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

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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.

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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

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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.

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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).

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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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