CHAPTER -6 CHEMICAL STANDARDS

6.1 Estimation of Total Phenolics

Phenols, the aromatic compounds with hydroxyl groups are widespread in plant
kingdom. They occur in plant parts. Phenols are said to offer resistance to diseases
and pests in plants. Phenols include array of compounds like tannins, flavonols, etc.
Total phenol estimation can be carried out with the Folin-Ciocalteau reagent. (Makkar
et al., 1993,)

Principle:
Phenols react with phosphomolybdic acid in Folin-Ciocalteau reagent in alkaline
medium and produce blue coloured complex (molybdenum blue) which was measured
spectroscopically at 765nm against a reagent blank after an hour.

Materials:
 70% Acetone extract, methanolic extract, hydro alcoholic extract.
 Folin-Ciocalteau reagent
 20% Sodium carbonate solution
 Gallic acid (as a standard): Stock solution -100 mg/ml.

Preparation of test solution:

1 gm of leaves powder was grounded with 10-time volume of 70% Acetone with the
help of pestle and mortar. The homogenate was centrifuged at 6,000rpm for 20 min.
The supernatant was saved. The residue was re-extracted with 5-times the volume of
70% Acetone centrifuged and pooled the supernatants. The supernatant was
evaporated to dryness to obtain residue which was (10mg) dissolved in 10ml of
distilled water (1000µg/ml) of the above solution was pipetted out 2.5ml in a 25ml
of volumetric flask gives final concentration upto (100 µg/ml.) from this different
dilutions are made.

Procedure:
1. Various concentrations (10 µg/ml-50 µg/ml) of gallic acid were prepared from
stock solution and (2.5-12.5) ml was transferred in each 25ml volumetric flask.
2. 1.5 ml of Folin-Ciocalteau reagent was added to test tubes containing standard
solutions and test solution.

62
CHAPTER -6 CHEMICAL STANDARDS

3. After 3 min, 4ml of 20% Sodium carbonate solution was added to test tubes
containing standard solutions and test solution.
4. The volume was made upto 25ml with distilled water.
5. The solutions were mixed thoroughly and absorbance was measured at 765 nm
against a reagent blank after an hour.

Table: 20. Estimation of Total phenolics.

Concentration Absorbance
Sr.no.
(MG/ML) (NM)
1. 10 0.256
2. 20 0.362
3. 30 0.468
4. 40 0.569
5. 50 0.640
6. 70 % Acetone Ext. 0.218
7. Methanolic Ext. 0.296
8. Aqueous Ext. 0.193

Gallic acid calibration curve

0.7
0.6
Absorbance (nm)

0.5
0.4 Gallic acid
0.3 Standard
0.2
0.1
0
1 2 3 4 5
concentration (mcg/ml)

r2 = 0.9962
Graph 1: Calibration curve for gallic acid

63
CHAPTER -6 CHEMICAL STANDARDS

Result:

The amount of total phenolics in Bridelia retusa Leaves was found to be 0.979-1.10
% w/w.

6.2 Estimation of Total Tannins.

Tannins and tannin like substances are widespread in nature and are probably present
in all plant materials. These are polyphenolic compounds divided into two main
groups-hydrolysable and condensed. Hydrolysable tannins contain a polyhydric
alcohol usually, if not always, glucose esterified with gallic acid or with
hexahydroxydiphenic acid. Condensed tannins are mostly flavan-3-ol (catechin) and
these cannot be hydrolysed to simple components. Total tannins were determined by
two methods which are as follows (Anon, 2002)

1) Determination of Total Tannins by Titrimetric Method:

Reagents

1. Indigosuphonic acid solution-

Dissolve Indigo carmine AR (1.0 g) in concentrated suphuric acid AR (50 ml).
continue adding small quantities of the above mixture to chilled water in a
volumetric flask taking care to mix the content of the flask thoroughly. Make
up the volume with water.
2. (0.1 N) Potassium permanganate solution-
Weigh accurately about 15.8 g of KMnO4 and dissolve in 900 ml distilled water.
Then heat for half hour and filter. Make the volume up to 1000ml.
Procedure:
1. Weigh accurately about 1 g of powdered drug and introduce in a (250ml) glass
stoppered flask, add water (100ml), shake for 1 hour and keep overnight.
2. Allow the solid material to settle and filter the liquid through a 12 cm diam. filter
paper, discarding first 20 ml of the filtrate.
3. Transfer 10 ml of the filtrate to a conical flask (1.0 liter), add water (750 ml) and
Indigosuphonic acid solution (25 ml).

64
CHAPTER -6 CHEMICAL STANDARDS

4. Finally titrate with 0.1 N potassium permanganate solution and shake vigorously
till a golden-yellow end- point (T2) is reached. Perform a blank determination (T1)
and make necessary correction.
Factor-
Each ml of 0.1 N potassium permanganate is equivalent to 0.004157 g of total
Tannins.

Calculation-

Total tannins can be calculated with the help of following formula.

[(T2-T1) x actual normality x 0.004157 x 1000]

Quantity of total tannins (%) = ----------------------------------------------------------
W x 0.1

Where, W = the weight of plant material in g.

Result:
The percentage of total tannins in Bridelia airyshawii leaves was found to be 18.73
% w/w.

6.4 Determination of Crude fiber content (Khandelwal K.R., 2000)

Procedure
5gm of accurately weighed fat free and moisture free bark powder was transferred to a
500ml beaker and to it 200ml of boiling sulphuric acid (0.255N) was added. The
mixture was boiled for about 30 minutes. The volume of the mixture was kept
constant with addition of water at frequent intervals. At the end of this period, mixture
was filtered through a muslin cloth and the residue was washed with hot water till it
becomes acid-free. The residue was transferred to the same beaker and 200ml of
sodium hydroxide (0.313N) was added to it. The mixture was boiled for 30minutes;
water was added to the mixture at-times to keep the volume constant. At the end of
this period, mixture was filtered through a muslin cloth and the residue was washed

65
CHAPTER -6 CHEMICAL STANDARDS

with hot water until it becomes free from the alkali. The residue was washed with
water and transferred to a crucible. The residue was dried overnight at 80-100 0C and
weighed (We). The crucible was heated in muffle furnace at 600 0C for 3 hours, cooled
and weighed (Wa).
We-Wa=Crude fibres.

Total crude fibre was calculated by the formula:

Result:
The crude fiber content of leaves of Bridelia airyshawii was found to be 1.7% w/w.

6.5 Determination of Ellagic Acid 70% Acetone Extract From Leaves

of Bridelia airyshawii. (Hagerman And Inoue 1998)

Reagents –
1. Sulphuric acid (2N): weigh accurately 5.4 ml of sulphuric acid
10 mg of Bridelia retusa (70% Acetone Extract) in 2N sulphuric Acid (1ml) were put
into constricted Vial seal the vial and frozen. The Vial were Vaccum walled and
heated for 24 hours at 100oc vial were cooled opened and the filtered the content made
upto 10ml with pyridine with mixture in dry test tube .then 1.1ml of pyridine and 1ml
of sample was quickly mixed after 0.10ml of HCL and mixing. The sample was
brought to 300c sodium nitrite (NaNO2) in water was added the absorbance at 538nm
was immediately recorded. After 36min incubating period at 30 oc the absorbance was
again recorded. The difference between the initial absorbance and the absorbance at
36min (at 538nm) was proportional to ellagic acid concentration. The measured
absorbance obeys the relationship for hydrolysed sample (Ellagic Acid Content)

66
CHAPTER -6 CHEMICAL STANDARDS

Absorbance of Sample = (0.0074 X [C X mg of Ellagic Acid] – 0.0296

Result
1. The (Hydrolysed) Ellagic Acid Content of Leaves of Bridelia airyshawii as found
to be 0.9 % w/w.
2. The (Free) Ellagic Acid Content of Leaves of Bridelia airyshawii was found to be
0.2 % w/w.

6.6: Estimation of Protein- Precipitable Phenolics (Makkar et. al)

Methods for quantification of tannins may be based on the chemical properties of
tannins or their capability to bind substrates, particularly proteins. Tannins are the
naturally occurring polyphenolics compounds of high enough molecular weight to
form a complex with proteins.

Principle
It is based on the formation of tannin -protein complexes (tannins in the plant extracts
and the protein, bovine serum albumin). Tannins present in the complex are
determined by using ferric chloride assay for total phenolics. Iron forms a complex
with the phenols to give pink colour or chromophore which is measured
spectriphotometrically.

Materials:
70% acetone extract.
Acetate buffer (Ph 4.8-4.9 0.2M)
Sodium Dodesyl Sulphate (1% w/v)
SDS-Triethanolamine (TEA) (1% SDS (w/v) and 7% Triethanolaamine (v/v)).
Ferric chloride reagent (0.01M in 0.1M HCL).
Glacial acetic acid.
BSA solution

67
CHAPTER -6 CHEMICAL STANDARDS

Reagents:
Acetate buffer : (Ph 4.8-4.9 0.2M) pipette out 11.40 ml of glacial acetic acid to about
800 ml of distilled water. Adjust ph of this solution to 4.8-4.9 with 4 N sodium
hydroxide solution, and bring the final volume to 1 litre. To it add 9.86 g of sodium
chloride to make its concentration 0.17M.
Sodium Dodesyl Sulphate (1% w/v) : dissolve 1 g of Sodium Dodesyl Sulphate in
100 ml of distilled water.
SDS-Triethanolamine (TEA) (1% SDS (w/v) and 7% Triethanolaamine (v/v)in
distilled water) solution : to 7 ml of Triethanolamine add 93 ml of distilled water and
dissolved 1g of SDS in this solution.
Ferric chloride reagent (0.01 M ) for making 0.1M HCL dilute 4.2 ml of concentrated
HCL (37%) to 500 ml with distilled water. Dissolved 0.81 g ferric chloride in 500 ml
of 0.1 M HCL. Filter and store the content in brown bottle.
Glacial Acetic acid
BSA solution : Dissolved 100 mg BSA in 100 ml of acetate buffer.

FORMATION OF TANNIN (PHENOLICS) IN TANNIN-PROTEIN COMPLEX

To 2ml of BSA solution add 50% methanol and increasing level of tannin containing extract
to make 3ml.
Preparation tannin-protein complex

Sr.no BSA 50% Methanol Extract

1 2 ml 0.95 ml 0.05 ml
2 2 ml 0.90 ml 0.10 ml
3 2 ml 0.85 ml 0.15 ml
4 2 ml 0.80 ml 0.20 ml
5 2 ml 0.75 ml 0.25 ml
6 2 ml 0.70 ml 0.30 ml

This may vary according to amount of tannins in sample. vortex the contents. Aloe the
mixture to stand in refrigerator (40c) overnight. Centrifuge at about 3000rpm for 10
min Remove the supernant carefully without disturbing precipitate. Add 1.5 ml of 1%
SDS solution to the precipitate and vortex it to dissolve the precipitate.

68
CHAPTER -6 CHEMICAL STANDARDS

DETERMINATION OF TANNIN (phenolics)

In Tannin – protein complex Take an aliquot of 1 ml of above dissolved complex.
add 3 ml of SDS-TEA solution. Then add a 1 ml portion of the ferric chloride reagent.
Record the absorbance at 510 nm after 15-30 min. convert the absorbance to tannic
acid equivalent using a standard curve. Multiply the values obtained by 1.5 to
obtained tannins in the complex. Draw the linear regression line between tannins
precipitated as tannic acid equivalent and mg leaf (in aliquot taken for the assay). The
slope of the curve (mg of tannic acid precipitated /mg of leaf, let it be X) represent
protein- Precipitable phenolics in the sample.

Result : The protein Precipitable phenolics of leaves of Bridelia airyshawii was found
to be 1.46 %w/w

69