Lab manuals for studying of Medicinal Plants
Developed by
Sanjeet Kumar
Ravenshaw University
Phytochemical Screening
Preparation of Plant extracts
Solvent extract was prepared using percolation method from 5gm of leaf powder which was macerated in solvent (50 ml) for 12 hrs in refrigerator. After 12 hrs sample was filtered and residue was again macerated in same solvent and for each solvent process was repeated thrice. Aqueous extract was prepared separately by taking the powder in distilled water followed by filtration. Filtrate were dried and concentrated to get semisolid mass.
Phytochemical assays
Test for Tannin
0.7 ml of the extracts was dissolved in 50ml of distilled water and was heated for 10 minutes. After cooling few drops of 1% ferric chloride was added. Colour of sample was changed from yellow to green and dark green precipitate was observed.
Test for Saponin
5 ml of extract was dried and to it was add 1ml of Ethyl acetate. Ethyl acetate was removed and add distilled water and mixture was shaken vigorously and observed for persistent foam which lasted for at least 15 minutes.
Test for Flavonoids
Few amount of leaf extract was taken in a flask and dissolved in 10% NaOH. Few drops of HCl was added. Yellow colour turned to colourless.
Test for Terpenoid
1ml of extract was mixed with 400 μl chloroform. Then the mixture was added by drop of sulphuric acid. A reddish brown interface indicates terpenoid present.
Test for Alkaloids
Few quantity of the each portion was stirred with 5 ml of 1% aqueous HCl on water bath and then filtered. Of the filtrate, 1 ml was taken individually into 2 test tubes. To the first portion, few drops of Dragendorff’s reagent were added; occurrence of orange-red precipitate was taken as positive.
Test for Phenolic compounds
Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenolic compounds.
Test for Steroids
0.2 g of each portion, 2 ml of chloroform was added, the solution was cooled well in ice followed by the addition of conc. H2SO4 carefully. Two layers developed of red and green indicated the presence of a steroidal ring.
Physiochemical parameters
Total ash %
Apparatus required:
(i) Muffle furnace (ii) Crucible (iii) Electronic balance (iv) Tongs
Procedure:
1. A clean, dry crucible of Pt or Ni or silica was taken.
2. The blank weight of crucible was taken (w1).
3. 2-4gms of sample (w) was weighed accurately in the crucible.
4. The sample was ignited with crucible inside the muffle furnace at 600-650°C for 1-2hours until it was white in colour indicating the absence of carbon.
5. The crucible was taken out and cooled inside desiccators and weighed.
6. It was dried inside a water bath, then on a hot plate.
7. Ignition was done inside the muffle furnace up to a constant weight.
8. The residue was allowed to cool inside desiccators for 30 mints and then weighed.
Initial weight of crucible = X gm
Sample weight =Y gm
Final weight of crucible with sample = Z gm
Total Ash % = Z – X / Y × 100
Total Fiber %
Apparatus:- 1lt beaker, round bottom flask, hot plate, muffle furnace, vacuum pump, Hot air oven, Watt man filter paper 541, crucible.
Procedure:-
1. 2gms of fat free sample was weighed in a1lt beaker.
2. 200ml of 0.255N Sulphuric acid solution was added and was kept for 2 hours at the hot plate at 90°C.
3. During digestion the flask was shaken at every 10mins and then the residue was filtered with the help of vacuum pump and Watt man filter paper541 which was previously wetted.
4. The filter paper was washed in coldwater and hot water till it became acid free.
5. Then the residue was transferred to the same beaker and 200ml of 0.313N NaOH solution was added to it and was boiled for 1hour by keeping it over the hot plate at 50°C.
6. The flask was shaken at every 10mins and the residue was filtered by the same process with the help of vacuum pump and 541 filter paper.
7. The filter paper was washed with hot and cold water till it became alkali free.
8. The residue on the filter paper was dried for 1.5hours inside the hot air oven at 105°C.
9. Then the sample was kept in the desiccators for 30mins and was then weighed.
10. The filter paper with residue was taken in a pre-weighed silica crucible and was placed inside the muffle furnace at 600-650°C.
11. It was kept inside the desiccators for 30mins and was then weighed.
Total Fiber (%) = [(Final wt.of filter paper and beaker)-(Blank wt.of filter paper and beaker)]
[(Final wt.of crucible)- (Blank wt.of crucible)] x 100
Sample weight
Total Magnesium (mg / 100 gm)
Apparatus Required
Muffle furnace, Electronic balance
Silica dish, volumetric flask
Reflux condenser
Pipette
Procedure
1. Take 2 Gms of sample in a preweighed silica dish.
2. Heat in a muffle furnace for 30 minutes.
3. Reflux the ash with 100 ml of 1:1 HCL for 30 minutes then cool and filter.
4. Make up to 250 ml in a volumetric flask.
5. Pipette out 25 ml aliquot of this and dilute to 50 ml.
6. Add 10 ml ammonia buffer solution of PH10 and mix well.
7. Add 50 mg of hydroxylamine hydrochloride and mix well.
8. Add 0.1 grams of eriochrome black-T indicator directly.
9. Then titrate with 0.1M EDTA.
10. Each ml of 0.1M EDTA is equivalent to 2.432 mg of magnesium.
Total Calcium (mg / 100 gm)
Apparatus Required
Ø Muffle furnace
Ø Silica dish
Ø Reflux condenser
Ø Volumetric flask
Ø Burette
Procedure:
Ø Take 2 grams of sample in a preweighed silica dish.
Ø Heat in a muffle furnace for 30 minutes.
Ø After ashing reflux the sample with 100 ml of 1:1 HCL for 30 minutes then cool and filter.
Ø Make up the filtrate to 250 ml in volumetric flask with distilled water.
Ø Add KOH till the PH is between 12 to 14.
Ø Then add 50 mg hydroxylamine hydrochloride and shake well until the solid dissolves.
Ø Add 0.1 gm patrons and readers indicator and titrate with 0.01M EDTA.
Ø The end point is change of color from wine red to pure blue.
Ø Each ml of 0.01M EDTA is equivalent to 0.4008 mg of calcium.
Nutritional Parameters
Protein estimation (%)
Apparatus:- Kjeldhal flask, receiving condenser, connecting tube, Bunsen burner, tripod strand, measuring cylinder, funnel, beaker, glass beads, heater, pipette.
Procedure: - Kjeldhal method was completed in three steps:
1. Digestion
2. Neutralization
3. Titration
Digestion:-
1. About 1g sample was taken and transferred to a Kjeldhal flask.
2. 5g anhydrous Na2SO4 & 0.2g CuSO4 was added as catalyst.
3. 25 ml concentrated H2SO4 was added to it and some glass beads is added to avoid pumping.
4. It was digested in a heater for 2h at a temperature of 800 c-1000c, till the solution turns green.
5. Digestion converse any nitrogenous food into ammonium sulphate (NH4SO4) and other organic matter in CO2 and H2O
Neutralization:-
1. About 300ml distilled water was added to the Kjeldhal flask.
2. In another flask 20 ml 4% H3BO3 solution along with 2-3 drops of mixed indicator was added.
3. The conical flask held was connected to receiving condenser, the tip of which should emerge into the boric acid solution flask.
4. Then the digestion flask was connected to a receiving flask by a tube called connective tube.
5. 30-40ml of 50% NaOH solution is added in Kjeldhal flask using funnel by which NH4SO4 get converted into NH4.
6. The NH4 gas thus form move out from the digestion flask and concentrated into the receiving flask which content excess of H3BO3.
7. The low pH of the solution in the receiving flask converts the NH4 gas in NH4 and simultaneously converts the H3BO3 to H2BO3 (borate ion).
Titration:-
About 150ml of H3BO3 solution is collected, then N2 content is estimated by titrating it against 0.1N H2SO4 solution using suitable mixed indicator, to determine the end point of the solution.
% of nitrogen= Burette reading x 0.0014 / Sample weight x 100
% of protein = % of nitrogen x 6.25
Total Fat estimation (%)
Apparatus: - Soxhlet unit, Watt man filter paper I, round bottom flask, electronic balance
Procedure:-
1. 2-5gms of sample was taken in a Wattman filter paper I and was transferred into another filter paper which was then converted into a thimble.
2. Then the blank weight of the round bottom flask was taken; then 150ml of petroleum ether was added into the Soxhlet unit just to pass a circulation.
3. Then the thimble was put in the Soxhlet unit and 50ml of petroleum ether was added to cover the thimble.
4. The Soxhlet unit was fitted properly and was run for 4-6 hours at a temperature of 70°C.
5. After 6hours the thimble was taken out from the extractor and petroleum ether was collected till the last drop carefully.
6. The flask was heated at a temperature of 100°C for 1.5 hours inside the oven.
7. It was then cooled in the desiccators for 1/2hours and was weighed immediately.
Total Fat (%) = Final weight-Initial weight x 100 / Sample weight
Carbohydrate content (N.F.E) =
100 - (% of moisture+ % of ash+ % of total protein + % of total fat + % of total fiber)
(Where, N.F.E. = Nitrogen Free Extract)
ENERGY
Gross Energy = (Fat x 9) + (Protein x 4) + (Carbohydrate x 4.1)
Metabolic Energy = 85% of Gross energy
Carbohydrates are the important components of storage and structural materials in the plants. They exist as free sugars and polysaccharides. The basic units of carbohydrates are the monosaccharides which cannot be split by hydrolysis into simpler sugars. The carbohydrate content can be measured by hydrolyzing the polysaccharides into simple sugars by acid hydrolysis and estimating the resultant monosaccharides.
Procedure
Standard Graph preparation
· First ten test tubes were taken including “blank” and the working standard was pipetted out from 0 to 0.9ml.
· Then volume was made up to 1ml with distilled water.
· Fresh 4ml of Anthrone reagent was added to each test tube at cooling condition.
· The test tubes were heated for 8minutes in a boiling water bath.
· Was then rapidly cooled and the colour change from green to dark green was observed spectrophotometrically at 630nm.
· Finally a graph was plotted taking glucose concentration on X-axis and Optical density on Y-axis.
Extraction of carbohydrate-
· Three replicas of 0.1 gm sample were taken and grinded with addition of 5ml of 2.5N HCL.
· Then grinded solution was then transferred to test-tubes and hydrolyzed by keeping it in boiling water bath for 3 hours.
· Then sample was then neutralized with solid sodium carbonate until the effervescence ceases.
· Then, centrifugation was performed for three times at 3000rpm for 20 mins.
· Finally the supernatant was collected in vials and volume was made up to 100ml with distilled water.
· Stored in a refrigerator for further use.
Estimation of carbohydrate-
· First seven test tubes were taken including blank and two test tubes for each replica.
· 0.1 and 0.2ml of sample was taken in each test tube except blank.
· The volume was made up to 1ml by addition of distilled water.
· 4ml of Anthrone reagent was added in each test tube in cooling condition.
· The test tubes were then heated for 8minutes in a boiling water bath.
· Was then rapidly cooled and the colour change from green to dark green was observed spectrophotometrically at 630nm.
· The amount of carbohydrate present in the sample was calculated from the standard graph.
Estimation of Proteins
Methods
0.1N NaOH., 2% Na2Co3, 1% CuSo4, 2 % Sodium Potassium Tarterate, Folin Phenol, BSA
Preparation of Standard Curve
a) 2% Na2Co3 (0.1 NaOH ) - Solution A
b) 1 % CuSo4 (in distilled water ) - Solution B
c) 2 % Sodium Potassium Tertarate ( in distilled water ) – Solution B2
d) Reagent C = 1ml B1 + 1ml B2 + 48 ml A
Procedure
1. Take BSA stock solution of concentration 1.2 mg/ml into 10 test tubes ranging 100- 1000 µg/ ml; make up vol. up to 1 ml by adding distilled water.
(Plus take another tube as blank reference filled in 1 ml of methanol only without BSA).
2. Add 4 ml of Reagent C, allow to stand for 10 minutes.
3. To these test tubes, add 0.5 ml of Folin phenol and allowed to stand for 30 minutes.
4. Measure O.D. in spectrophotometer at the wavelength 690 nm and / or 750 nm.
5. Prepare a graph putting these O.D. values in the Y axis and concentration of BSA in the X axis.
This graph is the standard curve for estimation of Protein present in the test sample.
Thin Layer Chromatography
Preparative TLC
Glass plate was cleaned with ethyl acetate then left for 15 min. 3 gm of silica gel was taken in a beaker and 15 ml water was added. Then slurry was poured over the glass plate and was allowed to dry, than heated by hot plate for few minute to activation of TLC plate.
Mobile phase
Chloroform: Methyl alcohol = 9:1
Rƒ values
The behavior of an individual compound in TLC is characterized by a quantity Known as Rƒ and is expressed as a decimal fraction. The Rƒ is calculated by dividing the distance the compound traveled from the original position by the distance the solvent travelled from the original position (the solvent front).
Qualitative analysis of alkaloids
It is used in qualitative analysis of alkaloids in control phase of both pharmaceutical formulations and vegetable drugs. TLC has been used for the isolation and determination of alkaloids in toxicology where the 30-60 minute runs give a great advantage in comparison to the 12-24 hours required for paper chromatography. Purine alkaloids have been separated by TLC on silicic acid, silica gel and aluminium oxide. The spots are visualized by spraying first with an alcoholic iodine-potassium iodine solution followed by 25% HCl- 96% ethanol.
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