4.1) Parameters and sampling frequency

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1 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. 49 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

2 Biochemical Enzymatic Sugars Phenol Starch Protease Oxidase Invertase Total Reducing Non-reducing Catalase Peroxidase ) 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 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/cm 2 was calculaed as:- Weight of intact leaf- initial weight of leaf Dust content (mg/cm 2 ) = Total surface area of leaf (cm 2 )

3 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. 51 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, 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.

4 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 o C until constant weight was obtained and the final weight was taken (W2 g). 52 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).

5 4.3) Method of measuring sugar (total, reducing and nonreducing), 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 (Cu 2 O). 53 The amount of Cu 2 O formed can be increased by adding arsenomolybdic acid which in turn is reduced to lower oxides of molybdenum by Cu 2 O. 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 H 2 SO 4 was added and heated at 49 0 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

6 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. 54 Preparation of reagents: 1.80%Ethanol: 80ml Ethanol was diluted up to 100ml DW. 2.1N Sulphuric acid (H 2 SO 4 ): 2.77ml conc. H 2 SO 4 (95-98%) was diluted up to 100 ml with DW N 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 Na 2 CO 3, 12.5gm Na-K-tartrate, 10gm NaHCO 3, 100gm Na 2 SO 4 were dissolved one by one and final volume made up to 50ml with DW. Nelson B: 15gm CuSO 4.7H 2 O 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. H 2 SO 4 was added to it. 3gm Sodium arsenate was dissolved in 25ml DW and both solutions were mixed. It was incubated at 35 0 C for overnight before use of it.

7 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 55 Supernatant I Residue +10ml 80% ethanol Centrifuged at g for 10 minutes Supernatant II Residue Supernatant I+II Total sugar Reducing sugar 1 ml aliquot + 1ml aliquot + 1 ml 1 N H 2 SO 4 1ml Nelson Somogyi s reagent Incubated in water bath at 49 0 C for 30 min. Add 1-2 drop of methyl red indicator Add 1N NaOH drop wise for neutralization (Colour change Pink to Yellow) Incubated in boiling water bath for 20min Add 1ml Arsenomolybdate Final vol. made upto 20ml with D.W 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.

8 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). 56 Procedure: 100mg plant material was weighed and homogenate with 10ml 80% ethanol. It was centrifuged at ,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% Na 2 CO 3 and 0.5ml Folin-Ciocalteau s reagent. It was boiled for 10minutes at C 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 % Sodium carbonate (Na 2 CO 3 ):20gm Na 2 CO 3 was dissolved into 100ml DW. 2. Folin-ciocalteau s reagent (1N): Commercially available reagent (2N) was diluted with an equal volume of DW.

9 CHART4.3 (b): FLOW CHART FOR TOTAL PHENOLS 100mg plant material crushed in 10ml 80% ethanol Centrifuge at ,000 g for 10mins Supernatant 1 residue + 10ml 80% ethanol 57 Centrifuge Supernatant 2 + Supernatant 1 residue discarded 1ml aliquot + 1ml 20% Na 2 CO 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.

10 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 I 2 KI 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 I 2 KI 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. 58 Preparation of reagents: 1.80%Ethanol: 80ml Ethanol was diluted up to 100ml DW % Potassium hydroxide (KOH): 700 mg KOH was dissolved into 100ml DW %acetic acid: 20ml glacial acetic acid was diluted up to 100ml with DW. 4. I 2 KI 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.

11 CHART4.3(c): FLOW CHART FOR STARCH 100mg plant material crushed in 10ml 80% ethanol Centrifuge at ,000 g for 10mins Supernatant 1 residue + 10ml 80% ethanol 59 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 I 2 KI 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.

12 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 4 0 C refrigerated centrifuge. Preparation of reagents: 1. Phosphate buffer (0.1M, ph=7): Phosphate A: 0.2 M dibasic sodium phosphate (35.61g Na 2 HPO 4.7H 2 O was dissolved up to 1,000ml with DW. Phosphate B: 0.2 M monobasic sodium phosphate (31.21g NaH 2 PO 4.2H 2 O was dissolved up to 1,000ml with DW.) Phosphate buffer: 61ml Phosphate A and 39ml Phosphate B were diluted up to 100ml DW. 60 CHART: FLOW CHART FOR ENZYME EXTRACTION Take 1gm plant material Grind it in 10ml phosphate buffer Centrifuge at 10,000 rpm for 15mins at 4 0 C 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.

13 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): 61 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 % Trichloro acetic acid (TCA): 20gm TCA was dissolved in 100ml DW. 4. Lowry s reagent:

14 Lowry A: 2% Na 2 CO 3 in 0.1N NaOH. Lowry B: 0.5% CuSO 4 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 62 1ml enzyme extract 1ml DW 2ml DW 1ml Phosphate Buffer 1ml 1% Casein Incubated for 1hr 1ml 20% TCA (Trichloro Acetic Acid) Incubate for 1hr. Centrifuge Supernatant 1ml Supernatant Residue (Discarded) 5ml Lowry Reagent

15 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. 63 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 H 2 O 2 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 H 2 O 2 and 1ml enzyme aliquot. It was incubated at room temperature for 1min. Futher reaction was stopped by addition of 10ml 20% H 2 SO 4. This mixture was titrated against 0.01N KMnO 4 to estimate the residual H 2 O 2 until a faint pink colour persisted for at least 15secs. This enzyme activity was expressed as amount of enzyme break down by H 2 O 2 /min/gm plant material. Preparation of reagent:- 1) Phosphate buffer (0.1M, ph=6.8): Phosphate A: 0.2M Monobasic sodium phosphate.

16 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 (H 2 SO 4 ): 2ml conc. H 2 SO 4 was diluted upto 100ml DW. 3) 0.01N Potassium permanganate (KMnO 4 ): mg KMnO 4 was dissolved upto 100ml DW. 64 4) 0.1M Hydrogen peroxide (H 2 O 2 ): 3.041ml H 2 O 2 (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 0.1MH 2 O 2 Incubated at room temperature for 1min Added 10ml 2% H 2 SO 4 Titrated against 0.01N KMnO 4 to estimate the residual H 2 O 2 until a faint pink colour persisted for at least 15sec Expressed enzyme activity as amount of ml enzyme broke Down by H 2 O 2 /minute/gm plant material.

17 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 H 2 O 2 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 NaH 2 PO 4.2H 2 O was dissolved upto 1,000ml with DW. Phosphate B: 0.2 M dibasic sodium phosphate (53.65g Na 2 HPO 4. 7H 2 O 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. 65 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) O.D. at 420 nm 0.5ml H 2 O 2 Incubate for 2min O.D. read at 420nm

18 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 (Cu 2 O). The amount of Cu 2 O 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. 66 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 (Na 2 SO 4 ) 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

19 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 M 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 (Na 2 SO 4 ) (5%): 5g Na 2 SO 4 was dissolved in 100ml D.W. 4. Nelson somogyi s reagent: Nelson A : 12.5 gm Na 2 CO 3, 12.5 gm Na-k tartrate, 10g NaHCO 3, 100g Na 2 SO 4 were dissolved one by one and final volume was made up to 500 ml DW. Nelson B :15g CuSO 4.7H 2 O 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 H 2 SO 4 was added to it. 3g sodium arsenate was dissolved in 25 ml DW and both solutions were mixed. It was incubated at 37 0 c over night before use of it. 67

20 CHART4.4 (d): FLOW CHART FOR INVERTASE Test Standard Blank 1ml enzyme extract 1ml DW 2ml DW 1ml Sucrose 68 1ml citrate Buffer (0.5 M, ph 5.4) Incubate for 1 hr 2ml alcohol + 2ml 5% Sodium Sulphate (Na 2 SO 4 ) 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

21 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 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

22 r = (dx. dy) (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, 70 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,

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