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					Application of Herbs to Functional Foods

        Functional foods have been defined as foods ingredients that provide a
        heath benefit beyond the traditional nutrient value of the food.

        With the growing interest in herbal products and an attitude toward
        wellness in the population, it is only natural that food manufacturers would
        turn to herbals as a new source of functional ingredients.

Herbal medicine

        Today, highly purified extracts and powders are put into capsules and

Herbals as ingredients in functional foods

       Numerous issues are raised when considering botanical ingredients as food
       additives, including regulatory requirements, safety, and identity, in
       addition to efficacy.

       Stability of botanical ingredients in foods that must be processed by heat,
       air, or pressure.
Nervous System

 Gingko biloba

    in Europe for the treatment of cerebral disturbance and circulatory

    use for people with Alzheimer’s disease and other form of dementia

    Active compound – Gingkolides : flavonoid glycoside, diterpene lactones
                           (inhibitors of platelet-activating factor and thus have
                           consequences on circulation, blood coagulation and inflammation)

    improve microvalsculature insuficiency

    increase in blood flow to the brain

    efficacious in treating tinnitis and vertigo

    Adverse Effect – headache , GI disturbances, skin allergy
Nervous System

 St. Jhon’s wort (Hypericum perforatum)

 precribed antidepressant in Europe

 antiviral and anticancer properties

 Active compound – Hypericin, pseudohypericin, flavonoids, naphthodianthroms

 Adverse Effects – photosensitivity, dose-related serotonergic symptoms,
                  dizziness, sedation
Nervous System

 Kava kava (Piper methysticum)

     used kava beverages as natural intoxicants

     Active components – Kavalactones
                           (sedative and intoxicating effects,
                            effect benzodiazepine or GAVA-binding site)

     Adverse effect – Yellow discoloration of the skin,
                   dose-related liver metabolic abnormalities
  Nervous System

valerian root (Valeriana officalis)

  mild sedative and sleep aid

  anxiolytic and antispasmodic activity

  Extract of valerian have affinity for GAVA receptor

  Active components – valepotriates, valerenic acid, sesquiterpenes

  Adverse effect – headache
    Heart and circulation

     Hawthorn plant (Crataegus oxucantha)

angia, hypertension, arrhythmia, and congestive heart failure

effective in preventing lipid deposits in the liver and hear, increasing low-density
lipoprotein(LDL) – receptor activity and reducing cholesterol synthesis

Active components – flavonoid, procyanidins, triterpene saponins, cardiactive amines

Adverse effect – dose related sedation, hypotension, arrhythmia
Immune system

 Echinacea (Echinacea purpurea)

  immunostimulating capabilities

  treating the common cold, flu, cough, and brochitis

  Active component – Echinosides, caffeic and ferulic acid, glycoprotein,

  Adverse effect – Allergy

  expect to enhance killing of the invading organism

  echinacea affects the phagocytes, long term ingestion of echinacea may
  potentionally do more harm than good

  increased reactivity of the phagocytic system may result in generation of
  more free radical
Digestive system

   Peppermint oil (Mentha piperita)

 improvement in the symptoms associated with IBS(Irritable Bowell Syndrome)

 Active components – Menthol

 acted as a calcium antagonist

 relaxation of gastointestinal smooth musclem, thus possibly promoting bowel
Digestive system

   Ginger (Zingiber officinale))

       promote gastrointestinal well-being

       prevent postoperative nausea

       A ginger extract increased stomach mobility

       Active components – Gingerols and gingerdiols, volatile oil
 Respiratory system

Licorice (Glycyrrhiza glabra)

act as an expectorant

loss of body potassium

Active components – Glycyrrhetic acid, triterpene saponins, flavonoids,

Adverse effect – High blood pressure due to sodium, water retention, and
                 potassium loss
Urinary system

 Saw palmetto (Serenoa repens)

treatment of benign prostatic hyperplasia (BPH)

The enlargement of the prostate is through to occur via the action of

DTH is derived from testosterone via an alpha reductase enzyme

Treatment for this condition uses drugs designed to inhibit the alpha
reductase enzyme

Saw palmetto berries contain compounds that inhibit the alpha reductase enzyme
Urinary system

 Cranberry ( Vaccinium macrocarpon)

 act as an antiseptic while at the same time preventing bacterial adherence
 to the epithelial cells of the urinary tract
Musculoskeletal system

 Feverfew ( Toncetum parthenuum)

  as a prophylactic treatment for migraine headaches

  act as analgesics and anti-inflammatory agents to reduce swelling and pain

  Active components – Sesquiterpene lactones

  mechanism by which feverfew worked was by inhibition of 5-lipoxygenase
  and cyclo-oxygenase enzyme activities
Flavonoids as
I. General backgroud on
phytochemicals as antioxidant
    Oxidant reaction of free radicals are thought to contribute
     to many health problems
-   Free radical: molecules with unpaired electrons
    Antioxidant are agent which restrict the deleterious effect
     of oxidant reaction
-   Direct effect: eliminating certain free radicals
-   Indirect effect: preventing radicals formation
    Endogenous antioxidants: enzymes
    Exogenous antioxidants: consumed in the diet
     - vitamine E, phytochemicals (flavonoids) <Flavonoid structure>
    Flavonoids
-   one of the major classes of phytochemicals
-   a family of polyphenols
-   be capable of exerting antioxidant effect in humans
    II. Possible antioxidant
    mechanisms of flavonoids
    Potential antioxidant actions of
- at least six different possible
  antioxidant mechanisms for
A. Direct radical scavenging
- direct radical scavenging
  as a single mechanism
- involved more than one type of
  reaction within an oxidant process
☞ Prevention of the formation of
    free radicals: indirect
    (Mechanisms B~F)
 II. Possible antioxidant
 mechanisms of flavonoids
B. Downregulation of radical production
 - Include downregulation of the production of superoxide radical
  and hydrogen peroxide, a precursor of free radicals
●   accomplished through downregulation of protein kinase C
 - protein kinase C: trigger secrection of superoxide and hydrogen
 - protein kinase C mediation : soy constituent genistein
 - genistein: belongs to the flavonoid class known as the isoflavones,
              is a classic inhibitor of protein kinase C in vitro

C. Elimination of radical precursors (hydrogen peroxide)
 - Flavonoids could inhibit production of superoxide and
   hydrogen peroxide
 - Flavonoids directly react elimination of radical formation
II. Possible antioxidant
mechanisms of flavonoids
D. Metal chelation
- some transition metals(iron): catalytically form reactive free
- Flavonoid structures have the chemical properties to chelate
  these metals in state where radical generation is inhibited,
  form complexes which actually eliminate radicals

E. Inhibition of xanthine oxidase
- act as antioxidants by inhibiting prooxidant enzymes
- Inhibition of xanthine oxidase produce superoxide radicals

F. Elevation of endogenous antioxidants
- elevate body concentration of endogenous antioxidant which
  themselves eliminate free radicals or their precursors
    III. Evidence that flavonoids act as
   Experimental animal studies
- the point at issue: whether an
  antioxidant effect is a primary or
  secondary effect of flavonoids
- ex) Table 8.2-C
 If a carcinogen is given to a rat..
 flavonoids inhibit tumor development
 through ① antioxidant effect
            ② some other action
- ② some other action:
  ex) inhibition of cytochrome P 450-
   mediated carcinogen activation
  : limit to produce tumors but would
   not really be an antioxidant effect
   of an flavonoids
III. Evidence that flavonoids act as
     Experimental animal studies
    - flavonoid-induced increase in measures of serum antioxidant
     capacities determined ex vivo
    - indicate that ingested flavonoids can impact radical-scavenging
     potential in an intact animal
    This rat study...
    - the difference was nearly 2.5-fold
    - designed to give a large response
    - a high dose of catechin                            (Total antioxidant status)

    - the basic diet was intended to
      minimize the presence of phyto-
      chemicals in the diet
    - Flavonoids have the potential to
      exert a major influence on TAS values
    III. Evidence that flavonoids act as
   Human studies
- still limited in scope
- so far examine flavonoid-rich foods rather than isolated flavonoids
Ⅳ. Flavonoids and lipoprotein
   Lipoprotein oxidation
- particularly useful for study of flavonoid
 ① both lipoprotein oxidation and flavonoid intake are thought to
   be relevant to cardiovascular disease
 ② many flavonoids are potent inhibitors of lipoprotein oxidation
   when added to the lipoprotein in vitro
- the results for stuidies on lipoprotein oxidation and flavonoid
   intake have varied greatly – differences in the study design
Ⅳ. Flavonoids and lipoprotein
   Design variable for studies of flavonoid intake effect
    on lipoprotein oxidation
A. Type of flavonoids
- Could be very important but does not seem to be the whole answer
    ex) tea flavonoid
B. Whole foods vs. flavonoid concentration (two hypotheses)
- ① combination of whole-food ingredients may be more effective
    than just a single flavonoid or even just the flavonoid fraction of
    food ex) vit.C+flavonoids/flavonoid alone
 ② absorption and metabolism of the flavonoids may depends on
    other food components ex) with/without alcohol in red wine
G. Background diet of the test subjects
- may be important in either enhancing or restricting a flavonoid
  intervention effect ex) catechin producing a large rise in TAS,
                          no effect on LDL+VLDL oxidation
Ⅴ. Evidence for specific
antioxidant mechanisms of
Ex) Various flavonoids have produced antioxidant
   actions against radicals generated and detected
   in a number of ways (in vitro)

   General methods
- include enzyme systems, nonenzymatic organic chemical
  reactions, metal-catalyzed events

   Detection methods
- include direct electron spin resonance(ESR) measurements
  of free radical disappearance, injury to cultured cells and cell
  organelles, diminished generation of oxidant products, and
  inhibition of oxidation of target molecules
  (lipid, lipoprotein, liposome or DNA)
Ⅴ. Evidence for specific
antioxidant mechanisms of

   Possible antioxidant action of Flavonoids

- The ability to suppress cellular production of radicals and their
  precursors such as hydrogen peroxide
- demonstrated in vitro using isolated phagocytes or cell organelles
- this action is harder to assess in vivo

    ●   One relevant indirect measurement

- measurement of plasma myeloperoxidase during oxidant stress
- secretion of myeloperoxidase by phagocytes
 → superoxide and hydrogen peroxide secretion (at the same time)
- myeloperoxidase ↑, superoxide & peroxide production ↑( in vivo)
Ⅴ. Evidence for specific
antioxidant mechanisms of
   Many flavonoids have chemical structures that allow
    chelating of transition metals (iron and copper)

- inhibit metal-catalyzed formation of free radicals
- protect against iron-induced damage to cultured cells

   Flavonoids can block iron-induced injury in rodents

- Flavonoids actually diminish the concentration of radicals
    detectable by ESR spin tapping

⇨    these results could be simply due to flavonoids
    eliminating the radicals after they are formed
Ⅴ. Evidence for specific
antioxidant mechanisms of

   Flavonoids and metals interaction

- have an additional antioxidant effect other than inhibiting
  metal-catalyzed radical formation
- Flavonoids-metals complexes can be catalyst that actually
  eliminates radicals
  ex) demonstrated in vitro with a rutin-copper complex
      - much more potent than rutin alone in eliminating
        superoxide radical and preventing lipid peroxidation
    ex) Flavonoids-copper complexes
      - can act antioxidant catalysts in vivo
Ⅴ. Evidence for specific antioxidant
mechanisms of flavonoids
    Another Possible antioxidant action of Flavonoids

    elimination of radical precursor(hydrogen peroxide)
    -block hydrogen peroxide-induced oxidation of biomolecules or
     cell cytotoxicity
    - protect biomolecules or cells by eliminating radicals after they
      are formed from hydrogen peroxide

     exert antioxidant actions by inhibiting the activities of the
     prooxidant enzyme xanthine oxidase (in vitro)
    - flavonoid concentrations used to inhibit xanthine oxidase
    - can be raised in terms of flavonoid effects on xanthine oxidase
    - two form: dehydrogenase (doesn’t generate superoxide radical)
                  oxidase (does generate superoxide radical): contributor
    - Flavonoids inhibit conversion to the oxidase form
Ⅴ. Evidence for specific
antioxidant mechanisms of
   Antioxidant action of Flavonoids
- flavonoids can act as andioxidants by elevating body contents
  of endogenous antioxidants or by preventing their depletion by
  certain stress states
- flavonoid- endogenous andioxidant interaction involves the
  antioxidant enzyme superoxide dismutase I (=Cu-Zn SOD)
- Superoxide Dismutase (SOD), an antioxidative enzyme,
  catalyzes the dismutation of superoxide radicals (O2-) into
  hydrogen peroxide (H2O2) and molecular oxygen (O2), making
  it a key enzyme in the defense against oxidative stress.
- In vivo SOD is being studied as a means to reduce tissue and
  cellular damage.
- In vitro SOD has been linked to the suppression of apoptosis
  in rat and mice cell lines.
 Ⅴ. Evidence for specific
 antioxidant mechanisms of
   Cu-Zn SOD
- A published paper indicates that
 in mice, inclusion of green tea in
 the drinking water increase colon
 levels of Cu-Zn SOD
- A similar result for Cu-Zn SOD
  activities in rat using synthetic
  version of the green tea flavonoid
 The flavonoid silymarin
  - increase lymphocyte SOD 1
 Some flavonoids may be able to
   prevent inactivation of SOD 1 in
   body site during an oxidant stress
 Ⅴ. Evidence for specific
 antioxidant mechanisms of
Flavonoids can also affect concentrations of antioxidant glutathione
 - chronic ingestion of synthetic catechin has no effect on liver
   glutathione contents but nearly doubles these values in lung
 Catechin ingestion partially
   protected against lung lipid
   peroxidation caused by
   diethylmalate (fig 8.2)
 flavonoid effects on the antioxidant
   enzymes glutathione peroxidase
   and glutathione reductase
  - Both activities are increased
    in mouse skin by feeding the
    isoflavone genistein
  - reductase enzyme activities:
     increase in intestine
  - both enzyme activities:
    increase in livers
Ⅵ. Prooxidant effects of
    Under certain circumstances… generate oxidant reactions

     -   damage the biological molecules

    Large issue
    - evidence for prooxidant actions of flavonoids primarily for
      studies done in vitro
    - whether prooxidant actions can occur in vivo
    - if flavonoids continue to gain attention for possible health-
       promoting effects, the issue of prooxidant actions must also
       be addressed
Ⅶ. Summary

    Flavonoid antioxidant actions
      can impact human health
Cruciferous Vegetables
and Cancer Prevention
       Elizabeth H.Jeffery and Vickie Jarrell
I.     Introduction
II.    Cancer Prevention by Cruciferous Vegetables
       A. Epidemiological Studies                               B.
       Laboratory Animal Studies
III.   Chemical Profile of Cruciferous Vegetables
IV.    Mechanisms of Chemoprevention
       A. Induction of Detoxification                             B.
       Inhibition of Activation                            C.
       Inhibition of Cell Proliferation and Apoptosis
V.     Development of Cancer Preventative Agents from Cruciferous
       Vegetables                                         A.
       Phenylethyl Isothiocyanate                            B.
       Indole-3-Carbinol                                    C.
       Sulforaphane and Sulforaphan Analogues
VI.     Bioactive Components Other Than Isothiocyanates
        A. Crambene                                            B. S-
        Methyl Cysteine Sulfoxide                         C.
VII.    Safety of Cruciferous Vegetables
VIII.   Impacting the American Diet
IX.     Summary
I.         Introduction

    30% of all cancers are considered to have a dietary component.
    Numerous purified dietary components have been shown to be
     mutagenic and are considered by many to be chemical
     initiators of carcinogenesis.
    Diet rich in fruits and vegetables is associated with a
     decreased risk for a number of different cancers.
    Evaluating the effect of cruciferous vegetables have shown an
     inverse relationship between intake of cruciferous vegetables
     and cancer incidence.
II.         Cancer Prevention
            by Cruciferous Vegetables

       In 1982. National Research Council : Consumption of
        cruciferous vegetables is associated with a reduction in the
        incidence of cancer at several sites in humans.
       A 1996 review of seven cohort studies : Inverse association
        between ¹crucifer ingestion and stomach cancer, ²cabbage and
        cauliflower ingestion and lung cancer, ³broccoli ingestion and
        all cancers.
       Review of 87 case-control studies : 67% described an inverse
        association between crucifers and all cancers.
       Intake of crucifers, and no other vegetable type examined, was
        inversely related to risk for bladder cancer.
       A diet rich in broccoli, cabbage, or a mixture of cruciferous
        vegetables is able to decrease one’s risk for cancer.
      ∴ Crucifers decrease the risk for cancer.
II.          Cancer Prevention
             by Cruciferous Vegetables
       Evaluated the effect of cruciferous vegetables on cancer, mostly
        by adding powdered, freeze-dried crucifers to the diets of
        laboratory animals administered chemical carcinogen.
       Cruciferous vegetables do protect against carcinogenesis.
     1. Mammary tumor formation by DMBA ← broccoli or cabbage : mammary
        tumor formation was inhibited.
     2. Mammary cancer incidence by N-methylnitrosourea ← 5 or 10% cabbage :
        decreased incidence of mammary cancer.
     3. Aflatoxin-induced hepatocarcinogenesis ← 25% cabbage : diminished
     4. Dimethylhydrazine-induced tumorigenesis ← cabbage diet : inhibited
     5. Placed under the skin of immune-deficient mice ← mammary tumor cells ←
        collard greens and cabbage : diminished the appearance of pulmonary
     6. Measure of nitropropane-induced DNA oxidative damage ← water extract of
        Brussels sprouts, or fed 3g of Brussels sprouts/day : appearance of urinary
        8-oxo-guanine was decreased.
III.     Chemical Profile
         of Cruciferous Vegetables

   Crucifers are rich sources of a number of a vitamins, including
    several carotenoids(β-carotene, lutein, zeaxanthin), vitamins
    C, E, K, as well as sterol
   Crucifers contain quite high concentrations of S-methyl
    cysteine sulfoxide which can break down to release volatile
    sulfur compounds
IV.        Mechanisms of

     Watternberg treated rats with a single dose pf benzyl
      isothiocyanate, either 24,4, or 2h before, or 4h after, giving the
      carcinogen DMBA.
      Treatment with benzyl isothiocyanate 4h before exposure to the
      carcinogen was significantly less effective than 2h prior to the
      carcinogen(DMBA), which decreased the incidence of mammary
      tumors by 77%.
     A single does of benzyl isothiocyanate could prevent initiation,
      within a narrow window of time.
     When administered benzyl isothiocyanate in the diet starting 1 week
      after DMBA treatment, fewer breast tumors developed.
     Anticarcinogenic effects of isothiocyanates are unlikely to be due to
      a single machanism, or even limited to a single stage of

A.    Induction of detoxification
B.    Inhibition of activation
C.    Inhibition of cell proliferation and apoptosis
IV.        Mechanisms of

•       Aliphatic glucosinolate hydrolysis products cause an
        upregulation of several phaseⅡ detoxification enzymes,
        including quinone reductase and several glutathione-S-
•       Indole-3-carbinol, causes an upregulation of both these
        phaseⅡ enzymes and the phaseⅠ cytochrome P450
        enzymes CYP1A1/2.
•       Glucosinolate hydrolysis products upregulate through at
        least two response elements in the genes of detoxification
        enzymes :
     1. Bifunctional inducers – xenobiotic response element(XRE),
        arylhydrocarbon response element(AhRE)
     2. Monofuctional inducers – antioxidant response element(ARE)
IV.      Mechanisms of

     Glucosinolate breakdown products may protect against
      initiation of cancer not only by induction phaseⅡ
      detoxification enzymes, but also by inhibiting CYP-
      dependent activation of precarcinogens.
     Both phenylethyl isothiocyanate(PEITC) and sulforaphane
      have been found to inhibit the phaseⅠ enzymes, CYP2E1
     Benzyl-isothiocyanate was cause the destruction of CYP2E1
      during metabolism.
IV.       Mechanisms of

     After myrosinase-dependent hydrolysis, most of the
      glucosinolate hydrolysis products were capable of inhibiting
     Using the human undifferentiated colon cancer cell line HT29,
      diindolyl methan and sulforaphan decreased cell viability by
      approximately 50%.
     A study in HeLa cells reported that allyl isothiocyanate, benzyl
      isothiocyanate, and PEITC all caused cell cycle arrest at G2/M.
     Similarly, a study in HepG2 cells showed G2/M arrest following
      treatment with the nitrile crambene.
IV.      Mechanisms of

     Tumor formation and growth may be controlled through a
      decrease in cell proliferation via arrest of the cell cycle
      and/or an increase in apoptosis or programmed cell death.
     Isothiocyanate-induced apoptosis was associated with an
      increase in caspase3.
     JNK(c-Jun N-terminal kinase), which lies upstream of
      caspase3 in apoptotic cascade, was also activated by
     When JNK activation was blocked, isothiocyanate-induced
      apoptosis was also suppressed.
     PEITC was found to induce apoptosis through a p53-
      dependent pathway.
     Overexpression of Bcl-2, was found to suppress PEITC-
      induced JNK activation and block PEITC-induced apoptosis.
V.      Development of Cancer
        Preventative Agents from
        Cruciferous Vegetables

    Gluconasturtiin is blocked the metabolic activation of NNK, a
     major carcinogen in tobacco.
    PEITC had an inhibitory effect on CYP2E1, effectively
     blocking the bioactivation of NNK. Also, PEITC induced a
     number of phaseⅡ enzymes
V.      Development of Cancer
        Preventative Agents from
        Cruciferous Vegetables

    Indole-3-carbinol has been found to inhibit the development
     of tumors in the forestomach, stomach, mammary gland,
     uterus, tongue, and liver of rodents.
    In three breast cancer cell lines, indole-3-carbinol caused
     a >50% inhibition in growth, with an increase in the
     quiescent cell fraction(G0/G1 phase of the cell cycle), and a
     doubling of the apoptotic rate.
    A number of studies in rodents and humans show that
     indole-3-carbinol cause an increase in 2-hydroxylation of
     estrogen, increasing the ratio of 2:16 hydroxylated products.
    When human breast cells were grown in medium containing
     indole-3-carbinol, the increase in the ratio of 2:16
     hydoxylated estrogens was inversely proportional to the
     growth of the cells.
V.      Development of Cancer
        Preventative Agents from
        Cruciferous Vegetables

    Sulforaphane decreases mammary tumor incidence when
     administered to rats before and during administration of
     DMBA, and inhibits neoplastic nodule formation in mouse
     mammary gland cultures.
    The high potency of sulforaphane, <0.6㎛ causing a doubling
     of quinone reductase in mouse hepatocyte culture.
VI.      Bioactive Components
         Other Than Isothiocyanates

     Most nitriles are relatively toxic, but fortunately are also
      relatively unstable.
     One nitrile, crambene is both stable and bioactive.
     Crambene is a monofunctional inducer, causing upregulation
      of quinone reductase and glutathione transferase while
      having no effect on CYP1A1/2 levels.
     Given both crambene and indole-3-carbinol together, total
      upregulation of quinone reductase was significantly greater
      than expected when adding together their individual effects.
VI.      Bioactive Components
         Other Than Isothiocyanates

     The odor of crucifers is, like the onion family, mostly
      associated with sulfur compounds.
     Crucifers contain high levels of SMCSO.
     When rats were administered a diet containing 4% SMCSO,
      they exhibited anemia and splenic hypertrophy, which
      reversed upon removal of SMCSO from the diet.
VI.         Bioactive Components
            Other Than Isothiocyanates

       Oltipaz, caused an increase in colonic glutathione-S-
        transferase and quinone reductase.
       Particularly contaminated by aflatoxins, and are seen to
        have an associated high risk for liver cancer.       Healthy
        adult(234) were divided into three groups.
    Given either a placebo/ 500㎎ oltipraz once weekly/ 125㎎ oltipraz daily
        after 1 month urinary analysis revealed that intermitternt
        high dose inhibited phaseⅠbioactivation of aflatoxin, while
        daily low-dose oltipraz increased phaseⅡ detoxification of
        bioactivated aflatoxin.
       Not only does oltipraz alter aflatoxin metabolism and protect
        against aflatoxin-induced liver cancer, but the unsubstituted
        dithiolethione found in cruciferous vegetables is more potent than
        oltipraz in cell culture.
VII.      Safety of Cruciferous

   Genotoxic potency – Brussels sprouts>cauliflower>cabbage, kohlrabi,
    broccoli>turnip, black radish
   If cruciferous vegetables are exposed to nitrate or nitrite under acid
    conditions, such as ingestion of unwashed fresh vegetables
    contaminated by nitrite fertilizers, or ingestion of pickled crucifers,
    mutagenic N-nitrose compound may be generate.
   A hemolytic syndrome, Brassica anemia or kale poisoning, is due to
    the presence of SMCSO.
   Cabbage is considered goitrogenic when consumed in excess.
   Thiocyanate ions inhibit iodine uptake sufficiently to cause iodine-
    deficiency goiter.
VIII.    Impacting the American Diet

   Fresh, freeze-dried, and cooked vegetables appear to have
    some ability to upregulate detoxification enzymes.
   Potency is enhanced when the crucifers are homogenized or
    chopped and allowed to stand prior freezing.
   Feeding cooked cabbage and Brussels sprout to humans cause
    an increase in detoxification enzymes, and decreased oxidative
    DNA damage.
   Cooking typically destroys the hydrolyzing enzyme,
    myrosinase, but apparently did not destroy the ability of
    crucifers to upregulate detoxification enzymes in the human.
   While 200 to 300g of cooked vegetable are effective in
    upregulating detoxification enzymes in humans.
   One meta-analysis of epidemiological data suggested that as
    few as 10g/day could have a significant effect on reducing the
    risk for cancer.
IX.       Summary

   Crucifer contain glucosinolates.
   Upon hydrolysis, glucosinolates yield a number of breakdown
    products, mostly isothiocyanates, that are biologically active.
   Several of isothiocyanate derivatives, phenylethyl
    isothiocyanate, sulforaphane, and indole-3-carbinol, have
    been decrease tumor incidence when incorporated into the
   Isothiocyanates have also increase or upregulate several
    detoxification enzymes.
   Crucifers may act through inhibition of cytochrome P450-
    dependent bioactivation of carcinogens resulting in decreased
    initiation of cancer and, through cell cycle arrest and apoptosis,
    resulting in decreased progeression of tumor growth.
Carotenoids, Metabolism and Disease
    Richard M.Faulks and Susan Southon
I.    Introduction
II.   Main Dietary Carotenoids
III. Carotenoid Absorption
IV. Approaches to Measurement of Absorption
V.    Distribution and Metabolism of Carotenoids
VI. Cardiovascular Disease and Cancer
VII. Other Metabolic Issues Regarding Carotenoids
I. Introduction:

                    CAROTENOIDS – What are they ?
- a class of highly unsaturated yellow to red pigments- occurring in plants and
animals, mammalian species, humans have no ability to synthesize.
-are all-trans (E) form C40 polyenes formed from eight 5-carbon isoprenoid
-the all-trans structure is subject to isomerization giving a cis-configuration (Z
form) at various positions on the polyene backbone.
-are apolar lipophylic molecules and no solubility in water but in organic
solvents and to some extent in fats and oils.
II. Main dietary carotenoids:
III. Carotenoid absorption:

-The carotenoids are hydrophobic, cannot soluble in the aqueous environment of
the gastrointestinal tract.

-To be absorbed at the enterocyte brush border, carotenoids dissolved in lipid and
lipid+bile salt systems.

-Carotenoids have to transfer to the lipid phase, therefore if the food containing
carotenoids in the presence of fats, the absorption of carotenoid must be easier.

-Some diseases avoid the absorption of carotenoid: cystic fibrosis, celiac disease,
vitamin A deficiency, gut parasites, etc…
IV. Approaches to measurement of absorption:

          A.Metabolic Balance Techniques:
   +The chronic dosing method:
-depend on an equilibrium between intake and excretion.
-fecal markers are collected at the beginning and end of the balance period: 5-8 days.

   +The single acute dosing method:
-the diet needs to be carotenoid-free for 5 days before and during the test period.
-collect feces until no carotenoid can be found in the test dose.

* Feces are the only significant excretory mechanism of nonabsorbable carotenoids.
          B.Ileostomy Mass Balance:

-Apply to those who have undergone ileostomy, the colon has been surgically
removed and the terminal ileum brought to a stoma on the abdominal wall.

-The digesta can be recovered after every 2h and all the residue from a test breakfast
can be recovered in 12h.

-Test meals of both isolated carotenoid and food are given to an overnight fasted
volunteer at breakfast.

The added advantage:
          -can obtain an excretion profile.
          -knowing a time span of the absorption.
C.Gastrointestinal Lavage Technique:

-Using “Colyte” to wash out the entire gastrointestinal tract -> receive a clear rectal
-Volunteers are permitted only water, diet soft drinks for the next 24h.
-all the effluent is collected and pooled with the effluent collected in the following day.
-the carotenoid recovered in the stool is subtracted from that fed to obtain an absorption

          # Advantage:
-standard the residue time of carotenoids in the gastrointestinal tract.
          # Disadvantage:
-can only be applied to healthy individuals.
-give an underestimation due to the use of the “Colyte”.
-the effect of the degradation and loss of unabsorbed carotenoids.
D. Plasma and Plasma Fraction Concentration Methods:
- The administration of an acute or chronic dose of isolated carotenoid, or carotenoid-
     containing food.
- Following the changes in plasma concentration of the carotenoid of interest.
          1.Acute Doses:

-The first peak in plasma concentration is due to the carotenoid present in the newly
    absorbed chylomicrons.

-The second peak result from an increase in the plasma lipid following a meal,
    providing the lipoprotein and triglyceride needed to transport the carotenoid in
    the plasma.
          # Disadvantage:
-cannot determine absolute absorption.
          # Advantage:
-can compare different doses and foods.
-derive some information regarding the relative absorption by comparison with
a standard dose (the isolated carotenoid).
          2. Chronic Dosing:
-Chronic dosing with supplements or foods can be carried out until the plasma
concentration reaches a plateau.
           *small dose (<= 15mg):
-The comparison assume there is a linear relationship between the dose and the
plasma excursion.
           *large dose (>15mg):
-The large doses exceed the capacity of the body’s normal handling mechanism
-> lead to change the relative ratios of pool sizes -> lead to nonlinear responses.
          # Disadvantage:
-Absolute absorption cannot be measured.
          # Advantage:
-The data can compare between isolated compounds and foods, and between different
E.Plasma Triglyceride Rich Lipoprotein Fraction:
-Using AUC (Area Under the plasma Curve) to calculate the rate of the absorption,
disposal, and overall absorption.
Some reports:
           1. 11% central cleavage, and 17% eccentric cleavage.
          2. central cleavage: 3.9% (male); 2.5% (female).
 =>both assume a cited chylomicron remnant half-life of 11.5 min.

Another report:
-half-life 2.5-7.9 min for the clearance of chylomicron triglyceride, rather than those
of chylomicron remnant clearance.
        # Advantage:
-Chylomicrons present in fasting plasma are few and devoid of carotenoids.

          # Disadvantage:
 -The plasma has to be ultracentrifuged to separate the lipoprotein classes, and
cannot separate the chylomicron fraction free of the other LDLs,VLDL.
F. Isotope Methods:
~*~Using 14C and 3H:
-To determine the duration, extent of absorption of -carotene and the degree of
conversion to retinol.
-Radiolabeled -carotenoid was absorbed: 8.7-16.8 %
-> most recovered as the retinyl esters -> largely converted to retinol.
~*~Using -[13C]:
-To study the metabolism of -carotene in humans.
-Draw blood sample to collect: -carotene, retinol and retinyl ester.
-> then using high-performance liquid chromatography (HPLC) to exam: the
purification, separation, quantity.
-Receive 64% 13C entered the plasma as retinyl ester, 21% as retinol, 14% as -[13C]
~*~Using -carotene-d8:
 (alternative to -[13C] because not subject to gas chromatography).
-Receive carotenoid dose 22%: 17.8% as carotenoid, 4.2% as retinoid.
V.Distribution and Metabolism of carotenoids:
-Once the carotenoid is inside the enterocyte -> transported to the serosal side, packaged
    with triglyceride and protein into chylomicrons -> excreted into the mesenteric
    lymphatics -> enters the subclavian vein via the thoracic duct.
-Using AUC for carotenoid concentration in chylomicrons; chylomicron remnant’s half-
     life: 11.5 min.
          A. Distribution:
 -The carotenoids are reexported by the liver in VLDLs (very low density lipoprotein)
-> then metabolized to low-,intermediate-high density lipoprotein.
 -The carotenoids are distributed in the body with body fat containing about 80% and
    liver 10% of the total body load.
 -The retina of the eye contains high levels of the xanthophylls, lutein, and zeaxanthin
    in association with binding protein.
-Carotenoderma (yellowing of the skin): those who consume large quantities of
carrots, particularly if they have a low body mass index (BMI).
-Consumption of high levels of lutein, zeaxanthin, and cantaxanthin, which are
accumulated in the eye lead to protein.
-Human serum contain about 34 carotenoids such as: -carotene, 13-or 9-cis--
carotene, lutein, zeaxanthin, -cryptoxanthin, lycopene, 5-cis-lycopene …
-The carotenids with retinol potential can be cleaved in the enterocyte, liver,
kidney…with suitable enzyme: 15,15’--carotenoid dioxygenase (EC
Central cleavage: producing 2 molecules of retinal which is reduced to retinol
or oxidized to retinoic acid.
Eccentric cleavage: producing apocarotenals in which the conjugated polyene
is shortened by -oxidation to one molecule of retinoic acid.
*retinol equivalent: 6:1(wt:wt) or 3:1 (a molar ratio).
-one -ionone ring.

*retinol equivalent: 12:1(wt:wt).
D.Antioxidant Reaction of Carotenoids:
1*Singlet energy transfer:
                       Carotenoid + light -> 1Carotenoid
           1Carotenoid   + Chlorophyll -> Carotenoid + 1Chlorophyll
-The process of photosynthesis.
-First, carotenoids absorb the light ->become singlet excited state.
-The excited carotenoids then pass the energy to chlorophyll -> produce singlet
excited chlorophyll.
2*Triplet enery transfer:
                            Molecule + light -> 3 Molecule
               3Molecule     + Carotenoid - 3Carotenoid + Molecule
                       3Carotenoid    -> Carotenoid + Heat
-The process prevents the production of damaging radicals.
-Molecules absorb the light -> make molecules triplet excited states.
-Carotenoid (polyene structure) absorb the excited energy from excited molecules ->
to become triplet excited state.
-After that, carotenoid decay exothermically -> to be ground state.

3* Singlet [0] quenching:
                         1O +   Carotenoid -> 3O2 + 3Carotenoid
                            3Carotenoid   ->Carotenoid + Heat
      1O :   extremely active state; 3O2: ground state.

-Carotenoids can quench the active state oxygen -> to produce triplet excited
carotenoid. -> then become a ground state by decaying exothermically.
E.Reaction with Radical Species:
-The carotenoids are antioxidant because they have polyene backbone -> terminate
free radical reactions.
-(1) the free radical joins onto the polyene chain to produce a much less reactive free
-(2) electron transfer from the carotenoid to the free radical to produce a less reactive
charged carotenoid radical.
-(3) donation of a hydrogen molecule to the free radical to produce a stable
carotenoid radical.

F.Erythropoitic Protoporphyria:
--carotene, cantaxanthin reduce photosensitivity in erythropoietic protoporphyria,
although there is a risk of cantaxanthin crystals forming in the eye.
G. Age Related Macular Degeneration: (AMD)
-The retina of the eye contains: 2 xanthophylls, lutein and zeaxanthin.
-Their functions: quench singlet oxygen, inactive triplet-excited molecules ->
reducing the oxidative stress on the eye proteins.
    Characteristic of AMD:
-Older subjects with low densities of macular pigment have impaired visual
-whereas those with normal pigmentation have similar visual sensitivity to younger
-Some evidences: Lutein can increase the density of macular pigment but the impact
on AMD is unknown.
VI. Cardiovascular disease and cancer:
Carotenoids can:
(1) break free radical reactions that oxidize unsaturated lipids.
(2) protect DNA from free radical attack.
=> be central to the initiation and progression of atherogenesis, cancer.
The disease is caused by the production of oxidized LDL because of impaired
    antioxidant status.
-LDL are oxidized by fatty acid hydroperoxides.
-The oxidized LDL is taken up by monocytes -> infiltrate the artery wall.
-> differentiate into macrophage -> produce foam cells -> form fatty streaks.
-**- Carotenoids can prevent the formation of lipid hydroperoxides by breaking the
     free radical propagation of lipid oxidation.
-However, this mechanism is unknown clearly.
-Carotenoids have been shown to have some beneficial effects to cancer initiation,
progression and proliferation at some sites.
-The carotenoid can suppress the proliferation of cells with chemically induced
neoplastic changes: removing neoplastic foci develop.
-No such protection is seen in carotenoid-treated cells subjects to irradiation or in
cell treated with carotenoid after carcinogen- induced production of neoplastic foci.
-Carotenoids are effective agents in the scavenging of free radicals -> to protect
DNA from damage by base corruption (oxidation, deletion, adduct formation).

VI.Other metabolic issues regarding carotenoids:
-There is very little information about the non-provitamin A carotenoids or the
provitamin A carotenoids that are not metabolized to retinol.
  Chemoprevention of colonic aberrant
crypt foci in Fischer rats by sulforaphane
      and phenethyl isothiocyanate
         Main content:
I.Abstract (1)
III.Description of this experiment (3)
IV.Results (4)
V.Discussion (5)
                  I. Abstract
  ACF: aberrant crypt foci
  AOM: azoxymethane
  BITC: benzyl isothiocyanate
  CYP: cytochrome P450
  GST: glutathione S-transferase I
  TCs: isothiocyanates
  NAC: N-acetylcysteine
  PEITC: phenethyl isothiocyanate
  SFN: sulforaphane
  GSTM1: glutathione S-transferase M1
  GST: glutathione S-transferase
Epidemiological studies find that:
The consumption of broccoli is reduced risk of colon
cancer in individuals with the (GSTM1) glutathione S-
transferase M1 null genotype.
GSTs are involved in excretion and elimination of (ITCs)
ITCs are major constituents of broccoli and watercress.
ITCs may play a role in protection against human colon
This article have shown that SFN and PEITC can inhibit
colon cancer.
II. Introduction
 What is Aberrant crypt foci (ACF)
 Aberrant crypt foci (ACF) have been identified in the
 methylene-blue stained mucosa of the human colon.
 Some lines of evidence suggest that ACF may be
 precursors of colon cancer

  Figure 1 : The formation of aberrant crypt foci (ACF)
Structures of SFN and PEITC and their NAC conjugates.

(SFN) sulforaphane is present at broccoli.

(PEITC) phenethyl isothiocyanate is present at watercress.

Their corresponding N-acetylcysteine (NAC) conjugates is


ITCs are principal constituent of broccoli and watercress,

PEITC- NAC belong to ITC        Watecress
                                                100g (wet weight) of
                                                broccoli or watercress
                                                can contain from 50 to
                                                200 µmol SFN or PEITC

   Figure 3 :     Structures of SFN and PEITC and their NAC conjugates.
Several laboratory animal studies have shown that:

PEITC is a potent chemopreventive agent for cancers of
the breast, lung.

However, there is only limited information about its
protective effect against colon cancer.

ITCs as chemopreventive agents in a variety of tumor
models, it is possible that SFN and PEITC would protect
against colon tumor development.

So we determine whether SFN and PEITC can inhibit the
formation of aberrant crypt foci (ACF) induced by
azoxymethane (AOM) in Fischer 344 rats.
III. Description of this experiment
Groups of six male F344 rats were treated with AOM
(azoxymethane) subcutaneously (15 mg/kg body wt)
once weekly for 2 weeks. (*)

SFN and PEITC and their NAC conjugates were
administered by gavage either three times weekly for 8
weeks (5 and 20 µmol, respectively) after AOM dosing
(post-initiation stage) or once daily for 3 days (20 and 50
µmol, respectively) before AOM treatment (initiation

The bioassay was terminated at week 10 after the second
AOM treatment. All animals were killed by CO2
The colons were excised, fixed in buffered formalin and
processed for microscopic examination.

ACF(aberrant crypt foci) were recorded using a standard
procedure described previously.

 ACF were distinguished from the surrounding normal
crypts by their increased size, significantly increased
distance from lamina to basal surface of cells and the
easily discernible percryptal zone.

 For statistical analysis, means were compared among
the groups using one-way analysis of variance followed
by Fisher's protected t-test.
IV. Results
Table I. Effects of SFN and PEITC on the formation of aberrant crypt foci induced (ACF) by AOM
     Treatment groupa           Dose of         Average         Number of aberrant crypt foci
                                  ITC         body weight                  (ACF)
                               compounds                         >Four             Total
                                 (µmol)                          crypts
                                              termination                             *
1      AOM                         -              310              52               153
2      AOM->SFN                    5              301           30(42)b,c         103(33)d
3      AOM->SFN-NAC                20             297            31(40)c          116(24)e
4      AOM->PEITC                  5              306            27(48)c          100(35)d
5      AOM->PEITC-NAC              20             313            38(27)f          113(26)e

6      SFN->AOM                    20             310            35(33)f          109(29)e
7      SFN-NAC->AOM                50             304            44(15)           120(22)c
8      PEITC->AOM                  20             307            35(33)f          115(25)c
9      PEITC-NAC->AOM              50             303           74(-42)c          198(-29)e

10     SFN                          5             309               0                 0
11     SFN-NAC                     20             312               0                 0
12     PEITC                        5             333               0                 0
13     PEITC-NAC                   20             301               0                 0
14     Control                     -              320               0                 0
aITC    compounds were administered during the post-
initiation phase in groups 2–5 and during the initiation
phase in groups 6–9.

bPercentage      inhibition compared with group 1.

cSignificantly   different from group 1 (P < 0.01).

dSignificantly   different from group 1 (P < 0.0001).

eSignificantly   different from group 1 (P < 0.001).

fSignificantly   different from group 1 (P < 0.05).

Table I (*)
                                         No significant differences in
                                         body weights were seen in
                                         any of the treated groups
                                         compared with the control
Figure 5: Growth curves of the treated
and the control groups during the
bioassay.                                Indicating that doses of
                                         ITCs and the conjugates
                                         used did not cause overt
                                         toxicity (Figure 5 ).
The bioassay was terminated on week 10 after the second
AOM dosing and ACF were quantified.    Table I
SFN, SFN-NAC, PEITC and PEITC-NAC all significantly
reduced the formation of total ACF from 153 to 100–116
and multicrypt foci from 52 to 27–38 during the post-
initiation treatment.
However, only SFN and PEITC were effective during the
initiation phase, reducing the total ACF from 153 to 109–
115 and multicrypt foci from 52 to 35.
The NAC conjugates were inactive as anti-initiators against
AOM-induced ACF.
These findings provide important laboratory evidence for a
potential role of SFN and PEITC in the protection against
colon cancer.
V. Discussion

These results, together with those from our earlier studies,
suggest that ITC conjugates render their inhibitory activity
in part by deconjugation to the parent ITCs.

Although the mechanism of inhibition of ACF at the post-
initiation phase is not currently clear.

Studies have shown that PEITC and related ITCs induce
p53-dependent or c-Jun kinase-mediated apoptosis in
cultured tumor cells
More recently it has been reported that SFN induces cell
cycle arrest in HT29 human colon cancer cells.The
growth arrest, followed by cell death via apoptosis.

Pretreatment of animals with PEITC and other related
ITCs blocked chemical-induced tumorigenicity by
inhibiting cytochrome P450 (CYP) enzymes responsible
for the activation of carcinogens, and consequently
reducing DNA damage .

All these activities could have contributed to the
inhibition of ACF formation during post-initiation.

 This article have shown that SFN and PEITC can inhibit
colon cancer.

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