Phytochemicals

I. Introduction: Phytochemicals are defined as bio active nutrient plant chemicals in fruits, vegetables,
grains, and other plant foods that may provide desirable health benefits beyond basic nutrition to reduce the
risk of major chronic diseases. It may stimulate the immune system, slow the growth rate of cancer cells, and
prevent DNA damage that can lead to cancer and other diseases as described in the following section
suggesting that many phytochemicals are antioxidants protecting the cells of the body from oxidative damage
from water and food.
II. Preparation of Stock Plant Extract
How to extract samples:

a. Seeds
-1. Preheat the extraction buffers A and B to 60 #C in a water bath.
2. Add 400 ml of each buffer A and B into a 1.5 ml nuclease-free microfuge tube containing 100 mg of
seed flour. In case of leaf, grind 100 mg of tissue in 500 ml of buffer A using a mortar and pestle, then
pour the mixture into a 1.5 ml tube. Pipette 400 ml of buffer B into the same tube.
Note: Tissue Layer should be used for high-throughput extraction and to prevent cross-
contamination

3. Vortex the mixture for 10 s to mix thoroughly. Incubate the tubes at 60 #C for 30 min in a water bath
and invert after every 10 min to homogenize.
4 Cool down the tubes at room temperature (RT) and add RNase A (25 mg/ml). Invert the tubes for 4–5
times and incubate at 37 #C for 20 min in an incubator.
5. Add 400 ml of CIA and vortex for 5 s to form an emulsion.
6. Centrifuge at 13,000 g for 10 min at RT to separate the organic and aqueous phases.
Note: If the aqueous layer is not transparent then repeat the step 5.
7. Carefully transfer the aqueous (transparent) phase using a micropipette into a new tube.
Note: Wide-bore tips should be used to prevent mechanical damage to DNA.
8. Add 2/3 vol of isopropanol and 1/10 of 3.5 M NaAc (pH 5.2).

9. Close the tubes tightly, gently invert for 5–6 times and then precipitate the DNA by incubating at
#20 #C for 15 min.
Note: To increase the precipitation of the DNA, the tube may be incubated for overnight.
10 . Centrifuge at 13,000 g for 5 min to pellet the precipitated DNA.
11 .Carefully remove the supernatant without disturbing the pellet and add 400 ml of 70% (v/v) chilled
(#20 #C) ethanol. Dislodge the pellet by flicking with a finger.
12. Centrifuge at 13,000 g for 5 min and discard the supernatant by decanting.
13. Remove the ethanol residuals by drying the DNA pellet on a heat block.
Note: Do not over dry the pellet because it will make it difficult to dissolve.
14. Dissolve the DNA pellet in 40 ml of nuclease-free water.
Note: TE buffer should be used to dissolve the DNA pellet intended for long storage.
B. Leaves
Procedure:
Reagents needed
1. Preheat 5ml CTAB (add 10µl mercaptoethanol to each 5ml CTAB) in a blue-topped 50ml centrifuge
tube at 60-65 o C. Remove and discard midribs, and wrap laminae in aluminium foil and freeze in liquid nitrogen.
0.5 – 1.0 gm tissue/5ml CTAB

(Can store leaf material after liquid Nitrogen – 1-2 days at –20 or –80 for longer periods)

2. Gently crumble leaf tissue over cold pestle of liquid nitrogen. Grind frozen leaf with one spatula of fine sand
add 0.5 spatula of PVPP powder after grinding.

3. Scrape powder into dry tube and add pre-heated buffer and mix gently. Avoid leaving dry material around rim
of tube. Adjust CTAB volume to give a slurry-like consistency, mix occasionally.

4. Incubate for 60 min at 60 o C

5. Add equal volume of chloroform/iso-amyl alcohol (24:1), Mix for about 3min, then transfer contents to narrow
bore centrifuge tubes. Balance by adding extra chlor/iso. Spin 5,000rpm for 10min (ensure correct tubes used),
brake off. (For extra pure DNA isolation - spin and retain supernatant before chloroform extraction).

6. Remove supernatant with wide-bore pastette (cut off blue tip) to clean tube, repeat chloroform extraction
once. Supernatant should be clear, though may be coloured.

7. Precipitate DNA with 0.66 vol. of cold isopropanol - can leave overnight. Spool out or spin down DNA, 2min at
2,000rpm.

8. Transfer to 5ml wash buffer for 20min.

9. Dry briefly and resuspend in 1ml T.E. (can be left overnight)

10. Add 1µl 10mg/ml RNAse to each 1ml T.E./DNA mixture and incubate for 60min at 37 o C. (If RNase in the
sample doesn't matter – stages 11 and 12 may be omitted)

11. Dilute with 2 volumes TE and add 0.3vol 3M Sodium acetate

(pH 8) + 2.5 vol cold 100% ethanol,

12. Spool DNA out. Air dry and re suspend in 0.5 to 1ml TE or water (takes time) and freeze until required.
 C. Fruits
Procedures:

1.Add fruit into extraction solution in the zipper bag. Close bag and squeeze out air.

2.Crush the fruit thoroughly for 5 minutes. CAREFUL don’t break the bag!

3.Place the bags into the hot water bath for about 10-15 minutes, making sure the fruit solution is fully beneath the
water line. Occasionally shake the bag to evenly distribute the heat.

4.Move the “mashed” bags of fruit solution into the ice bath for 1 minute. Remove and carefully mix the fruit solution
again. Repeat this procedure 5 times.

5.Tape the cheese cloth over the beakers. Filter the fruit mixture through the cheese cloth. Combine solutions from all
student groups at this point. Let the solution drain 5 minutes.

6.Using the large transfer pipettes, approximately 2 ml of the fruit solution into a test tube, one for each pair of
students.

7.Add approximately 2 ml of ice-cold ethanol to each tube by dropping it slowly down the side of the test tube, allowing
it to rest on top of the fruit mixture. Do not agitate the solution.

8.Let the solution sit for two minutes without disturbing it. The DNA will appear as transparent, slimy, white mucus
which can be spooled up with the wood applicator stick.

III. A. Some Phytochemicals and their Benefits

Phytochemicals Health Benefits Examples/Sources

1. Allyl Sulfides Garlic and Onions
Can keep your brain sharp by helping
prevent age-related changes in the
gut bacteria that are linked to
memory problems

2. Capsaicin Usefulness in regulation of blood Different hot pepppers

sugar levels.
3. Carotenoids Can protect you from disease and Bell peppers
enhance your immune system Broccoli
Cantaloupe
Carrots
Mangoes
Oranges
Spinach
4. Lycopene A powerful antioxidant with many Tomatoes
health benefits, including sun Watermelon
protection, improved heart health Pink guava
and a lower risk of certain types of Papaya
cancer.
5. Lutein -Suppress inflammation Kiwi fruit
-Defend against free radicals and Grapes,
oxidative stress Spinach,
-Enhance the sharpness of your Orange juice
vision Zucchini (or vegetable marrow)
-Improve your visual contrast Different kinds of squash.
sensitivity
 -Reduce glare impairment
-Protect eye tissue from sunlight
damage
-Reduce cell loss and death related to
eye disease
-Protect your eyes from harmful blue
light
-Convert light signals into electrical
signals in your retina and aid in the
transmission of those signals to the
visual cortex in your brain
6. Ellagic acid It removes toxins from your body and Strawberries
protects against harmful molecules Rapaberries
known as free radicals. Blackberries
Cherries
Walnuts
7. Isoflavones Protection against age-related Legumes seeds
diseases including cardiovascular
disease, osteoporosis, hormone-
dependent cancer and loss of
cognitive function.
8. Quercetin Fighting free radicals. Apples,
Reducing inflammation Onions,
Reducing the risk of cancer Parsley,
Preventing neurological diseases Sage,
Relieving allergy symptoms Olive oil,
Preventing infections Grapes
Reducing the risk of heart disease
Lowering high blood pressure
9. Resveratrol Has antioxidant and anti- Grapes juice
inflammatory properties to protect Wine
you against diseases like cancer, Peanut
diabetes, and Alzheimer's disease Cocoa

10. Sulforaphane It may protect your DNA Broccoli

It may slow tumor growth Cabbage
It neutralizes toxins Kale

B.

Phytochemicals Description Function/Uses Examples/Sources

1. Alkaloid Any of a class of naturally
occurring organic nitrogen-
containing bases. Alkaloids
have diverse and important
physiological effects on
humans and other animals
They may act as reservoirs Bacteria
for protein synthesis. They Fungi
may act as protective Plants and Animals
substances against the
animal or insect attacks.
Like hormones, they may
function as plant stimulants
or regulators in activities
like growth, metabolism
and reproduction.
2. Anthroquinone An aromatic organic Laxatives and antimicrobial Plants
compound. and anti-flammatory Fungi
agents. Lichens
Insects
3. Flavonoids Any of a class of non- Help regulate cellular Citrus fruit
nitrogenous biological activity and fight off free Berries
pigments extensively radicals that cause Apples
represented in plants. oxidative stress on your Legumes
Flavonoids are water- body
soluble phenolic
compounds (having a –OH
group attached to an
aromatic ring) and are
found in the vacuoles of
plant cells.
4. Saponins A large family of Plant defence against Legumes
structurally related disease and herbivores and Kidney beans
compounds containing a possibly as allelopathic Lentils
steroid or triterpenoid agents in competitive
aglycone (sapogenin) interactions between
linked to one or more plants.
oligosaccharide moietieS.
5. Tannins Complex chemical Responsible for decreases Wood
substances derived from in feed intake, growth rate, Leaves
phenolic acids (sometimes feed efficiency, net Buds
called tannic acid) metabolize energy, and Stems
protein digestibility in Fruits
experimental animals Seeds
Roots
6. Polyphenols Secondary metabolites that Improve digestion, brain Black beans
are common in plant function, and blood sugar White beans
kingdom levels, as well as protect
against blood clots, heart
disease, and certain
cancers
7. Terpenoids A large and diverse class of The largest class of plant Tea
naturally occurring organic secondary metabolites, Thyme
chemicals derived from the with basic functions in Cannabis
5-carbon compound growth and development, Spanish sage
isoprene, and the isoprene and more specialized roles Citrus Fruits
polymers called terpenes. in plant–environment
interactions,
resistance/tolerance to
environmental stresses,
and defense against
predators and pathogens.
8. Cyanogenic Glycosides Chemical compounds Defense chemicals Almonds
contained in foods that Sorghum
release hydrogen cyanide Cassava
when chewed or digested. Lima beans
Stone fruits
Bamboo shoots
References:

https://www.le.ac.uk/biology/phh4/dnaiso.htm

https://www.apsnet.org/edcenter/disimpactmngmnt/labexercises/PlantBiotechnology/Pages/Activity1.aspx