ESTIMATION OF TOTAL CARBOHYDRATE

The total carbohydrate content was estimated by the method of Hedge and Hofreiter, 1962.

Reagents 1. Glucose stock standard: 100 mg of glucose was dissolved in 100 ml of water in a
standard flask.

2. Working standard: 10 ml of the stock was diluted to 100 ml. 1.0 ml of this solution contains 100µg
of glucose.

3. Anthrone reagent: 0.2% anthrone was dissolved in ice cold concentrated sulphuric acid. Prepared
fresh before use

4. 2.5 N HCl.

Procedure

weighed 100mg of the sample into a boiling tube, hydrolysed by keeping it in a boiling water bath for
three hours with 5.0 ml of 2.5 N HCl and cooled to room temperature. Neutralized it with solid
sodium carbonate until the effervescence ceas made up the volume to 100 ml and centrifuged,
collected the supernatant and take 0.2 to 1.0 ml for analysis. Prepared the standards by taking 0.2-1.0
ml of the working standards. 1.0 ml of water serves as a blank made up the volume to 1.0 ml in all the
tubes with distilled water, then added 4.0 ml of anthrone reagent, heated for eight minutes in a boiling
water bath, cooled rapidly and read the green to dark green colour at 630 nm.

Calculation

A standard graph was drawn by taking the concentration of glucose on X axis and spectrophotometer
reading on Y axis. From the graph the concentration of glucose in the sample was calculated.

ESTIMATION OF PROTEIN BY LOWRY’S METHOD

Principle: The blue colour developed by the reduction of the phosphomolybdic phosphotungstic
components in the Folin –ciocalteau reagent by the amino acids tyrosine and tryptophan present in the
protein plus the colour developed by the biuret reaction of the protein with the alkaline cupric tartrate
are measured in the Lowry’s method.

Reagents: i. Folin –ciocalteau reagent (reagent D

ii. 0.5% Copper Sulphate (CuSO4.5H2O) IN 1% potassium sodium tartrate (Reagent B).

iii. Alkaline copper solution.: Mix 50ml of A and 1ml of B prior to use (Reagent C)
iv. Protein Solution (Stock Standard): Weigh accurately 50mg of bovine serum albumin (fraction V)
and dissolve in distilled water and make up to 50ml in a standard flask.

v. Working Standard Solution: Dilute 10ml of the stock solution to 50ml with distilled water in a
standard flask. 1.0ml of this solution contains 200µg protein.

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 pestle and mortar in 5-10mL 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 1.0ml of the working standard into a series
of test tubes.

2. Pipette out 0.1 ml and 0.2 ml of the sample extract in two other test tubes.

3. Make up the volume to 1.0 ml in all the test tubes. A tube with 1.0ml of water serves as the blank.
4. Add 5.0 ml of reagent C to each tube including the blank. Mix well and allowed to standing for
10mins.

5. Then add 0.5 ml of reagent D, Mix well and incubate at room temperature in the dark for 30min,
blue colour is developed. Take the reading at 660nm.Draw a standard graph and calculate the amount
of protein in the sample.

ESTIMATION OF TOTAL PHENOLS

The amount of total phenols in the plant tissues was estimated by the method proposed by Mallick
and Singh (1980).

Principle Phenols react with phosphomolybdic acid in Folin-Ciocalteau reagent to produce a blue-
coloured complex in alkaline medium, which can be estimated spectrophotometrically at 650nm
Reagents 1. Ethanol (80%) 2. Folin-Ciocalteau reagent (1N) 3. Sodium carbonate (20%) 4. Standard
gallic acid solution (100μg/ml in water)

Procedure

The sample (0.5g) was homogenized in 10X volume of 80% ethanol. The homogenate was
centrifuged at 10,000rpm for 20 minutes. The extraction was repeated with 80% ethanol. The
supernatants were pooled and evaporated to dryness. The residue was then dissolved in a known
volume of distilled water. Different aliquots were pipette out and the volume in each tube was made
up to 3.0 ml with distilled water. Folin- Ciocalteau reagent (0.5ml) was added and the tubes were
placed in a boiling water bath for exactly one minute. The tubes were cooled and the absorbance was
read at 650nm in a spectrophotometer against a reagent blank. Standard gallic acid solutions (0.2-1ml)
corresponding to 2.0-10μg concentrations were also treated as above. The concentration of phenols is
expressed as mg/g tissue.

Determination of Flavonoids
The approach was used to calculate the phenol content (Jia et al., 1998). Before collecting the
supernatant, 100 mg of material is weighed, homogenised with 2 milliliters of methanol, and
centrifuged at ten thousand rpm for 10 minutes. Up to 1.5 ml of supernatant was created
using distilled water, and 75 l of 5% NaNO2 was added. After waiting 5 minutes, 150 l of
10% AlCl3 was introduced to the mixture. It took five minutes for the combination to reach
room temperature. The absorbance was determined in relation to a reagent blanks with the
addition of 0.49 ml of 1M NaoH at 510 nm. For express the findings, milligrammes of rutin
equivalents (RE) per gramme of extract were employed.