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Sulforaphane GlucosinolateMonograph

IntroductionIntake of broccoli sprouts, a rich source of the

glucosinolate glucoraphanin, has been associatedwith decreased incidence, multiplicity, and tumorgrowth in animal cancer models.'^ In 1992, PaulTalalay, MD, and colleagues at Johns HopkinsUniversity identified the isothiocyanate, sulfora-phane, a biologically active metabolite of glucora-phanin, as the compound in broccoli responsiblefor many of its health benefits."* Since that time,more than 500 studies have been conducted on themechanisms and biological activity of sulforaphaneand its precursor, glucoraphanin.^ Glucoraphanin,also referred to as sulforaphane glucosinolate(SGS), is the most potent naturally-occurringinducer of phase 2 detoxification enzymes'*'^ and isan indirect, long-acting antioxidant.' 'Sulforaphane also exhibits broad-spectrumantimicrobial activity against numerous gram-positive and -negative bacteria,'" most notablyHelicbacter pylori." In addition, sulforaphanepossesses anti-inflammatory activity: it inhibitscytokine production in preclinical and clinicalstudies.'^" Sulforaphane's multiple moleculartargets and promising early research have lead to15 clinical trials currently underway to assess itseffects on various cancers, cardiovascular disease,upper airway inflammation, radiation dermatitis,and vascular health.'^

BiochemistryGlucoraphanin is a glucosinolate found in high

concentrations in the Mariner variety of broccoli{Brassica olercea itlica) and other members of theBrassica family."^ All glucosinolates are comprised

of a basic structure consisting of a -D-thioglucosegroup, a sulphonated oxime group, and an aminoacid-derived side chain.'^ Glucosinolates must beenzymatically hydrolyzed to their associatedisothiocyanate to become active.'" Sulforaphane(molecular formula C^H j^NOS^ is the biologicallyactive isothiocyanate produced when glucora-phanin is metabolized by the enzyme myrosinase(Figure 1)."

PharmacokineticsGlucoraphanin in broccoli is enzymatically

hydrolyzed by myrosinase, an enzyme compart-mentally separated from glucoraphanin in plantcells. Myrosinase is released when the plant ischewed or processed.^" Heating broccoli partiallydenatures and inactivates myrosinase, leaving theglucoraphanin at least partially intact. In the gut ofhealthy individuals any intact glucoraphanin isthen metaboHzed by myrosinase-producingbacteria.^' Because broccoli sprout or seed extractstaken orally contain no myrosinase to hydrolyzethe glucoraphanin, transformation to sulforaphanemust be carried out by the gut microflora.^'' Inindividuals with compromised intestinal flora andlow myrosinase activity, it is unclear if glucora-phanin exerts the same systemic effects asobserved in individuals with normal intestinal

Research in humans indicates approximately 74percent of sulforaphane from broccoli extract isabsorbed in the jejunum.''* After absorption,sulforaphane is metabolized via the mercapturicacid pathway.^ ''^ '^ Although this pathway involves acomplex interplay between the liver, small

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Figure 1. Glucoraphanin Metabolism

Glucose-CH2OH Myrosinase N=C=S

Glucoraphanin(Sulforaphane glucosinate)

Sulforaphane

Key words: sulforaphane,broccoli, glucoraphanin,SGS, isothiocyanate. Crucera,detoxification, detox,phase 2, phase II,H. pylori, Helicobacter,Brassica, cruciferous,chemoprotection,chetnoprotectant,anticarcinogen, carcinogen,cancer, inflammation,Gilbert's syndrome,anti inflammatory,antioxidant, Nrf2

intestine, and kidneys, the liver is thought to bethe primary site of activity and is the site ofsulforaphane conjugation to glutathione.Sulforaphane-glutathione conjugates are subse-quently converted to cysteinyl-glycine, cysteine,and N-acetykysteine conjugates in the kidneys orgut and then cycled back to the liver for acetylation.Of these conjugates, sulforaphane-N-acetylcysteineis the most prevalent.^^

Upon absorption into the bloodstream, sulfora-phane readily accumulates in tissue and exertsanticarcinogenic effects. In one human study, asingle 200 \iM dose of sulforaphane from broccolisprouts yielded peak plasma concentrationsbetween 0.943 and 2.27 fimol/L at one hour postfeeding; the half life of sulforaphane was 1.77 0.13 hours.^^ A pilot study in eight healthy womenundergoing reduction mammoplasty demonstrateda broccoli sprout extract containing 200 pMsulforaphane given orally one hour prior to tissueremoval resulted in average tissue uptake of 1.45 1.12 pmol/mg in the left hreast and 2.00 1.95pmol/mg in the right breast. Both detoxificationenzyme genes for NADH quinone reductase(NQOl) and heme oxygenase-1 (HO-1) weremeasureable in the excised breast tissue, indicatingcancer blocking activity after sulforaphane con-sumption.^ ** Research in mice has also demon-strated colonie tissue uptake of sulforaphane afteroral dosing, accompanied by a reduction inadenoma formation.^^ Excretion of sulforaphaneconjugates in the urine is via first-order kineticswith metabolites being cleared from the bodywithin 72 hours of dosing.''^-"'

Mechanisms of ActionIndirect Antioxidant and Carcinogen Detoxification

Sulforaphane is a pleotropic molecule and apotent inducer of numerous nuclear factor ery-throid-derived 2 (Nrf2)-dependent phase 2enzymes involved in xenobiotic detoxification.Enzymes induced by sulforaphane include theantioxidant response element (ARE) targets:NQOl," y-glutamylcysteine synthetase (GGCS),^ ^HO-1,^ = glutathione transferases (GST),'' glucuro-nosyl transferases,^" and epoxide hydrolases.^''These enzymes are regulated by the Nrf2 transcrip-tion factor, which upon release from the Kelch-likeECH-associated protein 1 (KEAPl), binds to AREsites in the enzymes' genes and upregulatescarcinogen detoxification.^'^'" Other Nrf2-mediatedeffects of sulforaphane include inhibition of LDLoxidation,""^ inhibition of dopamine oxidation,^''improvement of age-related THl immunity viarestoration of redox equilibrium,"" and reduction ofoxidative stress caused by electrophilic carcino-gens."' Sulforaphane also modulates phase 1cytochrome p450 (CYP) enzymes by decreasingCYPlAl, CYP2B1/2, and CYP3A4 activity, therebyinhibiting the activation of procarcinogens andpreventing the generation of DNA adducts duringthe initiation stage of cancer.''^ The overall neteffect on phase 1 and 2 enzymes is an increase inmetabolism and detoxification of chemicalcarcinogens."^

Other Chemopreventive MechanismsSulforaphane exerts a direct effect on human

cancer cells post-initiation. Research has demon-strated sulforaphane directly inhibits cell cycleprogression, primarily via G^ M arrest,"" "^ andinduces apoptosis of cancer cells via caspase

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activation, resulting in reduced tumor weight andvolume both in vitro and in animal cancermodels.''^'"' In human tissue samples, reductions inhistone acetylation correlate with increased cancergrade and risk of cancer recurrence.'''' Studies showsulforaphane directly inhibits histone deacetylase(HDAC), which correlates with induction of G^ Mcell cycles arrest and apoptosis."*" Sulforaphane alsoappears to upregulate apoptosis in cancer cells bymodulating nuclear factor kappaB (NFKB) activity"*^and increasing mitochondrial reactive oxygenspecies, causing disruption of mitochondrialmembrane potential and release of cytochrome C "And finally, sulforaphane potently inhibits angio-genesis and metastasis of tumors by reducingmicrocapillary formation and inhibiting cellmigration.'"' These effects were associated withdown regulation of angiogenesis factors, includingvascular endothelial growth factor (VEGF)." Figure2 summarizes the tumor inhibition effects ofsulforaphane.

Miscellaneous MechanismsSulforaphane's anti-inflammatory effects have

been attributed to inhibition of pro-inflammatorysignaling molecules and cytokines'-' such as NFKB,prostaglandin E2, and nitric oxide." Sulforaphanealso appears to reduce upper airway inflammationvia increased phase 2 enzyme detoxification of airpollutants and pollen, apparently via decreasedcellular oxidative stress, inhibition of inflamma-tory cytokine production, and decreased tissueinflammation.''' In vitro research has also shownsulforaphane inhibits the production of interleukinand tumor necrosis factor-alpha (TNF-a) inrheumatoid T cells.^ ^ Sulforaphane exhibitsbroad-spectrum antimicrobial activity, inhibitingthe growth of several gram-positive and -negativebacteria, including E. coli 0157:H7, Helicobacterpylori, Salmonella, Shigella, Staphylococcus aureus,Streptococcus pyogenes, Pseudomonas aeruginosa, andCryptococcus neo

Figure 2. Sulforaphane Mechanisms of Tumor Inhibition

Sulforaphane

Induction of Phase 2Response

AntibioticH. Pylori

IndirectAntioxidant

Effects

Variable Effectson Phase I

Cell CycleArrest &

Apoptosis

Anti-Inflammatory

Tumor Inhibition

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Clinical IndicationsCancerPreclinical and Animal Research

Numerous in vitro studies in human colon,leukemia, pancreatic, lung, and skin cancer celllines have demonstrated sulforaphane's inhibitoryeffects on cell cycle arrest,* -^^ ^^^ and research inhuman bladder" and prostate'"' cell lines hasshown it increases apoptosis. Sulforaphane's abilityto disrupt tubulin polymerization and inhibitmitosis has also been demonstrated in animalmodels of breast cancer.^ '^^ ^ Inhibition of histonedeacetylase and increased apoptosis in humancolon, prostate, and kidney cell lines has also beenreported.'''

In a pilot study involving three healthy volun-teers (ages 18-55), a single daily dose of 68 gBroccoSprouts (approximately 105 mg sulfora-phane) significantly inhibited HDAC activity inperipheral blood mononuclear cell cultures threeand six hours following consumption, suggestingsulforaphane may induce cell cycle arrest andapoptosis in humans.^^

In mice with experimentally induced prostatecancer, 6 |jmol sulforaphane by oral gavage threetimes weekly from age six weeks onward decreasedpulmonary metastasis incidence by 50 percent andmultiplicity by 63 percent. Prostate tissue samplesrevealed decreased cellular proliferation andincreased apoptosis when compared to controlmice.'"'' Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in awide variety of cancer cells. In a mouse model ofprostate cancer, tumor-bearing male mice weregiven sulforaphane (40 mg/kg), TRAIL (15 mg/kg)+ sulforaphane (40 mg/kg), TRAIL alone (15 mg/kg), or vehicle at varying intervals for four weeks.Although either sulforaphane or TRAIL alonedecreased tumor growth, the combination ofsulforaphane and TRAIL was more effective,suggesting sulforaphane may have a potentiatingeffect on TRAIL. The sulforaphane-TRAIL combina-tion also activated several caspases and was moreeffective at inhibiting markers of angiogenesis andmetastasis than either agent alone.''" Sulforaphanegiven to female breast cancer-bearing, non-obese,diabetic/severe combined immunodeficient (NOD/SCID) mice at a daily dose of 50 mg/kg for twoweeks eliminated breast cancer stem cells in vivoand halted tumor growth.''^

Clinical StudiesThe first direct observation of sulforaphane's

inhibitory effect on cancer in humans wasobserved in 200 healthy adults (ages 25-65) fromthe Jiangsu Province of China, a region with a highrate of hepatocellular carcinoma due to excessivedietary aflatoxin exposure and chronic hepatitis Binfection. The primary end-point of this blinded,placebo-controlled trial was to determine ifdrinking daily broccoli sprout infusions (containing400 |imol glucoraphanin) for two weeks couldreduce urinary excretion of aflatoxin DNA adducts

- indicators of DNA damage. A highly significantinverse association was observed for excretion ofdithiocarbamates (isothiocyanate metabolites ofglucoraphanin) and aflatoxin-DNA adducts inindividuals treated with broccoli sprout infusions.An average of approximately 12 percent (range1-45 percent) of the administered dose of broccolisprout glucoraphanin was excreted as dithiocarba-mates, with significant variability in excretion rates.The reason for this variation may be due todifferences in enteric microflora composition, someindividuals possibly having less myrosinase.Genetic polymorphisms of the glutathioneS-transferase enzyme involved in glucoraphaninmetabolism may also be partially responsible.''*

Cardiovascular Disease and HypertensionGlucoraphanin and sulforaphane afford cardio-

vascular protection via their antioxidant andanti-inflammatory properties, resulting in reducedoxidative stress, improvement in lipid profiles, anddecreased blood pressure. A phase 1 trial involving12 cigarette smokers (six men and six women)investigated whether consuming 100 g freshbroccoli sprouts daily (glucoraphanin/sulforaphanecontent not specified) for one week impactedoxidative stress markers and cholesterol values.Cholesterol levels, plasma amino acids, naturalkiller cell activity, serum coenzyme QIO, andmarkers of oxidative stress - plasma phosphatidyl-choline hydroperoxide (PCOOH), urinary 8-iso-prostane, and urinary 8-hydroxydeoxyguanosine

- were measured pre- and post-treatment. Afteronly one week of broccoli sprout intake, all subjectsdemonstrated decreased serum total- and LDL-cholesterol levels and reductions in all oxidativestress markers; females also had significantlyincreased HDL-cholesterol levels.^^

Animal research supports these findings. Studieson male and female spontaneously hypertensiverats on a glucoraphanin-enriched diet (equivalentto 27.3 |imol sulforaphane per g dried sprouts)

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showed decreased oxidative stress, lower bloodpressure, and less renal and central nervous systeminflammation in kidney and spinal cord tissuewhen compared to animals on glucoraphanin-free

Upper Airway InflammationAirborne diesel exhaust particles appear to

exacerbate lung and cardiovascular diseases byinducing oxidative stress.*' Sulforaphane inhibitscytokine production in human airway epithehalcells exposed to diesel extract via induction ofphase 2 enzyme genes NQOl and glutathione-S-transferase Ml. ' ' In the first study to demonstrateoral sulforaphane upregulation of phase 2 antioxi-dant enzyme expression in the human airway, Reidlet al administered BroccoSprouts* homogenates(BSH) to 57 healthy adult volunteers (average age34) in a single-blind, dose-escalation (25, 50, 75,100,125,150,175, and 200 g), three-day trial.Analysis demonstrated a sulforaphane content of0.283 nmol/mL BSH - the 175- and 200-mg dosesdelivering 89 and 102 |imol sulforaphane, respec-tively. Control subjects received a 200 g dose ofalfalfa sprouts, containing negligible amounts ofsulforaphane. Baseline nasal lavage and bloodsamples were collected from all participants andassessed for phase 2 enzyme expression. Subjectswere assessed again one day after final dosing.Significant increases in glutathione-S-transferases,HO-1, and NQOl were observed with the 200-gBSH dose compared to placebo. All doses were welltolerated and without serious side effects, althoughfour subjects reported mild gastrointestinal eventsthat did not require treatment.'''

Helicobacter pylori InfectionThe role of Helicobacter pylori in development of

stomach cancer is well established.^' Animalresearch shows sulforaphane given to humangastric xenograft-bearing mice at a daily dose of1.33 mg (equivalent to a 100-mg daily dose inhumans) is strongly bacteriocidal and eradicates H.pylori.^' Yanaka et al subsequently demonstratedglucoraphanin-rich three-day old hroccoli sprouts(6 (imol glucoraphanin/g) given to H. pylori-infected female mice reduced gastric bacterialcolonization, decreased mucosal TNF-OC andinterleukin-l expression, decreased gastricinflammation, and prevented gastric atrophy.These effects were not observed in Nrf2-depletedmice, indicating the important role of Nrf2-dependent phase 2 enzyme induction bysulforaphane.''

In a human arm of the Yanaka study, 48 H.py/ori-infected patients were divided into a broccolisprout treatment group (n=25) or an alfalfa sproutplacebo group (n=23). Those in the broccoli sproutgroup received 70 g sprouts daily, containing 6Hmol glucoraphanin/g, for eight weeks.Glucoraphanin feeding decreased breath testurease levels, H. pylori antigen, and pepsinogens Iand II - markers of gastric colonization andinflammation. These results indicate broccolisprouts as a source of glucoraphanin improve H.pylori infection sequelae and enhance chemopro-tection from H. py/on-induced stomach tumors.'^Two other clinical trials demonstrated the bacterio-cidal'''' and chemoprotective properties of sulfora-phane in individuals with H. pylori infection.'^

Gilbert's SyndromeGilbert's syndrome is characterized by genetic

polymorphisms in the UDP-glucuronosyltransferase (UGT) enzymes, theprimary one being UGT1A1*28, which is involvedin bihrubin glucuronidation. UGT polymorphismscan manifest as benign unconjugated hyperbilirubi-nemia, associated with reduced hepatic conjuga-tion, and may increase cancer risk in this popula-tion. In an observational study of 191 nonsmok-ing volunteers (ages 19-40) consuming 0-4 serv-ings of cruciferous vegetables daily, there was astatistically significant inverse association betweenthe UGTIAI gene/Cruciferae interaction and total,direct, and indirect bilirubin measurements.Sulforaphane from cruciferous vegetables has beenshown to induce UGTIAI activity, resulting ingreater bilirubin conjugation and clearance andpossibly mitigating the increased cancer risk.''

Rheumatoid ArthritisRheumatoid arthritis (RA) involves a tumor-like

expansion of the synovium characterized byhyperproliferation of synoviocytes, infiltration of Tand B cells, and increases in interleukin (IL) -6, -8,and -17. RA treatment involves suppression ofsynoviocyte proliferation and cytokine produc-tion.'" Due to the "tumor-like" attributes ofsynoviocytes and their role in RA progression,Kong et al investigated the effect of sulforaphaneon synoviocyte apoptosis in a mouse model of RA.Sulforaphane was administered peritoneally tomale mice at concentrations of 12.8, 63.8, and318.8 mg/mL/kg every other day for five weeks.Sulforaphane decreased synoviocyte survival up to51 percent compared to baseline, significantlydecreased IL-17 and TNF-a, and repressed the

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proliferative response in polymorphonuclear cellsto baseline levels. Histological examinationrevealed less inflammation, synovial hyperplasia,and bone destruction in mice treated with sulfora-phane compared to the control group.^^

Macular DegenerationOxidative stress in the retinal pigmented

epithelial (RPE) cell layer is associated withage-related macular degeneration, the leadingcause of hlindness in the elderly.^' In vitro andanimal research demonstrates that sulforaphaneprotects RPE cells from photo-oxidative damage;the degree of protection correlated with basallevels of glutathione and NADH quinonereductase.^"^"

Neurological ConditionsIn vitro and animal research indicates sulfora-

phane treatment of various neuronal cell lines(neuroblastoma, astrocyte, and primary corticalneurons) protects against neuronal injury causedby oxidative stress and inflammation. This isaccomplished via activation of Nrf2/ARE-mediateddetoxification enzymes and results in increasedintracellular glutathione levels and reduced rates ofapoptosis.**' '*'' These studies indicate sulforaphanemay protect against the types of neuronal injuryfound in Parkinson's and Alzheimer's diseases.

Side Effects and ToxicitySeveral studies have been conducted to assess

the safety of sulforaphane in humans. A random-ized, placebo-controlled, double-blind studyshowed broccoli sprout extracts were withoutsignificant side effects at doses of 25 and 100 i^molglucoraphanin for seven days.**^ Another random-ized, placebo-controlled study involving 200healthy adults consuming broccoli sprout infusionsdaily for two weeks (400 i^ mol or approximately175 mg glucoraphanin) showed no adverseeffects.*"" In a dose escalation safety study, broccolisprout extracts containing sulforaphane doses ashigh as 340 nmol were topically applied threeconsecutive times to forearm skin. Researchersreported significant induction of phase II enzymeactivity in biopsied tissue without any adversereactions.**^

DosageBased on available research, typical dosage for

broccoli sprout and seed extracts is 50-100 mgsulforaphane glucosinolate daily in divided doses.

Warnings and ContraindicationsSulforaphane and glucoraphanin from broccoli,

broccoli sprouts, and broccoli seeds has a goodsafety profile with no known contraindications ordrug interactions.

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