49

4.1) Parameters and sampling frequency

At the height of two to three meters, fully expanded mature leaves were

collected from each plant in the polythene bags and transported to the

laboratory. The leaf samples were collected on seasonal basis and this

frequency was strictly maintained throughout the year (November 2009 to

October 2010).

The following investigations were carried out in all the five plants Ficus

religiosa, Ficus benghalensis, Ficus glomerata, Azadirachta indica and

Polyalthia longifolia.

Dust fall on leaves was studied seasonally (winter, summer and rainy).For

photosynthetic study the chlorophyll pigments (Chlorophyll a, chlorophyll b,

and total chlorophyll) were studied and biochemical changes in leaves (Starch,

phenols, and sugars- total, reducing and non-reducing) were studied on seasonal

basis. Air pollution tolerance index and enzymatic activities i.e protease,

catalase, peroxidase and invertase were studied in winter season.

Experiment

Physiological Metabolism

Dustfall Chlorophyll APTI Biochemical Enzymatic

50

Biochemical Enzymatic

Sugars Phenol Starch Protease Oxidase Invertase

Total Reducing Non-reducing Catalase Peroxidase

4.2) Method of measuring dust fall, chlorophyll pigments (total, a,

and b) and air pollution tolerance index (APTI).

A: Measurement of dust falls on the leaves

From each plant, ten matured leaves were collected in the separate polythene

bags during winter, summer and rainy from November 2009 to October 2010.

Leaves were collected at the height of three to four meters from all the sites.

For dust fall measurement, the method of Dry technique described by Das and

Pattanayak (1997) was followed. In this technique first the intact leaf was

weighted (in mg) then dust particulates from leaf surfaces were gently collected

with the help of camel hair brushes and the weight of leaf was measured again.

The amount of dust – deposition in mg/cm2 was calculaed as:-

Weight of intact leaf- initial weight of leaf

Dust content (mg/cm2) = -----------------------------------------------------

Total surface area of leaf (cm2)

51

B: Measurement of chlorophyll pigments

The chlorophyll pigments in the leaves were estimated following the method of

Arnon (1949).

The fully expanded leaves from all the sites were collected in the poly-thene

bags and transported to the laboratory. The leaves were washed out thoroughly

with distilled water. Three replicates were used for each plant.

Weighted fresh leaf material was homogenized and extracted thrice in chilled

80% acetone (v/v). The volume of the acetone extract was made up to a known

one and the optical density was read at 645nm and 663nm wavelengths on a

spectrophotometer. The concentration of the chlorophyll pigments was

calculated using the following formula and the results are expressed in mg/g

fresh weight.

Chlorophyll a = [(12.7 X OD at 663) – (2.69 X OD at 645)] X dilution factor

Chlorophyll b = [(22.9 X OD at 645) – (4.68 X OD at 663)] X dilution factor

Total chlorophyll = [(20.2 X OD at 645) – (8.02 X OD at 663)] X dilution

factor.

C: Measurement of Air Pollution Tolerance Index

Air pollution tolerance index (APTI) was determined by the method given by

Singh and Rao, 1983. The samples were estimated for Leaf-extract pH (Singh

and Rao, 1983), relative moisture content (Wealtherly, 1965), total chlorophyll

(Arnon, 1949) and ascorbic acid (Abida begum et al., 2010). The fully

expanded leaves from all the sites were collected in the poly-thene bags and

transported to the laboratory. The leaves were washed out thoroughly with

distilled water. Three replicates were used for each plant.

52

Estimation of Leaf-extract pH (Singh and Rao, 1983): 0.5 g of leaf material was

ground to paste and dissolved in 50 ml of distilled water and Leaf-extract pH

was measured by using calibrated digital pH meter.

Relative moisture content (Wealtherly, 1965): Estimation of relative moisture

content: Fresh leaf samples collected from the study area and were brought

immediately to the laboratory and washed thoroughly. The excess water was

removed with the help of filter paper. The initial weight of samples were taken

(W1 g) and kept in oven at 600 oC until constant weight was obtained and the

final weight was taken (W2 g).

Total Chlorophyll content was measured by the method of Arnon (1949) as

mentioned above.

Ascorbic acid content (AA) (mg/g) was measured using spectrophotemetric

method. 1 g of the fresh foliage was put in a test-tube, 4 mL oxalic acid - EDTA

extracting solution was added, then 1 mL of orthophosphoric acid and then 1

mL 5% tetraoxosulphate(VI) acid added to this mixture, 2 mL of ammonium

molybdate was added and then 3 mL of water. The solution was then allowed to

stand for 15 minutes. After which the absorbance at 760 nm was measured with

a spectrophotometer (Abida Begum and Krishna, 2010).

APTI given as:

APTI = [AA (T + P) + R]

10

(Where AA is the ascorbic acid in mg/g, T is the total chlorophyll in mg/g, P is

pH of leaf sample and R is relative water content in mg/g).

53

4.3) Method of measuring sugar (total, reducing and non-

reducing), phenol and starch.

A: Measurement of Sugars (Nelson, 1944)

Principle: Monosaccharide readily reduces oxidizing agents such as Ferric

cyanide, hydrogen peroxide or cupric ions (Cu++

). In such reactions the sugar is

oxidized at carbonyl group and the oxidizing agent becomes reduced glucose or

other sugars capable of reducing oxidizing agents are called reducing sugars.

Thus by measuring the amount of oxidizing agent that is reduced by a sugar

solution, it is possible to estimate the sugar concentration. The method involves

the reduction of cupric ions (Cu++

) to cuprous ions (Cu+) which in alkaline

solution and forms yellow cuprous hydroxide, which in turn is converted by

heat of the reaction to insoluble red cuprous oxide (Cu2O).

The amount of Cu2O formed can be increased by adding arsenomolybdic acid

which in turn is reduced to lower oxides of molybdenum by Cu2O. The

coloured complex produced is known as molybdenum blue. The intensity of the

colour is related to the concentration of the reducing sugars in the sample.

Procedure: 100mg plant material was weighed and homogenate with 10ml

80% ethanol. It was centrifuged for 10rpm for 10minutes. Supernatant 1 was

collected; while 10 ml 80% ethanol was added again to the residue, centrifuge it

and supernatant 2 was mixed with supernatant 1. Residue was discarded.

Total sugars: To 1ml alcoholic aliquot, 1ml 1N H2SO4 was added and heated

at 490 C in water bath for 30 minutes for hydrolysis of the mixture. 1-2 drop of

methyl red indicator was added. 1N NaOH was added drop wise for the

neutralization (colour was to yellow from pink). 1ml Nelson Somogyi’s reagent

was added to it and the tube was kept in boiling water bath for 20 minutes.

After cooling of the test tube, 1ml arsenomolybdate was added and final

54

volume was made up to 20ml with DW. O.D. was noted at 540nm. Blank was

prepared in the same manner.

Reducing sugars: To 1ml alcoholic aliquot, Nelson Somogyi’s reagent was

added and kept in boiling waterbath for 20min. After cooling of the test tube,

1ml arsenomolybdate was added and final volume was made upto 20ml with

DW. O.D. was noted at 540nm. Blank was prepared in the same manner.

Non-reducing sugar= Total sugar – Reducing sugar.

The result was expressed as mg/gm plant material.

Preparation of reagents:

1.80%Ethanol: 80ml Ethanol was diluted up to 100ml DW.

2.1N Sulphuric acid (H2SO4): 2.77ml conc. H2SO4 (95-98%) was diluted up to

100 ml with DW.

3. 0.1N Sodium hydroxide (NaOH): 4gm NaOH was dissolved up to 100ml

with DW.

4. Methyl red indicator (1N): 0.1gm Methyl red powder was dissolved in 5ml

0.02M NaOH and final volume was made up to 250ml with DW.

5. Nelson Somogyi’s reagent:

Nelson A: 12.5gm Na2CO3, 12.5gm Na-K-tartrate, 10gm NaHCO3, 100gm

Na2SO4 were dissolved one by one and final volume made up to 50ml with

DW.

Nelson B: 15gm CuSO4.7H2O dissolved up to 100ml with DW.

Nelson Somogyi’s reagent: 50ml Nelson A and 1ml Nelson B were mixed.

6. Arsenomolybdate reagent: 25gm Ammonium molybdate was dissolved in

450ml DW, 21ml conc. H2SO4 was added to it. 3gm Sodium arsenate was

dissolved in 25ml DW and both solutions were mixed. It was incubated at 350C

for overnight before use of it.

55

CHART- 4.3(a): FLOW CHART FOR TOTAL SUGARS AND

REDUCING SUGARS

100 mg plant material was weighed

Homogenized with 10 ml 80% ethanol

Centrifuged at 5,000- 10,000rpm for 10 minutes

Supernatant I Residue +10ml 80% ethanol

Centrifuged at 5000-10000g for 10 minutes

Supernatant II Residue

Supernatant I+II

Total sugar Reducing sugar

1 ml aliquot + 1ml aliquot +

1 ml 1 N H2SO4 1ml Nelson Somogyi’s reagent

Incubated in water bath Incubated in boiling

at 490 C for 30 min. water bath for 20min

Add 1-2 drop of methyl red indicator Add 1ml Arsenomolybdate

Add 1N NaOH drop wise Final vol. made upto 20ml

for neutralization with D.W

(Colour change Pink to Yellow) OD was noted at 540 nm

1ml Nelson Somogyi’s reagent

Incubate for 20mins in boiling water bath

Add 1ml Arsenomolybdate

Final volume was made up to 20ml with D.W.

O.D. at 540nm.

56

The readings were compared with a standard which was prepared by using

different concentration of glucose. The results were expressed as mg/g plant

material.

B: Measurement of Total phenols (Bray et al., 1954)

Principle: Estimation of phenols using Folin- Ciocalteu’s reagent is based on

the reaction between phenols and an oxidizing agent phosphomolybdate which

results in the formation of a blue complex (Bray et al., 1954).

Procedure: 100mg plant material was weighed and homogenate with 10ml

80% ethanol. It was centrifuged at 5000-10,000rpm for 10minutes. Supernatant

1 was collected; while 10 ml 80% ethanol was added again to the residue,

centrifuge it and supernatant 2 was mixed with supernatant 1 and used for

estimation. Residue was discarded.1ml alcoholic aliquot was mixed with 1ml

20% Na2CO3 and 0.5ml Folin-Ciocalteau’s reagent. It was boiled for 10minutes

at 1000C in water bath. Final volume was made up to 20ml with DW and O.D.

was noted at 660nm. PPT were filtered or centrifuged before reading. Blank

was prepared in the same manner.

The result was expressed as mg/gm plant material.

Preparation of reagents:

1. 80%Ethanol: 80ml Ethanol was diluted up to 100ml DW.

1. 20% Sodium carbonate (Na2CO3):20gm Na2CO3 was dissolved into 100ml

DW.

2. Folin-ciocalteau’s reagent (1N): Commercially available reagent (2N) was

diluted with an equal volume of DW.

57

CHART4.3 (b): FLOW CHART FOR TOTAL PHENOLS

100mg plant material crushed in 10ml 80% ethanol

Centrifuge at 5000-10,000 g for 10mins

Supernatant 1 residue + 10ml 80% ethanol

Centrifuge

Supernatant 2 + Supernatant 1 residue discarded

1ml aliquot + 1ml 20% Na2CO3

0.5 ml Folin-Ciocalteau’s reagent

Boil in water bath for 10mins

Final volume made up to 20ml with DW

Ppt. filtered

O.D. at 660nm

The readings were compared with a standard which was prepared by using

tannic acid. The results were expressed as mg/g plant material.

58

C: Measurement of Starch (Chinoy, 1939)

Principle: The plant material is treated with aqueous sodium hydroxide in the

cold to dissolve the starch. This dissolved starch reacts with I2KI and gives a

coloured product and the starch content is determined by calculated with the

standard curve.

Procedure: 100mg plant material was weighed and homogenate with 10ml

80% ethanol. It was centrifuged for 10minutes. Supernatant 1 was collected;

while 10 ml 80% ethanol was added again to the residue centrifuge it and

supernatant 2 was mixed with supernatant 1 and removed. Residue was used for

starch estimation. The residue was dissolved in 20ml 0.7% KOH and boiled for

gelatinization for 40 minutes. It was centrifuged after cooling and 1ml aliquot

(Supernatant), 0.5ml 20% acetic acid; 1ml citrate buffer (0.05M, pH 5.0) and

1ml I2KI were added and incubated at room temperature for 10minutes. O.D.

was taken at 600nm. Blank was prepared in the same manner.

The result was expressed as mg/gm plant material.

Preparation of reagents:

1.80%Ethanol: 80ml Ethanol was diluted up to 100ml DW.

2. 0.7% Potassium hydroxide (KOH): 700 mg KOH was dissolved into 100ml

DW.

3. 20%acetic acid: 20ml glacial acetic acid was diluted up to 100ml with DW.

4. I2KI Solution: 200mg iodine crystal and 2gm KI were dissolved up to 100ml

with DW.

5. Citrate buffer: (0.05M, pH 5.0)

Citrate X: 0.1M Citric acid (2.19gm Citric acid was dissolved into 100ml DW.)

Citrate Y: 0.1M Sodium Citrate (2.94gm Sodium Citrate was dissolved into

100ml DW.)

Citrate buffer: 20.5ml Citrate X and 29.5ml Citrate Y were dissolved up to

100ml with DW.

59

CHART4.3(c): FLOW CHART FOR STARCH

100mg plant material crushed in 10ml 80% ethanol

Centrifuge at 5000-10,000 g for 10mins

Supernatant 1 residue + 10ml 80% ethanol

Centrifuge at 5,000-10,000g for 10min

Supernatant 2 + Supernatant 1 residue

Discarded

Dissolved in 20ml 0.7% KOH and

boiled for gelatinization for 40mins.

Cooled and centrifuged

1ml aliquot

0.5ml 20% acetic acid + 1ml citrate buffer + 1ml I2KI

Incubate at room temperature for 10mins

O.D. at 600nm

The readings were compared with a standard which was prepared by using

starch. The results were expressed as mg/g plant material.

60

4.4) Method of measuring enzyme activity (Protease, Catalase,

Peroxide, Invertase).

Method for enzyme extraction: Grind 1gm plant material in 10ml phosphate

buffer. Centrifuge the extract at 10,000 rpm for 15minutes at 40C refrigerated

centrifuge.

Preparation of reagents:

1. Phosphate buffer (0.1M, pH=7):

Phosphate A: 0.2 M dibasic sodium phosphate (35.61g Na2HPO4.7H2O was

dissolved up to 1,000ml with DW.

Phosphate B: 0.2 M monobasic sodium phosphate (31.21g NaH2PO4.2H2O was

dissolved up to 1,000ml with DW.)

Phosphate buffer: 61ml Phosphate A and 39ml Phosphate B were diluted up to

100ml DW.

CHART: FLOW CHART FOR ENZYME EXTRACTION

Take 1gm plant material

Grind it in 10ml phosphate buffer

Centrifuge at 10,000 rpm for 15mins at 40C

Use supernatant for enzyme activity.

A: Measurement of Protease activity (Cruz et al., 1970)

Principle: Protease hydrolyses protein into its constituent amino-acids. By

estimating the amount of protein hydrolyzed in a solution, the activity of the

protease enzyme can be known.

61

The Folin- Lowry assay used for protein estimation was used here for

determining the activity of the protease enzyme. Protein reacts with Folin

reagent to give a coloured complex. The intensity of the colour depends upon

the amount and type of aromatic amino-acid produced due to hydrolyses by the

enzyme. The type of amino-acid varies for different proteins.

Procedure: 3 test tube, 1ml enzyme aliquot, 1ml phosphate buffer (0.2 M,

pH=7.0 and 1ml 1% casein solution were mixed. It was incubated at room

temperature for 60 min. 1ml 20% Trichloro acetic acid (TCA) was added to it.

Standard tube had 1ml DW, 1ml Phosphate buffer, 1ml Trichloro acetic acid

(TCA) and 1ml casein. Blank has 2ml DW, 1ml phosphate buffer and 1ml

Trichloro acetic acid (TCA). All test tubes were incubated at room temperature

for 60 minutes. All the 3 test tubes were centrifuged and residue was discarded.

1ml aliquot (supernatant) was mixed with 5ml Lowry ‘C’ and incubated at

room temperature for 10 min. 0.5 ml Folin - Ciocalteau’s reagent was added in

each and again incubated for 10 minutes at room temperature. O.D of blank was

related with zero setting, standard gave the reading of casein, while test gave

reading of reduced amount of protein at 600nm. Subtraction of control and test

gave protease activity.

Result was expressed as mg/protein reduced/gm plant material.

Preparation of reagent:

1. Phosphate buffer (0.2M, pH=7):

Phosphate A: 0.2M Monobasic sodium phosphate.

Phosphate B: 0.2M Dibasic sodium phosphate.

Phosphate buffer: 39ml phosphate A was mixed with 61ml phosphate B.

2. 1% casein solution: 1gm casein was dissolved in 5ml 1N NaOH and final

volume was made up to 100ml with DW.

3. 20% Trichloro acetic acid (TCA): 20gm TCA was dissolved in 100ml DW.

4. Lowry’s reagent:

62

Lowry A: 2% Na2CO3 in 0.1N NaOH.

Lowry B: 0.5% CuSO4 in 1% of Na-K-tartrate.

Lowry C: 50ml Lowry A was mixed with 1ml Lowry B.

5. Folin-Ciocalteaus reagent (1N):

Commercially available reagent (2N) was diluted with an equal volume of DW.

CHART4.4 (a): FLOW CHART FOR PROTEASE

Test Standard Blank

1ml enzyme extract 1ml DW 2ml DW

1ml 20% TCA (Trichloro Acetic Acid)

Incubate for 1hr.

Supernatant Residue

(Discarded)

1ml Supernatant

5ml Lowry Reagent

1ml Phosphate Buffer

1ml 1% Casein

Incubated for 1hr

Centrifuge

d

63

Incubated for 10minutes

Add 0.5ml Folin Reagent

Incubated for 10min

O.D. at 600

The readings were compared with a standard which was prepared by using

different concentration of tyrosine. The results were expressed in mg tyrosine

liberated/hr/mg protein.

B: Measurement of Catalase activity (Chance and Maehly, 1955)

Principle: The enzyme catalase is an endogenous antioxidant present in all

aerobic cells helping to facilitate the removal of hydrogen peroxide. The

enzyme consists of 4 subunits of the same size, each of which contains a heme

active site to accelerate the decomposition of H2O2 to water and oxygen.

Procedure: This activity was done by titration method. Reaction, mixture was

prepared by mixing 3ml of phosphate buffer (0.1M, pH= 6.8), 1ml 0.1M H2O2

and 1ml enzyme aliquot. It was incubated at room temperature for 1min. Futher

reaction was stopped by addition of 10ml 20% H2SO4. This mixture was titrated

against 0.01N KMnO4 to estimate the residual H2O2 until a faint pink colour

persisted for at least 15secs. This enzyme activity was expressed as amount of

enzyme break down by H2O2/min/gm plant material.

Preparation of reagent:-

1) Phosphate buffer (0.1M, pH=6.8):

Phosphate A: 0.2M Monobasic sodium phosphate.

64

Phosphate B: 0.2M Dibasic sodium phosphate.

Phosphate buffer: 51ml phosphate A and 49ml phosphate B were diluted

upto 200ml DW.

2) 2% Sulphuric acid (H2SO4): 2ml conc. H2SO4 was diluted upto 100ml

DW.

3) 0.01N Potassium permanganate (KMnO4): 158.04mg KMnO4 was

dissolved upto 100ml DW.

4) 0.1M Hydrogen peroxide (H2O2): 3.041ml H2O2 (100v/v, 30%) was

diluted upto 1000ml DW.

CHART4.4(b): FLOW CHART FOR CATALASE

1ml Enzyme aliquot

+ 3ml phosphate buffer (0.1M, pH=6.8)

+ 1ml 0.1MH2O2

Incubated at room temperature for 1min

Added 10ml 2% H2SO4

Titrated against 0.01N KMnO4 to estimate the residual H2O2

until a faint pink colour persisted for at least 15sec

Expressed enzyme activity as amount of ml enzyme broke

Down by H2O2 /minute/gm plant material.

65

C: Measurement of Peroxidase activity (George, 1955)

Procedure: 1ml enzyme aliquot was mixed with 1ml phosphate buffer (0.1M,

pH= 6.4) and 1ml 20mM guaiacol. Optical density was noted at 420nm. 0.5ml

H2O2 was added and reading was noted after 2 mins. Calculation was done

expressed as O.D difference/minute/gm plant material. Blank was prepared in

the same manner.

Preparation of the reagent:

1) Phosphate buffer (0.1 M, 6.4= pH):

Phosphate A: 0.2 M monobasic sodium phosphate (27.8g

NaH2PO4.2H2O was dissolved upto 1,000ml with DW.

Phosphate B: 0.2 M dibasic sodium phosphate (53.65g Na2HPO4.7H2O

was dissolved upto 1,000 with DW.

Phosphate buffer: 73.5ml Phosphate A and 23.5ml Phosphate B were

diluted upto 200ml DW.

2) 20mM Guaiacol: 0.22ml Guaiacol was upto 100ml with DW.

CHART4.4(c): FLOW CHART FOR PEROXIDASE

Test Blank

1ml Enzyme + 1ml DW +

1ml Phosphate buffer + 1ml Phosphate buffer +

1ml Guaiacol (20mM) 1ml Guaiacol (20 mM)

0.5ml H2O2

Incubate for 2min

O.D. read at 420nm

O.D. at 420 nm

66

The results of enzyme activity were expressed as O.D. difference/min/mg

protein.

D: Measurement of Invertase (Hatch and Glasziou, 1963)

Principle:Invertase is an enzyme which hydrolyses disaccharides into

monosaccharide. Sucrose is the substance that gets reduced to monosaccharide

that is reducing sugar which can be produces by invertase and can also be

measured.

Monosaccharide acts as an oxidizing agent and oxidizes Cu++

. The sugar that

reduces the oxidizing agent is known as reducing sugar. The method involves

reduction of cupric ions (Cu++

) into cuprous ion (Cu+) which is alkaline in

nature and forms yellow cuprous hydroxide, which in turn is converted by heat

of the reaction to insoluble red cuprous oxide (Cu2O). The amount of Cu2O is

dissolved in arsenomolybdate and forms a coloured complex. The developed

colour is related to the concentration of the reducing sugars and is measured

using the spectrometer.

Procedure: 3 test tubes were taken for this activity (1) test (2) control (3)

blank.1ml enzyme aliquot, 1ml 0.1M sucrose solution in citrate buffer (0.1M,

pH=5.4) and 1ml citrate buffer were added to the test tube.1ml DW, 1ml

sucrose solution and 1ml citrate buffer were added to the control tube.2ml DW

and 1ml citrate buffer were added to the blank tube. All test tubes were

incubated at room temperature for 60minutes. After the incubation, 2ml

absolute alcohol and 2ml 5% Sodium sulphate (Na2SO4) were added in all 3t.t.

All test tubes were again incubated in boiling water bath for removal or

evaporation of alcohol (10-20 minutes). Impurities were filtered or centrifuged

and each test tube had left 5ml solution. 1ml solution aliquot and 1ml Nelson

somogyi’s reagent were mixed and kept in boiling water bath for 20 minutes.

1ml arsenomolybdate was added to each after cooling. Final volume was made

67

up to 20ml with DW. O.D. was noted at 540nm.Blank was related with zero

setting, control gave the reading of sucrose, while test gave reading of reduced

sucrose. Subtraction of control and test gave Invertase activity.

Result was expressed as glucose reduced/gm plant material.

Preparation of the reagent:

1. Citrate buffer (0.1 M, pH=5.4):

Citrate A: 0.1M citric acid (21.01gm citric acid was dissolved up to 1000ml

with DW.)

Citrate B: 0.1M Sodium citrate (29.41gm Sodium citrate was dissolved up to

1000ml with DW.)

Citrate buffer: 16ml citrate A and 34ml citrate B were mixed.

2.0.1M sucrose in citrate buffer: 3.42gm of sucrose was dissolved up to 1000ml

with citrate buffer (0.1 M, PH-5.4)

3. Sodium sulphate (Na2SO4) (5%):

5g Na2SO4 was dissolved in 100ml D.W.

4. Nelson somogyi’s reagent:

Nelson ‘A’: 12.5 gm Na2CO3, 12.5 gm Na-k tartrate, 10g NaHCO3, 100g

Na2SO4 were dissolved one by one and final volume was made up to 500 ml

DW.

Nelson ‘B’:15g CuSO4.7H2O was dissolved up to 100ml with DW.

Nelson Somogyi’s reagent: 50 ml Nelson ‘A’ and 2ml Nelson ‘B’ was mixed.

5. Arsenomolybdate reagent:-

25gm ammonium molybdate was dissolved in 450ml DW, 21 ml concentrated

H2SO4 was added to it. 3g sodium arsenate was dissolved in 25 ml DW and both

solutions were mixed. It was incubated at 370c over night before use of it.

68

CHART4.4 (d): FLOW CHART FOR INVERTASE

Test Standard Blank

1ml enzyme extract 1ml DW 2ml DW

Incubate for 1 hr

2ml alcohol + 2ml 5% Sodium Sulphate (Na2SO4)

Incubate in boiling water bath for evaporation of alcohol (10-20 minutes)

Impurities were filtered or centrifuged

1ml solution aliquot + 1ml Nelson Somogyi’s reagent

Incubation in boiling water bath for 20 minutes

Add 1ml AMR (Arsenomolybdate reagent)

Prepare final volume 20ml with D.W.

O.D. at 540nm

1ml Sucrose

1ml citrate Buffer (0.5 M, pH 5.4)

69

The O.D was compared with a standard which was prepared using different

concentration of glucose. The results of enzyme activity were expressed as mg

glucose released/hr/mg protein.

4.5 STATISTICAL RESULT

It is essential to understand the relationship between different parameter

when the study is completed.

4.5.1 Relationship between the parameters (Correlation)

Any relationship between the two variable is known as correlation. If

one variable increases or decreases with a corresponding increase or decrease of

the other variable, a direct positive correlation exists between the two variables.

If one variable decrease with an increase in the other variable, then there is a

negative or inverse correlation. There are two different methods to study

correlation

Graphic method

It is the simplest method of showing the relationship between two

variable. In this one variable is represented on X-axis and other variable on Y-

axis on graph paper. Data corresponding to X and Y axis were plotted in form

of dots. And then estimated lines joining first and last points was drawn on the

graph paper to find out correlation.

Correlation coefficient

The graphic method indicates the existence of a correlation. But it is not

possible to calculate the extent or degree of relationship using these graph.

So,this was calculated by using following formula

70

∑ (dx . dy)

r =

√∑(dx)2 . ∑(dy)

2

Where,

r is the correlation coefficient,

x and y are the two variable

dx is the deviation from the x-mean of the x variable,

dy is the deviation from the y mean of the y variable,

∑ (dx . dy) is the sum of the products of the deviations,

∑ (dx)2 is the sum of the squares of the deviations of the x variable,

∑ (dy)2 is the sum of the squares of the deviations of the y variable,