*2.3.1. Soxhlet extraction method*

An extract is prepared using the soxhlet extraction method. In this method, "thimble" made up of cellulose or cloth placed put up the material to be extracted is placed in a central compartment, lower compartment connected with a siphon device and side-arm both. The solvent is placed in the lower compartment, and a reflux condenser is attached above the central sample compartment. It is made sure that all the components of the setup are assembled together with appropriate contents to complete the apparatus [12]. For the extraction procedure, three different solvents were used, one after another, they were methanol, hexane and distilled water, respectively. Each extraction procedure took around 6 h. For each extraction, 230 ml of the solvent was placed in the lower compartment. A sample of 25 g of the fruit

**Figure 2.** *Opuntia elatior* Mill. fruits.

powder was placed in a porous thimble and kept in the middle compartment. For the procedure, the solvent in the lower container is heated to its boiling temperature (different solvents have different boiling temperature to maintain), and reflux condenser vapor passes through the side arm up. The thimble containing the material to be extracted using the vapor liquefies and drips. Here, central compartment extract gradually collects from warm water percolates through the material and the wall of the thimble. The entire liquid in the central compartment flows through the side tube and back into the lower solvent container of the height of the extract reaches to the top of the siphon. The extract removed in a petri dish and left aside to evaporate. After evaporation of solvent, the leftover extract was filled in the eppendorf tube. This process is then repeated with the other solvents.

**Method:** (1) From 30 ml of stock solution, take 1 ml of sample and make volume of up to 4 ml with distilled water. (2) Add 6 ml of biuret reagent to it and mix well. (3) Heat the mixture at 37°C for 10 min. (4) Cool the tubes and read the absorbance at 520 nm against a reagent blank. (Prepare similarly with 4 ml of distilled water). (5) Draw the standard graph by pipetting out 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ml, of standard protein solution into a series of test tubes and make volume of up to 4 ml with distilled water in each. Carry out steps 2 to 4. (6) Calculate

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Carbohydrates are first hydrolyzed into simple sugars using hydrochloric acid to form furfural. This compound condenses with anthrone to form a green colored complex which can be

**Procedure:** (1) From 30 ml of stock solution, take 0.5 ml of test sample in a test tube. (2) Make up volume of 1 ml with distilled water then add 4 ml of Anthrone reagent. (3) Heat the tubes in a boiling water bath for 10 min, the color changes from blue to green after boiling. (4) Prepare the working standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 ml, where '0' serves as blank. (5) All the tubes including the sample tubes by adding distilled water and make up the total volume to 1 ml. (6) After adding the 4 ml of anthrone reagent. (7). Ten minutes of heat is provided in a boiling water bath. (8) After the tube is cooled rapidly and read the absorbance at 620 nm the green to dark green color. (9) The plotting concentration of the standard on the X-axis versus absorbance on the Y-axis and draw the standard graph. (10) The amount of carbohydrates present in the sample tube find out from the

**Preparation of standard solution:** 5 mg of ascorbic acid was taken and dilute in 5 ml of dis-

**Procedure:** (1) Take 25 ml of test sample and add 25 ml of 20% Meta phosphoric acid. (2) Dilute it to 100 ml with distilled water. (3) Take 10 ml of aliquot from the above solution and add 2.5 ml of acetone. (4) Titrate it with 0.05% dye solution till a pink color persist for 15 s (V1). (5) For the standard readings take 0.05 g of vitamin C (ascorbic acid) (A). (6) Add 60 ml of 20% Metaphosphoric acid and dilute it to 50 ml with distilled water. (7) Titrate the known volume of the above solution with 0.05% dye solution till a pink color persist for

A mg = Std. vitamin C taken; V1 ml = Vol. of dye taken to titrate the sample; V2 ml = Vol. of dye taken to titrate std. vitamin C; B = Total vol. of std. solution taken which is to be titrated

Amount of ascorbic acid (mg/100ml) <sup>=</sup> (<sup>A</sup> mg)<sup>X</sup> (*V*2)X<sup>250</sup> <sup>X</sup><sup>100</sup> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ (V1 ml)<sup>X</sup> (B)<sup>X</sup> ( *wt of Sampled* )

against 0.05% dye; 250 = Total vol. of std. solution made after dilution.

the protein content in the sample using a standard graph.

*2.4.5. Estimation of carbohydrate by Anthrone method*

measured by using colorimetrically at 620 nm.

graph calculate.

15 s (V2).

Calculation:

*2.4.6. Determination of vitamin-C*

tilled water to give concentration of 1 mg/1 ml.
