to evaporation. The solvent depends on the type of
metabolite required, since GHPs can be found in both polar as well as nonpolar
phase of solvents [54]. In general, the crushed plant material is first added into
hexane for defattening of the material. The time required for defattening depends on
the laboratory developed method, but usually 4–24 h incubation in solvent is
sufficient for this purpose. For ensuring the proper defattening process, after the
initial incubation, the solvent is replaced with fresh hexane and the same is kept for
24 h. Following incubation, both the solvents are combined, filtered through 0.22 μm
filter, and evaporated at 30 C under vacuum using rotary evaporator. The defattened
solvent often contains a number of GHPs and hence is also stored for further
analysis. After the initial defattening, other solvents relevant to the experiment in
hand are used for the extraction of other GHPs. But usually, methylene chloride
(DCM) is the most commonly used solvent for the extraction of GHPs. The
concentrated extracts are stored in a vial at – 40 C until further use [55].
In another extraction method by Fahey et al. [56], triple solvents, viz., dimethyl-
sulfoxide, dimethylformamide, and acetonitrile were used. The method involved the
homogenization of the plant material followed by the addition of equal volume of the
three solvents. The temperature of this mixture was kept at a low temperature of
–50 C to prevent the escaping of GHPs. The triple solvent may also be replaced
with dual solvents or more according to the experimental requirements. The GHPs
with the specific polarity will be absorbed in the respective solvent and can be
separated using separating funnel and dried under vacuum at 30 C to obtain
the GHPs.
In another set of experiments by Arora et al. [55], the plant material was
homogenized using distilled water and the extracting solvent was added to it. The
extraction solvents were varied in different methods (methylene chloride, diethyl
ether, and ethyl acetate). The GHPs were extracted thrice with the extraction solvents
and the solvents were pooled and passed through anhydrous sodium sulfate. The
extraction solvents were dried using three different drying conditions, viz. air drying,
nitrogen gas drying, and rotary evaporator drying. The experiment showed that
dichloromethane was the most effective extraction solvent and rotary evaporation
as the most efficient drying condition.
4.2 Hot Extraction Methods
The hot extraction methods in contrast to the earlier discussed method requires a
higher temperature for the extraction of GHPs from different plant materials. Since
the GHPs are volatile in nature, therefore, hot extraction methods are much suitable
392 R. Arora et al.
Fig. 2 Soxhlet apparatus, where (a) Soxhlet apparatus and (b) heating mantle
for the extraction of these valuable secondary metabolites. There are two basic
methods for hot extraction of GHPs. The first being Soxhlet extraction in which
the crushed plant material (seeds, leaves, roots, etc.) is added in the Soxhlet
apparatus (Fig. 2). The desired solvent such as methylene chloride or 80% methanol
is added in the round bottom flask. The flask is fitted with Soxhlet apparatus and is
heated at temperature ranging from 30 C to 50 C using heating mantle for a time
period of 3–10 h. The saturated solvent containing GHPs thus obtained is collected
and concentrated under vacuum using rotary evaporator [57, 58].
In the other hot extraction method known as hydrodistillation, only one solvent
is used, i.e., distilled water. The crushed plant material is added in a round bottom
flask and is mixed with distilled water. The flask is fitted with a Clevenger’s
apparatus and the mixture is heated using heating mantle until boiling (Fig. 3).
The temperature is reduced to 60 C to continue the boiling process for 2–3 h. The
condensed volatile GHPs are collected in the outer pipe. Following the completion
of the protocol, the extract and water is removed and the GHPs are obtained using
either methylene chloride or diethyl ether as solvent [55]. The significance of this
method for isolation of GHPs leads to the development of a number of modifica-
tions for improving this method for obtaining higher yield in less time as discussed
below.
Microwave assisted hydrodistillation is a modification of traditional hydrodis-
tillation in which the heating equipment was changed from heating mantle to a
microwave. As per the method given by Wei et al. [59], a microwave was modified to
keep the setup including a flat bottom flask and a Clevenger’s apparatus inside it
(Fig. 4). The microwave was operated at a power of 577 W and for a time period of
37.5 min. The water present in the extract was removed by passing the mixture
through anhydrous sodium sulfate. Although this modification provided no improve-
ment in the yield or composition of the extract, but a significant decrease in the
extraction time was observed.
Fig. 3 Clevenger’s apparatus

used in hydrodistillation
method
4.3 Other Extraction Methods
Apart from the above mentioned extraction methods involving the conventional hot
and cold extraction methods, a number of new alternatives have also been performed
to obtain the improved yield and composition. Among these, supercritical fluid
extraction is an advanced technique involving the use of supercritical fluids having
properties of both liquid as well as gas (Fig. 5). The most common supercritical fluid
used by the researchers is CO2, owing to its low cost, nontoxicity, odorless, and
colorless properties. An important aspect of using this fluid is that it can be modified
to gas or liquid as per extraction requirements. A comparative extraction of GHPs
using supercritical extraction by CO2 revealed an improvement in the composition
and amount [60].
The GHPs may also be extracted in an indirect way by an initial extraction of
intact GSLs. This method involves the pulverization of plant material followed by
the deactivation of myrosinase enzyme using liquid nitrogen or boiling water. The
intact GSLs thus obtained are extracted using different solvents, viz. methanol, ethyl
acetate, and methylene chloride. The intact GSLs thus obtained are desulfated in
394 R. Arora et al.
Fig. 4 Setup for microwave assisted hydrodistillation
Fig. 5 Equipment for

supercritical fluid extraction
Fig. 6 Indirect extraction of

glucosinolate hydrolytic
products using DEAE
sephadex column
DEAE sephadex column using an external supply of either artificial or natural

myrosinase enzyme (Fig. 6). An important advantage of this method is that the
type of GHPs required may be modified as per our requirement by changing the
column conditions (acidic, basic, or neutral pH) and temperature (low, room tem-
perature, or high) [61].
5 Glucosinolate Hydrolytic Products Analysis Methods
The GHPs extracted by the above mentioned methods can be utilized for a number of
biological activities such as antifungal, antibacterial, herbicidal, and anticancer
properties. Before utilizing it for any purpose, the analysis of its composition and
quantity of each compound is necessary to analyze the synergistic or antagonistic
effect of each compound. The analytical methods thus employed range from the
most common and traditional methods like paper and thin layer chromatography to
much advanced microchip analysis.
5.1 Chromatography Methods
The chromatographic methods involve the analysis of different components of the

extract on the basis of polarity and size.
5.1.1 Paper Chromatography

Paper chromatography (PC) is the oldest method during early 1900s for the analysis
of GHPs. The thiourea formed following the reaction amid ammonia and
isothiocyanates is detected and separated by paper. While the hydrophobic compo-
nents are analyzed using water saturated chloroform, the hydrophilic components on
the other hand involved the mixture of butanol, toluene, and water as mobile phase.
The low reproducibility of GHP analysis using this method makes it a difficult
choice for analysis [62].
396 R. Arora et al.
5.1.2 Thin Layer Chromatography

Thin layer chromatography (TLC) is much reliable and repeatable method for the
analysis of GHPs. The method uses silica or cellulose gel formed on glass plate as a
stationary phase, while different combination of solvents as per the polarity are used
as mobile phase. TLC can be used for both qualitative as well as quantitative analysis
of GHPs. The repeatability, reproducibility, and the ability to analyze a large number
of samples at the same time make this method highly useful for the initial analysis of
GHPs [63].
5.1.3 High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is the analytical technique mostly
preferred for the analysis of GHPs since late 1900s. The accurate results along with
an ease of reproducibility and repeatability makes it an idle analytical method till
date. The variation in the mobile phase, flow rate, and the column used helps in
analyzing almost all the combination of GHPs present in different extracts (Fig. 7).
The different compounds present in the extracts are analyzed using different detec-
tors such as photodiode array and the much important mass spectrometer
(MS) [64]. The MS attachment makes it easier for the identification of a compound
without the presence of any standard by detecting the molecular mass of compound.
New and improved versions of HPLC are regularly being formed having all the
advantages of the regular HPLC, while solving its limitations. Some of these
improved HPLC instruments are ultra high performance liquid chromatography
(UHPLC), hydrophilic interaction liquid chromatography (HILIC), and high speed
counter-current chromatography (HSCCC) [65, 66].
Fig. 7 High performance liquid chromatography equipment

5 SFN, TRAIL, and Cancer Stem Cells

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5 SFN, TRAIL, and Cancer Stem Cells

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364 S. Banerjee and S.B.

5 SFN, TRAIL, and Cancer Stem Cells

5.1 Prostate Cancer

An extension to the above report in support of SFN-sensitizing prostate cancer cells

following sulforaphane-only treatment and the combination with TRAIL further

5.2 Pancreatic Cancer

To address the need to eliminate oncogenically transformed CSC in pancreatic

6 Sulforaphane Combination with Other Natural Agents

Against the backdrop of foregoing thought-provoking reports from laboratory inves-

effect of cruciferous vegetables in relation to the risk of developing prostate

7 Sulforaphane and Clinical Trials

Given the exceptional inciting laboratory research conclusions projecting SFN as a

Table 1 Clinical trials with sulforaphane in cancer (http://www.clinicaltrials.gov)

8 Conclusions and Perspective

Based on the above overwhelming evidence supporting preclinical cognizance of

metabolism of sulforaphane from standard and high-glucosinolate broccoli. Am J Clin Nutr

35. Pawlik A, Wiczk A, Kaczynska A, Antosiewicz J, Herman-Antosiewicz A (2013)

Rohit Arora, Sakshi Bhushan, and Saroj Arora

R. Arora (*) • S. Bhushan (*) • S. Arora (*)

# Springer International Publishing Switzerland 2017 383

Glucosinolates (GSLs) are class of plant secondary metabolites that contain

Biosynthesis of glucosinolates has been widely studied for exploring the

1. Side chain elongation

process of isomerization involved in the shifting of the hydroxyl group and

The formation of biologically important hydrolytic products such as isothiocyanates,

Scheme 2 Process of biosynthesis of glucosinolates

3.1 Bactericidal/Antibacterial Activity

of GHPs to inactivate various intracellular enzymes, thus resulting in oxidative

3.2 Antifungal Activities

3.3 Insecticidal Activity

In addition to the above phenomenon, Brassica members have shown immense

3.4 Antimutagenic Activity

3.5 Antiproliferative Activity

4 Glucosinolate Hydrolytic Products Extraction Methods

4.1 Cold Extraction Methods

4.2 Hot Extraction Methods

Fig. 3 Clevenger’s apparatus

4.3 Other Extraction Methods

Fig. 4 Setup for microwave assisted hydrodistillation

Fig. 5 Equipment for

Fig. 6 Indirect extraction of

DEAE sephadex column using an external supply of either artiﬁcial or natural

5 Glucosinolate Hydrolytic Products Analysis Methods

5.1 Chromatography Methods

The chromatographic methods involve the analysis of different components of the

5.1.1 Paper Chromatography

5.1.2 Thin Layer Chromatography

5.1.3 High Performance Liquid Chromatography

Fig. 7 High performance liquid chromatography equipment

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R. Arora () • S. Bhushan () • S. Arora (*)