1. Internship
On
MOLECULAR STUDIES AND PHYTOCHEMICAL
ANALYSIS OF SOME IMPORTANT PLANTS OF THE
REGION
Submitted to:
Dr. Shashikiran Nivas
Research Coordinator
Laboratory of Applied Biology
Dr. W. Küppers Biotech Unit
St. Aloysius College
Mangalore – 575 003
Karnataka, India
By:
Ms. Alisha Annie Maben
Department of Biotechnology
BITS Pilani Dubai University, DIAC
Dubai, U.A.E.

2. 2
ACKNOWLEDGEMENTS
Firstly I would like to express my heartfelt gratitude to Dr. Leo D’souza, Director of the
Laboratory for providing me with an opportunity to apply and understand the concepts of
molecular studies and phytochemical analysis in a practical atmosphere.
I am also thankful to Dr. Shashikiran Nivas, Research Coordinator for guidance and
concerted efforts throughout the duration of my internship.
I would also like to thank Ms. Pallavi B. Naik and Ms. Alifha Severes, Research Fellows of the
Institution and Ms Chitra Pinto, Student Scholar for having offered me their valuable
assistance in my project work.
Lastly, I acknowledge the company and cooperation of Mr. Shivananda and Mr Sachin
Patavardhan during this period.

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TABLE OF CONTENTS
1. AIMAND OBJECTIVES 5
2. INTRODUCTION 6
2.1. ZINNIA 6
2.2. DNA EXTRACTION 7
2.3. CTAB METHOD FOR PLANT DNA EXTRACTION 8
2.4. ALOE VERA 9
2.5. PHYTOCHEMICALS IN ALOE VERA 10
2.5.1. ALKALOIDS 11
2.5.2. TEREPENOIDS 11
2.5.3. PHENOLICS 11
2.5.3.1. FLAVONOIDS 11
2.5.3.2. TANNINS 11
2.5.4. SAPONINS 12
2.5.5. POLYURONIDES 12
2.5.6. REDUCING SUGARS 12
3. REVIEW OF LITERATURE 13
3.1. CTAB METHOD OF DNA EXTRACTIONS FOR ZINNIA PLANT 13
3.2. PHYTOCHEMICAL ANALYSIS OF ALOE VERA 13
4. MATERIAL AND METHODS 15
4.1. DNA EXTRACTIONS BY CTAB METHOD 15
4.1.1. Materials 15
4.1.2. Chemicals 15
4.1.3. PROTOCOL 1: With SDS (sodium dodecyl sulphate) 17
4.1.4. PROTOCOL 2: With Liquid Nitrogen 18
4.2. PHYTOCHEMICAL TESTS 19
4.2.1. Materials 19
4.2.2. Chemicals 19
4.2.3. Preparation of Extracts 19
4.2.4. Test for the presence of Alkaloids 20
4.2.4.1. Picric Acid Test 20
4.2.4.2. Tannic Acid Test 20
4.2.4.3. Mayer’s Test 20

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4.2.4.4. Bayer’s Test 21
4.2.5. Test for the presence of Terepenoids 21
4.2.5.1. Test for Monoterpenes 21
4.2.5.2. Test for Sesquiterpenes 21
4.2.5.3. Hirshonn Reaction 21
4.2.5.4. Test for Coumarins 21
4.2.6. Test for the presence of Phenolics 21
4.2.6.1. Follin’s Reagent Test(for polysaccharides) 21
4.2.6.2. Vanillin Test(for tannins) 21
4.2.6.3. Ferric Chloride Test(for tannins) 22
4.2.6.4. Shinoda Test (for flavonoids) 22
4.2.6.5. Lead Acetate Test (for flavonoids) 22
4.2.7. Test for the presence of Saponins 22
4.2.8. Test for the presence of Polyuronides 22
4.2.9. Test for the presence of Reducing sugars 22
5. RESULTS AND DISCUSSIONS 23
5.1. DNA EXTRACTION 23
5.2. PHYTOCHEMICAL TESTS 25
6. SUMMARY 28
7. REFERENCES 29

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1. AIM AND OBJECTIVES
Aim:
 To learn the methodology of DNA extraction protocols using the various
CTAB methods.
 To analyse phytochemicals in Aloe vera plant.
Objectives:
 To vary the CTAB extraction protocol on the parameters of the cell lysis
agent used and the concentration of the extraction buffer. And
accordingly determine the protocol with a good quality yield of DNA for
Zinnia.
 To establish the available benefits of the Aloe vera plants through the
phytochemicals present in it and also look for further potential uses.

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2. INTRODUCTION
In this study the Zinnia plant has been utilized for DNA extractions by the CTAB method with
varying protocols. The DNA obtained has been subjected to electrophoresis for the analysis
of its quality and also quantified using a spectrophotometer. Further, aqueous and
methanolic extracts of Aloe vera sample were obtained for phytochemical analysis to test
the presence/absence of alkaloids, terepenoids, phenolics, tannins, flavonoids, saponins,
polyuronides and reducing compounds.
2.1. ZINNIA
Zinnia is an annual and perennial plant of the family Asteraceace. Their habitat
ranges from native to scrub and dry grassland areas. The plant generally grows
upto 10-100cm tall. Most of the species have solitary long stemmed flowers but
some have a lax habit with spreading stems that mound over the surface of the
ground. These plants possess sessile leaves which grow opposite to each other.
The morphology of the leaf ranges from linear to ovate and the coloration varies
from pale to middle green. The flowers have a variety of appearances from single
to row of petals, to a dome shape with the colours white, chartreuse, yellow,
orange, red, purple and lilac.
Zinnia plants are highly valued for their aesthetic beauty. They are popularly used
to ornament gardens as come in diverse shapes and attractive colours. An added
advantage is that these plants can withstand the hot temperatures of summer
and it can be readily cultivated from seeds. Prerequisites for the optimal growth
of the plant are fertile, humus rich and well- drained soil, in an area with full sun.
A number of species of Zinnia are prevalent flowering plants and interspecific
hybrids are appearing more frequently. Their varied habits allow for uses in
numerous parts of a garden and their tendency to draw butterflies and humming
birds is seen as desirable.
Fig.1. Zinnia plants growing in the garden of St. Aloysius College, Mangalore,
Karnataka, India

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2.2. DNA EXTRACTION
DNA (deoxyribonucleic acid) is a macromolecule referred to as a nucleic acid. It is
the carrier of genetic information essential for the production of other cell
components and for the reproduction of life. It has a twisted double helix
conformation which comprises of long strands of alternating sugars and
phosphate groups, lined with nitrogenous bases (adenine, thymine, guanine and
cytosine). It is organised into structures called chromosomes and is housed
within the nucleus of our cells.
DNA extractions are procedures in molecular biology conducted to separate
Nucleic acids from plant cells which is then followed by the Polymerase chain
reaction (PCR). This facilitates in amplifying a specific sequence of DNA of
interest for the purpose of genetic engineering. Such techniques now allow
scientists to produce variable and improved traits of plants either for enhanced
aesthetic value or to generate higher productivity in plant crops.
The outline of DNA extraction in general, would be:
 Lysis of cells enclosing the DNA of interest –
This is often done by sonicating the sample. Vortexing with phenol is
helpful in breaking of the proteinaecous cell wall. The addition of SDS
detergent is necessary in removal of lipid membranes. Liquid nitrogen (-
170⁰C) is another alternative which freezes the tissue. After maceration
with liquid nitrogen a finely powdered sample is obtained that increases
the surface area of extraction and is DNAse free due to the extremely low
temperature.
 Precipitation of Protein-
Cellular and histone proteins are degraded by the addition of appropriate
proteases. This is achieved by using a salt such as ammonium or sodium
acetate. When the sample is vortexed with phenol-chloroform and
centrifuged the proteins will remain in the organic phase and can be
discarded carefully. The DNA settles at the interface of the two phases.
 Precipitation of DNA-
Cold ethanol or isopropanol is added to the DNA and centrifuged, to
assist in the precipitation of DNA. As DNA is insoluble in alcohol it comes
out of the solution and the alcohol serves as a wash to cleanse it off the
previously added salts.

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 Purification of DNA-
The resultant DNA pellet is washed with 70% ethanol and then
centrifuged for the retrieval of the pellet.
 Storage of DNA-
After decanting the alcohol, the pellet is air dried and re-suspended in TE
(Tris – EDTA) buffer. It can be refrigerated for storage and further use.
 Purity and yield of DNA-
The presence and quality of DNA can be confirmed by electrophoresing
on an agarose gel containing Ethidium bromide (EtBr) as a fluorescent dye
that intercalates within the DNA and helps in it analysis with the UV trans-
illuminator.A photo of the gel gives us a clear idea about the nature
(sheared or intact), shape (coiled, linear etc) and size (in kbp – kilo base
pair) of the DNA fragment.
Further the yield and purity of the DNA sample is analysed using the
spectrophotometer. DNA absorbance readings are taken at 260nm since
this is the wavelength at which DNA absorbs the maximum amount of
light.
The concentration of DNA sample is calculated using the formula:
Concentration (µg/ml)= A260 reading × dilution factor × 50µg/ml
The yield is obtained by:
DNA yield (µg) = DNA concentration × total sample volume (ml)
The purity of the DNA obtained is calculated using the A260/A280
absorbance ratio. A ratio of around 1.8 represents a pure DNA sample.
2.3. CTAB METHOD FOR PLANT DNA EXTRACTION
A good quality of DNA is the foremost requirement for any of its downstream
applications hence a highly reliable protocol is essential for the extraction of
DNA.
It is frequently observed that mature plants contain high concentrations of
polysaccharides, polyphenols, proteins and other secondary metabolites which
deteriorates the quality of the DNA extracted and makes it unfit for amplification
by PCR methods. Hence CTAB (cetyltrimethyl ammonium bromide) method for
DNA extraction is preferred for plants as it effectively eliminates the DNA
contaminants.

9. 9
The CTAB extraction buffer contains sodium chloride, TrisHCl, EDTA and CTAB.
Here sodium chloride provides positive ions to neutralise the negative charges in
the nucleic acids thus aggregating the nucleic acid molecules together. TrisHCl
provides the apt pH for the DNA extraction and increases the cell membrane
permeability. EDTA is a chelating agent that chelates the divalent metal ions and
prevents the magnesium mediated aggregation of nucleic acids with proteins and
with each other. And CTAB is the cationic surfactant preferred for the highly
polymerised DNA from the plant material. This detergent simultaneously
solubilizes the plant cell wall and lipid membranes of internal organelles. Once
this happens the proteinase in CTAB frees the DNA by destroying the histones
and releasing the DNA. This is permitted to take place at 65⁰C to optimise the
enzyme activity in CTAB.
β-mercaptoethanol is added along with the extraction buffer and is designed for
plant DNA isolation as it’s a strong reducing agent which can remove tannins and
polyphenols often present in the crude plant extract. It also helps in denaturing
proteins by breaking the disulphide bonds between the cysteine residues.
Polyvinylpyrrolidone (PVPP) is added to remove the phenolic compounds from
plant DNA extracts. PVPP forms hydrogen bonds with the phenolic compounds.
It’s exceptionally good at absorbing polyphenols during DNA purification; else
these polyphenols deactivate the proteins if not removed and therefore inhibit
many downstream reactions like PCR. Extended chloroform – isoamyl alcohol
treatment followed by RNase treatment yields a good quantity of DNA which is
free of protein and RNA. The problems of DNA degradation, contamination, low
yield due to irreversible binding of phenolic compounds and co-precipitation of
polysaccharides with DNA were avoided by this method.
2.4. ALOE VERA
Aloe vera is classified as a succulent plant which has the capacity to thrive in arid
regions. The plant generally grows upto 80-100cm in height. The thick green
leaves of Aloe vera grow in a whorl formation and it doesn’t possess a typical
stem. The leaves are lined with spiky edges which act as the plants defence
mechanism from pests. This plant has the potential of surviving in temperature
extremities as long as its roots are not destroyed.
It has found its use in traditional medicine, in dietary supplements and also in
commodities, whereas the gel has been used in yoghurts, beverages and
desserts. The regions of Aloe vera which are frequently utilized are the bitter

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exudate which functions as a natural drug for its cathartic effect and is widely
employed as a bittering agent in alcoholic beverages and as a laxative. And the
inner pure gel which when prepared appropriately can be used in medicinal
products or cosmetics.
Fig.2. Aloe vera plants growing in the garden of St. Aloysius College, Mangalore, Karnataka,
India
2.5. PHYTOCHEMICALS IN ALOE VERA
Aloe vera is known to produce at least 6 antiseptic agents such as lupeol, salicylic
acid, urea nitrogen, cinnamonic acid, phenols and sulphur. All of these
constituents are recognized as antiseptics as they exterminate or inhibit the
bacteria, fungus and viruses, elucidating the potency of the plant to alleviate
internal and external infections.
Two active pain killers found in the juice of the plant are lupeol and salicylic acid.
It contains at least three anti-inflammatory fatty acids, cholesterol, campesterol
and β-sitosterol. These are extremely effective in cure of skin abrasions, allergic
reactions, rheumatoid arthritis, rheumatic fever, acid indigestion, ulcers, and
Inflammatory conditions of organs like stomach, small intestine, colon, liver,
kidney and pancreas. β-sitosterol is also aninfluential anti-cholesterol which
reduces the levels of the lethal cholesterol and proves beneficial for heart
patients.
Pharmacologically Aloe vera is seen to promote immunity and also work as a
system detoxifier. It is recommended in adjuvant therapy with antibiotics,
NSAIDS (Non-steroidal Anti-inflammatory drugs) and chemotherapy to eliminate
drug induced adverse effects. It is used as an alternative medicine for conditions

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like type II diabetes, eye disorders, tumours, spleen enlargement, bronchitis and
asthma. It also functions in the form of a dietary supplement in pre and post-
operative patients, postmenopausal women and in cases of osteoporosis
(Rajeshwari R. et al; 2012).
2.5.1. ALKALOIDS
Alkaloids are heterocyclic, nitrogen containing compounds, usually with
potent activity and bitter taste. They are of limited distribution in the
plant kingdom. The various groups have diverse clinical properties.
Tropane alkaloids have anticholinergic activity and are usede medically to
reduce smooth muscle spasms, hypersecretion and pain. Isoquinoline
alkaloids are used to inhibit various conditions as pain, cancer cells and
bacteria and stimulation of bone marrow leucocytes as well as myocardial
contractility.
2.5.2. TEREPENOIDS
These compounds are lipophilic and tend to volatilise readily. They have
strong odours and flavours. Their actions vary greatly, a range of which
have been utilised in herbal remedies. Of particular importance are
antineoplastic, antibacterial, antiviral effects as well as gastrointestinal
stimulation.
2.5.3. PHENOLICS
Plant phenolic compounds range from simple, low molecular weight,
single aromatic ringed compounds to large and complex polyphenols. The
plant phenolic compounds are salicylic acid, phytoalexins,
furanocoumarins, anthocyanins, flavonols, hydrolysable tannins,
condensed tannins.
2.5.3.1. FLAVONOIDS
Flavonoids consist of a central three ring structure and can occur as
glycosides. All compounds contain phenol groups involved in an effect
as general antioxidant. Other actions are diverse wherein several
structures reduce inflammation or carcinogenicity.
2.5.3.2. TANNINS
Tannins are indiscriminately bind to proteins and larger tannins are
used as astringents in cases of diarrhoea, skin bleedings and
transudates. Condensed tannins are polymers formed by the

12. 12
condensation of flavans. They do not contain sugar residues. Such
tannins have a potent free radical scavenging activity.
Hydrolysable tannins are tannins hydrolysed by weak bases and weak
acids to produce carbohydrate and phenolic acids. These tannins
which include gallic and ellagic acids are inhibitors of HIV replication.
They have also shown potential antibacterial and anticancer effects.
2.5.4. SAPONINS
Most saponins (soap forming compound) occur as glycosides. The
aglycone consists of either pentacyclictriterpenoids or teteracyclic
steroids. The saponin glycosides hydrophobic aglycone, which gives a
emulsifying properties can be used as detergents. Saponins show immune
modulating and antineoplastic effects. A common in vitro effect is
haemolysis of red blood cells.
2.5.5. POLYURONIDES
Polyuronides are polysaccharides, which uronic acids contain. The uronic
acids are always in pyranose before and give the macromolecules acidic
character and the ability of water to store and thus a gel to form.
2.5.6. REDUCING SUGARS
A reducing sugar is any sugar that either has an aldehyde group or is
capable of forming one in solution through isomerism. The aldehyde
functional group allows the sugar to act as a reducing agent, important in
the browning of many foods.

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3. REVIEW OF LITERATURE
3.1. CTAB METHOD OF DNA EXTRACTIONS FOR ZINNIA PLANT
Zinnia plant is renowned for its aesthetic and diverse features and is widely used
as an ornamental plant. This plant has exquisite colours and shapes which are the
traits that dictate the consumer’s interest and fascination towards plants used
for decorations. Thus the traits like colour and the morphology of the flowers
play a crucial role in the commercial success of Zinnia in the market. The
inheritance of colour in flowers is a wide arena for research as it varies in
different species. Colour can be a dominant, recessive, additive in nature or
could arise due to a combination of factors. Plants can also possess two or more
genetic defects or alterations to make them white or non coloured. The same
concept is also applicable for differences in petal shapes and sizes amongst
varying species of Zinnia. Hence DNA extractions are conducted on ornate plants
like Zinnia to be able to study genetic variations in the plant (Gultom T. et al;
2012).
Generally for plants belonging to the family of Asteraceace, the CTAB method of
DNA extraction (1987 Doyle and Doyle method) is performed on the plant leaves
and flowers. 0.1-0.5g of the leaf/flower sample was taken, dehydrated and
macerated using 3% of CTAB buffer as a detergent in a 1.5ml eppendorf tube. Or
else the same amount of sample is macerated to fine powder with liquid
nitrogen in a mortar and pestle and transferred into a 1.5ml eppendorf tube to
which 2% of pre-warmed CTAB buffer is added. The buffer in either case has a
pinch of PVPP and 0.4-1% of β-mercaptoethanol added just before use. The
solution is incubated at 65⁰C for 45mins and is mixed by inversion every 15mins;
500µl of chloroform- isoamyl alcohol was added to every tube and mixed for
1min. samples were centrifuged at 4⁰C for 10mins at 12000rpm. The supernatant
was transferred into an eppendorf tube and the chloroform-isoamyl alcohol
wash was conducted thrice. Now to the supernatant cold isopropanol is added to
ensure the precipitation of the DNA. Mix by inversion and centrifuge at
12000rpm for 10mins. Now the DNA pellet would be adhered to the eppendorf
tube and the supernatant is discarded. The pellet is washed with 70% ethanol, air
dried and re-suspended in 100µl of TE buffer with 5µl of ribonuclease in each
tube. The solution is incubated at 37⁰C for 1 hour, stored at -20⁰C (Aline Borges
et al; 2009).
3.2. PHYTOCHEMICAL ANALYSIS OF ALOE VERA
Plants through generations have proven to be a vital source of medicines. The
World Health Organization gives statistics stating that 80% of the world’s

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population still use herbal remedies for their treatment. Aloe Vera is known to be
a popular folk medicine through the eras. Aloe vera as an alternative medicine
has been used to treat skin ailments, gastrointestinal disorders, for wound
healing, antiulcer and diabetes. Currently the plant is being used for
nutraceuticals, cosmetics and skin care. Hence it is vital to analyse the
phytochemical compounds in Aloe vera (S. Arunkumar et al; 2009).
The aqueous and the ethanolic extracts were obtained from the cold extraction
procedure. 100g of fresh samples were weighed into conical flasks and the
respective solvent was added into the flasks and left for 48hours in a shaker. The
mixtures were filtered by applying vacuum pressure and the filtrates were
concentrated using rotator evaporator and subjected for the various studies
(Omale James et al; 2010).
Existing literature reports that phytochemical analysis of Aloe vera extracts
shows the presence of tannins, flavonoids, alkaloids and terepenoids (Ejoba
Raphael; 2012). Ferric chloride test was conducted to confirm the presence of
tannins. Lead acetate test was done for the presence of flavonoids. Mayer’s and
Wagner’s reagent was used to test for alkaloids. For terepenoids the extract was
dissolved in equal amounts of chloroform and evaporated. After which
concentrated sulphuric acid was added and heated for 2mins. A greyish colour
precipitate confirmed the presence of terepenoids (K. Thu et al; 2013).

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4. MATERIAL AND METHODS
4.1. DNA EXTRACTIONS BY CTAB METHOD
4.1.1. Materials
Autoclaved and deionised water, micropipettes with tips, cut tips,
eppendorf tubes and stand, spatulas, petri plates, mortar and pestles,
sterilised blades, measuring cylinder, glass rods, ice box, gel tray with well
gate, power supply and electrophoresis chamber.
4.1.2. Chemicals
 1.5 % CTAB extraction buffer (200ml)
 1.0M TrisHCl
Take 50ml of sterilised water and dissolve 31.52g of TrisHCl. Ensure
that the solutions pH = 8.
 0.5M EDTA
Take 75ml of sterilised water and dissolve 37.22g of EDTA. Ensure
that the solutions pH = 8.
 1.5% CTAB
Take 10 ml of sterilised water and dissolve 3g of CTAB. Ensure that
the solutions pH = 8.
 1.5% NaCl
Take 10 ml of sterilised water and dissolve 3g of NaCl. Ensure that
the solutions pH = 8.
Makeup the volume of the extraction buffer upto 175ml and then
check the pH = 8, after which the volume is made upto the required
200ml.
 2.5% CTAB extraction buffer
The above mentioned procedure is followed the only change being that
2.5%CTAB is used in place of 1.5% CTAB.
For 2.5 % CTAB, take 10 ml of sterilised water and dissolve 5g of CTAB.
Ensure that the solutions pH = 8.
 3.0M Sodium Acetate (100ml)
Take 75ml of sterilised water and dissolve 24.60g of sodium acetate.
Ensure that the solutions pH = 5.2 and then make the volume upto 100
ml. Store at 4⁰C.
 0.5M Tris EDTA buffer (200ml)

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For 0.5 M of TE buffer 10mM TrisHCl and 1mM EDTA is required. Take
100 ml of sterilised water and dissolve 0.1211 of Tris HCl. Take 100ml of
sterilised water and dissolve 0.372g of EDTA. Both the solutions are
mixed and ensure that the pH = 8. Store at 4⁰C.
 Chloroform-isoamyl alcohol (50ml)
Chloroform- isoamyl alcohol is prepared in the ratio 24:1. Hence 48ml of
chloroform, is dissolved in 2ml of isoamyl alcohol to obtain the required
solution. Store at 4⁰C.
 70% Ethanol (100ml)
Take 30ml of sterilised water and dissolve 70ml of ethanol in it. Store at
-20⁰C.
 15% SDS (sodium dodecyl sulphate)
Take 100ml of sterilised water and dissolve 15g of SDS. The solution is
heated till a clear solution appears.
 Liquid nitrogen
 β-mercaptoethanol
 PVPP (Polyvinylpyrrolidone)
 Isopropanol (ice cold)
 RNase A solution
 Tris saturated phenol
 Tris : Borate : EDTA (TBE Buffer)
For 5X TBE buffer solution, 54g of Tris base, 27.5g of Boric acid and 20ml
of EDTA is taken and the volume is made upto 500ml with sterilised
water. Ensure that the solutions pH = 8.
 Ethidium bromide (EtBr) (10mg/ml)
10mg of EtBr was dissolved in sterilised water and the volume was made
upto 1ml. Store the solution in amber coloured bottle and maintain at
4⁰C.
 Gel loading dye
 0.8% Agarose gel
To make 50 ml of 0.8% agarose gel, 50 ml of 0.5X TBE buffer was taken
and to it 0.4g of agarose was added. It was heated till a clear solution
was obtained. When the temperature of the agarose gel is about 50⁰C,

17. 17
25µl of EtBr was mixed into it and the gel was cast into the tray with the
wells positioned prior to pouring.
4.1.3. PROTOCOL 1: With SDS (sodium dodecyl sulphate)
1. The DNA extraction is performed by the Doyle and Doyle CTAB method
with minor modifications.
2. Maintaining cold conditions throughout is a must in DNA extractions
hence all the micro tips, cut micro tips, glass rods, spatulas and
eppendorfs are preserved in cold conditions at -20⁰C.
3. Weigh 1-2g of the Zinnia leaf samples and wash with distilled water
then 70% ethanol followed by distilled water again. Dry the sample to
remove moisture.
4. The leaf is finely chopped using a sterilised blade and transferred into
an eppendorf tube. To this tube 500µl of 15% SDS is added. A glass rod
is used for maceration of the sample within the tube. Maceration is
completed when the sample shows colour change and gives a slurry
appearance.
5. Add 750µl of CTAB buffer (warmed for half an hour at 56⁰C) along with
20µl of β-mercaptoethanol and a pinch of PVPP. Mix by inversion.
6. The tubes are now placed in a water bath at 56⁰ for 25mins. (mix every
5mins)
7. After the tubes come to room temperature, add 700µl of chloroform-
isoamyl alcohol and mix by inversion for 20mins.
8. Centrifuge at 3000rpm at 4⁰C for 25mins.
9. The supernatant is collected. To it are added 2/3rd volume of
isopropanol (cold) and 1/10th volume of 3.0M sodium acetate. Mix by
inversion and let it stand for 10mins at 4⁰C.
10. Centrifuge at 3000rpm at 4⁰C for 15mins.
11. Collect the pellet and to it add 500µl of 70% ethanol and shake well by
inversion.
12. Centrifuge at 3000rpm at 4⁰C for 15mins. (Repeat twice)
13. Allow the pellet to air dry. Then place it in the hot air oven at 56⁰C for
15mins.
14. Dissolve the pellet in 500µl of 0.5M TE buffer. And store at 4⁰C.
15. The next day thaw the DNA sample and add 10µl of RNase A solution,
centrifuge at 10⁰C for 5mins at 3000rpm.
16. Place the tubes in the water bath at 65⁰C for 1.5 hours.
17. Once the tubes come to room temperature add equal volumes of tris
saturated phenol and chloroform-isoamyl alcohol. Mix by inversion.

18. 18
18. Centrifuge at 3000rpm at 4⁰C for 25mins.
19. The supernatant is collected and to it add equal volume of chloroform-
isoamyl alcohol. Mix by inversion.
20. Centrifuge at 3000rpm at 4⁰C for 25mins.
21. The supernatant is collected. To it are added 2/3rd volume of
isopropanol (cold) and 1/10th volume of 3.0M sodium acetate. Mix by
inversion and let it stand for 10mins at 4⁰C.
22. Centrifuge at 4600rpm at 4⁰C for 15mins.
23. Collect the pellet and to it add 500µl of 70% ethanol and shake well by
inversion.
24. Centrifuge at 6000rpm at 4⁰C for 15mins. (Repeat twice)
25. Allow the pellet to air dry. Then place it in the hot air oven at 56⁰C for
15mins.
26. Dissolve the pellet in 500µl of 0.5M TE buffer. And store at 4⁰C.
27. 12.5µl of the DNA sample is mixed with 12.5µl of the gel loading buffer
and is run on 0.8% agarose gel (containing 0.5X TBE buffer and
25µlEtBr). The voltage is set at 75V. After 30 – 45mins the gel is
observed under the UV transilluminator and a gel image is recorded.
28. For quantification of the DNA sample. 20µl of DNA is diluted in TE
buffer to make up a volume of 3ml after which absorbance readings
are taken in a spectrophotometer at 260nm and 280nm respectively
and the purity of the DNA is determined by their ratio.
4.1.4. PROTOCOL 2: With Liquid Nitrogen
 Maintaining cold conditions throughout is a must in DNA extractions
hence all the micro tips, cut micro tips, glass rods, mortar and pestles,
spatulas and eppendorfs are preserved in cold conditions at -20⁰C.
 Weigh 1-2g of the Zinnia leaf and flower sample and wash with distilled
water then 70% ethanol followed by distilled water again. Dry the sample
to remove moisture.
 Now roughly cut the sample and place it in the mortar with a pinch of
PVPP and to this sufficient liquid nitrogen is added and the sample is
quickly macerated until fine powder is obtained. The sample is rapidly
transferred into an eppendorf to which 750µl of CTAB buffer (warmed for
half an hour at 56⁰C) along with 20µl of β-mercaptoethanol is added. Mix
by inversion.
 After this the above mentioned protocol is to be followed from step 6
onwards.

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4.2. PHYTOCHEMICAL TESTS
4.2.1. Materials
Conical flasks, sterilised blades, blotting paper, pre-weighed petridishes,
test tubes with rack, micropipettes with micro tips, glass pipette and
beaker.
4.2.2. Chemicals
 Autoclaved water
 Methanol
 DMSO (Dimethyl Sulfoxide)
 Picric Acid
 Tannic Acid
1g of Tannic acid is dissolved in 1ml of ethanol and diluted with water to
10ml.
 Mayer’s Reagent
135.8mg of Mercuric chloride is dissolved in 6ml of water and poured into
a solution of 500mg of potassium iodide in 2mlof water. Add sufficient
water to make the volume upto 10ml of solution.
 Solid potassium permanganate
 10% of Vanillin Reagent (in ethanol)
 Concentrated Sulphuric acid
 Concentrated Hydrochloric acid
 Trichloroacetic acid
 10% Sodium hydroxide (in distilled water)
 Follin’s Reagent
 10% Ferric chloride (in distilled water)
 10% Lead acetate (in distilled water)
 Metallic magnesium
 Ethanol
 Fehling’s solution A
 Fehling’s solution B
4.2.3. Preparation of Extracts
The Aloe vera plants were taken and washed under running water till it
was clean from all the soil sediments. The root, stem and leaves were
separated and about 2-3g of each sample was placed into a closed pouch
made of blotting paper.

20. 20
For aqueous extracts, 3 conical flasks of 250ml capacity were taken with
100ml of autoclaved water and in them were placed the root, leaf and
stem sample of the Aloe vera respectively. The flasks were closed with
cotton plugs.
For methanolic extracts, 3 conical flasks of 250ml capacity were taken
with 100ml of methanol and in them were placed the root, leaf and stem
sample of Aloe vera respectively. The flasks were immediately closed with
cotton plugs as the solvent evaporates very quickly.
These flasks were kept in a shaking incubator at 27⁰C at 110 rpm for
48hours after which extracts were obtained. The methanolic extracts
were flash evaporated at 65⁰C whereas the aqueous extracts were
evaporated at 110⁰C after which they were transferred into petriplates of
8 ml capacity. The petriplates were allowed to dry completely till the
entire solvent evaporated.
Now the petriplates were post-weighed and the quantity of the extract
was determined. Taking the concentration of the extract as a standard
the sample was dissolved in appropriate volume of DMSO and stored.
4.2.4. Test for the presence of Alkaloids
4.2.4.1. Picric Acid Test
200µl of extract was taken in a test tube to it 100µl of picric acid was
added. The yellow crystalline precipitate confirms the presence of
alkaloids.
4.2.4.2. Tannic Acid Test
To 200µl of the extract in a test tube, 100µl of tannic acid was added.
The appearance of yellow crystalline precipitate confirms the
presence of alkaloids.
4.2.4.3. Mayer’s Test
To 1ml of extract add 2-3 drops of Mayer’s reagent. Cream or yellow
precipitate confirms the presence of alkaloids.

21. 21
4.2.4.4. Bayer’s Test
To 200µl of the extract, distilled water and potassium permanganate
is added in a test tube. Brown solution with a brown precipitate
confirms the presence of alkaloids.
4.2.5. Test for the presence of Terepenoids
4.2.5.1. Test for Monoterpenes
200µl of the extract is taken in a test tube to which 200µl of 10% Vanillin
reagent and 200µl of concentrated sulphuric acid are added. The
appearance of red colour confirms the presence of monoterpenes.
4.2.5.2. Test for Sesquiterpenes
To 200µl of the extract in a test tube, 100µl of concentrated sulphuric
acid was added. The appearance of red, brown, or blue colour confirms
the presence of sesquiterpenes.
4.2.5.3. Hirshonn Reaction
500µl of the extract was heated with 200µl of trichloroacetic acid. Red to
purple colour change is observed for a positive result.
4.2.5.4. Test for Coumarins
To 1ml of the extract, 1ml of 10% sodium hydroxide was added. The
presence of coumarins is indicated by the formation of yellow precipitate.
4.2.6. Test for the presence of Phenolics
4.2.6.1. Follin’s Reagent Test (for polysaccharides)
To 200 µl of extract in a test tube, 100 µl of Follin’s reagent was added.
The appearance of blue colour confirms the presence of polysaccharides.
4.2.6.2. Vanillin Test (for tannins)
To 200 µl of the extract in a test tube, 200µl of 10% vanillin in ethyl
alcohol and 200 µl of concentrated sulphuric acid are added. The
appearance of red colour confirmed the presence of tannins.

22. 22
4.2.6.3. Ferric Chloride Test (for tannins)
A portion of the extract (500 µl) was diluted with distilled water (2ml) in a
ratio of 1:4 and few drops of 10% ferric chloride were added to it. Blue or
green colour indicates the presence of tannins.
4.2.6.4. Shinoda Test (for flavonoids)
200 µl of the extract was heated in a water bath. A piece of metallic
magnesium and three drops of concentrated hydrochloric acid was
added. The appearance of red or orange colour confirms the presence of
flavonoids.
4.2.6.5. Lead Acetate Test (for flavonoids)
200 µl of the extract were taken in a test tube and to it few drops of 10%
lead acetate solution was added. The appearance of a yellow precipitate
confirms the presence of flavonoids.
4.2.7. Test for the presence of Saponins
The test tube containing 200 µl of the extract was taken with 500 µl of distilled
water for 2mins and shaken. The appearance of foam for at least 15mins
confirms the presence of saponins.
4.2.8. Test for the presence of Polyuronides
To 200 µl of extract in a test tube, 200 µl of ethanol was added. The appearance
of violet or blue precipitate confirms the presence of polyuronides.
4.2.9. Test for the presence of Reducing Sugars
To 200 µl of the extract in a test tube, 100 µl of Fehling’s solutions A and B were
added and kept in a hot water bath for 2mins. The appearance of brick red
precipitate confirmed the presence of reducing compounds.

23. 23
5. RESULTS AND DISCUSSIONS
5.1. DNA EXTRACTION
Below are the results of the extracted DNA run by electrophoresis
Fig.3. Gel documentation of the DNA extracted by SDS method.
Fig.4. Gel documentation of the DNA extracted by liquid nitrogen.
LANE 1: Zinnia leaf sample2C1TE
LANE 2: Zinnia leaf sample2C2TE
LANE 3: Zinnia leaf sample2C1
LANE 4: Zinnia leaf sample2C2
1 2 3 4
1 2
LANE 1: Zinnia leaf sample- ZF
LANE 2: Zinnia flower sample- ZL

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Table 1: Results of the DNA extractions by various CTAB protocols
Protocol Concentration
(mg/ml)
Purity Yield (mg)
1. SDS
2C1TE 5.13 1.9 0.0513
2C2TE 5.205 1.906 0.0520
2C1 4.185 2.021 0.0418
2C2 9.975 1.837 0.0975
2. LIQUID NITROGEN
ZF 1.815 2.283 0.0182
ZL 1.890 2.311 0.0189
The yield of DNA obtained from the SDS protocol is more than the protocol using liquid
nitrogen. But from comparison of the gel images it is evident that the genomic DNA
obtained from SDS is prone to shearing when compared to the DNA obtained from liquid
nitrogen. Also it is noteworthy that the DNA extracted with liquid nitrogen has an extremely
high purity which is preferable for RAPD analysis

25. 25
5.2. PHYTOCHEMICAL TESTS
Table 2: Results of the different phytochemical tests conducted on the aqueous and
methanolic Aloe vera extracts
Test Stem Extracts
(13.3mg/ml)
Leaf Extracts
(6.6mg/ml)
Root Extracts
(3.3mg/ml)
Aqueous Methanol Aqueous Methanol Aqueous Methanol
1. Alkaloids
Picric Acid - + - + - -
Tannic Acid - + - + - -
Mayers Test - + - + - +
Bayer’s Test - + + - - -
2. Terepenoids
Monoterepenes + - + - + -
Sesquiterepenes - + - + + +
Hirshonn Test - - - - - -
Coumarins - + + + - +
3. Phenolics
Follin’s Test - - - - - -
Vanillin Test + - + - + -
Ferric chloride
Test
- - - - - -
Shinoda Test - - - - - -
Lead Acetate
Test
- + + + - +
4. Saponins - - - - - -
5. Polyuronides - - - - - -
6. Reducing
compounds
- - - - - -

26. 26
Fig.5.Positive resultsforPicricAcidTest Fig.6. Positive ResultsforTannicAcidtest
Fig.7.Positive ResultsforMayer’sTest Fig.8. PositiveResultsforBayer’sTest
Fig.9.Postive ResultsforMonoterepenes Fig.10. Positive ResultsforSesquiterepenes
TEST FOR ALKALOIDS TEST FOR ALKALOIDS
TEST FOR ALKALOIDS TEST FOR ALKALOIDS
TEST FOR TEREPENOIDS TEST FOR TEREPENOIDS

27. 27
Fig.11.Positive ResultsforCoumarins
Fig.12.Positive ResultsforVanillinTest
Fig.13. Positive Results for Lead Acetate Test
The phytochemical analysis of Aloe vera shows the presence of alkaloids prominently in the
methanolic stem and leaf extracts. Terepenoids are equally obtained from all the different
extracts. Tannins were present in all the aqueous extracts. Flavonoids were prevalent in the
leaf extracts. Aloe Vera lacked saponins, polyuronides and reducing sugars. The results
obtained coincide with existing literature.
TEST FOR TEREPENOIDS
TEST FOR PHENOLICS
TEST FOR PHENOLICS

28. 28
6. SUMMARY
DNA extractions were carried out by the Doyle and Doyle CTAB method with minor
variations. The protocol was standardised for the Zinnia plant. Hence good DNA yield was
obtained by the SDS and liquid nitrogen methods. The DNA showed higher quality and
reduced shearing for the protocol with liquid nitrogen. Thus for further downstream
applications like RAPD analysis, it is preferred that the DNA is extracted with liquid nitrogen.
Similar protocols were also conducted with the Aloe vera leaves but the protocol was not
yet standardised and hence DNA wasn’t obtained.
Further phytochemical analysis was performed for the Aloe vera plant parts that are the
root, stem and leaves. Aqueous and methanolic extracts were obtained from the plant parts
by cold extraction method. After which they were subjected to a series of tests to check for
the presence/absence of alkaloids, terepenoids (monoterepenes and sesquiterepenes),
phenolics (tannins and flavonoids), saponins, polyuronides and reducing sugars.
Aloe vera displayed positive results for alkaloids, terepenoids, tannins and flavonoids. Hence
this potential property focuses its use in various pharmaceutical and cosmetic preparations.

29. 29
7. REFERENCES
1. Aline Borges, Marianna Siva Rosa, Gustavo H. R, Jurema R. Q, Eduardo de Andrade,
Elizabeth A. V.; 2009; CTAB methods for DNA extraction of sweet potato for
microsatellite analysis; Scientia Agricola (Piracicaba, Brazil); Vol. 66; Iss. 4; pg 529-
534.
2. R. Rajeswari, M. Umadevi, C. Sharmila Rahale, R. Pushpa, S. Selvavenkadesh, K. P.
Sampath Kumar, Debjit Bhowmik; 2012; Aloe vera: The Miracle Plant and its
Medicinal and Traditional uses in India; Journal of Pharmacognosy and
Phytochemistry; Vol. 1; Iss. 4; pg 34-38.
3. Tumiur Gultom, Aziz-Purwantoro, Endang Sulistyaningsih, Nasrullah; 2012;
Estimating of RAPD marker associated to color gene in Zinnia elegans JACQ; ARPN
Journal of Agricultural and Biological Science; Vol. 7; Iss. 11; pg 958-961.
4. S. Arunkumar, M. Muthuselvan; 2009; Analysis of Phytochemical constituents and
Antimicrobial Activities of Aloe vera L. against clinical pathogens; World Journal of
Agricultural Sciences; Vol. 5; Iss. 5; pg 572-576.
5. Omale James, Emmanuel T. Friday; 2010; Phytochemical composition, bioactivity and
wound healing pH of Euphorbia heterophylla (Euphorbiaceae) leaf extract;
International Journal on Pharmaceutical and Biomedical Research; Vol. 1; Iss. 1; pg
54-63.
6. Ejoba Raphael; 2012; Phytochemical constituents of some leaves extract of Aloe vera
and Azadirachta indica plant species; Global Advanced Research Journal of
Environmental Science and Toxicology; ARPN Journal of Agricultural and Biological
Science; Vol. 1; Iss. 2; pg 14-17.
7. K. Thu, Yin Y. Hon, Tin A. Khang, Ohn M. Tun; 2013; Study on phytochemical
properties, antibacterial activity and cytotoxicity of Aloe vera L.; World Academy of
Science Engineering, Technology; Vol. 7; Iss. 5; pg 114-118.