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Phytochemical analysis:

Alkaloid detection:

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powdered sample was dissolved in 2ml of dilute HCL and filtered, this extract
was subjected to varied alkaloidal reagents.

Dragendorff’s reagent:
1 or 2ml of Dragendorff reagent when added to the extract, results in red-orange
precipitates which is a positive test. (Tiwari.,2011 Willow)

Hager’s reagent:
Few ml of sample was mixed with few drops of the saturated solution of picric
acid. Prominent yellow precipitate confirms the alkaloid presence. (Bhandary; Tiwari;

Mayer’s reagent:
To a few ml of extract, two drops of Mayer’s reagent are added along the sides
of tubes. Yellow or white precipitate indicates the presence of alkaloids. (Pandey;
Banu, Maria)

Wagner’s reagent: Few
drops of Wagner’s reagent are added to few ml of filtrate along sides of the test
tube. Reddish-brown precipitate evident the presence of secondary metabolite
alkaloid. (Chanda; Maria)

Detection of phenolic compounds:

Ferric chloride test: To
the aqueous extract few drops of 5% ferric chloride solution is added. Presence
of phenolic compounds can be observed with dark green or bluish black colour. (Banu;
Jaradat; Tiwari)

Lead acetate test: The
extract wass dissolved in 3ml of 10% lead acetate solution. White precipitate can
confirm the presence of phenolics. (Banu; Pandey)

Tannin detection:

Gelatin test: 2ml
of 1% gelatin containing solution with 10% sodium chloride was added to the
sample extract. Appearance of white precipitate manifests the presence of
tannins. (Tiwari; Pandey; Bhandary)

Ferric chloride test:
5% of ferric chloride solution is added to 2ml of extract. A dark green or blue
colour disclose the tannin presence. (Rattana; Maria)

Flavonoid detection:

Alkaline reagent:
10 % of ammonium hydroxide solution is mixed with the sample extract. Flavonoid
detection can be done with yellow fluorescence. (Jaradat; banu; Bhandary)

Shinoda test:
1ml of ethanol and drops of conc. HCL was added to the extract in isopropyl
alcohol, red colour confirms the aurones, chalcones presence. If there is no
colour change formation of orange, red colouration in addition of metallic
magnesium indicates the flavones and flavonols presence. (Rattana; Jaradat; Sherani)

Sodium hydroxide test: Few
drops of 10% sodium hydroxide was added to extract in isopropyl alcohol to
obtain a yellowish red or orange or blue colour which confirms the flavones,
flavonols presence. (Maria; Sherani)

Detection of phytosterols:

Liebermann-Burchard reaction: To
the extract, add 2ml of acetic anhydride and few drops of concentrated. H2SO4
along the sides. Colour change to blue evident the steroidal compound presence.
(Boxi.,2010; banu; Kumar)

Detection of glycosides:

extract is hydrolysed with concentrated HCL and the filtrate is subjected for
the following test.

Modified Borntrager’s test: Add
3ml of chloroform to the hydrolysed extract and shaken. Chloroform layer was
separated and mixed with 10% ammonia solution. Formation of pink colour in
ammoniacal layer show the presence of anthranol glycosides (Tiwari; Banu)

Legal’s test:  Add sodium nitroprusside in pyridine and
sodium hydroxide to the extract. Colour change from pink to red reveal the
cardiac glycosides presence. (Banu; Tiwari)

Kellar- Kiliani:
2ml of glacial acetic acid with 1
drop of ferric chloride solution and 1ml of conc. H2SO4
were added to the extract. Brown ring formation confirms the presence of
cardiac glycosides. (Obianime and Uche.,2008; Rattana; Bhandary)

Terpenoid detection:

Salkowski’s test: 3ml
of concentrated sulphuric acid with 2ml of chloroform was added to the extract.
Presence of triterpenes can be observed with reddish brown colour.  (Obianime.,2008; Rattana; Sherani)

Copper acetate test:
To 2ml of extract few drops of copper acetate solution were added. Emerald
green colouration confirms the diterpenes presence. (Tiwari Pandey)

Saponin detection:

Froth test:
The plant sample is diluted with distilled water up to 20ml and shaken
vigorously. Foam formation suggests the saponin presence. (Adegoke.,2010;
Rattana; Sherani)

Test for Carbohydrates

Molisch test:
To few drops of extract 2 drops of alpha-naphthol solution and conc. sulphuric acid
is added to obtain a violet ring formation that confirms the presence. (Tiwari;
Boxi.,2010; Jaradat)

Barfoed’s test: The
filtrate was treated with equal volume of Barfoed’s reagent and kept in boiling
water bath for 2 mins. The test tube is then cooled, red colouration indicates
the presence. (Boxi.,2010)

Benedict’s test:
The extract was treated with Benedict’s reagent and heated. Reducing sugar
presence was observed with orange-red precipitate. (Jaradat;Banu; Bhandary)

Fehling’s reagent:
The extract is hydrolysed with dil. HCL and an equal volume of Fehling’s A
& B were added and heated. Red precipitate was observed. (Akinyemi;Tiwari)

Detection of proteins and amino-acids:

Millon’s test: 2ml
of extract is added with Millon’s reagent. White precipitate confirms the
protein presence (Jaradat;Banu)

Xanthoproteic test: To
few drops of extract conc. nitric acid is added to the sides of the test tube.
Yellow colour observed indicates the protein presence. (Tiwari)

Ninhydrin test:
0.25% w/v ninhydrin reagent was added to the extract and boiled for 1-2 min to
obtain blue colour. This indicates the amino-acid presence. (Bhandary)

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