For your 1st part of question, please study this review paper

Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept

Protocol for reducing suger

Extraction and Estimation of reducing sugars

Alcohol is highly effective in penetrating tissues and stopping the enzymatic activity. Boiling alcohol is more effective than cold alcohol. Hence, it is advisable that tissues used for analysis be extracted in boiling 80% alcohol.

Reagent

1. 80 % Alcohol

Procedure

1. Grind a known weight of material in boiling 80% ethyl alcohol (5-10 ml/g material) thoroughly in a mortar with pestle or in a blender for 5-10 min. And cool under cold water.

2. Pass the extract through two layers of cheese cloth.

3. Re-extract the ground material as in step 1.

4. Pool both the extracts together and filter through Whatman No.41 filter paper. Collect the filtrate, note the volume and store in sealed vials at 0-4C. The preserved alcohol extract and residue can be used for analysis of various compounds.

1.1.1. Estimation of reducing sugars

Sugars with the prescence of potentially free aldehyde or keto group are able to reduce metal ions under alkaline conditions. Such sugars are called as reducing sugars. Some of these are glucose, galactose, lactose and maltose. Reducing sugars may be estimated following either by Nelson-Somogyi method (Somogyi, 1952) or the Dinitrosalicylic acid (DNS) method (Miller, 1972).

Nelson Somogyi's method

Principle

The reducing sugars when heated with alkaline copper tartrate reduce the copper from the cupric to cuprous state and thus cuprous oxide is formed. When cuprous oxide is treated with arsenomolybdic acid, the reduction of molybdic acid to molybdenum blue takes place. The blue colour developed is compared with a set of standards in a colorimeter at 620 nm.

Reagents

1. Alkaline copper tartrate:

a). Dissolve 2.5 g of anhydrous sodium carbonate, 2 g of sodium bicarbonate, 2.5 g of potassium sodium tartrate and 20 g of anhydrous sodium sulphate in 80 ml water and make up to 100 ml.

b) Dissolve 15 g of copper sulphate in a small volume of distilled water. Add one drop of sulphuric acid and make up to 100 ml.

Mix 4 ml of (b) and 96 ml of solution (a) before use.

2. Arsenomolybdate reagent: Dissolve 2.5 g ammonium molybdate in 45 ml water. And 2.5 ml sulphuric acid and mix well. Then add 0.3 g disodium hydrogen arsenate dissolved in 25 ml water. Mix well and incubate at 37C for 24 to 48hours.

3. Standard Glucose solution (stock): 100 mg of glucose in 100 ml distilled water.

4. Working standard: Dilute 10 ml of stock solution to 100 ml with distilled water (100 g/ml).

Procedure

1. Weight 100 mg of the sample and extract the sugars with hot 80% alcohol twice (5ml each time).

2. Collect the supernatant and evaporate on water bath.

3. Add 10 ml of water and dissolve the sugars.

4. Pipette out aliquots of 0.1 or 0.2 ml of alcohol-free extract to separate test tubes.

5. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of the working standard solution into a series of test tubes.

6. Make up the volume in both sample and standard tubes to 2 ml with distilled water.

7. Pipette out 2 ml distilled water into a separate tube to serve as a blank.

8. Add 1 ml of alkaline copper tartrate reagent to each tube.

9. Place the tubes in boiling water for 10 minutes.

10. Cool the tubes and add 1 ml of arsenomolybdic acid reagent to all the tubes.

11. Make up the volume in each tube to 10 ml with water.

12. Read the absorbance of blue colour at 620 nm after 10 min.

13. From the graph drawn, calculate the amount of reducing sugars present in the sample.

Calculation

Absorbance corresponds to 0.1ml of test = x mg of glucose

10 ml contains =  x 10 mg of glucose/0.1 = % of reducing sugars

Reference

Sadasivam, S. and A. Manickam, (1992). In: Biochemical Methods for Agricultural Sciences, Wiley Eastern Limited. New Delhi. pp.5-6.

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