MATERIALS AND METHODS

Collection of sample
Collection of coconut (Cocos nucifera) and Aloe vera plant (Aloe barbadensis
miller)

Preparation of extract

Extraction of Aloevera gel

Fully grown Aloe vera leaves were collected and cleaned with distilled water.
The inner gel was made a colloidal solution after the green skin was removed.
The solution was filtered using sterile filter paper to obtain pure aloe vera gel.

Aloe Vera leaf

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Wash with water
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Trimming the tip and base
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Extract the pulp using sterile blade
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Filter the gel using Whatman no 1filter paper
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Pure aloe vera gel obtained

Virgin Coconut oil extraction

Traditional boiling method

Coconut oil is extracted by boiling coconut milk to evaporate the water, leaving
the oil behind. In order to extract approximately 14 L of coconut milk, the
processes last for an hour or until all the oils get separated from the milk.
Fully mature coconut
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Removal of husk
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Cut into halves
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Grating
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Coconut milk extraction
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Hot processing
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Virgin coconut oil
Test organisms
Bacterial strains were obtained from MLT department of Presentation College
applied science. Five microorganisms namely Klebsiella Pneumonia,
Escherichia Coli, Staphylococcus aureus, Bacillus and Pseudomonas were used
for evaluating the antimicrobial activity. All the organisms were identified by
Gram staining (Hans Christian Gram, 1884) and biochemical characterization.
Microscopic examination by Gram staining (Hans Christian Gram, 1884)
The bacterial colonies from nutrient broth were directly smeared on a clean
glass slide under aseptic condition and was allowed to air dry and subsequently
heat fixed. The smear was flooded with the Crystal Violet for a minute; washed
with the tap water; flooded with the gram's iodine mordant and was allowed to
stand for a minute with a subsequent wash using tap water. The stained smear
was decolorized with the 95% ethyl alcohol until crystal violet failed to wash
from the smear followed by a wash with the tap water and then the counter
stained with the suffering for 60 to 80 seconds. The smear was given a final
washing. The blot dried smears were observed under oil immersion (100x)
objective.

DETERMINATION OF ANTIMICROBIAL ACTIVITY

Preparation of bacterial culture

Each bacterial strain was inoculated into a separate sterile tube containing 5 ml
Sterile Mueller Hinton broth and incubated overnight.
Preparation of Mueller Hinton Agar media
Take 500ml dried conical flask, put these reagents like Suspend 7g of Mueller
Hinton agar powder and 4.25g Agar-Agar in 250 ml of distilled water.Mix and
dissolve them completely.Sterilize by autoclaving at 121°C for 15 minutes.Pour
the liquid into the petri dish and wait for the medium to solidify. Be sure that
you are preparing the agar in the clean environment to prevent any
contamination. Carefully measure the quantity of beef extract, Casein acid
Hydrolysate, Starch and Agar-Agar as per the quantity of medium to be
prepared. Store the Medium at low temperature in dust and contamination free
environment for later use. Carefully pour the liquid MHA medium in the Petri
plates so that its depth should not be less or more than 4 mm to get the error free
results.
Well diffusion method to identify antimicrobial activity
Plates were prepared using Mueller Hinton agar and were labelled. Organisms
were spread evenly in separate plates and well of 5mm was filled with virgin
coconut oil and Aloe vera plant extract in 50µl and 100µl concentration
individually and in combination. All plates were kept for incubation. After 24
hours of incubation, the diameter of the zone of inhibition is measured.
Inoculation of test plates
Sterile cotton swab is dipped into microbial suspension,the swab rotated several
times and pressed firmly on the inside the wall of the tube above the fluid level,
the dried surface of Mueller Hinton Agar medium contained in the plate
repeated by streaking two or more times rotating the plate approximately 60
each time to ensure an even distribution of the inoculums. As a final step the rim
of the agar also swabbed. The inoculums were left to dry at room temperature
with the lid closed.
 Reading the plates and interpreting the result
After 18 to 24 hours of incubation each plate is examined.if the plate was
satisfactory streaked and the inoculum was correct the resulting zone of
incubation will be a confluent lawn of growth if the individual colonies are
Apparent the inoculum was too light and the test must be repeated. The
diameter of the zone of complete inhibition is measured to the nearest whole
millimeter, using a scale which is held a few inches above a black surface non
reflecting background.

DETERMINATION OF PHYTOCHECHICAL ANALYSIS

Preliminary phytochemical screening

ALOE VERA

Materials and Methods

The plant extracts prepared were subjected to preliminary qualitative
phytochemical tests using standard methodologies

a .Tests for Proteins

Ninhydrin test: Boil 2 ml of 0.2% Ninhydrin solution with the entire plant
crude extract, appeared violet colour indicate the presence of proteins and
amino acids.

b. Tests for Carbohydrates

 Fehling’s solutions test: Boil a mixture of Fehling solutions A and B with equal
volumes were added to crude plant extract. A red colour precipitate indicated
the presence of reducing sugars.
 Benedict’s reagent test: Boil 2 ml of Benedict’s reagent with a crude extract, a
reddish-brown colour indicated the presence of the carbohydrates.
 Molisch’s solution test: Shake 2 ml of Molisch’s solution with crude plant
extract then add 2 ml of H2SO4 concentrated and poured carefully along the side
 of the test tube a violet ring appeared at the inter phase of the test tube indicated
the presence of carbohydrate.
 Iodine test: 2 ml of iodine solution mixed with crude plant extract. Purple or
dark blue colours prove the presence of the carbohydrate.

c. Test for Phenols and Tannins

Two milliliters of 2% solution of FeCl3 mixed with crude extract. Black or blue-
green colour indicated the presence of tannins and phenols.

d. Tests for Flavonoids

Alkaline reagent test: 2 ml of 2% NaOH solution was mixed with plant crude
extract, intensive yellow colour was formed, which turned into colourless when
added 2 drops of diluted acid to solution, this result indicated the presence of
flavonoids.

Test for Alkaloids

a. Wagner’s Test: Extracts were treated with Wagner’s reagent (Iodine in

Potassium Iodide). A brown/reddish precipitate indicates the presence of
alkaloids.
b. Hager’s Test: Extracts were treated with Hager’s reagent (saturated picric
acid solution). Presence of alkaloids is confirmed by the formation of
yellow coloured precipitate

e. Test for Saponins

Five milliliters of distilled water was added to crude plant extract in a test tube
and it was shaken vigorously. The foam formation indicated the presence of
saponins.

f. Tests for Glycosides

 Liebermann’s test: 2 ml of acetic acid and 2 ml of chloroform mixed with

entire plant crude extract. The mixture was then cooled and added
H2SO4 concentrated, green colour indicated the entity of aglycone steroidal part
of glycosides.
 Salkowski’s test: H2SO4 concentrated (about 2 ml) was added to the entire plant
crude extract. A reddish-brown colour produced indicated the entity of steroidal
aglycone part of the glycoside.
 Keller-kilani test A mixture of Acetic acid glacial (2 ml) with 2 drops of 2%
FeCl3 solution was added to the plant extract and H2SO4 concentrated. A brown
ring produced between the layers which indicated the entity of cardiac steroidal
glycosides.

g. Test for Steroid

Two milliliter of chloroform and concentrated H2SO4 were mixed with the entire
plant crude extract. In the lower chloroform layer produced red colour that
indicated the presence of steroids. Another test was performed by mixing 2 ml
of each of acetic acid with H2SO4 concentrated and crude extract with 2 ml of
chloroform. Green colour indicated the entity of steroids.

h. Test for Terpenoids

Two milliliter of chloroform was mixed with the plant extract and evaporated on
the water path then boiled with 2 ml of H2SO4 concentrated. A grey colour
produced indicated the entity of terpenoids.

f.Test for Terpenes

Copper acetate Test: Extracts were dissolved in water and treated with 3-4 drops
of copper acetate solution. Formation of emerald green colour indicates the
presence of diterpenes

Test for coumarins

To the extract in alcohol, a few drops of sodium hydroxide solution were added.
Dark yellow colour formation indicates the presence of coumarins.

Test for Anthraquinones

To 1ml of the extract add 1ml of 10% FeCl3 and 0.5ml of conc. HCl. Boil in a
water bath for few minutes. Appearance of a pink or deep red colour indicates
the presence of anthraquinones (Harborne J.B et al., 1973).

Test for Phlobatannins

1ml of the extract was boiled with 1% HCl. The formation of red precipitate
indicates the presence of phlobatannins.

VIRGIN COCONUT OIL

Alkaloid analysis

One mL of VCO sample was put into a test tube and the sample was dripped
with 2N sulfuric acid. Then the mixture was tested with Meyer, Wagner and
Dragendorff reagents. A positive result could be confirmed if the Meyer reagent
shows white or yellow deposits. The mixture with Wagner reagent would show
the formation of brown deposits if there were alkaloids in the sample.
Meanwhile, Dragendorff shows red or orange deposits if the sample contains
alkaloids.

Phenol/tanin analysis

About 1 mL of VCO sample is put into a test tube, 2 drops of FeCl3 reagent are
added to this test tube. If there was a change in colour to green or blue green
then the sample may contain phenol or tannin.

Analysis of flavonoids

About 1 mL of the VCO sample was put into a test tube and given 0.1 mg of
magnesium powder. Into the mixture, a 0.4 mL amyl alcohol (37% mixture of
hydrochloric acid and 95% ethanol with the same volume) and 4 mL alcohol
were being added. The mixture was then shaken and changes of colours were
observed, if red, yellow, or orange was formed on the amyl alcohol layer, the
sample could be confirmed to contain flavonoids

Saponin analysis
\About 1 mL of the VCO sample was inserted into the test tube, then 1 mL of
hot water was added. This mixture was shaken continuously for 30 s. After that
the mixture was rested to be then observed. If the foam lasted more than 10 min
and did not disappear when 1 drop of 2 N hydrochloric acid was added, it could
be confirmed that the simple contain saponins.

Steroid/terpenoid analysis

One mL of VCO sample was added to the test tube and added 2 mL of
chloroform. This mixture is then given 10 drops of glacial acetic acid and 3
drops of concentrated sulfuric acid. Positive results if, the mixture observed
changes colour to red and then changes to blue and green. The red colour
indicates the triterpenoid content