BIOACTIVE COMPOUNDS

FST 5104
 Bioactive Compounds

• Desirable constituents of Foods

• Compounds that are biologically active
• Capable of modulating metabolic processes and
resulting in the promotion of better health.
• Antioxidant activity, inhibition or induction of enzymes,
inhibition of receptor activities, antidiabetic,
anticancer and others
 Bioactive compounds

• Compounds with broad diversity of structures and

functionalities
• Ingredients for production of nutraceuticals, functional
foods or designer foods
• Phytochemicals, biopeptides, Fatty acids, functional
CH2O
• Some found in high concentration such as polyphenols
and carotenoids but others can only be found at very low
levels
• Structural diversity and complexity make chemical
synthesis unprofitable.
• Difficulties in screening and producing led to the
development of advanced technologies.
• The commonly used methods for their extraction are the
conventional liquid–liquid or solid–liquid extraction and
the advanced include pressurized-liquid extraction,
subcritical and supercritical extractions, and microwave-
and ultrasound-assisted extractions
 Sources Material
• Malaysia blessed with abundant flora and
fauna
• Plants-phytochemicals
• Microorganisms
– bacteria, fungus
• Marine organisms
• Animal products
 Biological Activity

• Cancers
• Antimicrobial
• Diabetes
• Heart diseases
• Aging
• Obesity
• Energy Production/ergogenic
• Boost immune system
• Depression
 Phytochemicals
• Most widely studied
• Biologically active chemical compounds
found in plants
• Non-nutritive components in the plant-based
diet that possess substantial anti-
carcinogenic and anti-mutagenic and other
biological properties
• Not nutrients like vitamins or minerals
• Believed to have health benefits especially
related to heart disease and cancer- chronic
diseases
 Cosmos caudatus (ulam raja)

Bioactive compounds in cosmos

caudatus are mainly flavonoids,
quercetin, catechin, rutin,
myricetin

1. Antidiabetic
2. Anti-Coronary heart
disease
3. Antioxidant,
antiobesity agent
4. Anticancer
5. Anti-aging
6. Immune system
 Phytochemicals classification

Allyl
sulphide-
(onion and
garlic)

Organosulfur

Phenolics
 Alkaloids

Caffeine Theobromine

Carotenoids
 Phenolics

• The most widely studied

• Phenolics, derivatives of benzene (cyclic derivatives of
polyphenols) with one or more hydroxyls, are the
products of secondary metabolism in plants, frequently
found attached to sugar (glycosides), thus water-
soluble.
• Provide essential functions in the reproduction and the
growth of the plants
• As defense mechanisms against pathogens, parasites,
and predators
• Contribute to the color, flavour of plants
 Phenolic acids

• Phenolic acids are mainly divided in to two sub-groups,

hydroxybenzoic and hydroxycinnamic acid
• Possess much higher in vitro antioxidant activity than well
known antioxidant vitamins

• Gallic acid p-coumaric

• Vanilic acid Caffeic
• Syringic acid ferulic
• Protocatechic acid Sinapic
 Phenolic Acid
• Hydroxybenzoic acids
possess a common
structure of C6-C1 and
derived from benzoic acid.
• Soluble (conjugated with
sugars or organic acids)
and bound with cell wall
fractions as lignin.
• As compared to
hydroxycinnamic acids,
hydroxybenzoic acids are
generally found in low
concentration in red fruits,
onions and black radish
etc.
• Hydroxycinnamic acids,
derived from cinnamic acid,
present in foods often as
simple esters with quinic
acid or glucose.
• The most abundant soluble
bound hydroxycinnamic acid
present is chlorogenic acid
(a combined from form of
caffeic and quinic acids).
• The four most common
hydroxycinnamic acids are
ferulic, caffeic, p-coumaric,
and sinapic acids.
•
 Biological effects of Phenolic acids

• Plant phenolics exhibit high antioxidant activity

• Found ubiquitously and well documented for other health protective
effects like antimicrobial, anticancer, anti-inflammatory, anti-
mutagenic etc
• Epidemiology evidence indicates that a diet rich in antioxidant fruits
and vegetables significantly reduces the risk of many oxidative
stress related diseases, cancers, diabetes and cardiovascular.
• The number and position of hydroxyl group in a particular phenolic
compound leads to the variation in their antioxidant potential
• Phenolic acids, possess phenol moiety and resonance stabilized
structure which causes the H-atom donation results in antioxidant
property through radical scavenging mechanism.
• Other mode of action is radical quenching via electron donation and
singlet oxygen quenching are also known for the antioxidant activity
of phenolic acids.
Functionalities of Phenolic Acids
 MOA of Phenolic acid

• Phenolic compounds are known as direct antioxidants, however they

also showed indirect antioxidant activity by inducing endogenous
protective enzymes
• Act as antioxidants (due to the reactivity of phenol moiety; hydroxyl
substituent on aromatic ring).
• Even though several mechanisms are known, but radical scavenging
via hydrogen atom donation is the main method.
• Substituents on the aromatic ring in phenolic acids affect the
stabilization of structure and consequently affect the radical-
quenching ability.
• Different phenolic acids therefore have different antioxidant activity,
depending on the structures
 Anti-diabetic Agent

• Phenolic acids influence the function of glucose and insulin

receptors
• They increase the expression of glucose transporter GLUT2
in pancreatic β-cells (produces the insulin) and promote the
translocation of GLUT4 through AMP activated protein
kinase pathways.
• Chlorogenic and ferulic acids proved the same transporter
stimulation mechanism and work as antidiabetic agents
• There is also inhibition of α-glucosidase and α-amylase
enzymes crucial for the conversion of dietary carbohydrates
into glucose. Delay digestion and absorption
 “French paradox”
• Research on flavonoids received an added impulse with the
discovery of the “French paradox”, the low cardiovascular mortality
rate observed in Mediterranean populations although their intake of
saturated fat and cholesterol (meat intake) was high.
• This is in association with high tomatoes, herbs, red wine
consumption and is related to a low incidence of atherosclerotic
diseases
• The association between flavonoid intake and the long term effects
on mortality was studied and it was suggested that flavonoid intake
is inversely correlated with mortality due to coronary heart disease,
cancer and chronic diseases
• Diet is a cornerstone of cardiovascular disease prevention.
 Flavonoids
• The largest class of phenolics and most widely studied
• Most common group in the human diet
• The first observation regarding their biological activities was
published in 1936
• Originally proposed to be required as vitamin, the term “vitamin P”
(also known as citrin) but was later dismissed
• Natural substances with variable phenolic structures found in fruits,
vegetables, grains, bark, roots, stems, flowers, and seeds
• More than 4000 varieties of flavonoids have been identified, many of
which are responsible for the attractive colors of flowers, fruit, and
leaves
• These natural products were known for their beneficial effects on
health long before flavonoids were isolated as the effective
compounds
 THEIR ROLES IN PLANTS
Secondary metabolites of plants

• contribute to the color and flavor

• protect against
• UV-radiation
• fungus, insect and pests
• signal for N-bound bacteria
• modify enzyme reactions
• 75% kaempferol and quercetin and 10% myricetin.

kaempferol quercetin myricetin

Flavonoids are diphenylpropanes, derived from aromatic amino
acids, phenylalanine, tyrosine and malonate with the basic
structure comprising the flavan nucleus (phenyl- γ-pyrone or
diphenylpropan 1-one), with 15 carbon atoms arranged in 3 rings
(C6-C3-C6), which are labeled A, C and B

Molecular
structure of the
flavone
backbone (2-
phenyl-1,4-
benzopyrone)

The A ring usually has hydroxylation pattern at the 5 and 7

position.
The B ring comes from the shikimate pathway and is usually
4’-, 3’4’-, or 3’4’5’-hydroxylated
 Different groups of Flavonoids

• All these structures had the phenyl group (outer ring)

attached where a flavone should, at the 2 carbon. Slight
modifications beyond this can change the molecule
A flavone can be subcategorized into:
• Flavones (Flavone backbone, double bond between Carbon 2
and 3)
• Flavonols (hydroxy group at the 3-carbon; between the
ketone and outer ring)
• Flavanols (lacking the ketone group)
• Flavanones (Flavone backbone without the double bond)
• Anthocyanins (Similar to Flavanols, protonated O, double
bond at 2 and 3)
 Molecular
structure of each group of flavonoid

2-phenylchromen-4-one
3-hydroxy-2-
Flavanol phenylchromen-4-one

Cyanidin, Pelargonidin, Delphinidin

quercetin, kaempferol, myricetin
Apigenin, luteolin
Hesperidin, Naringin, Naringenin
 PHYSIOLOGICAL ACTIVITY OF
FLAVONOIDS
“Nature's biological response modifiers" because of
strong experimental evidence of their ability to modify
the body's reaction.

The claims include:

-antioxidant and /or radical scavenger activity
• immunomodulating and anti-inflammatory effects
• anti-asthmatic and anti-allergic effects
• anti-viral and anti-bacterial and anti-fungal effects
• estrogen activity
• influencing effect on mutagenicity and
carcinogenicity
• hepatoprotective effect
• effects on circulation, modification of permeability
 Antioxidative effect
• The best-described property
• The flavonol and catechins the most powerful flavonoids for
protecting the body against reactive oxygen species
• Inhibit lipid peroxidation that cause cellular membrane damage.
• This cellular damage causes a shift in the net charge of the cell,
changing the osmotic pressure, leading to swelling and eventually
cell death.
• Free radicals attract various inflammatory mediators, contributing to
a general inflammatory response and tissue damage.
• The increased production of reactive oxygen species during injury
results in consumption and depletion of the endogenous scavenging
compounds.
 Direct scavenging of free radicals

• Flavonoids can interfere with different free radical–producing

systems, and increase the function of the endogenous antioxidants
• Oxidized by radicals, resulting in a more stable, less-reactive radical.
• Flavonoids stabilize the reactive oxygen species by reacting with the
reactive compound of the radical.
• Because of the high reactivity of the hydroxyl group of the
flavonoids, radicals are made inactive

Flavonoid(OH) + R• > flavonoid(O•) + RH

• Selected flavonoids can directly scavenge superoxides

• Other flavonoids can scavenge the highly reactive oxygen derived
radical called peroxynitrite.
• Epicatechin and rutin, quercetin are powerful radical scavengers
 IC50 of selected plants (DPPH)

Scientific name Common name IC50 (μg/mL)

Catechin 20.730.14a
Catechin
King of salad 21.310.13a
Cosmos caudatus
α-tocopherol 21.420.95a
α-tocopherol
BHA 22.150.02a
BHA
Betle 23.000.18a
Piper betle
Beluntas 29.280.47b
Pluchia indica
Henna 31.090.25bc
Lawsonia inermis
Bebuas 31.910.43bcd
Premna cordifolia
Curry 32.800.54cd
Murraya koennigii
Tenggek burung 34.400.17d
Melicope lunu
Lemuni hitam 40.060.42e
Vitex negundo
Pennywort 40.500.10e
Centella asiatica
 IC50 of selected plants

Kaduk 56.600.40f
Piper longum
False turmeric 61.490.32g
Curcuma xanthorrhiza
Mollucan spinach 63.120.11g
Gynura procumbens
Fingerroots 71.460.45h
Boesenbergia rotunda
King of bitter 74.100.41h
Andrographis paniculata
Sesban 74.210.32h
Sesbania grandiflora
Cekur 80.530.23i
Kaempferia galanga
Gendarussa 84.900.33j
Justicia gendarussa
Noni 85.200.22j
Morrinda citrifolia

Pereskio bleo Jarum 7 87.360.25jk

Talinum triangulare Talinum 87.500.27jk

Psophocarpus tetragonolobus Winged bean 89.590.17k

 Total phenolic compounds

5
DPPH radical scavenging

3
activity

1 R = 0.8613

0
0 200 400 600 800
Phenolic compounds (mg GAE/g)

Total flavonoids

4
DPPH radical scavenging

3
activity

1 R = 0.8430

0
0 1000 2000 3000 4000 5000 6000 7000
Total flavonoids content (mg/100 g)
 Anti-atherosclerotic effects

• Flavonoids have major influence on vascular system.

• Oxygen radicals can oxidize LDL, which injures the endothelial wall
and promotes atherosclerotic changes.
• Clinical studies have shown that flavonoid intakes protect against
coronary heart disease.
• Flavonoids in regularly consumed foods might reduce the risk of
death from coronary heart disease in elderly men.
• Oxidative stress and vascular damage are seen to play a key role in
dementia, and the intake of red wine is reported to prevent the
development of Dementia
• The intake of flavonoids was reported to be inversely related to the
risk of incident dementia
 Nitric Oxide

• Nitric oxide or nitrogen monoxide is an important

signaling molecule in humans
• Molecules responsible for transmitting information
between cells in the body.
• NO is involved in many physiological and pathological
processes within the mammalian body both beneficial
and detrimental
• Appropriate levels of NO production are important in
protecting organ from ischemic damage
• Nitric oxide contributes to vessel homeostasis by
inhibiting vascular smooth muscle contraction and
growth, platelet aggregation, and leukocyte adhesion
to the endothelium
 Sustained levels of NO
• However sustained levels of NO production result in direct tissue
toxicity and contribute to vascular collapse
• Chronic expression of NO is associated with various carcinomas and
inflammatory conditions including diabetes, multiple sclerosis,
arthritis and ulcerative colitis
• Atherosclerosis, diabetes or hypertension often show impaired NO
pathways
• Nitric oxide is also generated by phagocytes (monocytes,
macrophages, and neutrophils) as part of the human immune
response.
• Phagocytes are armed with inducible nitric oxide synthase (iNOS)
• Nitric oxide secreted as an immune response as free radicals and is
toxic to bacteria
• Nitric oxide can contribute to reperfusion injury when an excessive
amount produced during reperfusion (following a period of ischemia)
reacts with superoxide to produce the damaging oxidant
peroxynitrite

• ONO2−
 Superoxide, nitric oxide and peroxynitrite

• Nitric oxide reacts with free radicals, producing the highly

damaging peroxynitrite
• Formation of peroxynitrite in vivo has been ascribed to the
reaction of the free radical superoxide with nitric oxide
radical:

·O2− + ·NO → ONO2−

• Peroxynitrite is an oxidant and because of its strong

oxidizing properties, can damage molecules, including DNA
and proteins
• Peroxynitrite can directly oxidize LDLs, resulting in
irreversible damage to the cell membrane.
 Nitric-oxide and flavonoids

• Although nitric oxide is important in maintaining dilation of

blood vessels, higher concentrations of nitric oxide produced
by inducible nitric-oxide synthase in macrophages can result
in oxidative damages
• Nitric oxide is reported to be directly scavenged by
flavonoids
• Therefore, it has been speculated that nitric oxide
scavenging plays a role in the therapeutic effects of
flavonoids
• Several flavonoids, including quercetin, interfere with nitric-
oxide synthase activity
 Xanthine oxidase pathway

• Xanthine oxidase (XO) catalyzes oxidation of hypoxanthine

to xanthine and further catalyze the oxidation of xanthine to
uric acid, generates reactive oxygen species (ROS)
• Xanthine oxidase is a source of oxygen free radicals
• The xanthine oxidase pathway has been implicated as an
important route in the oxidative injury to tissues
• In the reperfusion phase (reoxygenation), xanthine oxidase
reacts with molecular oxygen, releasing superoxide free
radicals.
• Inhibition of xanthine oxidase has been proposed as a
mechanism for improving cardiovascular health
• Flavonoids inhibit xanthine oxidase activity
• Structure-function of flavonoids with the most XO-inhibitory
properties used to predict IC50 of xanthine oxidase
• The flavonoids found to have the lowest IC50 include:
apigenin (1.0 µM), luteolin (0.75 µM), kaempferol (2.5 µM),
quercetin (1.5 µM), and myricetin (1.5 µM)
• Luteolin was the most potent inhibitor of xanthine oxidase.
• Numerous natural products have been found to inhibit
xanthine oxidase in vitro or in model animals.
• Generally, planar flavones and flavonols with a 7-hydroxyl
group inhibit xanthine oxidase
• Quercetin inhibit xanthine oxidase activity, thereby resulting
in decreased oxidative injury
 CHELATING ABILITY OF FLAVONS AND
FLAVANONS

• 3’, 4’- dihydroxy configuration

Conditions of chelating • carbonyl group at the position 4

property: • free OH-groups at 3- and 5
positions
 Chelating ability of herbs

High chelating ability Moderate chelating ability

100 30 P.cordifolia
P.tetragonol
obus
L.inermis
P.betel 24
75

% chelating activity
% chelating activity

C.xanthorrhiz
P.bleo
18 a
S.grandiflora
50
T.triangulare
12 B.rotunda
citric acid
25 G.procumbe
6 ns
K.galanga
V.negundo
0 0
0 0.5 1 2 4 8 0 0.5 1 2 4 8
Concentration (mg/ml) Concentration (mg/ml)
 Background

• Estrogens, produced in the ovaries and testis, have many biological

effects in the body beyond that of the reproductive system
• Maintaining bone density by regulating the formation and resorption
of bone
• Lipid profile
• Protein synthesis
• GI tract motility
• Coagulation
• Play significant role in women’s mental health
• Sudden estrogen withdrawal, fluctuating estrogen, and periods of
sustained estrogen low levels correlates with significant mood
lowering
• Clinical recovery from postpartum, perimenopause, and
postmenopause depression
 Phytoestrogens

• Phytoestrogens are plant derived estrogen-mimics that are

capable of binding to the estrogen receptor (ER) and affect
estrogen mediated processes
• A number of these compounds have been identified in
legumes fruits, vegetables, and whole grains, seed oils
commonly consumed by humans
• Studies in humans, animals, and cell culture systems
suggest that dietary phytoestrogens play important role in
prevention of menopausal symptoms, osteoporosis, cancer,
and heart disease.
• Proposed mechanisms include estrogenic and anti-
estrogenic effects, induction of cancer cell differentiation,
inhibition of tyrosine kinase and DNA topoisomerase
activities, suppression of angiogenesis, and antioxidant
effects
 Phytoestrogens

Coumestrol
(clover, alfafa)

Estrogen

Genistein Daidzein Biochanin A

(legumes, alfafa)
 ISOFLAVONES

•Related to flavonoids

•Act as phytoestrogens

•Strong antioxidant

• Major isoflavones in soybeans are genistein and

daidzein

Isoflavone backbone (3-

phenyl-4H-1-benzopyr-4-
one)
 Isoflavones

• Possess both pro-estrogenic and anti-estrogenic

functions
• The structural similarity between isoflavones and
estrogen, as well as evidence indicating that estrogen
can improve or protect some aspects of cognitive
health
• The phenolic B-ring of isoflavones confers binding to
estrogen receptors (ER β > α, although at a lower
affinity relative to estradiol) and acts like natural
selective estrogen receptor modulators
 SOY ISOFLAVONES

• Soy isoflavones are the most studied phytoestrogens, consisting

of genistein, daidzein, glycitein and their respective glucosidic
conjugates (daidzin, genistin, and glycitin)
• Soybeans and soy foods contain approximately 0.2–1.6 mg of
isoflavones/gdw
• They have different binding affinities to ERs based on their
structures
• Genistein was reported to have higher affinities than other
derivatives
• Genistein has one-third the potency of estradiol when it
interacts with ERβ, and one thousandth of the potency of estradiol
when it interacts with ERα
• Genistein can induce similar responses in breast, ovarian,
endometrial, prostate, vascular, and bone tissues and cell lines as
estradiol
 Menopause

• Menopause is associated with mid-life, when many

women begin to experience signs and symptoms of
aging, such as increases in blood pressure, changes in
lipid profiles, loss of bone mass density, and diminished
memory and cognition.
• Decrease in the quality of life due to sleep deprivation,
hot flashes, mood swings, forgetfulness and difficulty in
concentrating
• These symptoms are linked to the declining and erratic
production of estrogen
• Many postmenopausal women feel that their cognitive
abilities decline during menopause, and some believe
that HRT may alleviate this decline
 Hormone replacement therapy

• Research suggests that HRT, may improve or protect memory

function and slow the progression of other symptoms, such as bone
loss
• However, studies have shown that HRT is not as safe or effective as
previously thought
• Many women are reluctant to accept HRT because of potential
health concerns including increased risk of breast cancer, stroke,
coronary heart disease, pulmonary embolism, and uterine bleeding
 Epidemiological studies

• Many epidemiological studies showed people who

consume high amounts of isoflavonoids in their diets have
lower rates of several cancers including breast, prostate
and colon cancer
• In Asia, where consumption is high, the rates of these
cancers are low
• Immigrants from Asia living in the US have an increased
risk of cancer compared to people living in Asia
• The increase in cancer rates is correlated with the length
of stay in the US and the exposure to the North American
diet
 Soy and Cognitive functions

• Although clinical data for investigating the effects of

soy isoflavones on the cognitive function are few and
do not show the same results, clinical trials indicate
that soy isoflavones might improve cognitive function
not only in postmenopausal and young adult women
but also in young adult men
 SOPHIA Study

• SOy and Postmenopausal Health In Aging (SOPHIA)

examined the effects of soy isoflavone
supplementation

(110 mg/day) on cognitive function of postmenopausal

women ( mean age- 60 years) for six months

• The study showed that category fluency, logical

memory and recall was higher in women receiving soy
isoflavone supplement than in controls.
• Study involving postmenopausal women receiving 60
mg of soy isoflavones per day or placebo for 12 weeks
showed significantly higher performance in the recall
of pictures and in sustained attention and ability to
plan tasks
• These women did not demonstrate any improvement
in menopausal symptoms, mood or sleepiness.
• This suggests a specific improvement in frontal lobe
functions and the cognitive improvements were not
the result of a better quality of life
• However, in a double-blind, randomized, placebo-
controlled trial of 202 healthy postmenopausal women
aged 60 to 75 years conducted in the Netherlands
showed no improvement of cognitive function in
postmenopausal women.
• The women were randomly assigned into two groups,
with one receiving 25.6 g of soy protein containing 99
mg of isoflavones and the other receiving only milk
protein on a daily basis, and cognitive function did not
differ significantly between the groups after 1 year.
 Study involving
Students
• A ten-week study demonstrated that diets high in soy increased
student’s long term and short-term memory
• Male and female students were assigned either a high (100mg/day)
or low (0.5 mg/day) isoflavone diet.
• At the beginning and end of the study period the subjects were
tested for a variety of cognitive skills.
• The students in the high isoflavone diet group had significant
improvements in short term and long term memory, and mental
flexibility
• The low isoflavone group had no change in these parameters.
• Since the low isoflavone diet was also low in soy products, it is
unclear whether the phytoestrogens are responsible for this effect.
 Curcumin
• Polyphenol from tumeric
• Management of oxidative stress
• Inflammatory conditions
• Metabolic syndrome
• Arthritis
• Hyperlipidemia
• Aid in management of exercise-induced inflammation
and muscle soreness
• Poor bioavailability, rapid metabolism and elimination
• Cancer
 Curcumin
• Studies have shown curcumin interfere with cell-signaling pathways, thus
suppressing the transformation, proliferation, and invasion of cancerous
cells
• In prostate cancer cell lines, curcumin inhibits cell growth and induces
apoptosis
• It aids in the management of oxidative and inflammatory conditions,
metabolic syndrome, arthritis, anxiety, and hyperlipidemia.
• Most of these benefits can be attributed to its antioxidant and anti-
inflammatory effects.
• Ingesting curcumin by itself does not lead to the associated health
benefits due to its poor bioavailability, which appears to be primarily due
to poor absorption, rapid metabolism, and rapid elimination.
• There are several components that can increase bioavailability. For
example, piperine is the major active component of black pepper and,
when combined in a complex with curcumin, has been shown to increase
bioavailability by 2000%.
 MOA of Curcumin
• The structure of curcumin is comprised of the central 1,6-
heptadiene-3,5-dione bearing two terminal phenolic rings.
• Curcumin’s effect on free radicals is carried out by several different
mechanisms.
• It can scavenge different forms of free radicals, such as reactive
oxygen and nitrogen species (ROS and RNS, respectively)
• it can modulate the activity of GSH, catalase, and SOD enzymes
active in the neutralization of free radicals
• it can inhibit ROS-generating enzymes such as
lipoxygenase/cyclooxygenase and xanthine hydrogenase/oxidase
• In addition, curcumin is a lipophilic compound, which makes it an
efficient scavenger of peroxyl radicals, therefore, like vitamin E,
curcumin is also considered as a chain-breaking antioxidant
 Diet in the Cancer
Process Dietary
Carcinogens,
heterocyclic
amines,
amines,
Procarcinogen
PAHs
B-vitamins, glutathione,
flavonoids Phase I metabolizing Folate Deficiency
enzymes Ultimate carcinogen
Isothiocyanate, selenium,
other phytochemicals Phase II metabolizing
enzymes Hypomethylation
P450 enzymes DNA adducts of P53
system

Obesity
Somatic alteration of
oncogenes,Tumour-
Physical Activity
 Protein suppressor genes and DNA Cucurmin
 Methionine repair genes
 Cholesterol Hormones & Growth
Factors Abnormal DNA & cell replication
Specific nutrients e.g. Redifferentiation
carotenoids, retinol, Apoptosis -3 fatty acids
flavonoids- quercetin
Precancerous lesions & dysplasia
Cucurmin
Cancer Sugar, hi
glycemix index

Metastasis
 Carotenoids

• Carotenoids are yellow, orange, and red pigments

synthesized by plants

• Plants’ secondary metabolites, function to absorb

light in photosynthesis, protecting plants against
photosensitization

• The five principal carotenoids found in human

plasma, as the result of ingesting plants, include
alpha and beta-carotene, beta-cryptoxanthin, lutein,
and lycopene, but over 600 have been identified to
date
Major Carotenoids in foods

lutein

Cryptoxanthin
 Carotenoids
• Role in plants:
– Carotenoids prevent formation of oxygen radical
• Role in human diet:
– Powerful antioxidants that prevent degenerative
effects
– Some convert to vitamin A-eyesight, growth,
development and immune response.

• α- and β-carotene → orange-colored roots, fruits,

vegetables

• Lycopene→red

• Low total carotenoids → white

• Rich source of carotenoids in crude palm oil

 Lycopene

• Sources include watermelon, tomatoes,

pink grapefruit, and guava

• It has been estimated that 80% of the

lycopene in the diet comes from tomatoes
and tomato products like tomato sauce,
tomato paste, and catsup

• Unlike alpha-carotene, beta-carotene, and

beta-cryptoxanthin, lycopene is not a
provitamin A carotenoid
 Lycopene: Function
• Studies have shown lycopene possess
superior antioxidant abilities in comparison
to other carotenoids

• Ability to quench singlet oxygen and prevent

oxidative damage to other molecules and
cellular structures because of its unique
structure of 11 conjugated double bonds and
no cyclic groups

• Researchers believe that lycopene may

influence the genesis of several chronic
diseases in a tissue-specific manner
 Lycopene: bioavailability

• Studies have shown that cooking tomato products

significantly increases lycopene bioavailability
compared to raw products, in the presence of a small
amount of oil or fat

• Fat soluble

• Cooking/processing release bound lycopene

• As little as 3-5 grams of fat in a meal appears

sufficient to ensure carotenoid absorption
 Absorption

• The absorption of carotenoids is limited to a range

of 10-30%

• The low absorption and high excretion rate is due

to that carotenoids are tightly bound to large
molecules, forming protein complexes in foods

• Separation of carotenoids from protein complexes

or dispersion of carotenoid aggregates occurs upon
heating these food items, which, increases their
bioavailability
 Factors that affect absorption
of Carotenoids
• Level of dietary fat intake
– Studies comparing carotenoid absorption rate with
a 40% fat diet to a 20% showed a significant
reduction in absorption

• Level of carotenoids ingested

– Intestinal absorption decreases significantly as
carotenoid intake increases
• Lycopene, being more lipophilic, are carried
exclusively by LDL, residing deep within the
core of the lipoprotein, lipid layer

• Beta-carotene probably protrudes into the

aqueous interface of the lipoprotein

• The location of the carotenoid within the

lipoprotein molecule may influence tissue
uptake
 Tissue Distribution

• Because of its lipophilic nature, lycopene tends to

accumulate in body tissues

• It predominates in prostate, liver, adrenal glands, and

testes, with lycopene accounting for up to 80% of total
carotenoid in these latter two tissues

• Lycopene concentration in the testes and adrenals is

up to 9X’s higher than in the tissue with the second
highest amount, the liver
 Biological Activity
• The general mechanism by which carotenoids
influence development of chronic disease is by
preventing oxidative damage in biological systems,
which includes damage to the cell membrane and
other structures, DNA molecules, lipids, and proteins

• This damage arises from interaction with free radicals

• From environmental toxins and air pollutants-
such as ozone and nitrogen dioxide
• Sunlight and Ionizing radiation
• Certain drugs, Cigarette smoke

PBRC 2005
 Prostate Cancer

• Strongest known link between lycopene and chronic

disease

• Health Professionals Follow-Up Study (HPFS),

suggested that frequent intake of tomato products or
lycopene, is associated with reduced risk of prostate
cancer
 Bladder, Cervical, Breast, Lung Cancers

• Researchers at the John Hopkins University reported a

study of 25,802 participants

• 35 cases of bladder cancer were diagnosed after the

12-year study period, with the risk increasing with
decreasing serum levels of both lycopene and
selenium

• Most studies show that lung and breast cancer are not
related to serum lycopene levels, although there is
study by the Harvard School of Public Health linking
breast cancer with lycopene
 Cardiovascular Disease
• Oxidative damage is believed to be the underlying
mechanism in the etiology of cardiovascular disease

• Oxidized LDL has been proposed as being instrumental

in atherogenesis

• Together with primary defense, carotenoids function as

a network to protect LDL against oxidation

• Its believed that once tocoferol (primary defense) is

depleted, carotenoids may become involved as a second
barrier, with LDL being oxidized only when the
carotenoids are destroyed
 Cardiovascular Disease
In Smokers

• Reactive oxygen species found in smoke will increase

LDL oxidation, thus smokers are at higher risk for
development of CVD

• A study of smokers indicated that increased plasma

concentrations of lycopene, ascorbic acid, and beta-
carotene were significantly inversely associated with
several parameters indicative of oxidative stress,
including oxidation of LDL
 Cruciferous vegetables

• The cruciferae are the family of plants that include the

various familiar members of the species Brassica
oleracea.

• Examples include: Broccoli, cabbage, cauliflower,

kale, Brussels sprouts
• Other examples also widely consumed include:
Oriental cabbage, arugula, watercress, radish and
wasabi
 Glucosinolates and Isothiocyanate

• A characteristic chemical property of cruciferous

plants is their high content of glucosinolates.

• Glucosinolates and their isothiocyanate are well-

known protectors against carcinogenesis, suggesting
that greater intakes of these vegetables may lower the
risk of several types of cancer.

Myrosinase

74 of 19
 Myrosinase

• When food is prepared or chewed,

or in response to plant injury by
predators, the enzyme myrosinase
is released.
• The enzyme responsible for
hydrolyzing glucosinolates to
isothiocynates- defense system
for the plants.
• In the absence of myrosinase
humans have the ability to
efficiently convert glucosinolates
to isothiocynates through the
action of microflora in the GI
tract.

Cabbage worm
 Overview

• At least 120 chemically distinct

glucosinolates have been identified in plants.
• Although the majority have been isolated
from crucifers, 15 other families of plants are
known to contain glucosinolates.
• Attempts have been made to assess human
glucosinolate consumption, some estimates
are as high as 300 mg/d.
Health Benefits Of
glucosinolates
•Anti-carcinogenic Activity
•Anti-tumorogenic Activity
•Inhibition of Metastasis
•Inhibition of Platelet Aggregation
 Diet in the Cancer
Process Dietary
Carcinogens,
heterocyclic
amines,
amines,
Procarcinogen
PAHs
B-vitamins, glutathione,
flavonoids Phase I metabolizing Folate Deficiency
enzymes Ultimate carcinogen
Isothiocyanate, selenium,
other phytochemicals Phase II metabolizing
enzymes Hypomethylation
P450 enzymes DNA adducts of P53
system

Obesity
Somatic alteration of
oncogenes,Tumour-
Physical Activity
 Protein suppressor genes and DNA Cucurmin
 Methionine repair genes
 Cholesterol Hormones & Growth
Factors Abnormal DNA & cell replication
Specific nutrients e.g. Redifferentiation
carotenoids, retinol, Apoptosis -3 fatty acids
flavonoids- quercetin
Precancerous lesions & dysplasia
Cucurmin
Cancer Sugar, hi
glycemix index

Metastasis
 Chemoprotective effects of
isothiocyanates

• Since the early 1960s, both natural and synthetic

isothiocyanates have attracted considerable and
growing attention as important and effective
protectors against chemical carcinogenesis in a
number of animal models.

• Even though only few glucosinolates have been

examined, some appear to be very effective in
inhibiting carcinogenesis.
Fruit and vegetable intake and incidence of
bladder cancer in a male prospective study

Relative P-value
Risk
Fruits and vegetables 0.75 0.25
Fruits 1.12 0.68
Vegetables 0.72 0.09
Yellow vegetables 1.01 0.50
Green leafy 0.99 0.81
vegetables
Cruciferous 0.49 0.008
vegetables
* The only significant reduction in relative
risk was observed with cruciferous
vegetables.
 Health Professionals Follow-up Study

• In the Health Professionals Follow-up Study, bladder

cancer was only weakly associated with low intake of
fruits and vegetables, but high intake (5 or more
servings/week) of cruciferous vegetables was
associated with a statistically significant 51%
decrease in bladder cancer.
 Prostate cancer

• Cohen et al. examined the relationship between fruit and

vegetable consumption and prostate cancer incidence in men
<65 y of age.
• High fruit consumption showed no effect on prostate cancer
incidence.
• High overall vegetable consumption was associated with
reduced risk.
• However, cruciferous vegetables were clearly protective
when risk was adjusted for total vegetable consumption and
other variables.

PBRC 82 of 19
 Resistant starch

Resistant starch refers to the portion of starch and starch

products that resist digestion as they pass through the
gastrointestinal tract.
RS is an extremely broad and diverse range of materials and
of different types
They are mostly defined according to their physical and
chemical characteristics
The general behavior is physiologically similar to that of
soluble, fermentable fiber
The most common results include increased fecal bulk and
lower colonic pH
 Types and Sources of resistant starch

• Found in grains, seeds and legumes and resists digestion because

bound within the fibrous cell walls and not accessible to the
enzymes
• Found in some starchy foods, including raw potatoes and green
(unripe) bananas. The starch granules are structured in a way
which prevents the digestive enzymes from breaking them down
• Formed when certain starchy foods, including potatoes and rice,
are cooked and then cooled. The cooling turns some of the
digestible starches into resistant starches via retrogradation.
• Man-made and formed via a chemical process, etherisation,
esterification or cross-bonding
• Oats are one of the most convenient ways to add resistant starch to your
diet.100 grams of cooked oatmeal flakes contain 3.6 grams of resistant
starch. Cooked oats cool for several hours — or overnight — could increase
the resistant starch
• Beans or legumes contain 1–5 grams /100 grams) after cooked
• Fava beans are an excellent source of resistant starch. When deep fried or
roasted, provide 7.72–12.7 grams /100-gram
• Raw Potato starch is one of the most concentrated sources of resistant
starch (80%)
 Health benefits of resistant starch

• Improved insulin sensitivity

• Reduced appetite and may aid weight loss by improving satiety
• Resistant starch has fewer calories than regular starch — two vs
four calories per gram.
• Feed the bacteria in your gut and increases the production of short-
chain fatty acids such as butyrate
• Short-chain fatty acids play a key role in gastrointestinal health.
• The way we prepare starch-containing foods affects their starch
content, as cooking or heating destroys most resistant starches.
• However, you can recapture the resistant starch content of some
foods by letting them cool after cooking.
 Characteristic Fibre component Description Benefits

• Lowers total and LDL

Water insoluble/Less Cellulose Main structural cholesterol
ermented component of plant cell • Reduces risk of heart
wall. Insoluble in disease
concentrated alkali, • Adds bulk to stool
soluble in concentrated • Alleviates
acid. constipation

Hemicellulose Cell wall • Absorption of water---

Soluble to some polysaccharides, which increase bulk---
contain backbone of β- Speeds the passage
extend
1,4 glucosidic linkages. of foods through the
Soluble in dilute alkali. digestive system
• Facilitates regularity–
gut health
• Inhibit carcinogenesis

Lignin Non-carbohydrate cell • Bind cholesterol, bile

wall component. salts, carcinogens-not
Complex cross-linked absorbed by the body-
phenyl propane polymer. --- lower cholesterol
Resists bacterial level (dual actions)
degradation. • Inhibit carcinogenesis

Chitin and chitosan Chitosan is a high

molecular weight,
linear, polycationic
heteropolysaccharide
consisting of two
monosaccharides: N-
acetyl-glucosamine and
D-glucosamine
 Characteristic Fibre component Description Benefits

Water soluble/Well Pectin Components of primary • Attracts water and turns

fermented cell wall with D- to gel during digestion,
galacturonic acid as trapping carbohydrates,
principal components. fats and slowing
Generally water soluble absorption of glucose
and gel forming • Lowers blood glucose
levels

Gums Secreted at site of • Balances intestinal pH and

plant injury by stimulates intestinal
specialized secretary fermentation production of
cells. Food and short-chain fatty acids
pharmaceutical use. • Reduce risk of colorectal
cancers
Resistant starch refers • stimulating the
Resistant Starch to the portion of starch proliferation of the
and starch products intestinal flora
that resist digestion as (microbiome)
they pass through the • trapping substances that
gastrointestinal tract. can be dangerous for the
human organism
(mutagenic and
carcinogenic

Mucilages Synthesized by plant, • stimulating the

prevent desiccation of proliferation of the
seed endosperm. Food intestinal flora
industry use, (microbiome), improve
hydrophilic, stabilizer. mental health, enhance
immune system
• Lowering of risk for
chronic diseases-Heart
 Mechanisms Involved

FOOD COMPONENTS

Bifidobacterium, Inhibit
Bacteriocides
Ruminococcus carcinigenesis

Reduce
inflammation
oligosaccharides

Butyrate (feed for

the microbes)
Lactic acid Preferred fuel for
the colon cells
propionate acetate
Rapidly absorbed
Other serotonin
bioactive
compounds
Oligosaccharides and modulation of microbiota

Oligosaccharides have been found to play significant role in the

modulation of microbiota.
Several such oligosaccharides, xylooligosaccharides (XOS),
mannooligosaccharides (MOS), and arabino-
xylooligosaccharides (AXOS), are derived from hemicellulosic
macromolecules such as xylan, mannan, and arabino-xylan,
respectively.
These oligosaccharides serve as substrates for the probiotic
production of health-promoting substances (short-chain fatty
acids, branched chain amino acids and other metabolites)

Modulation of the
microbiota

Health Benefits
 Bioactive peptides

• Peptides with biological activities, released

during protein hydrolysis, and play important
role in metabolic regulation and modulation, of
the body for health promotion and disease risk
reduction
• Specific proteins fragments that have a
positive impact on body functions or
conditions and may ultimately influence health
 Bioactive peptides

• They are released during enzymatic proteolysis

(gastrointestinal digestion, in vitro hydrolysis using
proteolytic enzymes) of proteins
• During food processing (cooking, fermentation,
ripening).
• Bioactive peptides are known for their ability to
inhibit protein-protein interactions due to their
small size and specificity.
• Nature remains the largest source of bioactive
peptides mostly, plants and animals and microbes
Protein-based functional
ingredients
 Variety of food sources

Sunflower
seed
Milk

Bioactive
Fish
peptides
Soy bean

Palm kernel
waste Green Beans,
legumes, wheat
 Milk is a rich source of
bioactive peptides

Antihypertensive Immunomodulating

Opioid
(pain Antimicrobial
reliever)

Antioxidant
Antithrombotic
 Angiotensin-converting enzyme
Inhibitor

Antihypertensive peptides have been found effective in

preventing/treating hypertension mainly by inhibiting the
Angiotensin-converting enzyme (ACE), which plays a key
role in the regulation of blood pressure and electrolyte
homeostasis (the equilibrium of water and salt in the
body.
ACE is a nonspecific dipeptidyl carboxypeptidase that
converts decapeptide angiotensin I into octapeptide
angiotensin II, which is known as a potent vasoconstrictor.
ACE can increase blood pressure by converting the
inactive decapeptide angiotensin I to the potent
vasoconstrictor angiotensin II.
Therefore, inhibition of ACE activity is considered to be a
useful therapeutic approach in the treatment of high blood
pressure.
 ACE-inhibitory peptides

• Generally short chain peptides with 2–12 amino acids and studies show
that large peptides cannot bind to active sites of ACE
• In some cases, the amino acid type could be more important than the
length of the peptide.
• This could be related to amino acid composition, since peptides with acidic
amino acids (Asp and Glu) may cause a net negative charge.
• The interaction of negatively charged peptides with ACE could chelate zinc
atoms, which are necessary for enzyme activity.
• Specific amino acid residues at the C- end and/or N- end like tyrosine,
phenylalanine, tryptophan, proline, lysine, isoleucine, valine, leucine, and
arginine in peptides has been reported have strong influence on ACE
binding
• The structure of ACE-inhibitory tetrapeptides has been expressed as Tyr
and Cys present at the first position; His, Trp, and Met at the second
position; Ile, Leu, Val and Met at the third position; and Trp at the fourth
position
• The tripeptides VPP and IPP, casein derivatives, have shown effects on the
inhibition of angiotensin-converting enzyme by suppressing hypertensive
activity
• Valine-Proline-Proline and Isoleucine-Proline-Proline
 Amino Acid Code
alanine ala A Isoleucine ile I

arginine arg R leucine leu L

asparagine asn N
lysine lys K
aspartic acid asp D
methionine met M

asparagine or asx B
aspartic acid phenylalan phe F
ine

cysteine cys C
proline pro P
glutamic acid glu E
serine ser S

glutamine gln Q threonine thr T

glutamine or glx Z
glutamic acid tryptophan trp W

tyrosine tyr Y
glycine gly G

histidine his H valine val V

 Generation of Biopeptides

HYDROLYSIS BY DIGESTIVE
ENZYMES

HYDROLYSIS BY PROTEOLYTIC
PROTEIN MICROORGANISMS
BASED FOOD
(PRECURSOR)

ACTIONS OF PROTEOLYTIC
ENZYMES DERIVED FROM
MICROORGANISMS OR PLANT
 Production and Processing of
Bioactive Peptides
• The most common methods to produce bioactive
peptides have been by enzyme hydrolysis of food
proteins or by fermentation.
• In few cases, water extracts of mushrooms and
some plant parts have been found to contain
bioactive peptides
• In this process, the protein material is subjected
to enzyme hydrolysis at a given temperature
and pH
• The use of enzymatic hydrolysis to produce
bioactive peptides is preferred than fermentation
due to the short reaction time, ease of scalability
and predictability.
 Enzyme hydrolysis

• A single or more than one proteases (purified

or crude) can be used to hydrolyze the protein
sources (alcalase, subtilisin, pepsin, papain
etc).
• Addition of the enzymes whether
simultaneously or sequentially would depend
on the optimal pH and temperature of the
enzymes
• No specific proteolytic enzymes are known to
produce specific bioactive peptides in foods
• Produce highest yield
• Optimization required (time, enzymes, pH,
temp)
 ACE Inhibitor
Antihypertensive peptides have been found effective in
preventing/treating hypertension mainly by inhibiting the
Angiotensin-converting enzyme (ACE), which plays a key
role in the regulation of blood pressure and electrolyte
homeostasis (the equilibrium of water and salt in the
body.
ACE is a nonspecific dipeptidyl carboxypeptidase that
converts decapeptide angiotensin I into octapeptide
angiotensin II, which is known as a potent vasoconstrictor.
 Antioxidant activity of Biopeptides

• Most antioxidant peptide-derived food sources have

molecular weight ranges of 500–1800 Da
• The antioxidant activity of peptides is determined by the
presence of amino acids in their primary structure.
• Most include sulfur-containing methionine and cysteine;
aromatics as tryptophan, tyrosine and phenylalanine, and
those containing the imidazole ring such as histidine
• Antioxidant LLPHH peptide derived from proteolytic
digestion of soy protein shows high antioxidant activity.
• Leu-leu-pro-his-his
• Studies demonstrated that cysteine and methionine
residues are essential in several enzymes which
constitute an important antioxidant defence mechanism.
• Peptides with residues of tryptophan, tyrosine or histidine
at the terminal carbon, present a strong free radical
scavenging.
 Antioxidant peptides

• Antioxidant peptides may exert strong synergistic effect

with some other antioxidants, such as phenolic
compounds.
• Conventional sources of peptides with antioxidant
activity include soy, rice, chickpea and wheat, have
important amino acid composition of PHH or aromatic
amino acids sequences
• With regard to potato, the presence of three biopeptides
(PGER, PDRR and PGERR) with an inhibitory effects on
the oxidation of linoleic acid of 55.3, 58.5 and 61.7%
• Antioxidant peptides that target the mitochondrial inner
membrane, has been developed potentially reduce
intracellular ROS, and may be beneficial in the treatment
of aging and diseases associated with oxidative stress
MOA of Antioxidant Amino Acids

The positive contributions

of SCAA (Cys and Met) to
the antioxidant activity of
the samples can be
attributed to their S-
groups, which are prone
to oxidation by the
reactive species leading
to the formation of stable
oxidation products,
cystine and methione
sulfoxide

The sulfhydryl group of

Cys also acts as a strong
reducing agent, hence its
strong positive
contribution in reducing
ferric ion
Aspartic acid (Asp) and glutamic acid (Glu) can
also donate their acidic hydrogen atoms near
neutral pH, and this may have resulted to their
strong contributions to the antioxidant
activities of the samples.
Similarly, Phe, Tyr and Try played a positive
role in H2O2-scavenging and this can be
attributed to susceptibility of its aromatic ring
to oxidation
Potential mechanisms of action of
antioxidant bioactive peptides
 Antimicrobial Peptides

• AMPs are promising in developing new antimicrobial drugs to

treat bacterial infections, antibiofilm agents, immune
modulators, anti-cancer agents and anti-inflammatories.
• Bioactive peptides increase the permeability through binding
to the bacterial wall and perforate the wall, thus exhibiting
their antimicrobial activity.
• Activity toward a broad spectrum of bacteria, and mode-of-
actions by disrupting their membrane, or by entering inside
bacterial cells to interact with intracellular components.
• AMPs generally target and bind bacterial membranes via
peptide-lipid interactions.
• Some AMPs kill bacteria by interacting with DNA or RNA, and
therefore interfere with their synthesis, replication, and
translational processes
 AMP Actions

• They can modulate the immune response

• demonstrate anti-cancer activity and
• inhibit or eradicate biofilms.
• kill bacteria directly, by either targeting a broad
spectrum of bacteria, or by being selective for Gram-
positive or Gram-negative bacteria.
• Also active against pathogenic species such as viruses,
fungi and parasites
 Nisin

• Nisin is an AMP produced by Gram-positive lactic acid

bacterium, Lactococcus lactis.
• Occurs in five natural variants, the two most common being
nisin A and nisin Z which differ by one amino acid in residue, 27-
histidine and 27-asparagine, respectively.
• It also has a low molecular weight of approximately 3.3 kDa
• The accepted antimicrobial mechanism of nisin is based on the
depolarisation of charged cell wall components, or
• hydrophobic interactions, allowing nisin to reach cell
membranes where it can bind to lipid II in the cell membrane,
resulting in the formation of aqueous transmembrane pores and
leakage of intracellular components.
• Nisin also cause cell wall lysis in certain bacteria
• Nisin is currently approved for use as a safe biological
preservative FDA and WHO due to its non-toxicity and its
bacteriolytic and bactericidal activities
• Nisin has also been explored as an antitumor agent
NISIN-Applications
• Lactoferrin is a peptide found in cow milk and human milk.
Colostrum contains about seven times more lactoferrin than
is found in milk produced later on.
• Acts against various Gram positive and Gram-negative
bacteria, yeast and fungi
• Disruption of normal membrane permeability.

Lactoferrin
 Other Activities
• Recent studies have shown that peptides derived from fish have
important physiological effects on obesity, anti-inflammatory
effects, and ability to reduce plasma levels of cholesterol and
triglycerides
• Supplementation with hydrolyzed fish protein in obese men
exhibited a decrease of weight and body composition markers at
doses of 1.4 and 2.8 g after 90 days
• Promising results have been found in clinical studies using short-
term diets containing peptides for treatment of obesity.
• Antiobesity formula was developed with soy proteins and
peptides and sugar-free drinks and diets containing protein
hydrolysates.
• Those drinks contain oligopeptides with three to six amino acid
residues obtained by enzymatic hydrolysis of soy protein
• Mechanism involved include that peptides of soy, potatoes, beans
and marine products suppress food intake and gastric emptying
by stimulating cholecystokinin (CCK), increasing antioxidant
activity and inducing satiety
 Anti-Diabetic Peptides

• BHs and BPs from food have been discovered

to impart their antidiabetic potentials through
one or more mechanisms
1. inhibition of digestive enzymes
2. inhibition of the enzyme- Dipeptyl peptidase IV
(DPP-IV). Highly selective DPP-IV inhibition
represents an effective and safe strategy for
therapy of type 2 diabetes
3. decrease in blood glucose levels and
4. increase in insulin uptake.
 Single vs multifunctional
peptides
• Food-derived bioactive peptides with single
activities have been well documented
• Few peptides with multiple functions have been
reported.
• Generation of a single peptide with multifunctional
bioactivities will be preferred
• Able to simultaneously give multiple health
benefits.
• Various food proteins using various different
enzymes have been studied to search for peptides
with multiple functions.
 Issues in Bioactive Peptide Development

• The extent of protein hydrolysis is an indispensable factor .

• Peptides released during fermentation is susceptible to further
hydrolysis as long as the enzymatic reaction process goes on
• May result in a decrease or loss of bioactivity (peptide
degradation)
• Peptides produced by microbial fermentation using wild microbes
are not reproducible.
• The microbes are live cells and their metabolic activities, the type
of enzymes and levels cannot be controlled. Therefore, the
quantities of specific bioactive peptides released after
fermentation cannot be guaranteed.
• Improved strains or genetically recombinant strains or pure
enzymes may help mitigate this challenge.
• The use of enzymes for protein hydrolysis is more expensive than
microbial fermentation.
 Effect of processing

• Food processing methods can significantly affect the

biological activity of bioactive peptides.
• Physical processing methods such as ultrasound, heat
and irradiation may affect the protein structure and
functions.
• Processing could also result in maillard reactions and
lead to the production of allergenic compounds
• Processing may increase the susceptibility of peptides to
gastrointestinal digestion and absorption
• It is important to determine the optimal conditions within
which proteins (and peptides) could be processed to
maintain or enhance their bioactivities.
• However, processing methods that may reduce the
activity of one peptide may enhance the activities of
others.
 Issues

• Most bioactive peptides have not been clinically

tested.
• The effect maybe different from specie to specie
and not to be extrapolated.
• The stability of the generated peptides is also
questionable.
• Most food derived peptides are easily degraded in
the gut and therefore do no exhibit
any resultant activity in the body when tested in
vivo.
• In this case, pure isolated peptides with good
activities could be stabilized against digestion by
encapsulation process.
• Such stabilization strategies would improve the
bioavailability of the peptides after consumption.
• Non Halal perception
 Omega-3 Fatty Acids
Alpha-linolenic acid (ALA)
Eicosapentaenoic acid (EPA)
Docosahexaenoic acid (DHA)

Omega- 6

PBRC 2005
 Omega-3 fatty acid

• Human can synthesize other omega-3 fatty acids from

ALA

• Eicosapentaenoic acid (EPA): 20:5n-3

• Docosahexaenoic acid (DHA): 22:6n-3

• These two are usually referred to as marine-derived

omega-3 fatty acids because they are abundant in
certain species of fish

• Whereas, ALA is considered a plant-derived omega-3

fatty acid
 EPA and DHA: Sources
Food Serving EPA (g) DHA (g) Amt providing
1 g of EPA +
DHA
Herring, Pacific, cooked 3 ounces 1.06 .75 42.5g

Salmon, chinook, cooked 3 ounces .86 .62 56 g

Salmon, Atlantic, cooked 3 ounces .28 .95 70 g

Oysters, Pacific, cooked 3 ounces .75 .43 70g

Salmon, sockeye, cooked 3 ounces .45 .60 85g

Trout, rainbow, cooked 3 ounces .40 .44 99

Tuna, white, packed in 3 ounces .20 .54 113g

water

Crab, dungeness, cooked 3 ounces .24 .10 254g

Shrimp, cooked 3 ounces .15 .12 311g

Cod, Pacific, cooked 3 ounces .09 .15 353g

Fish oil, menhaden 1 gram .13 .09 5 grams

Fish oil, salmon 1 gram .13 .18 3 grams

Benefits of Omega-3 fatty
acids

1. Decrease risk of arrhythmias which can

lead to sudden cardiac death
2. Decrease triglycerides levels
3. Decrease rate of atherosclerotic plaques
4. Lower blood pressure
5. Reduce the risk of Coronary Heart
Disease
6. Significantly reduces deaths from heart
disease
7. Increase apoptosis of cancer cells

©2009 David F. Lemire, D.C.

 Docosahexaenoic acid

• Found in very high concentrations in the

cell membranes of the retina

• Conserves and recycles DHA even when

omega-3 fatty acid intake is low

• Studies in animals indicate that DHA is

required for the normal development and
function of the retina
Impaired Visual and Neural Development

The last trimester of pregnancy is a critical period for

the accumulation of DHA in the brain and retina,
preterm infants are vulnerable to adverse effects of
insufficient DHA on visual and neural development

• Although preterm infants can synthesize DHA from

ALA, they can’t synthesize enough to prevent declines
in plasma and cellular DHA levels without additional
dietary intake

• Preterm infants fed formulas with DHA added had

significantly improved measures of visual function
compared to preterm infants fed DHA-free formulas in
5 out of 5 randomized controlled trials
 Eicosanoids
• Eicosanoids are signaling molecules made by oxidation of
20-carbon fatty acids.
• They exert complex control over many bodily systems,
mainly in inflammation or immunity, and as messengers in
the CNS.
• Eicosanoids are derived from either omega-3 (ω-3) or omega-
six (ω-6) fatty acids.
• The ω-6 eicosanoids are generally pro-inflammatory; ω-3s
are the opposite
• The amounts and balance of these fats in a person's diet will
affect the body's eicosanoid-controlled functions, with
effects on cardiovascular disease, triglycerides, blood
pressure, and arthritis.
• Anti-inflammatory drugs such as aspirin and
other NSAIDs act by down regulating eicosanoid synthesis.
Eicosanoid is biologically active lipid mediators (C20 fatty acids and their
metabolites), including prostaglandins, thromboxanes, leukotrienes and
other oxygenated derivatives, which exert their effects at very low
concentrations. They are produced primarily by three classes of enzymes,
cyclooxygenases (COX-1 and COX-2), lipoxygenases (LOX) and cytochrome
P450 epoxygenase.
 The Good and the Bad
Eicosanoids are hormone-like compounds formed in our cells, that have
the mission to send and receive messages to all body cell

1 126
Impact of linoleic acid high diet

Linoleic acid
 Excessive Omega 6 fatty acid Intake

A high consumption of omega-6 PUFAs, found in most

types of vegetable oil, increase the likelihood that
postmenopausal women to develop breast cancer.
Similar effect was observed on prostate cancer for men

Chronic excessive production of n−6 eicosanoids is

associated with heart attacks, thrombotic stroke,
arrhythmia, arthritis, osteoporosis, inflammation, mood
disorders, obesity, and cancer.
 Linolenic acid and
Coronary Heart Disease
In a prospective study of 43,757 male health
professionals followed for 6 years, a relatively small
increase in ALA intake (1% of total energy) was
associated with a 59% decrease in the risk of acute MI

• Women who consumed oil and vinegar salad dressings 5-6

times weekly had a risk of fatal CHD that was 54% lower
than those who rarely consumed, even after adjusting the
analysis for vegetable intake

• Although the evidence is limited, it is indicated that

increased ALA intakes may decrease the risk for CHD,
especially in populations with relatively low levels of fish
consumption
 CHD Treatment

• Results of randomized controlled trials in

individuals with documented coronary heart
disease suggest a beneficial effect of dietary
and supplemental omega-3 fatty acids

• Therefore, it has been recommended that

individuals with documented CHD consume 1
g/d of EPA and DHA combined
 Fish Consumption
And Sudden Cardiac Death
• Several studies have found inverse relationships
between fish consumption and sudden cardiac death

• In a prospective study, omega-3 fatty acid intakes

equivalent to two fatty fish meals per week were
associated with a 50% decrease in the risk of primary
cardiac arrest

• Plasma levels of EPA and DHA were found to be

inversely related to the risk of sudden death,
supporting the idea that omega-3 fatty acids are
partially responsible for the beneficial effect of fish
consumption and sudden cardiac death
 Fish Consumption
And Stroke
• A stroke is a result of impaired blood flow to a
region of the brain, which may be due to
obstruction of a blood vessel by a blood clot
(thrombotic or ischemic stroke) or the rupture of
a blood vessel (hemorrhagic stroke)

• Even though the effects of increased omega-3

fatty acid intake and the incidence of stroke have
not been studied as thoroughly as the relationship
with CHD, data suggest that increased fish intake
may decrease the risk of thrombotic or ischemic
stroke but not hemorrhagic
 Omega-3 and Cancer

• Marine-derived fatty acids have been found to

inhibit proliferation and promote apoptosis in
breast, prostate, and colon cancer cell lines
• Studies in animal models of cancer also indicate
that increased intake of EPA and DHA decreases
the occurrence and progression of mammary,
prostate, and intestinal tumors
• However, few human study demonstrated
significant inverse relationships between fish or
omega-3 fatty acid intake and the risk for breast,
prostate, or colorectal cancers
 Omega 3 Fatty Acids

• An increase in the consumption of fish and fish n-3

polyunsaturated fatty acids in industrialized countries
may contribute to lower breast and colorectal cancer
risks
• It is concluded that fish consumption is associated with
protection against the later promotional stages of
colorectal carcinogenesis,
but not with the early
initiation stage.
 Diet in the Cancer
Process Dietary
Carcinogens,
heterocyclic
amines,
amines,
Procarcinogen
PAHs
B-vitamins, glutathione,
flavonoids Phase I metabolizing Folate Deficiency
enzymes Ultimate carcinogen
Isothiocyanate, selenium,
other phytochemicals Phase II metabolizing
enzymes Hypomethylation
DNA adducts of P53

Obesity
Somatic alteration of
oncogenes,Tumour-
Physical Activity suppressor genes and DNA
 Protein
 Methionine repair genes
 Cholesterol Hormones & Growth
Factors Abnormal DNA & cell replication
Specific nutrients e.g. Redifferentiation
carotenoids, retinol, Apoptosis
-3 fatty
flavonoids- quercetin
acids
Precancerous lesions & dysplasia

Cancer Sugar, hi
glycemix index

Metastasis
 Diabetes Mellitus

• Cardiovascular diseases are the leading causes of

death in individuals with diabetes

• Hypertriglyceridemia (fasting serum TG of 200

mg/dl or higher) is a common lipid abnormality in
individuals with Type 2 diabetes

• A number of randomized controlled trials have

found that fish oil supplementation significantly
lowers serum triglyceride levels in diabetic
individuals
 Diabetes Mellitus
• Few control trials have examined the effect of fish
oil supplementation on cardiovascular disease
outcomes in diabetics

• One prospective study, following 5103 women

diagnosed with type 2 DM but free of
cardiovascular disease at the start of the study,
found decreased risks

• Those with higher fish intakes were associated

with significantly decreased risks of CHD over the
16 years of study, suggesting that increasing EPA
and DHA levels may be beneficial to diabetic
individuals, especially those with elevated serum
triglycerides
 Inflammatory Diseases
Rheumatoid arthritis
• Rheumatoid arthritis is the most common
systemic inflammatory rheumatic (joint)
disease

• Studies have been conducted to determine

the effects of omega-3 fatty acids on
rheumatoid arthritis

• Clinical benefits were observed at a minimum

dose of 3 g/day of EPA + DHA, and were not
apparent until at least 12 weeks of
supplementation
 Inflammatory Diseases
Rheumatoid arthritis

Some investigators report that patients

taking omega-3 fatty acid supplementation
were able to lower their doses of nonsteroidal
anti-inflammatory drugs (NSAIDS), but not all
findings on this issue were consistent
 Major Depression
And Bipolar Disorder
• Several small studies have found omega-3 fatty acid
levels to be lower in the plasma and fat of individuals
suffering from depression compared to controls

• In one study conducted, for 30 individuals, with bipolar

disorder, consuming large amounts of EPA (6.2 g/d)
and DHA (3.4 g/d), they had a significantly longer
period of remission than those on an olive oil placebo
over a 4 month period

• Patients who took the EPA + DHA supplement also

experienced less depression than those who took
the placebo
 Major Depression
And Bipolar Disorder

• Although these very limited pilot studies

produce somewhat optimistic results, larger
and long-term randomized trial are needed to
determine the efficacy of marine-derived
omega-3 fatty acid supplementation on major
depression
 Schizophrenia
• Schizophrenia is a chronic disabling brain disorder that
affects approximately 1% of the population

• A pilot study in 45 schizophrenic patients found

that the addition of 2 g/day of EPA to standard
antipsychotic therapy was superior to the addition of
a 2 g/day to DHA or a placebo in decreasing residual
symptoms
• Mice deprived of docosahexaenoic acid during
pregnancy, are more likely to produce pups that display
schizophrenia-like symptoms as adults
• Although limited evidence does suggest that EPA
supplementation may be a useful adjunct to
antipsychotic therapy in schizophrenic patients, larger
long-term studies addressing clinically relevant
outcomes are needed
Production of
Bioactives Biomass
Extraction

Crude Extract
Determination of Separation
Biological Activity (fractionations)

Extract Fractions

Isolation

Active Pure Compounds

Lead Compound
Identification
 Production of Targeted Bioactives

BIOMASS

EXTRACTION-SFE

BIOLOGICAL ACTIVITY PROFILING-NMR, GCMS

MULTIVARIATE ANALYSIS-PCA, PLS-DA

IDENTIFICATION
BIOMARKERS
 Extraction
Separation of species from a solid or liquid matrix
based on the existence of differences among relative
solubility of diverse compounds in a certain solvent.

solvent

extract

Extraction step allows for the separation of

substances enriched in compounds of interest.
Extract (depend on solvent):
Water, methanol,
Hexane or ethanol extract,
etc.
 Solvents of different polarities

• Polar solvents
methanol
ethanol
acetone

• Non Polar Solvents

Hexanes
toluene
dichloromethane
diethyl ether
Sonicated,
microwave, Water
PEF

Assisted
Solvent
Extraction
EXTRACTION
METHODS

SFE Accelerated
solvent
Solvent extraction- problem

▪The extraction techniques of the phenolic

components from vegetable matrices are
essentially executed with organic solvents
which pose different problems:

•The possible presence of solvent

residues in the final product

•The expensive disposal costs of the

solvents.
Supercritical fluid extractor
Crude Extract

• A complex mixture of unknown

components obtained from the biomass

• Separation of the mixture done by the

differences in physical or chemical
properties
 Crude extracts separation
• Plant extracts usually occur as a combination of various
type of bioactive compounds with different polarities,
separation remains a big challenge for the process of
identification
• A number of different separation techniques such as TLC,
column chromatography, flash chromatography,
Sephadex chromatography and HPLC, should be used to
obtain pure compounds.
• Pure compounds are then used for the determination of
structure and biological activity.
• Non-chromatographic techniques such as immunoassay,
which use monoclonal antibodies (MAbs), phytochemical
screening assay, Fourier-transform infrared spectroscopy
(FTIR), can also be used to obtain and facilitate the
identification of the bioactive compounds.
 Isolation of XTZ from C. xanthorrhiza Roxb.

Curcuma xanthorrhiza Roxb. (100 g)

75% CH3OH extract (11.2 g)

Fractionated with EtOAc

Aqeous layer
EtOAc layer (4.8 g) Fractionated with BuOH

BuOH layer (1.7 g) Aqueous layer (1.1 g)

Silica gel column chromatography, eluted with n-hexane: ethyl acetate (10:1, v/v)

Fr. II
Fr. I Fr. III Fr. IV Fr. V Fr. VI Fr. VII Fr. VIII

(0.5 g) acetylation and silica gel column chromatography

eluted with: n- hexane: ethyl acetate (15:1, v/v)

Compound IIa 14

4 Structure of xanthorrhizol
(0.4 g) deacetylation OH
5
3

Compound II 6 2
12
Xanthorrhizol, 0.2 g 1

7 9 11 Hwang JK, Shim JS, Baek NI, Pyun YR.

2000. Planta Med 66: 196-197.
15 8 10 13
 Biomass
Extraction

Crude Extract
Determination of
Biological Activity Separation
(in vitro)

Functional Ingredient

Development of Functional Foods with

targeted biological activity
 Separation Techniques
• Centrifugation
• Distillation
• Crystallization
• Filtration
• Chromatography
 HPLC

• HPLC is a versatile, robust, and widely used technique for

the isolation of natural products
• The technique is the main choice for fingerprinting study for
the quality control of herbal plants
• Natural products are frequently isolated following the
evaluation of a relatively crude extract in a biological assay
in order to fully characterize the active entity.
• The biologically active entity is often present only as minor
component in the extract and the resolving power of HPLC is
ideally suited to the rapid processing of such
multicomponent samples on both an analytical and
preparative scale.
 Bio-Activity Guided Separation
Crude extract showing
antibiotic activity

Characterization
by TLC

Separation by
column
chromatography

Assay for antibiotic

activity

Pure compounds
Characterization of Pure Compounds

Pure compounds

Assay against pathogenic

Structure determination
bacteria
•HO1H-NMR
OH OH
OH HO O
HO O
O OH OH O
HO O OH OH O

• 13C-NMR
O
O
O O
O O

Tetramycin B • 2D- NMR

HO OH
HO OH
NH2
NH2

Polyene Macrolides • Infrared spectroscopy

• UV spectroscopy
OH
OH OH
OH HO O
HO O

• Mass spectrometry
HO HO O OH OH OH OH O OH
O OH OH OH OH O OH
O
O
O
O
O
O OH NH2
OH NH2
OH
OH

Amphoteracin A
 QUIZ 3
• You are a researcher and Product Development Executive in a
famous Functional Food Company. Your CEO has requested
you to come up with a new product targeted for metabolic
syndrome, in particular, diabetes using local herbs.
• The CEO said that the product must appeal to both children
and the elderlies.

• i) You are required to write a detail report on how such food

was developed. Be specific on the procedures you used,
including the tests and analyses that were done. Biological
activity of the food developed and bioactive compounds
responsible must be supported by scientific data. (10 marks)
• ii) State the probable output and outcome of this project (5
marks)
QUIZ 1

• Identify one major

bioactive compound in
these foods.
• For each postulate its
biological effect