Materials and Methods
4. Materials and Methods
4.1. Collection of plant material
The plant material was collected flower market in Tirupathi and authentified
by [Link], Professor, Department of Dravyaguna, [Link] Medical
College, Tirupati.
4.2. Preparation of plant extract
The fresh petals of flower Carissa Carandas were shade dried. The dried
petals were grinded to get coarse powder. 250 gm of coarse powder was subjected to
cold maceration process using ethanol (70:30) as solvent. The extraction was
continued for 7days at room temperature with occasional shaking. Then the extract
was filtered, collected and concentrated at 70 oC on a heating mantle until a softy mass
obtained. It was then thoroughly air dried to remove all the traces of solvent and then
was subjected to freeze drying. The obtained plant extract was preserved in cold
condition i.e. below 0oC till the end of treatment period.
4.3. Preliminary Phytochemical Screening
Standard qualitative screening test of the extract was carried out for various
plant constituents. The crude extract was screened for the presence or absence of
secondary metabolites using standard procedures (Khandelwal, 2005).
4.3.1. Test for alkaloids
4.3.1. a) Preliminary test: A 100 mg of ethanolic extract was dissolved in dilute
hydrochloric acid. Solution was clarified by filtration. Filtrate was tested with
Dragendroff’s and Mayer’s reagents. The treated solutions were observed for any
precipitation.
4.3.1. b) Confirmatory test:Five grams of the ethanolic extract was treated with 40%
calcium hydroxide solution until the extract was distinctly alkaline to litmus paper,
and then extracted twice with 10 ml portions of chloroform. Chloroform extracts were
combined and concentrated in vacuum to about 5 ml. Chloroform extract was then
spotted on thin layer plates. Solvent system (n-hexane-ethyl acetate, 4:1) was used to
develop chromatograms and detected by spraying the chromatograms with freshly
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Materials and Methods
prepared Dragendroff’s spray reagent. The existence of alkaloids was established by
development of an orange colour spots.
4.3.2. Test for steroidal compounds
4.3.2. a) Salkowski’s test: 0.5 g of the ethanolic extract was dissolved in 2 ml
chloroform in a test tube. Concentrated sulfuric acid was carefully added on the wall
of the test tube to form a lower layer. A reddish brown colour at the interface
indicated the presence of a steroid ring (i.e. the aglycone portion of the glycoside).
4.3.2. b) Lieberman’s test: 0.5 g of the ethanolic extract was dissolved in 2 ml of
acetic anhydride and cooled well in an ice-bath. Concentrated sulfuric acid was then
carefully added. A colour change from purple to blue to green indicated the presence
of a steroid nucleus i.e. aglycone portion of’ the cardiac glycosides.
4.3.3. Test for phenolic compounds
4.3.3. a) To 2 ml of filtered solution of the ethanolic extract of Carissa Carandas of
the plant material, 3 drops of a freshly prepared mixture of 1 ml of 1% ferric chloride
and 1 ml of potassium Ferro-cyanide was added to detect phenolic compounds.
Development of bluish-green colour was confirmed for presence of phenolic
compounds.
4.3.3. b) The desiccated extract of 100 mg was liquefied in [Link] crystals of
ferric sulfate were added to the mixture. Formation of dark-violet color indicated the
presence of phenolic compounds.
4.3.4. Flavonoids
4.3.4. a) Test for free flavonoids: Five milliliters of ethyl acetate was added to a
solution of 0.5 g of the extract in water. The mixture was shaken, allowed to settle and
inspected for the production of yellow colour in the organic layer which is taken as
positive for free flavonoids.
4.3.4. b) Lead acetate test: To a solution of 0.5 g of the extract in water about 1 ml
of 10% lead acetate solution was added. Production of yellow precipitate is
considered as positive for flavonoids.
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Materials and Methods
4.3.4. c) Reaction with sodium hydroxide: Dilute sodium hydroxide solution was
added to a solution of 0.5 g of the extract in water. The mixture was inspected for the
production of yellow color which considered as positive test for flavonoids.
4.3.5. Test for saponins
4.3.5. a) Froth test: 0.5 g of the ethanolic extract was dissolved in 10 ml of distilled
water in a test tube. The test tube was stoppered and shaken vigorously for about 30
seconds. The test tube was allowed to stand in a vertical position and observed over a
30 minute period of time. If a “honey comb” froth above the surface of liquid persists
after 30 min. the sample is suspected to contain saponins.
4.3.6. Test for tannins
4.3.6. a) Ferric chloride test: A portion of the ethanolic extract was dissolved in
water. The solution was clear up by filtering process. 10% ferric chloride solution was
added to the clear filtrate. Bluish to black colour change was observed.
4.3.6. b) Formaldehyde test: To a solution of about 0.5 g of the extract in 5ml water,
3 drops of formaldehyde and 6 drops of dilute hydrochloric acid were added. The
bring about mixture was heated for 1 min and then subjected for cooling. The
precipitate formed (if any) was washed with hot water, warm alcohol and warm 5%
potassium hydroxide successively. Formation of massive precipitate which left a
residue coloured after it is washed it states the occurrence of phlobatannins
4.3.6. c) Test for Phlobatannins: Deposition of a red precipitate when an aqueous
extract of the plant part was boiled with 1% aqueous hydrochloric acid was taken as
an evidence for the presence of phlobatannins.
4.3.6. d) Modified iron complex test: To a solution of 0.5 g of the plant extract in
five milliliter of water a drop of 33% acetic acid and 1 g sodium potassium tartrate
was added. The mixture was warmed and filtered to remove any precipitate. A 0.25%
solution of ferric ammonium citrate was added to the filtrate until no further
intensification of colour is obtained and then boiled. Purple or blackish precipitates
which is insoluble in hot water; alcohol or dilute ammonia denotes pyrogallol tannin
present.
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Materials and Methods
4.3.7. Test for Anthraquinones
4.3.7. a) Test for free Anthraquinones (Borntrager’s test): The ethanolic extract of
the plant material (equivalent to 100 mg) was shaken vigorously with 10 ml of
benzene, filtered and 5 ml of 10% ammonia solution added to the filtrate. Shake the
mixture and the presence of a pink, red or violet color in the ammonia (lower) phase
indicated the presence of free Anthraquinones.
4.3.7. b) Test for O-Anthraquinones glycosides (Modified Borntrager’s test):For
combined Anthraquinones, 5 g of the plant extract was boiled with 10 ml 5%
sulphuric acid for 1 hour and filtered while hot. The filtrate was shaken with 5 ml
benzene; the benzene layer separated and half its own volume of 10% ammonia
solution added. The formation of a pink, red or violet color in the ammonia phase
(lower layer) indicated the presence of anthraquinone derivatives in the extract.
4.3.8. Test for Carbohydrates
The extracts were treated with 3 ml of alpha naphthol in alcohol and
[Link] acid was carefully added to side of the test tubes. Formation of a
violet ring at the junction of two liquids indicates presence of carbohydrates.
4.3.8. a) Fehling`s Test: Take both the Fehling`s solution A and Fehling`s solution B
and heat for 2 min. Presence of red to brown color indicates the Reducing sugars in
the test compound.
4.3.8.b) Benedict’s Test: To the sample benedict’s solution was added and heated,
appearance of reddish orange precipitate indicates presence of reducing sugars.
4.3.8. c) Barfoed`s Test: The sample were treated with Barfoed`s reagent and heated,
appearance of reddish orange precipitate indicates presence of reducing sugars.
4.3.9. Test for Proteins
4.3.9. a) Biuret`s Test: To the extracts copper sulphate solution followed by sodium
hydroxide solution, a violet color precipitates indicates presence of proteins.
4.3.9. b) Million’s Test: To the extracts million’s reagent was added, appearance of
pink color indicates presence of proteins.
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Materials and Methods
4.3.10. Test for Gums and Mucilage
The extracts were treated with 25 ml absolute alcohol and then the solution was
filtered. The filtrate was examined for its swelling properties.
4.3.11. Test for Glycosides
A pinch of the extract were dissolved in glacial acetic acid and few drops of
ferric chloride solution was added followed by the addition of [Link] acid,
formation of red ring at the junction of the two liquids indicates presence of
glycosides.
4.3.12. Test for Terpenes
The extracts were treated with tin and thionyl chloride, appearance of pink
color indicates presence of terpenes.
4.4. Experimental work:
Anti microbial activity:
Test Organisms Bacterial strains were obtained from National Chemical
Laboratories (NCL), Pune and Microbial Type Culture Collection (MTCC),
Chandigarh. The strains used for the present study were Staphycococcus aureus
(NCIM 2079), Bacillus subtilis (NCIM 2063), Escherichia coli (NCIM 2931, Proteus
vulgaris (NCIM 2027).
Procedure:
The antimicrobial activity of the extract was assessed by disc diffusion
method. Nutrient agar medium was prepared and sterilized by an autoclave. In an
aseptic room, they were poured into a petridishes to a uniform depth of 4 mm and
then allowed to solidify at room temperature. After solidification, the test organisms,
Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Proteus vulgaris were
spread over the media with the help of a sterile swab socked in bacterium and is used
for antibacterial study. The ethanolic extract residues were dissolved in dimethyl
sulfoxide (DMSO) to produce a concentration of 100, 250,500 µg/disc and used for
the study. Ofloxacin 5 µg/disc was used as the standard. Then the sterile filter paper
discs (6mm) having a capacity to hold 10 µl of extracts were immersed in definite
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Materials and Methods
concentration of plant extracts and placed over the solidified agar in such a way that
there is no overlapping of the zone of inhibition. Plates were kept at room temperature
for half an hour for the diffusion of the sample into the agar media. The organism
inoculated petridishes were incubated at 37 °C for 24 hours. After the incubation
period is over, the zone of inhibition produced by the samples and standard were
measured. All tests were performed in triplicate.
Antioxidant activity by DPPH method 20
Antioxidant behavior of the extracted compound was measured in vitro by the
inhibition of generated stable 2,2-diphenyl- 1-picrylhydrazyl (DPPH) free radical.
Methods vary greatly as to the generated radical, the reproducibility of the generation
process, and the end point that is used for the determination. The DPPH solution was
prepared by dissolving accurately weighed 22 mg of DPPH in 100 ml of ethanol.
From this stock solution, 18 ml was diluted to 100 ml with ethanol to obtain 100 μM
DPPH solutions.
Structural changes of DPPH during oxidation
Procedure:
The sample solution was prepared by accurately weighed 2.1 mg of each of
the compounds and dissolved in 1 ml of freshly distilled DMSO separately to obtain
solutions of 2.1 mg/ml concentration and the standard solution of was prepared by
accurately weighed 10.5 mg of α-Tocopherol and dissolved in 1 ml of freshly distilled
DMSO to get 10.5 mg/ml concentration.A different concentration of extract was
prepared by the addition of ethanolic solution of DPPH radical. The reaction mixture
was vortexed thoroughly and left in the dark at room temperature for 30 min. The
absorbance of the mixture was measured spectrophotometrically at 517 nm against the
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Materials and Methods
corresponding blank solution. The final concentration of the samples and standard α-
Tocopherol solutions used is 100 μg/ml. The percentage scavenging DPPH radical
inhibitions were calculated by using the following formula:
(|control|−|sample|)
DPPH radical scavenging activity (%)= ×100
|control|
Where, Abs control was the absorbance of DPPH radical and ethanol, Abs sample
was the absorbance of DPPH radical and sample/standard.
The scavenging activity was expressed in terms of IC50, the concentration of the
samples required to give a 50% reduction in the intensity of the signal of the DPPH
radical. The results were done at least in triplicate.
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