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The collected, cleaned and coarsely powdered of Clerodendrum phlomidis (Linn) was used for the extraction purposes. 1kg of powdered leaves was used. It was then extracted with various solvents from non polar to polar such as Petroleum ether, Chloroform, Ethyl acetate and Methanol. The solvents used were distilled before use. The extraction was carried out with various solvents by of hot soxhlet extraction for 72 Hrs. After each solvent extraction, the extracts were filtered through whattmann filter paper to remove any impurities is present.
PREPARATION OF EXTRACTS
a) Petroleum ether extract of leaves of Clerodendrum phlomidis (Linn).
About 1kg of dry coarse powder was extracted first 5 liters of Petroleum ether (60-80Â°C) in a hot soxhlet extraction using round bottomed flask for 72 Hrs. After completion of extraction the petroleum ether was filtered and concentrated to dry mass by vaccum distillation. A dark green colour residue (1.48 % w/w) was obtained. The extract was then stored in a desicator (Practical pharmacognosy. 1994).
b) Chloroform extract of leaves of Clerodendrum phlomidis (Linn).
The marc left after Petroleum ether extract, was dried and subsequently extracted with 4 liters of Chloroform (40-60Â°C) in a hot soxhlet extraction using round bottomed flask for 72 Hrs. After completion of extraction, it was filtered and the solvent was removed by distillation under reduced pressure. A yellowish green colour residue (0.86 %w/w) was obtained .The extract was then stored in a desicator.
c) Ethyl acetate extracts of leaves of Clerodendrum phlomidis (Linn).
The marc left after Chloroform extract, was dried subsequently extracted with 3 liters of methanol (40-60Â°C) in a hot soxhlet extraction using round bottomed flask for 72 Hrs. After completion of extraction, it was filtered and the solvent was removed by distillation under reduced pressure. A brown colour residue (0.63 %w/w) was obtained. The extract was then stored in a desicator.
d) Methanol extracts of leaves of Clerodendrum phlomidis (Linn).
The marc left after Ethyl acetate extract, was dried subsequently extracted with 2 liters of methanol (40-60Â°C) in a hot soxhlet extraction using round bottomed flask for 72 Hrs. After completion of extraction, it was filtered and the solvent was removed by distillation under reduced pressure. A dark brown colour residue (8.24 %w/w) was obtained. The extract was then stored in a desicator.
From the weight of the each extractive residue, the extractive values were calculated in percentage. All the above extracts were used for identification of constituents by phytochemical tests and for the pharmacological studies. The yields of various extract were shown in the Table No: 1.
EXTRACTIVE VALUES OF THE LEAVES OF
CLERODENDRUM PHLOMIDIS (LINN)
QUALITATIVE PHYTOCHEMICAL ANALYSIS
Qualitative chemical tests were carried out for all the extracts of leaves of Clerodendrum phlomidis (Linn) to identify the various phytoconstituents. The various tests and reagents used are given below and observations are recorded. (Table No.2)
Tests for carbohydrates:
To 2-3 ml of extract, added few drops of Î±-naphthol solution in alcohol, shaken and added concentrated H2SO4 form sides of the test tube was observed for violet ring at the junction of two liquids (Indian Pharmacopeia, vol II. 199).
1 ml Fehling's A and Fehling's B solutions was mixed and boiled for one minute. Added equal volume of test solution. Heated in boiling water bath for 5-10 min was observed for a yellow, then brick red precipitate.
Equal volume of Benedict's reagent and test solution in test tube were mixed. Heated in boiling water bath for 5 min. Solution may appear green, yellow or red depending on amount of reducing sugar present in test solution
Tests for Alkaloids
To the 1 ml of extract, add 1 ml of Mayer's reagent (Potassium mercuric iodide solution). Whitish yellow or cream colored precipitate indicates the presence of alkaloids.
To 1 ml of the extract, add 1 ml of Dragendroff's reagent (Potassium bismuth iodide solution). An orange-red precipitate indicates the presence of alkaloids.
To 1 ml of the extract, add 1 ml of Hager's reagent (saturated aqueous solution of picric acid). A yellow colored precipitate indicates the presence of alkaloids.
To 1 ml of the extract, add 1 ml of Wagner's reagent (Iodine in potassium iodide solution). Formation of reddish brown precipitate indicates the presence of alkaloids. (Kokate C.K et.al, 2007).
Tests for Glycosides
Hydrolysis of extract:
A minimum quantity of the extracts is hydrolyzed with hydrochloric acid for few minutes on water bath and the hydrolysate is subjected to the following tests.
Â¡). Legal's test:
To the hydrolysate 1 ml pyridine and few drops of sodium nitropruside solution added, then it is made alkaline with sodium hydroxide solution. Color change shows the presence of glycosides.
Â¡Â¡). Borntrager's test:
Hydrolysate is treated with chloroform and the chloroform layer is separated. To this, equal quantity of dilute ammonia solution is added. Color changes in the ammonical layer shows the presence of glycosides.
Bal jet's test:
A test solution observed for yellow to orange color with sodium picrate.
Keller Killiani test:
Dissolve the extract in acetic acid containing traces of ferric chloride and transfer to a test tube containing sulphuric acid. At the junction, formation of a reddish brown color, which gradually becomes blue, confirms the presence of glycoside.
Tests for Phyto Steroids
Small quantity of extract is dissolved in 5 ml of chloroform separately. The above obtained chloroform solutions are subjected to Salkowski and Liebermann - Burchard tests (Harbone. JB. 1973).
To the 1 ml of above prepared chloroform solution few drops of concentrated sulphuric acid is added. Formation of brown ring indicates the presence of phytosterols.
Liebermann - Burchard test:
The above prepared chloroform solutions are treated with few drops of concentrated sulphuric acid followed by 1 ml of acetic anhydride solution. A bluish green color solution shows the presence of phytosterols.
Tests for Flavanoids
To dried powder or extract added 5 ml 95% ethanol, few drops concentrated HCl and 0.5 g magnesium turnings. Pink color was observed (Quality Control of Herbal Drugs. 2002).
Ferric Chloride test:
Test solution with few drops of ferric chloride solution shows intense green color.
Alkaline reagent test:
Test solution when treated with sodium hydroxide solution shows increase in the intensity of yellow color, which becomes colourless on addition of drops of dilute acid.
Lead Acetate solution test:
Test solution with few drops of lead acetate solution (10%) gives yellow precipitates.
Test for terpenoids
Dissolve 2 to 3 granules of tin metal in 2 ml of thionyl chloride solution. Then add 1 ml of the extract into the test tube. The formation of a pink color indicates the presence of terpenoids.
5 ml of aqueous extract of each plant sample is mixed with 2 ml of CHCl3 in a test tube. 3 ml of concentrated H2SO4 is carefully added to the mixture to form a layer. An interface with a reddish brown coloration is formed if terpenoids constituent is present. (Journal of Medicinal Plants Research Vol. 3(2), pp.068).
Tests for Saponins
The extracts are diluted with 20ml of distilled water and then agitated in a graduated cylinder for 15minutes. Formation of foam layer indicates the presence of saponins. (Khandelwal K.R, 2007).
Added test solution to one drop of blood placed on glass slide. Haemolytic zone whether appeared was observed.
Tests for Proteins and Amino acids
To 3 ml test solution added 4% NaOH and few drops of 1% CuSO4 solution observed for violet or pink color (Practical Pharmacognosy. 1996).
Mixed 3 ml test solution with 5 ml Million's reagent, white precipitate. Precipitate warmed turns brick red or precipitate dissolves giving red color was observed.
Mixed 3 ml test solution with 1 ml concentrated H2SO4 observed for white precipitate.
3 ml test solution and 3 drops 5% Ninhydrin solution were heated in boiling water bath for 10 min. observed for purple or bluish color
Tests for Tannins and Phenolic compounds
To 2 - 3 ml of extract, add few drops of following reagents:
5% FeCl3 solution: deep blue - black color.
Lead acetate solution: white precipitate.
Gelatin solution: white precipitate.
Bromine water: decoloration of bromine water.
Acetic acid solution: red color solution.
Dilute iodine solution: transient red color.
Dilute HNO3: reddish to yellow color.
Test for Fixed Oils and Fats
Small quantity of the extract is placed between two filter papers. Oil stain produced with any extract shows the presence of fixed oils and fats in the extracts.
Few drops of 0.5N alcoholic potassium hydroxide are added to the extract with few drops of phenolphthalein solution. Later the mixture is heated on water bath for 1 - 2 hours soap formation indicates the presence of fixed oils and fats in the extracts.
Test for Gums and Mucilage's
Ruthenium red test:
Small quantities of extract are diluted with water and added with ruthenium red solution. A pink color production shows the presence of gums and mucilage's.
TABLE NO: 2
QUALITATIVE PHYTOCHEMICAL ANALYSIS OF EXTRACTS OF LEAVES OF CLERODENDRUM PHLOMIDIS (LINN)
TEST OF EXTRACTS
TANNINS & PHENOLIC COMPOUNDS
FIXED OILS & FATS
GUMS & MUCILAGES
PROTEINS & AMINO ACIDS
(+ ) = indicates presence, (-) = indicates absence
Based on qualitative analysis we have selected Ethyl acetate extract of clerodendrum phlomidis (Linn) leaves for further studies because Ethyl acetate extract is having more phytoconstituents when compared to all other extracts.