phytochemistry introduction -...
TRANSCRIPT
Chapter – VI
PHYTOCHEMISTRY
INTRODUCTION Phytochemistry is the chemistry (chemical analysis) of plant
products. The chemicals present in leaf powder are analysed chemically
by qualitatively and quantitively. Presence or absence of a chemical will
gives the criteria to evaluates the drug or to standardize the drug.
Estimation of particular chemical or element will also considered as
criteria. The quantitive analysis of elements / chemicals like nitrogen,
crude proteins, crude fats, crude fibers, carbohydrates etc. may fluctuate
with the age of the shrubs plants, season of collection, hence these values
are not considered as criteria. But their use in combination roughly gives
the idea. Their values with little variation should be accepted. The
quantitive analysis of a single chemical or element should not be
considered as strict criteria for standardization or evaluation. This species
no any information of phytochemical studies but other species Abutilon
indicum and Abutilon theophrasti very important of the medicinal plants
reported by various authors Ashok Kumar, (2011), Bagi, et. al. (1984,
1985), Dashputre and Naikwade, (2011), Deokule and Patale (2002),
Guno et. al., (2009), Guno, (2009), Kaushik, (2009), Khadabadi and
Bhajipale, (2010), Lakshmayya et. al., (2000), Yogesh et. al. (2010) and
also some chemical constitute and phytochemical investigation of genus
Abutilon are reported Gambhir, (1952), Gaind, (1976), Dhanalaksmi et.
al. (1990), Hussain, (2005), Karamat, (2003).
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In phytochemical investigation the sample of leaf powder obtained
from Abutilon ranadei species studied for following parameters:
MATERIAL METHODS
The leaf samples were collected from Torna fort Pune district
Maharashtra. The exact location of the plants were collected are given in
the form of longitude, latitude and altitude. The date of their collection
and field numbers are also provided (Table- 1).
The plants were collected for vegetative propagation these plants
removed leafs by handling without damaging the plants. The leafs were
collected in polyethylene bags and brought to the laboratory within 2-3
days. These were initially dried in shade and later in oven at 40oC till
constant weight, made in to fine powder and stored in sealed plastic
container for further analysis.
The morphological characters of the plants were studied in detail
and their herbarium sheets were prepared which were preserved in the
Herbarium of Department of Botany, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad.
Physical evaluation
1) DRY MATTER (DM)
Dry matter (DM) was calculated by weighing the sample after
drying to a constant weight in an Shade place or sunlight at 35 ± 5°C. For
this purpose, 100 g of sample was taken in a clean dry pre-weighed tray
and kept in shade place for 48 hours or more, till constant weight. Weight
of the dried sample was reported as percent dry matter (DM).
The dried samples were usually ground to a fine powder and stored
in sealed containers for further analysis.
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2) BULK DENSITY
The fine powder of leaf samples was filled in a cube of 1cm x 1cm
x 1cm, the heap of powder was removed by a scale to maintain
uniformity. The powder was removed from the cube and its weight was
taken. That weight was considered as bulk density mg/cm3.
3) CHEMICAL ANALYSIS
A) Quantitative analysis
NITROGEN (N)
The dry sample is digested with concentrated sulphuric acid
(H2SO4) in the presence of catalyst. During the digestion, nitrogenous
compounds are converted to ammonium sulphate ((NH4)2 SO4). It is then
made strongly alkaline with sodium hydroxide (NaOH). The released
ammonia (NH3) is distilled into boric acid (H3BO3) solution. The
ammonium tetraborate formed is then titrated against 0.035 N
hydrochloric acid for the determination of nitrogen (N).
Reagents
1) Concentrated H2SO4, AR grade, sp. gr. 1.85
2) Catalyst : A mixture of copper sulphate (CuSO4), potassium
sulphate (K2SO4) and Selenium dioxide (SeO2 ) in a ratio 1: 9:
0.02.
3) NaOH solution (40%): Dissolve 400g NaOH in 1000 ml of
distilled water.
4) Mixed indicator: Dissolve 300 mg bromocresol green and 200 mg
methyl red in 95% ethyl alcohol. Make the volume to 500 ml with
95% ethyl alcohol.
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5) Boric acid solution : 2% H3BO3 solution is made by dissolving 10
g H3BO3 in 480 ml of glass distilled water. To this about 5 ml
mixed indicator is added and the volume is made to 500 ml.
6) 0.035 N HCl : 25 ml of concentrated HCl is taken in a volumetric
flask and diluted upto 500 ml with distilled water. This serves as a
stock solution. To determine normality of this solution, 1 g
ammonium tetra borate ((NH4)3 BO3) is taken in 50 ml conical
flask. To it 10 ml glass distilled water and 2-3 drops of mixed
indicator are added. This is titrated with the stock solution of HCl
to calculate the normality using following equation:
1000
Normality of HCl = ----------------------------------
titration value (ml) x 190.72
After determining the normality of stock solution, 0.035 N Hcl is
prepared by appropriate dilution.
Procedure:
1) Digestion: Transfer carefully, accurately weighed 300 mg of dry
plant material in a Kjeldahls flask. Add a pinch of catalyst with
the help of spatula. Slowly add 7.5 ml concentrated sulphuric
acid (H2SO4). Heat the flasks gently on a digestion stand until the
fumes of H2SO4 are freely evolved. Increase heat until acid boils
vigorously and digest till the mixture is clear, i.e. apple green in
colour or colourless. During digestion care must be taken to
avoid particles of indigested carbon sticking on the sides of the
tube. Cool the contents of flask and use for the distillation. For
this purpose the digested material is made upto a volume of 50
ml in volumetric flask with distilled water.
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2) Distillation: This is usually carried out with the Markham's
steam distillation apparatus. Heat the steam boiler to produce
steam. Keep a 50 ml conical flask, containing 10 ml boric acid
solution, at the delivery end of the condenser. Tip of the
condenser should be just beneath the surface of H3BO3 solution.
Introduce 5 ml of previously diluted, digested sample into the
distillation flask through funnel. Close the funnel with ground
glass rod. Put 10 ml NaOH solution in the funnel and introduce it
slowly into distillation flask. The ammonia formed due to the
treatment of NaOH passes along with the steam and is absorbed
by H3BO3, at the condenser outlet to form ammonium tetraborate
((NH4)3BO4). This results into the change in colour of H3BO3
solution from pink to green. Continue distillation till the volume
becomes to about 20 ml. Titrate the ((NH4) 3BO4) with 0.035 N
HCl till pink colour reappears and record titration value.
Calculate strength of NH3, in the distillate using equation :
1 ml 0.035 N HCl = 0.5 mg of N
Calculate the amount of N for 50 ml of the sample, which will be
equivalent to that present in 300 mg of dry plant material, Compute
the N per cent in dry sample and record it as N % of dry matter
(DM).
CRUDE PROTEIN (CP)
On an average, most of the proteins have 16 % nitrogen in their
composition. Thus the amount of N content, when multiplied by 6.25,
gives the crude protein (CP) content of the sample.
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CRUDE FAT (CFat)
The fats present in the plant material are extracted in the solvent
consisting of chloroform (CHC13) and methanol (CH3OH). This is done
in soxhlet extraction assembly and after complete evaporation of the
solvent, the amount of extracted fat is measured.
Reagents
1) Solvent: Chloroform + methanol (2:1)
Procedure:
Weigh 2 g dry plant material and transfer it into a thimble prepared
with Whatman filter paper No. 1. Plug the mouth of thimble with fat free
absorbent cotton. Take clean, dry 250 ml receiver flask from the soxhlet
assembly and add the solvent to it just to reach the level of the neck.
Introduce the thimble with sample into the soxhlet. Assemble the
apparatus and place it on heating mantal with temperature controlling
device. Fit water condenser at the top of the soxhlet. Extract the fat for 8
hours at 60°C. When the extraction is over, remove the thimble from
soxhlet. Assemble the apparatus again and heat to recover most of the
solvent from the receiver flask. When the receiver flask contains about 25
ml solvent along with the extracted fat, disconnect the receiver flask.
Transfer the solvent in a clean, previously weighed beaker with rinsing
for 2 to 3 times. Evaporate the solvent completely and dry it in a hot air
oven at 95°C, cool in a dessicator and weigh. Measure the amount of fat,
extracted per 2 g of the sample, and calculate the amount of Cfat as
percent of dry matter (DM).
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CRUDE FIBRE (CF)
Crude fibre (CF) is determined as that fraction remaining after
digestion with dilute solutions of sulphuric acid (H2SO4) and sodium
hydroxide (NaOH) under carefully controlled conditions. The major part
of it contain carbohydrates and it is a valuable parameter in deciding the
nutritive quality of animal feed.
Reagents:
1) 1.25 % H2SO4: Dissolve 5 ml con. H2SO4 in 395 ml distilled
water.
2) 2.5% NaOH: Dissolve 5 g NaOH in 100 ml distilled water and
make the volume to 200 ml with distilled water.
3) 70% ethyl alcohol.
Procedure:
Transfer 2 g defatted sample to a 500 ml spoutless beaker and add
200 ml 1.25% H2SO4 to it. Break up the lumps with the help of glass rod
having a rubber policeman. Cover the beaker with a conical flask, half
filled with cold water, which serves as water condensor. Boil for 30
minutes and make up any loss in volume during the boiling with hot
distilled water. Filter through Whatman filter paper No. 54 by washing
the residue several times with hot distilled water. Take out the residue
back in the beaker with 100 ml water and to it add 100 ml 2.5% NaOH.
Boil for 30 minutes as earlier. Filter through previously weighed
Whatman filter paper No. 54. Wash the residue several times with hot
water and lastly with 70% alcohol. Dry it over night at 100°C to a
constant weight. Cool and weigh. Incinerate the residue along with filter
paper in a crucible at 600±20°C for 2 hours in a muffle furnace until all
the carbonaceous matter is burnt. Cool the crucible in a desiccator and
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weigh. Record the loss in weight as crude fibre (CF) and calculate the
amount of CF on DM basis.
TOTAL ASH
The residue after incineration of sample at 550-600°C is known as
ash. For this purpose the sample is subjected to a high temperature upto
600°C and then the ash content is determined. During ignition to such a
high temperature all organic compounds decompose and pass off in the
form of gases, while the mineral elements remain in the form of ash.
Procedure:
Take 2 g oven dry sample in a previously weighed vitrosil silica
crucible. Heat it on hot plate for about 30 minutes, till the sample is
sufficiently charred and turns black. Replace the lid of the crucible and
keep it in muffle furnace. Allow the temperature to raise upto 600°C and
keep it constant for 2 hours. Remove the crucible on cooling and transfer
directly to desiccator, cool and weigh immediately. Find out the weight of
ash, obtained per 2 g of sample, and calculate the ash content as per cent
of dry matter (DM).
WATER SOLUBLE ASH (WSA)
The ash was boiled for 5 minutes with 25 ml of distilled water.
Insoluble matter was collected in ashless filter paper and washed with hot
water, ignited and weighed. Weight of the insoluble matter was
subtracted from the weight of ash. The difference in weight represents the
water soluble ash. Percentage of water soluble ash was calculated with
reference to the air dried drug.
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ACID INSOLUBLE ASH (AIA)
Reagents:
1) 5 N Hydrochloric acid (HCl): Dilute 41.7 ml concentrated HC1 to
100 ml with distilled water.
Procedure :
Add 50 ml of 5N HC1 to the ash obtained in crucible as above.
Heat the mixture for 30 minutes in hot water bath. Allow to cool and
filter through Whatman filter paper No. 42. Wash the filter paper with
water until the washings are free from acid. Dry the filter paper along
with acid insoluble portion of ash in an oven at 100 °C overnight.
Transfer it to desiccator and weigh. Determine AIA per unit weight of the
sample used for ashing and calculate it as per cent of dry matter.
The filtrate obtained during the determination of AIA, is collected
and made to the volume upto 100 ml. This acid soluble portion of ash is
stored for the determination of the minerals like calcium (Ca) and
phosphorus (p).
CALCIUM (Ca)
Acid soluble ash fraction of the plant material is used for
determination of calcium (Ca). For this purpose the Ca in an aliquat is
precipitated as calcium oxalate. The precipitate is then dissolved in acid
and the content of oxalate ions determined titrimetrically with potassium
permanganate (KMnO4).
Reagents:
1) Methyl red indicator: Dissolve 1 g methyl red in sufficient
alcohol to make 1 litre solution.
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2) Ammonium oxalate ((COO.NH4). H2O) solution: Dissolve 6 g
of ammonium oxalate in sufficient distilled water to make 100
ml solution.
3) 2 N sulphuric acid (H2SO4): Dilute 5.6 ml concentrated H2SO4
(AR grade) to 100 ml with distilled water.
4) 0.01 N Potassium permanganate (KMnO4): Dissolve 316 mg
KMnO4 in distilled water and dilute it to the volume of 1 litre.
Keep the solution in glass stoppered bottle and store in dark.
Procedure:
An aliquat (25 ml) of the acid soluble ash portion is diluted to
about 150 ml with distilled water. Few drops of methyl red are added and
the mixture is neutralised with ammonia (NH3) solution till the pink
colour changes to yellow. The solution is heated to boiling and 10 ml
ammonium oxalate solution is added. The mixture is allowed to boil for a
few minutes. Glacial acetic acid is then added till distinctly pink colour
reappears. The mixture is then kept aside for 12 to 24 hours at room
temperature. When the precipitate of calcium oxalate settles down, it is
filtered through Whatman filter paper No. 42. The precipitate is washed
several times with water, to make it free from acid. It is then transferred
in a small beaker by piercing a hole in the filter paper and by pouring
over it about 15 ml 2 N H2SO4. This is heated to above 40°C and titrated
against 0.01 N KMnO4 solution until the first drop, which gives the
solution a pink colouration persisting for at least 30 seconds.
The amount of Ca is calculated using an equation :
1 ml of KMnO4 = 0.2004 mg of Ca
The per cent Ca on DM basis is then calculated on the basis of the
amount of sample used for preparing / estimation ash, the volume to
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which acid solution of ash is diluted and the volume of the aliquat taken
for the precipitation of calcium.
PHOSPHORUS (P)
The acid soluble portion of ash is diluted and treated with
molybdate solution. The phosphomolybdic acid formed is then reduced
by the addition of 1, 2, 4 - Aminonephthol sulfonic acid (ANSA) reagent
which produces blue colour. The intensity of the colour, which is
proportional to the amount of phosphorus present, is measured using
colorimeter.
Reagents :
1) 10 N H2SO4: Carefully add 200 ml concentrated H2SO4 (36 N)
to 520 ml of distilled water.
2) Molybdate solution: Dissolve 25 g of ammonium molybdate in
20 ml. of distilled water. Transfer it to a volumetric flask
containing 500 ml. of 10 N H2SO4 and bring the final volume to
1 litre using more distilled water. Mix well and store in brown
bottle.
3) Aminonaphtholsulfonic acid (ANSA) reagent: (a) 15% sodium
bisulphite (NaHSO3): Take 30 g reagent grade NaHSO3 in a
beaker. Add 200 ml of distilled water and stir to dissolve, (b)
20% sodium sulphite (Na2SO3): Dissolve 20 g of reagent grade
anhydrous Na2SO3 in distilled water and dilute to 100 ml. Filter
if necessary, (c) ANSA reagent: Take 195 ml of 15% NaHSO3,
solution in a beaker. Add 500 mg of 1, 2, 4 -
aminonaphtholsulfonic acid, and mix thoroughly. To this add 5
ml of 20% Na2SO3 followed by thorough mixing. If the solution
is not complete, add more Na2SO3, 1 ml at a time, with shaking
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but avoid in excess. Transfer this ANSA reagent to a brown-
glass bottle and store in cold.
4) Standard phosphorus (P) solution: Dissolve exactly 351 mg
pure dry monopotassium phosphate (KH2PO4) in 500 ml of
distilled water and transfer to a 1 litre volumetric flask. Add 10
ml of 10 N H2S04, dilute to the mark with water and mix. Five
ml of this solution contains 0.4 mg phosphorus.
Procedure:
Take 0.5 ml acid soluble portion of ash in a test tube (the amount
of this may be modified depending on the phosphorus content). Dilute it
to a volume of 10 ml with distilled water. Simultaneously take a blank
containing only 10 ml distilled water. Add 1 ml molybdate solution to
each test tube and mix, then add 0.4 ml ANSA reagent and again mix.
Allow to stand for 5 minutes and read the optical density (O.D.) at 660
mµ using colorimeter by setting it to zero with the blank.
Establish the O. D. of standard phosphorus solution by preparing a
standard graph containing 0 to 1 ml standard phosphorus solutions in
series of test tubes. Determine the amount of phosphorus in an aliquat
with the help of standard graph and calculate the phosphorus content in
the plant sample considering its amount taken for ashing, volume of the
acid soluble ash and amount of aliquat used for the reaction.
GROSS ENERGY (GE)
The determination of gross energy (GE) of feed and food products
is 'a technique frequently employed in nutritional investigations. A
method described below for the determination of GE employ the
oxidation of sample with a solution of potassium dicromate (K2Cr2O7) in
H2SO4. Energy value is obtained by dividing the amount of 1.5 N
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K2Cr2O7 required to oxidise 1 g of material by a factor depending on the
protein content. This technique gives the results in good agreement with
those obtained by Bomb calorimetry.
Reagents :
1) 1.5 N K2Cr2O7: Dissolve 73.5 g K2Cr2O7 in distilled water and
make the volume to 1 litre.
2) 0.15 N sodium thiosulphate (Na2S2O3) solutions: Dissolve 37.5
g Na2S2O3 in water and dilute it to 1 litre.
3) Potassium iodide (KI) solution: 100 g of KI and 32 g of sodium
bicarbonate (NaHCO3) are dissolved in distilled water and
diluted to 500 ml.
Procedure:
Introduce exactly 50 mg dry sample conical flask of 250 ml
capacity. Add 8 ml of 1.5 N K2Cr2O7 followed by 16 ml concentrated
H2SO4. Simultaneously prepare a blank for each set. Mix well the
contents of the flask and set aside for 90 minutes with intermittent
shaking. Dilute the oxidised solution with distilled water, cool and make
upto 100 ml.
Withdraw a 10 ml aliquat from each flask and to it add 4 ml of KI
solution. Store in dark for 30 minutes, dilute with 20 ml distilled water
and titrate the liberated iodine with 0.15 N Na2S2O3 solution using starch
as an indicator. The excess dicromate present is calculated from the
titration figure and substracted from blank value to obtain the quantity of
1.5 N K2Cr2O7 used in the oxidation.
Determine the amount of 1.5 N K2Cr2O7 required for oxidation of 1
g sample and calculate the GE in KCal per g of sample using following
equation:
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ml 1.5 N K2Cr2O7 used to oxidise 1 g sample
GE (Kcal/g DM) = ------------------------------------------------------------
(23.39 – 0.069 P + 0.000226 P2)
Where P is the crude protein (CP) content in the sample expressed
as per cent of dry matter (DM).
POTASSIUM (K)
The acid soluble portion of ash was diluted and feed to flame
photometer atomizer.
Chemicals:
10 mEq/litre (1 mEq/litre = 39 ppm).
Dissolve 0.746 gms of pure dry KC1 in a litre of glass distilled water,
1) 200 mEq/litre (1 mEq/litre - 23 ppm) NSL. Dissolve 11.69 gms of
pure dry NaCl in a litre of glass distilled water.
Procedure:
Take 1 ml. of acid soluble portion, of ash in a measuring cylinder.
Dilute it to a volume of 25 ml with distilled water. Simultaneously feed
distilled water to atomizer and adjust the set F.S. control Aspirate the
standard mixed solution 1.7/0.8 mEq per litre on Na/K solution and wait
at least for 30 sec. Adjust set F.S. Control of Na side for a read out of
170 and that at the K-side for read out of 80. Repeat steps 4. 5, 6 and 7
(Flame photometer manual modi 127) until the reading are stabilized the
unit now stands calibrated. The pressure is 0 to 10 mEq/1 and power 230
V + 10% 50 Hz to be maintained. Now feed sample solution to the
atomizer to get the relative concentration wait at least for 30 sec before
taking the next reading.
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Establish the reading of standard stock solution of potassium by
preparing a standard graph containing 0.01 to 0.08 ml standard potassium
solution in series of reading.
Determine the amount of potassium on aliquat with the help of
standard graph and calculate the potassium content in plant sample
considering its amount taken for ashing volume of the acid soluble ash
and amount of aliquat used for the reaction.
TOTAL CARBOHYDRATES
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
monosaccharide’s which cannot be split by hydrolysis into more simpler
sugars. The carbohydrate content can be measured by hydrolysing the
polysaccharides into simple sugars by acid hydrolysis and estimating the
resultant monosaccharide’s.
Reagents:
- 2.5 N HCl
- Anthrone Reagent: Dissolve 200 mg anthrone in 100 ml of ice cold
95% H2SO4, Prepare fresh before use.
- Standard Glucose: Stock solution – Dissolve 100 mg glucose in
100 ml distilled water. Working standard – Dilute 10 ml of stock
solution to 100 ml with distilled water. Store in refrigerator after
adding a few drops of toluene.
Procedure
1 Weigh 100 mg of the sample into a boiling tube.
2 Hydrolyse by keeping it in a boiling water bath for three hours with
5 ml of 2.5 N + HCl and cool to room temperature.
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3 Neutralise it with solid sodium carbonate until the effervescence
ceases.
4 Make up the volume to 100 ml and centrifuge.
5 Collect the supernatant and take 0.5 ml aliquat for analysis
6 Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 ml of the
working standard. '0' serves as blank.
7 Make up the Volume to 1 ml in all the tubes including the sample
tubes by adding distilled water.
8 Then add 4 ml of anthrone reagent.
9 Heat for eight minutes in a boiling water bath.
10 Cool rapidly and read the green to dark green colour at 630 nm.
11 Draw a standard graph by plotting concentration of the standard on
the X-axis versus absorbance on the Y-axis.
12 From the graph calculate the amount of carbohydrate present in the
sample tube.
Calculation
Amount of carbohydrate present in l00 mg of the sample
mg of glucose
= ---------------------------------- x 100 (mg/100 mg)
Volume of test sample
PROTEIN
Protein can be estimated by different methods as described by
Lowry and also by estimating the total nitrogen content. No method is
100% sensitive. Hydrolysing the protein and estimating the amino acids
liberated can give exact quantification of protein. The method developed
by Lowry et al is sensitive enough to give a moderately constant value
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and hence largely followed. Protein content of enzyme extracts is usually
determined by this method.
Reagents:
— 2% Sodium Carbonate in 0.1N Sodium Hydroxide (Reagent A)
— 0.5% Copper Sulphate (CuSO4.5H2O) in 1% potassium sodium
tartrate
(Reagent B)
— Alkaline Copper solution: Mix 50 ml of reagent A and l ml of regent
B prior to use (Reagent C)
— Folin-Ciocalteau Reagent (reagent D) —Reflux gently for 10 hours a
mixture consisting of l00g sodium tungstate (Na2WoO4.2H2O), 25 g
sodium molybdate (Na2MoO4.2H2O), 700 ml water, 50 ml of 85%
phosphoric acid, and 100 ml of concentrated hydrochloric acid in a 1.5 l
flask. Add l50 g lithium sulfate, 50 ml water and a few drops of bromine
water. Boil the mixture for 15 min without condenser to remove excess
bromine. Cool, dilute to 1 L and filter. The reagent should have no
greenish tint. (Determine the acid concentration of the reagent by titration
with 1 N NaOH to a phenolphthalein end-point.)
— Protein Solution (Stock Standard)
Weigh accurately 50 mg of bovine serum albumin (Fraction V) and
dissolve it in distilled water and make up to 50 ml in a standard flask.
— Working Standard
Dilute l0 ml of the stock solution to 50 ml with distilled water in a
standard flask. One ml of this solution contains 200 µg protein.
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Procedure
Extraction of Protein from sample
Extraction is usually carried out with buffers used for the enzyme assay.
Weigh 500mg of the sample and grind well with a mortar and pestle in 5-
10 ml of the buffer. Centrifuge and use the supernatant for protein
estimation.
Estimation of Protein
1. Pipette out 0.2, 0.4, 0.6, 0.8 and l ml of the working standard into a
series of test tubes.
2. Pipette out 0.l ml and 0.2 ml of the sample extract in two other test
tubes.
3. Make up the volume to l ml in all the test tubes. A tube with l ml of
water serves as the blank.
4. Add 5 ml of reagent C to each tube including the blank. Mix well
and allow to stand for 10 min.
5. Then add 0.5 ml of reagent D, mix well and incubate at room temp
in the dark for 30 min. Blue colour is developed.
6. Take the readings at 660 nm.
7. Draw a standard graph and calculate the amount of protein in the
sample.
REDUCING SUGAR (RS)
The majority of methods for the determination of glucose are based
upon the ability of glucose in hot alkaline solution to reduce certain
metallic ions of which the cupric and ferric cyanide ions are most
commonly used. The following method was used for estimating water-
soluble reducing sugars.
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Procedure:
Transfers 2ml of the bark extract to a folin-Wu-sugar tube
graduated at 25 ml and to other similar tubes add 2 ml of standard sugar
solutions containing 0.2 to 0.4 mg of glucose respectively. To each tube
add 2 ml of the alkaline copper solution. The surface of mixture must
now have reached the constricted part of the tube. Transfer the tubes to
rapidly boiling water bath and heat for 8 minutes. Cool in running water
without shaking. To each tube add 2 ml of phosphomolybdic acid
reagent. After about 1 minute dilute to the mark with water and mix. It is
essential that adequate attention be given to this mixing because the
greater part of the blue colour is formed in the bulb of the tube. Transfer
the solution to suitable container and determine the O.D. at 420 mµ,
setting the photometer to zero density with a blank obtained by treating
2ml of water with alkaline copper reagent heating etc. Just as in the
analysis of the bark filtrate.
Reagents:
1) Standard sugar solution:
These standard sugar solution should be in hand (a) a stock
solution -1 per cent glucose made up in saturated benzoic acid solution
(b) a solution containing 2 mg of sugar in 1 ml (20 ml of stock solution
diluted 100 ml with water) (c) solution containing 0.2 and 0.4 of sugar in
2 ml made by dilution of (b) with water. The dilute standards are best
made up fresh a couple of times a week, Merck’s highest purify dextrose
is satisfactory.
Alkaline Copper Solution:
Dissolve 40 g of pure anhydrous sodium carbonate in about 400 ml
of water and transfer to a litre flask, add 7.5 g of tartaric acid and when
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the latter has dissolved add 4.5 gm of cystallised copper sulphate. Mix
and make up to a volume of 1 litre, if the chemicals used are not pure a
sediment of cuprous oxides may form in the course of 1 or 2 weeks.
If this solution happens remove the supernatant reagent with a
siphon or filter through a good quality filter paper. The reagent seems to
keep indefinitely. To test for the absence of cuprous copper in the
solution the deep blue colour of the copper should almost completely
vanish. In order to foreset all improper use of this reagent attention
should be called to the fact that if contains extremely little alkali, 2 ml by
filtration (using the falling of the blue copper filtration colour as
indicator) requiring only about 1.4 ml of normal acid.
3) Phosphomolybdic acid solution:
To 35 gm of molybdic acid and 5 gm of sodium tungstate add 200
ml of 10 per cent sodium hydroxide and 200 ml of water. Boil vigorously
for 20 to 40 minutes so as to remove nearly the whole of the ammonia
present in the molybdic acid. Cool, dilute to about 350 ml and add 125 ml
of concentrated (85%) phosphoric acid. Dilute to 500 ml.
For higher values of percentage glucose or with deeper cuvettes
carry out the analysis using less filtrate plus water to 2 ml and correct the
calculations accordingly.
TOTAL SUGAR
For total sugar 50 ml of the sample extract was acid hydrolised by
boiling with 5 ml 1 N HCL cooled and then 5 ml 1 N NaOH added and
followed the procedure of Reducing sugar.
EXTRACTIVE VALUES
Different plant species would obviously have different chemical
profile. Chemicals present in plant material could be dissolved in
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different solvent for the purpose of different analysis. There fore seven
solvents water, alcohol, benzene, petrolium ether, acetone, methanol,
chloroform were selected to determine the soluble substance.
Determination of Water - Soluble Extractive
10 gm of air dried bark, coarsely powdered was macerated with
100 ml of distilled water in a closed flask, for twenty four hours, shaking
frequently. Solution was filtered and 25 ml of Filtrate was evaporated in a
tarred flat bottom shallow dish, further dried at 100°C and weighed. The
percentage of water soluble extractive was calculated with reference to
the air dried bark.
Determination of Methanol-soluble extractive:
10 gm of air dried bark, coarsely powdered was soaked with 100
ml of methanol in a closed flask for twenty four hours with frequent
shaking. It was filtered rapidly, taking precautions against loss of
methanol. 25 ml of filtrate was then evaporated in the tarred flat bottom
shallow dish, dried at 100oC and weighed. The percentage of alcohol-
soluble extractive was calculated with reference to the air dried drug.
Determination of Alcohol - Soluble Extractive
10 gm of air dried bark, coarsely powdered was soaked with 100
ml of alcohol in a closed flask for twenty four hours with frequent
shaking. It was filtered rapidly, taking precautions against loss of alcohol.
25 ml of filtrate was then evaporated in a tarred flat bottom shallow dish,
dried at 100°C and weighed. The percentage of alcohol soluble extractive
was calculated with reference to the air dried bark.
Determination of Benzene-soluble Extractive:
10 gm of air dried drug, coarsely powdered was soaked with 100
ml of Benzene in a closed flask for twenty four hours with frequent
120
shaking. It was filtered rapidly, taking precautions against loss of
Benzene. 25 ml of filtrate was then evaporated in the tarred flat bottom
shallow dish, dried at 100oC and weighed. The percentage of Benzene-
soluble extractive was calculated with reference to the air dried drug.
Determination of Petroleum Ether - Soluble Extractive
10 gm of air dried bark, coarsely powered was macerated with 100
ml of ether in a closed flask for twenty four hours with frequent shaking.
It was filtered rapidly, taking precautions against loss of ether. 25 ml of
filtrate was then evaporated in a tarred flat bottom shallow dish, dried at
100°C and weighed. The percentage of ether soluble extractive was
calculated with reference to the air dried drug.
Determination of chloroform-soluble:
10 gm of air dried drug, coarsely powdered was soaked with 100
ml of chloroform in a closed flask. For twenty four hours with frequent
shaking. It was filtered rapidly, taking precaution against loss of
chloroform. 25 ml of filtrate was then evaporated in a tarred flat bottom
shallow dish, dried at 100oC and weighed. The percentage of chloroform
soluble extractive was calculated with reference to air dried drug.
Determination of acetone-soluble Extractive:
10 gms of air dried drug, coarsely powdered was soaked with 100
ml of acetone in a closed flask for twenty four hours with frequent
shaking. It was filtered rapidly, taking precautions against loss of acetone.
25 ml of filtrate was then evaporated in the tarred flat bottom shallow
dish, dried at 100oC and weighed. The percentage of acetone-soluble
extractive was calculated with reference to the air dried drug.
121
Determination of tolune Extractive:
10 gms of air dried drug, coarsely powdered was soaked with 100
ml of tolune in a closed flask for twenty four hours with frequent shaking.
It was filtered rapidly, taking precautions against loss of tolune. 25 ml of
filtrate was then evaporated in the tarred flat bottom shallow dish, dried at
100oC and weighed. The percentage of tolune-soluble extractive was
calculated with reference to the air dried drug.
Determination of Di ethyl ether -soluble:
10 gm of air dried drug, coarsely powdered was soaked with 100
ml of Di ethyl ether in a closed flask. For twenty four hours with frequent
shaking. It was filtered rapidly, taking precaution against loss of Di ethyl
ether. 25 ml of filtrate was then evaporated in a tarred flat bottom shallow
dish, dried at 100oC and weighed. The percentage of Di ethyl ether-
soluble extractive was calculated with reference to air dried drug.
Determination of Hexene- Soluble Extractive
10 gm of air dried bark, coarsely powered was macerated with 100
ml of Hexene in a closed flask for twenty four hours with frequent
shaking. It was filtered rapidly, taking precautions against loss of Hexene.
25 ml of filtrate was then evaporated in a tarred flat bottom shallow dish,
dried at 100°C and weighed. The percentage of Hexene soluble extractive
was calculated with reference to the air dried drug.
Qualitative analysis
ALKALOIDS
Alkaloids comprise the largest single class of secondary
metabolites. They are basic plant products having a nitrogen containing
heterocyclic ring system and high pharmacological activity, often used a
122
criterion in classification of only those groups of plants which contain
them. The presence of various types of alkaloids are used effectively in
classifying various taxa (Gibbs, 1974).
Alkaloids, as a rule are insoluble in water but soluble in organic
solvents. But their salts are soluble in water and insoluble in organic
solvents. Alkaloids are normally extracted from plants into weakly acids
(1N HCl or 10% acetic acid) or acidic alcoholic solvents and are then
precipitated with concentrated ammonia. They are also extracted into any
organic solvent after treating plant materials with a base. The bases free
the alkaloids and make them soluble in organic solvents. From the
organic solvents, the alkaloids are extracted into acidic solutions and
tested with specific reagents.
FLAVONOIDS
Flavonoids are polyphenols which include all the C6-C3- C6
compounds related to a flavone skeleton. The flavone may be considered
as consisting of (i) a C6-C3 fragment (phenyl propane unit) that contains
the 'B' ring and (ii) a C6 fragment with 'A' ring. Both these units are of
different biosynthetic origin. The flavonoids are subdivided on the basis
of oxidation level of C3 fragment of the phenyl propane unit, as
anthocyanidins, flavones, flavonols, chalcones, and aurones etc.
(Geissman, 1962). These pigments sometimes completely replace the
carotenoids as the yellow flower/ fruit pigments. Anthocyanidins are the
purple/ blue pigments while chalcones and aurones are yellow in colour.
Flavonols and flavones, though classified as colorless flavonoids, are
responsible for the white, cream, or ivory colors of the flowers.
Flavonoids have been one of the most exploited phytochemical
characters for classification of plants. The data on flavonoids are
123
incorporated with that from other disciplines during classification of
angiosperm (Dahlgren, 1980; Cronquist, 1981).
Different groups of flavonoids can successfully be correlated with
phyllogenetically significant morphological characters. Flavonols,
especially quercetin and myrcetin, as well as proanthocyanidins
characteristically occur in primitive woody plants, and they gradually
disappear from more advanced herbaceous families (Bate-Smith, 1962).
Flavonols which are phylogenetic markers are characteristic of leaves of
woody plants, being replaced by flavones in the leaves of herbaceous
species. Flavones appeared late during the course of evolution and
therefore are found mostly in advanced taxa. O-Methylation of flavones
is another advanced feature. Substitution of an extra hydroxyl group in
the 'A' ring of flavonoids seem to follow a similar pattern i.e. woody
plants have 8-hydroxy flavonols (e.g. Gossypetin) while herbaceous taxa
elaborate 6-hydroxy flavones (e.g. Scutellarein) (Harborne and Williams,
1971).
Most of the flavones and flavonols occur as water soluble
glycosides in plants. They are extracted with 70% ethanol or methanol
and remain in the aqueous layer, following partition of this extract with
solvent ether. Due to phenolic nature of flavonoids they change in colour
when treated with bases (especially Ammonia) and thus are easily
detected in chromatograms or in solutions. Flavonoids contain conjugated
aromatic systems and thus show intense absorption bands in UV and in
the visible regions of the spectrum. Single flavonoid aglycone may occur,
in a plant, in several glycosidic combinations and for this reason it is
considered better to examine the aglycones present in hydrolysed plant
extracts (Harborne, 1984).
124
Normally the flavonoids are linked to sugar by O-glycosidic bonds.
which are easily hydrolysed by mineral acids. But there is another type of
bonding in which sugars are linked to aglycones by c-c bonds. The latter
group of compounds, known as c-glycosides (glycoflavones) are
generally observed among flavones. They are resistant to normal methods
of hydrolysis and will remain in the aqueous layer when hydrolysed
extract is obtained with ether in order to remove aglycones.
Biflavones are flavone-dimers, mostly of apigenin and its
methoxylated derivatives, in which the two monomers are linked by c-c
linkages 8-8' (cupressoflavone), 5'-8' (amentoflavone). 5'6"
(robustaflavone) or 3-8" (hinokiflavone) or 4'-3" (ochnaflavone). In
addition to the flavone dimers, flavone-flavanone dimers (agathisflavone
and rhusflavone) also are reported, they occur as glycosides The
biflavones have a very restricted distribution reported from most of the
Gymnosperms except the Pinaceae, a few Pteridophytes (Psilotum.
Selaginella) and a few angiosperms (the Anacardiaceae, Caprifoliaceae,
Casuarinaceae). Their omnipresence in Gymnosperms may be correlated
with the primitive nature of the group and therefore in angiosperms the
biflavones are considered to be a primitive feature. The concept is not
questioned and it is suggested that the biflavonoids developed
independently in Gymnosperms and Angiosperms.
PHENOLICS
Phenolic compounds include a wide range of plant substances
which possess in common an aromatic ring bearing one or more hydroxyl
substituents. They include a wide range of organic compounds and are of
frequent distribution in plants. Much work has been done particularly by
Bate - Smith and Harborne on systematic distribution of these
125
compounds. They indicated utility of phenolics as reliable chemical
marker.
Phenolics are divided into two groups. Simple benzene derivatives
(Hydroxy benzoic acids, Cinnamic acids, Coumarins etc.) and
Flavonoids, which have C15 skeleton. Quinines and phenolic acids
belonging to first group and flavones, flavonols to the second group.
Pheonlic compounds have been selected for chemical screening of the
bark samples during present work.
SAPONINS
Saponins are glycosides which form emulsions with water and
possess marked haemolytic properties. They possess steroidal or
triterpenoid aglycones. The steroidal saponins are common in monocots,
while the triterpenoid saponins are found in dicots. Their taxonomic value
is less at higher levels of hierarchy although they may be used as useful
chemical characters at lower levels.
TANNINS
Tannins are polyphenols of high molecular weight which have the
property of combining with protein, forming water insoluble and non-
putrescible leather. Based on their reaction with mineral acids two main
types of tannins are recognized, the condensed tannins and the
hydrolysable tannins. The condensed tannins, which polymerize on
hydrolysis, universally occur in Ferns and Gymnosperms and are
widespread among the woody angiosperms. In contrast, hydrolysable
tannins, which get broken up to simpler units on acid treatment, are
limited to dicotyledonous plants and are found in a relatively few
families. Tannins are correlated well with other primitive characters and
thus the presence of these compounds is considered primitive. Between
126
the two groups, the hydrolysable tannins are advanced. The highly
advanced, herebaceous taxa are generally devoid of these compounds.
Condensed tannins or flavolans can be regarded as being formed
by the condensation of catechin or gallocatechin molecules and flavon-3,
4-diols to form dimers and higher oligomers with carbon-carbon bonds
linking one flavan unit to the next by 4-8 or 6-8 linkage. The name
proanthocyanidins is used alternately for condensed tannins because, on
treatment with hot acids, some of the carbon-carbon linking bonds are
broken and anthocyanidins are released. This property is used for the
detection of condensed tannins. Hydrolysable tannins are mostly
gallotannins and ellagitannins depending on whether gallic acid or ellagic
acid is present esterified with glucose. They yield the corresponding
phenolic acid and glucose on hydrolysis.
Thin layer chromatography:
Materials : Apigenin, Lupiol , Stigmasterol ( Sigma Aldrich )
TLC(Merck ), Methanol, Hexane Chlolroform, Ethyl acetate, ( Rankem ),
Methods:
Collection of samples:
The leaves Abutilon ranadei were collected from different
localities of Western Ghats. The materials were identified with the help
of floras and kept in BAMU herbarium Aurangabad. (MS) India. The
surfaces of collected materials were washed thoroughly by distilled water
and kept for air dry.
Extraction: The materials of the plants were pulverized into fine powder
by mixture. The fine powders of the samples were soaked in methanol
and kept overnight for extraction at 15 º c .The samples were centrifuge at
127
6000 rpm for 15 minutes above extraction process repeated until the
residues become colorless. The methanol of supernatants was removed by
rotary evaporator. The concentrated residues were kept at – 20 ºc for
further experiment
Fractionations: The residues of the samples dissolved in 10 % Methanol
and the fractionated by Chloroform and Ethyl acetate. Each fractions
were concentrated by rotary evaporator and residues dissolved in
methanol
ANTIMICROBIAL ACTIVITY
Antibacterial study
Organisms used for Antibacterial study:
Gram Positive
Bacillus licheniformis (PCSIR-B-252)
Bacillus subtilis (PCSIR-B-248)
Micrococcus luteus (NRRL-B-287)
Nocardia asteroids (NRRL-178)
Gram Negative
Escherichia coli (PCSIR-B-67)
Proteus mirabilis (ATCC 29245)
Salmonella typhimorium (ATCC 14028)
Standard Antibiotics
Benzyl penicillin, Ampicillin and Streptomycin were taken as
positive control (standard antibiotics) for bacterial species. The
concentration of the reference antibiotics was 1µg/mL. Distilled water
was used as negative control against all the species.
Sample Preparation
128
Extracts were made from air-dried coarsely powdered materials of leaf
powder of the plant [A.ranadei; leaves (2.0 kg), The plant parts were de
waxed separately with n-hexane at room temperature for 24 h. After
filtration, the hexane was removed in a rotary evaporator under reduced
pressure yielding the respective crude semisolid extracts. The defatted
residues were dried and macerated in methanol thrice at room
temperature for 10, 6, and 4 days. After filtration the three extracts were
combined and the solvent was removed in a rotary evaporator under
reduced pressure yielding the respective semidried crude extracts. Each
residual extract (leaves, stems and roots) was re dissolved in distilled
water to 1000, 2000, 3000, and 4000 µg/ml concentrations. Seed oil was
dissolved in DMSO.
Preparation of Nutrient broth
Nutrient broth (0.8 g) was dissolved in 100mL distilled water by
heating. Allow pH to 7.4 and then sterilized it in an auto clave at 121°C
for 15 minutes.
Preparation of Nutrient agar
Nutrient broth (0.8 g) was dissolved in 100mL distilled water by heating.
Then added 1.3g of nutrient agar in it and heated till clear solution was
prepared. Allow the pH to 7.4 and then sterilized it in an auto clave at
121°C for 15 minutes.
Preparation of Inoculum
Stock slants of bacterial culture were taken and a loop full of
culture was added to the sterilized slants in the test tube. The cultures
were incubated at 37°C for 24 hours. After that a loop full from these
cultures was transferred to conical flask of freshly prepared nutrient broth
129
and incubated for 24 hours at 37°C in a shaker. These cultures served as
inoculum.
Preparation of Petri – dishes
The bacterial species were maintained on nutrient agar slants. Molten
nutrient agar (20mL) was poured into sterilized Petri dishes as a basal
layer. Plates were inoculated with 5mL inoculum of the respective
organism. Put lids on the dishes. Allowed them to cool and solidify. The
agar core 4mm was then removed from the set agar at four peripheral
positions. The holes were aseptically filled with the extract
concentrations and reference standards. After keeping the Petri dishes in
the flat position for one hour, the incubation period was allowed to
proceed for 24hours at 37°C for bacterial cultures. The diameters of the
clear zones around the wells were observed and recorded. There were
triplicates for each dilution and standards. The results were recorded in
Table-20.
Measurement of mic
The minimum inhibitory concentration (MIC) was reported as the lowest
concentration of the compound capable of inhibiting the complete growth
of the bacterium being tested. MIC was determined graphically as an
extraplotation of linear relationship to zero value and results were
tabulated in Table-21.
RESULT AND DISCUSION
Plants were sampled on ten separate harvest dates. Significant
differences did not occur for any tissue dry weights. Unless otherwise
noted, values for tissue concentrations presented were significantly
different.
130
Physical evaluation:
1. DRY MATTER
The per cent dry matter ranged from as low as 65.5 % in
Abutilon ranadei leaf. The dry matter content varied with species as well
as organs of the same species under investigation. (Table-17).
2. BULK DENSITY Abutilon ranadei leaf powders had 579 mg/ cm3 bulk density
present (Table-17). In general the bulk density of leaf powder was higher.
3. CHEMICAL ANALYSIS
Quantitative analysis
NITROGEN
Nitrogen content in Abutilon ranadei leaf was with 3.33 % nitrogen,
(Table-17). Thus in general leaves of this plant were rich in nitrogen
content.
CRUDE PROTEIN The protein content in Abutilon ranadei leaves was 18.16 µ g/ml protein
was recorded (Table-17).
CRUDE FAT
Abutilon ranadei leaf it was recorded as 25.0 % (Table-17).
CRUDE FIBER
The leaf of Abutilon rnadei was 13.06 % crude fiber reported (Table -17).
TOTAL ASH
In Abutilon ranadei leaf the ash conents were 1.8 % reported. In general
the leaves were with high ash content as they were rich in nutrients
(Table17).
131
WATER SOLUBLE ASH
Abutilon ranadei the leaf was with 10.40 % water soluble ash.(Table -17).
Thus almost samples were found to be rich in water soluble inorganic
nutrients and minerals.
ACID SOLUBLE ASH
The percentage of acid soluble ash was reported from Abutilon ranadei
leaf 2.52 % (Table-17).
ACID INSOLUBLE ASH
The percentage of acid insoluble ash was reported from Abutilon ranadei
leaf 0.28 % (Table-17).
CALCIUM
The per cent calcium in the leaf of Abutilon ranadei was 5.5% reported.
(Table-17).
PHOSPHORUS
The phosphorus was recorded in Abutilon ranadei leaf 0.65 % percent of
phosphorus(Table -17).
MAGNESIUM
The per cent magnesium in the leaf of Abutilon ranadei was 0.93%
reported. (Table-17).
SULPHUR
The per cent sulphur in the leaf of Abutilon ranadei was 0.26% reported.
(Table-17).
GROSS ENERGY:
The gross energy ranges from 0.2593 Kcal which was expressed in
Abutilon randei leaf (Table -17).
132
POTASSIUM
The potassium percentage in the Abutilon ranadei leaf was 0.06
% recorded of potassium (Table-17).
TOTAL CARBOHYDRATES
The percentage of carbohydrate in the Abutilon ranadei leaf powder has
15.04 % percent carbohydrate (Table-17).
TOTAL REDUCING SUGAR
The percentage of reducing sugar 1.68 % was occurred in the stem of
Abutilon ranadei reducing sugars occur (Table -17).
TOTAL SUGAR
Abutilon ranadei leaf was 2.75 % total sugar recorded (Table -17).
Some elements were recorded by Tetali (2004). These elemnets are
follows and also mentioned table no. 19. Iron (Fe) 590, Manganese (Mn)
40, Zinc (Zn) 116, Copper (Cu) 22, Molybdenum (Mo) 1, Boron (B) 51
and Sodium (Na) 1984 ppm
Qualitative analysis
ALKALOIDS
Alkaloids present in only methanol extract in leaf sample of
Abutilon ranadei (Table-18).
FLAVONOIDES
Flavonoides present in leaf extract of all the solvent of Abutilon
ranadei (Table-18).
GLYCOSIDES
Glycosides present in only methanol extract in leaf sample of
Abutilon ranadei (Table-18).
133
PHENOLICS
Phenolics present in leaf extract of all the solvent of Abutilon
ranadei (Table-18).
SAPONIN
Saponin was present in all solvents of leaf extract of Abutilon
ranadei (Table-18).
STERIODES
Steriodes absent in all solvents of leaf extract Abutilon ranadei
(Table-18).
STEROLS
Sterols absent in all solvents of leaf extract Abutilon ranadei
(Table-18).
TANNINS
Tannins present in all solvent of leaf extract of Abutilon ranadei
(Table-18). GUMS
Gums present in the solvent of leaf extract of Abutilon ranadei
except in petroleum ether (Table-18).
Extractive value in Methanol
The Abutilon ranadei was a highest leaf extractive value of
Methanol solvent 15.5 %. The lowest extractive value was found in the
leaf sample of Petrolium ether 1.0 % (Table-19). Leaf samples extractive
values of ten solvents shown in table 19.
134
Table –17. Chemical composition of Abutilon ranadei leaf extract.
Sr. No. Parameter % mg/cm3 mg/ml K/cal ppm 1 Dry Matter (DM) 65.5 - - - - 2 Bulk Density - 579 - - - 3 Nitrogen (N) 3.33 - - - - 4 Crude protein (CP) - - 18.16 - - 5 Crude Fat (C Fat) 25.0 - - - - 6 Crude Fiber (C Fiber) 21.5 - - - - 7 Total Ash 1.8 - - - - 8 Water soluble ash (WSA) 10.40 - - - - 9 Acid soluble ash (ASA) 2.52 - - - - 10 Acid insoluble ash (AIA) 0.28 - - - - 11 Calcium (Ca) 5.5 - - - - 12 Phosphorus (P) 0.65 - - - - 13 Magnesium (Mg) 0.93 - - - - 14 Sulphur (S) 0.26 - - - - 15 Gross Energy (GE) - - 0.2593 - 16 Potassium (K) 2.08 - - - - 17 Carbohydrates 15.04 - - - - Protein 18 Reducing Sugar (RS) 1.68 - - - - 19 Total Sugar (TS) 2.75 - - - - 20 Iron (Fe) - - - - 590 21 Manganese (Mn) - - - - 40 22 Zinc (Zn) - - - - 116 23 Copper (Cu) - - - - 22 24 Molybdenum (Mo) - - - - 1 25 Boron (B) - - - - 51 26 Sodium (Na) - - - - 1984
135
Table -18. Preliminary Phytochemical screening of leaf Extract of Abutilon randei Woodr. & stapf.
Sr. No. Test Methanolic extract
Ethanolic extract Petrolium ether extract
1 Alkaloides - - -
2 Flavonoids + + +
3 Glycosides + - -
4 Phenolic compounds + + +
5 Saponins + + +
6 Steroids - - -
7 Sterols - - -
8 Tanins + + +
9 Gums + + -
136
Table -19. Extractive values of different solvents of Abutilon ranadei leaf extract
Sr. No. Solvent Extractive value %
1 Water 9.0
2 Methanol 15.5
3 Alcohol 4.5
4 Benezene 2.0
5 Petrolium ether 1.0
6 Chloroform 2.0
7 Acetone 5.5
8 Tolune 5.5
9 Di ethyl ether 5.5
10 Hexene 1.5
SE 0.94
CD 2.13
137
Thin Layer Chromatography (TLC)
Detection of Apegenin, Lupeol and Stegmasterol in methanolic extracts of
leaves of Abutilon ranadei
The apegenin, lupeol and stegmasterol are pharmaceutically very important
compounds and they are treated as a drug against many disorders. Lupeol showed
different medicinal properties such as antiprotozoal, antimicrobial,
antiinflammatory, antitumor and chemopreventive (Margareth et. al. 2009) and it
also an effective against models of prostate and skin cancers (Prasad et. al. 2008 ;
Nigam et. al. 2007 ; Saleem et. al. 2004). Also Apigenine possess monoamine
transporter activator property (Zhao et. al., 2010). The active principles in Abutilon
ranadei their medicinal property have been not studied. But other species
Pentacyclic Triterpene and flavonoid components in genus Abutilon isolated from
the leaves and their activity against bacteria (both Gram positive and Gram
negative) have been studied Muhammad et al. (2009); Muhit et al. (2010);
Paszkowski, W. L. and R. J. Kremer. (1573). In present study we have detected
three bioactive constituents such as Apegenin, Lupeol and Stegmasterol in
methanol extracts of leaves of Abutilon ranadei.
TLC was carried out on Merck silica gel 60 F254 plates (20 cm x 20cm).
Aliquots of a standard copmpounds in methanol ranging from 5 – 25 µg were
applied as spots at the origin on a plate. The Apigenin developed with toluene:
ethanol: formic acid (90:25:10) (Nikolova et. al., 2004). The Lupeol (Attarde et.
al., 2008), and Stigmasterol (Anjoo and Ajay, 2011) developed with toluene: ethyl
acetate: formic acid (9: 1: 0.1) in a pre-saturated chromatographic chamber.
Developed plates were dried in a stream of hot air (hair dryer) and visualized under
UV light. Apigenin showed blue fluoresces at 254-366 nm. For Lupeol and
Stigmasterol TLC plates were kept on iodine vapour in a chamber and brown
colored spots of Lupeol and Stigmasterols observed by naked eyes. The extracted
fractions (Benzene, Chloroform, Petrolium ether and Aqueous) from plant
138
materials applied on TLC with standard aliquots and same process carried out as
we discussed for standard compounds.
Detection of Apigenin.
Apigenin (4, 5, 7-trihydroxyflavone) is a flavones that is the aglycone of
several glycosides. It is a yellow crystalline solid that has been used to dye wool.
Medicinally it is very important because it inhibits pancreatic cancer cell
proliferation (Michael et. al., 2006). We have prepared methanol extract and
fractionated into different solvent with decreasing polarity. For the assessment of
compound we have developed TLC with standard marker (Apigenin). The apigenin
is a less polar compound and our result indicated that the apigenin extracted in
Benzene and Petrolium ether fractions. (Table –A-I). The some spots from these
fractions were traveled equal distance of standard apigenin (RF value = 0.5) and
observed faint yellowish by naked eyes. Under UV light it was clearly observed in
dark bluish spots. We have analyzed four fractions prepared from leaves, Abutilon
ranadei. The (Table-A-I) clearly indicates that apigenin available in leaves of
Abutilon ranadei. It is highly soluble in Benzene and Petrolium ether but it also
extracted in chloroform and water in case of leaves of Abutilon ranadei. (Table –A-
I).
Detection of Lupeol
Lupeol is a pharmacologically active triterpenoid found in a variety of
plants. We have analyzed the presence of Lupeol in all four fractions extracted
from leaves of Abutilon ranadei. The fractions such as Benzene, Chloroform,
Petrolium ether and water were used for qualitatively analysis of Lupeol with
standard marker. Our result indicated that the lupeol is a polar compound because
we have observed mostly in Benzene and chloroform less in water and totally
absent in petroleum ether (Table – A-II). The Rf value of lupeol was calculated and
it was 0.46. According to standard marker the lupeol is whitish in coloured and it
was not observed under UV light at 254-366 nm. It was observed brownish colored
139
when it kept under iodine vapors. By table no. A-II the lupeol available in leaves of
Abutilon raandei.
Detection of Stigmasterol
Stigmasterol is one of a group of plant sterol soluble mostly in organic
solvents and contains one alcohol functional group and insoluble in water.
Medicinally is used as the precursor of vitamin D3 and it also possesses potent
antioxidant, hypoglycemic and thyroid inhibiting properties (Panda et. al., 2009).
We have detected qualitatively this molecule from Abutilon ranadei. It was whitish
colored compound and it observed brownish colored when kept under iodine vapor.
The Rf value of stigmasterol was 0.45 when tlc developed in toluene: ethyl acetate:
formic acid (9: 1: 0.1) mobile phase. By table no. 25, 26 and 27 stigmasterol
detected in the plants and it mostly appeared in ethyl acetate and chloroform
fractions. Results observe mostly indicate the stigmasterol Petrolium ether and Water and
absent the Benzene and Chloroform showing table no. A-III.
Table–A. Detection of chemicals by TLC of Abutilon ranadei.
Apigenin Present ( + ), Apigenin Absent ( - ),Lupiol Present ( + + ), Lupiol Absent ( - - ), Stigmasterol Present ( +++ ) , Stigmasterol Absent ( - - - ).
Plant name
Plant part
Extracted Solvent Fraction
Name of Standard Used
Apigenin Lupiol Stigma sterol Abutilon ranadei
Leaves Benzene ( + ) ( ++ ) ( --- )
Chloroform ( - ) ( ++ ) ( --- )
Petrolium ehter ( + ) ( -- ) ( + + +)
Water ( - ) ( ++ ) ( +++ )
140
ANTIMICROBIAL ACTIVITY
Antibacterial Activity of plant extracts
The extractive values of different solvents were tabulated in Table 17. The
methanolic extract of A. ranadei was found to produce significant (P<0.001) anti
bacterial activity, than the other extracts, against the gram positive organisms like
Bacillus subtilis, Staphylo- coccus aureus, Sarcina leuka, Bacillus megatherium
and gram negative organisms like Escherichia coli, Pseudomonas aerugenosa,
Proteus vulgaris, Shigella sonnie, when compared with the standard antibiotics,
Penicillin potassium and Streptomycin sulphate are tabulated in Table 20 and 21.
The petroleum ether extract did not produce any significant antibacterial activity
(P>0.05) when compared with standards. Similar results of some articles of
Abutilon indicum antimicrobial and antibacterial activities, Aderotimi and Samue,
(2006), Adisakwattana et. al., (2009), Arulsamy, (2009), Attia, (1973), Dharmesh
et. al. (2010), Mehta, (1997) and Abutilon theophrasti Kremer, (1986).
The results of the agar-well diffusion method showed that the crude
methanolic extracts of A. ranadei. exhibits antimicrobial activity against the gram
positive organisms such as B. Subtilis, S. Aureus, S. Leuka, B. Megatherium and
gram negative organisms E. coli, P. Aerugenosa, P. Vulgaris, S. Sonnie with a
maximum diameter of zone of inhibition ranging from 23.3 mm followed by 19.4
upto and 17.1 mm, 20.5 mm, 21.4 mm, 20.0 mm, 23.5 mm and 21.1 mm
respectively. It has produced a comparable activity similar to the standard
antibiotics taken for the study.
Further, this study suggests that the isolation of the active principle
responsible for the activity will reveal one or more novel antibacterial agents.
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Table – 20. Antibacterial Activity of Leaf Extracts of Abutilon ranadei Gram Possitive Organisms
Table – 21. Antibacterial Activity of Leaf Extracts of Abutilon ranadei Gram Negative Organisms
Conclusion
Phytochemical study i.e. qualitative and quantitative analysis of chemicals
criteria can be used as to standardize the drug which used in preparation of
medicine. The phytochemical study of the plants part Abutilon ranadei having no
evaluation of medicinal properties. Therefore, economic use depends partially on
the quantitative and qualitative aspects of there organic reserves, specially during
these studies, leaves of A. ranadei were subjected to proximate analysis. The
results obtained were; dry matter (65.5%), bulk density (579mg/cm3), nitrogen
(3.33%), crude protein (18.16mg/ml), crud fat (25.0%), crude fiber (21.5%), total
ash (1.8%), water soluble ash (10.40%), acid soluble ash (2.52%), acid insoluble
Sr. No.
Name of organism
Agar-well Diffusion (Zone of Inhibition in mm) Pet. ether
Acetone Hexene Methanol Water Penicillin
1 B. Subtilis -- 10.0 11.0 23.3 12.5 24.1
2 S. Aureus -- 13.5 10.2 19.4 19.5 23.0
3 S. Leuka 10.2 9.0 7.2 17.1 11.3 23.6
4 B. Megatherium 10.8 8.0 11.0 20.5 13.0 22.5
SE 3.03 1.20 0.90 1.29 1.84 0.35
CD 9.65 3.81 2.87 4.09 5.86 1.11
Sr. No.
Name of organism
Agar-well Diffusion (Zone of Inhibition in mm) Pet. ether Acetone Hexene Methanol Water Streptomycin
1 E. Coli -- 12.2 13.5 21.4 11.0 23.0
2 P. Aerugenosa -- 11.4 10.2 20.0 12.2 23.4
3 P. Vulgaris -- 13.5 10.2 23.5 10.3 22.5
4 S. Sonnie 10.0 11.0 11.0 21.1 10.2 24.3
SE 2.50 0.55 0.78 0.73 0.46 0.38
CD 7.95 1.75 2.48 2.33 1.47 1.21
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ash (0.28%), calcium (5.5%), phosphorus (0.65%), magnesium (0.93%), sulphur
(0.26%), Gross Energy (0.2593K/cal), potassium (2.08%), carbohydrates
(15.04%), reducing sugar (1.68%), total sugar (2.75%). The some elements was
also analyzed by (Tetali et al. 2004) iron (590ppm), manganese (40ppm), zinc
(116ppm), copper (22ppm), molybdenum (1ppm), boron (51ppm) and sodium
(1984ppm).
The antimicrobial activities of the crude extracts of leaves plant parts of A.
ranadei were checked against three Gram-negative bacteria, four Gram-positive
rods using agar diffusion method. These primary phytochemical studies of the
Abutilon ranadei.
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