Method for detecting crude polysaccharide in immunity enhancing product

Abstract

The invention discloses a method for detecting crude polysaccharide in an immunity-enhancing product, and relates to the technical field of detection, the immunity-enhancing product is taken and ground, water is added for mixing, freezing and heating drying are carried out, ultrasound is started while drying is carried out, pre-sample powder is obtained, water is added for boiling, then cooling is carried out, amylase and maltase are sequentially added for enzymolysis, enzyme deactivation and drying are carried out; sample powder is obtained; the method comprises the following steps: selecting a mixed solvent to extract sample powder, then adopting a basic cupric reagent to precipitate glucan, and then carrying out phenol-sulfuric acid reaction to obtain a to-be-detected solution; and calculating the crude polysaccharide content of the to-be-detected liquid according to the standard curve. According to the method, firstly, the sample is pretreated, that is, the sample is dissolved in water and then frozen, and then ultrasonic treatment is combined, so that sample particles are loosened and expanded, and subsequent dissolution in a solvent is facilitated. In addition, starch and dextrin are sequentially subjected to enzymolysis to form glucose, and then the glucose is precipitated, so that the influence of starch and dextrin auxiliary materials on polysaccharide is reduced, and the detection precision of crude polysaccharide is greatly improved.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for detecting crude polysaccharides in an immunity enhancing product. Background Art

[0002] With the continuous improvement of living standards, people begin to advocate various health foods, hoping to use their effects of strengthening physical fitness and improving immunity to prevent diseases and improve health levels. In particular, there are more and more products that enhance immunity, and crude polysaccharides are one of the effective ingredients, which are attracting more and more attention. From a pharmacological point of view, polysaccharides have anti-tumor, anti-viral, immunity-enhancing, antioxidant, anti-cardiovascular disease and other effects. Therefore, the content of crude polysaccharides is one of the indicators that need to be detected. In the prior art, most of them use chromatographic methods to directly analyze the monosaccharide components in polysaccharides, and use indirect methods (phenol-sulfuric acid method, anthrone-sulfuric acid method) to analyze the polysaccharide content.

[0003] Since the purity of crude polysaccharides is not high enough and they are often affected by dextrin or starch, resulting in inaccurate content in subsequent detection, it is necessary to improve the sample pretreatment and extraction steps. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a method for detecting crude polysaccharides in an immunity enhancing product.

[0005] The technical solution of the present invention is as follows:

[0006] A method for detecting crude polysaccharides in an immunity enhancing product comprises the following steps:

[0007] S1: Pretreatment of immunity-boosting products;

[0008] Grind the immunity enhancing product, add water and mix, then freeze at -20 to -10°C for 20 to 30 minutes to obtain ice particles, then heat the ice particles to 20 to 40°C and dry them, while starting ultrasound to obtain pre-sample powder;

[0009] Add water to the sample powder and boil it, then cool it down and add amylase to perform enzymolysis and inactivate the enzyme, then continue to add maltase to perform enzymolysis and inactivate the enzyme, and dry it to obtain the sample powder;

[0010] S2: taking sample powder, extracting with a mixed solvent to remove interfering components such as monosaccharides, oligosaccharides, glycosides and alkaloids, and obtaining a first sample solution;

[0011] S3: taking the first sample solution, using a basic divalent copper reagent to precipitate dextran, and then reacting with phenol-sulfuric acid to obtain a test solution;

[0012] S4: Use the standard solution to draw a standard curve, measure the absorbance value of the test solution at a wavelength of 400-600nm, and calculate the crude polysaccharide content of the test solution based on the standard curve.

[0013] As a preferred embodiment of the present invention, in step S1, the frequency of ultrasound is 20-30 kHz.

[0014] As a preferred embodiment of the present invention, in step S1, the enzymatic hydrolysis temperature is 30-60°C, and the enzymatic hydrolysis time is 5-20 min.

[0015] As a preferred embodiment of the present invention, in step S2, sample powder is taken, a mixed solvent is added, mixed and centrifuged at 2800-3500r / min for 3-6min, and the supernatant is discarded; the residue is washed with ethanol, centrifuged and the supernatant is discarded, the operation is repeated 3-4 times, the residue is dissolved with water and the volume is fixed, and mixed to make the first sample liquid.

[0016] As a preferred embodiment of the present invention, in step S2, the mixed solvent comprises choline chloride, amyl alcohol and chloroform in a volume ratio of 1:1:3-5.

[0017] As a preferred embodiment of the present invention, in step S2, the mixed solvent contains 10-20 wt % of water.

[0018] As a preferred embodiment of the present invention, in step S3, the extraction is carried out by ultrasonic extraction, specifically, ultrasonic extraction is performed 3-5 times at 100-150W and 30-60°C, each time for 10-20 minutes.

[0019] As a preferred embodiment of the present invention, a first sample solution is taken, a sodium hydroxide solution and a divalent copper reagent solution are added, the solution is boiled in a boiling water bath, the solution is cooled and then centrifuged, the supernatant is discarded, the residue is washed with a washing solution and then centrifuged, the supernatant is discarded, and the process is repeated three times. The residue is dissolved with a sulfuric acid solution and transferred to a 50 mL volumetric flask, diluted to the mark with water, and mixed to obtain a second sample solution; the second sample solution is precisely aspirated, placed in a colorimetric tube, a phenol solution is added, and the solution is mixed on a rotary mixer, concentrated sulfuric acid is carefully added, and the solution is carefully mixed again, the solution is boiled in a boiling water bath, and the solution is cooled to obtain a test solution.

[0020] As a preferred embodiment of the present invention, in step S4, the method of the standard curve is as follows:

[0021] Measure 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of glucose standard application solution and place them in colorimetric tubes respectively, add 2 ml, add 1 ml of phenol solution, and 10 ml of concentrated sulfuric acid, mix well, boil in water bath for 2 min, cool to room temperature, measure the absorbance at 485 nm with a 1 cm colorimetric dish, and draw a standard curve.

[0022] The beneficial effects of the present invention are as follows: a method for detecting crude polysaccharides in an immunity-enhancing product of the present invention. Since most of the existing immunity-enhancing products are capsules, the contents of the capsules mostly contain starch or dextrin, which affects the true content of crude polysaccharides. Therefore, starch or dextrin needs to be further detected before detection. The present invention first pre-treats the sample, i.e., first dissolves it in water, then freezes it, and then combines it with ultrasound to make the sample particles loose and puffed, which is more conducive to subsequent dissolution in a solvent. In addition, before the determination, starch and dextrin are enzymatically hydrolyzed into glucose in sequence by enzymatic hydrolysis, and then precipitated with anhydrous ethanol, which reduces the influence of starch and dextrin auxiliary materials on polysaccharides and greatly improves the detection accuracy of crude polysaccharides. At the same time, the present invention uses a mixed solvent to extract the sample, which can effectively remove the interfering components of monosaccharides, oligosaccharides, glycosides and alkaloids, so that the measurement of polysaccharide content is more accurate. DETAILED DESCRIPTION

[0023] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0024] The high molecular weight substances with a molecular weight greater than 10,000 in the sample are precipitated in 800 mL / L ethanol solution and separated from water-soluble monosaccharides and oligosaccharides. The polysaccharides with a glucan structure are selectively precipitated from other high molecular weight substances using an alkaline divalent copper reagent. The polysaccharides are then reacted with phenol-sulfuric acid and their content is determined by carbohydrate colorimetry. The color intensity is proportional to the glucan content in the crude polysaccharide, and the crude polysaccharide content in the food is calculated based on this.

[0025] instrument:

[0026] Spectrophotometer;

[0027] Centrifuge;

[0028] Rotary mixer;

[0029] Reagents: Unless otherwise specified, all reagents used in this method were of analytical grade and the water used was distilled water.

[0030] Ethanol solution (800mL / L): Add 80mL of anhydrous ethanol to 20mL of water and mix well.

[0031] Sodium hydroxide solution (100 g / L): Weigh 100 g of sodium hydroxide, dissolve it in water and dilute it to 1 L, mix well and set aside.

[0032] Copper stock solution: weigh 3.0 g CuSO4·5H2O and 30.0 g sodium citrate, dissolve in water and dilute to 1 L, mix well and set aside.

[0033] Copper reagent solution: Take 50 mL of copper stock solution, add 50 mL of water, mix well, add 12.5 g of solid anhydrous sodium sulfate, and dissolve it. Prepare fresh before use.

[0034] Detergent: Take 50 mL of water, add 10 mL of copper reagent solution and 10 mL of sodium hydroxide solution, and mix well.

[0035] Sulfuric acid solution (100mL / L): Add 100mL of concentrated sulfuric acid to about 800mL of water, mix well, and dilute to 1L after cooling.

[0036] Phenol solution (50g / L): Weigh 5.0g of refined phenol, dissolve it in water and dilute it to 100mL, mix well. The solution can be stored in a refrigerator for one month.

[0037] Dextran standard stock solution: accurately weigh 0.5000 g of dextran (wt 500000) standard dried to constant weight, dissolve in water and make up to 50 mL, mix well, and store in a refrigerator. The dextran concentration in this solution is 10 mg / mL.

[0038] Dextran standard working solution: Accurately pipette 1.00 mL of dextran standard stock solution into a 100 mL volumetric flask, add water to the mark, mix well, and store in a refrigerator. The dextran concentration in this solution is 0.1 mg / mL.

[0039] Preparation of standard curve:

[0040] Accurately pipette the following dextran standard solution: 0.0, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00 mL (equivalent to 0.0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10 mg of dextran), place them in 25 mL colorimetric tubes, add water to 2.0 mL, add 1.00 mL of 50 g / L phenol solution, mix well on a rotary mixer, carefully add 10.0 mL of concentrated sulfuric acid, mix well again, boil in a boiling water bath for 2.0 min, cool, and use a spectrophotometer at a wavelength of 485 nm, with the reagent blank solution as the reference, and a 1 cm colorimetric dish to measure the absorbance value. Draw a standard curve with the dextran concentration as the horizontal axis and the absorbance value as the vertical axis.

[0041] Find out the mass of glucan from the standard curve, calculate the crude polysaccharide content in the sample, and perform a sample blank experiment at the same time.

[0042] Calculation results:

[0043]

[0044] Where: X-crude polysaccharide content in the sample (in terms of glucan), mg / 100g;

[0045] W1-the mass of dextran in the sample test solution, mg;

[0046] W2-mass of dextran in sample blank solution, mg;

[0047] M-sample weight, g;

[0048] V1-total volume of sample extract, mL;

[0049] V2-volume of sample extract used to precipitate crude polysaccharides, mL;

[0050] V3-volume of crude polysaccharide solution, mL;

[0051] V4-volume of crude polysaccharide solution used to precipitate dextran, mL;

[0052] V5-total volume of sample solution, mL;

[0053] V6 - Volume of sample solution for determination, mL.

[0054] The immunity enhancing product used in the following examples is Epimedium, Rhodiola and Ginseng Capsule.

[0055] Example 1

[0056] A method for detecting crude polysaccharides in an immunity enhancing product comprises the following steps:

[0057] S1: Pretreatment of immunity-boosting products;

[0058] Grind the immunity enhancing product, add water and mix, then freeze at -20°C for 25 minutes to obtain ice particles, then heat the ice particles to 30°C and dry them, while starting ultrasound to obtain pre-sample powder;

[0059] Add water to the sample powder and boil it, then cool it down and add amylase to perform enzymolysis and inactivate the enzyme, then continue to add maltase to perform enzymolysis and inactivate the enzyme, and dry it to obtain the sample powder;

[0060] S2: taking sample powder, extracting with a mixed solvent to remove interfering components such as monosaccharides, oligosaccharides, glycosides and alkaloids, and obtaining a first sample solution;

[0061] S3: taking the first sample solution, using a basic divalent copper reagent to precipitate dextran, and then reacting with phenol-sulfuric acid to obtain a test solution;

[0062] S4: Use the standard solution to draw a standard curve, measure the absorbance value of the test solution at a wavelength of 485nm, and calculate the crude polysaccharide content of the test solution based on the standard curve.

[0063] In step S1, the frequency of ultrasound is 25 kHz.

[0064] In step S1, the enzymolysis temperature is 50° C. and the enzymolysis time is 15 min.

[0065] In step S2, sample powder is taken, mixed solvent is added, mixed and centrifuged at 3000r / min for 5min, and the supernatant is discarded; the residue is washed with ethanol, centrifuged and the supernatant is discarded, the operation is repeated 3 times, the residue is dissolved with water and fixed to volume, and mixed to prepare the first sample solution.

[0066] In step S2, the mixed solvent comprises choline chloride, amyl alcohol and chloroform in a volume ratio of 1:1:3.

[0067] In step S2, the mixed solvent contains 10 wt% of water.

[0068] In step S3, the extraction is carried out by ultrasonic extraction, specifically, ultrasonic extraction is performed 4 times at 150W and 35°C, each time for 15 minutes.

[0069] Take the first sample solution, add sodium hydroxide solution and divalent copper reagent solution, boil in a boiling water bath, centrifuge after cooling, discard the supernatant, wash the residue with washing solution and centrifuge, discard the supernatant, repeat 3 times, dissolve the residue with sulfuric acid solution and transfer to a 50mL volumetric flask, add water to dilute to the scale, mix well, and obtain the second sample solution; accurately pipette the second sample solution, place it in a colorimetric tube, add phenol solution, mix well on a rotary mixer, carefully add concentrated sulfuric acid, mix well again, boil in a boiling water bath, cool, and obtain the test solution.

[0070] Example 2

[0071] A method for detecting crude polysaccharides in an immunity enhancing product comprises the following steps:

[0072] S1: Pretreatment of immunity-boosting products;

[0073] Grind the immunity enhancing product, add water and mix, then freeze at -10°C for 30 minutes to obtain ice particles, then heat the ice particles to 30°C and dry them, while starting ultrasound to obtain pre-sample powder;

[0074] Add water to the sample powder and boil it, then cool it down and add amylase to perform enzymolysis and inactivate the enzyme, then continue to add maltase to perform enzymolysis and inactivate the enzyme, and dry it to obtain the sample powder;

[0075] S2: taking sample powder, extracting with a mixed solvent to remove interfering components such as monosaccharides, oligosaccharides, glycosides and alkaloids, and obtaining a first sample solution;

[0076] S3: taking the first sample solution, using a basic divalent copper reagent to precipitate dextran, and then reacting with phenol-sulfuric acid to obtain a test solution;

[0077] S4: Use the standard solution to draw a standard curve, measure the absorbance value of the test solution at a wavelength of 485nm, and calculate the crude polysaccharide content of the test solution based on the standard curve.

[0078] In step S1, the frequency of ultrasound is 30 kHz.

[0079] In step S1, the enzymolysis temperature is 60° C. and the enzymolysis time is 10 min.

[0080] In step S2, sample powder is taken, mixed solvent is added, mixed and centrifuged at 3000r / min for 5min, and the supernatant is discarded; the residue is washed with ethanol, centrifuged and the supernatant is discarded, the operation is repeated 3 times, the residue is dissolved with water and fixed to volume, and mixed to prepare the first sample solution.

[0081] In step S2, the mixed solvent comprises choline chloride, amyl alcohol and chloroform in a volume ratio of 1:1:3.

[0082] In step S2, the mixed solvent contains 10 wt% of water.

[0083] In step S3, the extraction is carried out by ultrasonic extraction, specifically, ultrasonic extraction is performed 4 times at 150W and 35°C, each time for 15 minutes.

[0084] Take the first sample solution, add sodium hydroxide solution and divalent copper reagent solution, boil in a boiling water bath, centrifuge after cooling, discard the supernatant, wash the residue with washing solution and centrifuge, discard the supernatant, repeat 3 times, dissolve the residue with sulfuric acid solution and transfer to a 50mL volumetric flask, add water to dilute to the scale, mix well, and obtain the second sample solution; accurately pipette the second sample solution, place it in a colorimetric tube, add phenol solution, mix well on a rotary mixer, carefully add concentrated sulfuric acid, mix well again, boil in a boiling water bath, cool, and obtain the test solution.

[0085] Example 3

[0086] A method for detecting crude polysaccharides in an immunity enhancing product comprises the following steps:

[0087] S1: Pretreatment of immunity-boosting products;

[0088] Grind the immunity enhancing product, add water and mix, then freeze at -15°C for 25 minutes to obtain ice particles, then heat the ice particles to 30°C and dry them, while starting ultrasound to obtain pre-sample powder;

[0089] Add water to the sample powder and boil it, then cool it down and add amylase to perform enzymolysis and inactivate the enzyme, then continue to add maltase to perform enzymolysis and inactivate the enzyme, and dry it to obtain the sample powder;

[0090] S2: taking sample powder, extracting with a mixed solvent to remove interfering components such as monosaccharides, oligosaccharides, glycosides and alkaloids, and obtaining a first sample solution;

[0091] S3: taking the first sample solution, using a basic divalent copper reagent to precipitate dextran, and then reacting with phenol-sulfuric acid to obtain a test solution;

[0092] S4: Use the standard solution to draw a standard curve, measure the absorbance value of the test solution at a wavelength of 485nm, and calculate the crude polysaccharide content of the test solution based on the standard curve.

[0093] In step S1, the frequency of ultrasound is 25 kHz.

[0094] In step S1, the enzymolysis temperature is 40° C. and the enzymolysis time is 15 min.

[0095] In step S2, sample powder is taken, mixed solvent is added, mixed and centrifuged at 3000r / min for 5min, and the supernatant is discarded; the residue is washed with ethanol, centrifuged and the supernatant is discarded, the operation is repeated 3 times, the residue is dissolved with water and fixed to volume, and mixed to prepare the first sample solution.

[0096] In step S2, the mixed solvent comprises choline chloride, amyl alcohol and chloroform in a volume ratio of 1:1:3.

[0097] In step S2, the mixed solvent contains 10 wt% of water.

[0098] In step S3, the extraction is carried out by ultrasonic extraction, specifically, ultrasonic extraction is performed 4 times at 150W and 35°C, each time for 15 minutes.

[0099] Take the first sample solution, add sodium hydroxide solution and divalent copper reagent solution, boil in a boiling water bath, centrifuge after cooling, discard the supernatant, wash the residue with washing solution and centrifuge, discard the supernatant, repeat 3 times, dissolve the residue with sulfuric acid solution and transfer to a 50mL volumetric flask, add water to dilute to the scale, mix well, and obtain the second sample solution; accurately pipette the second sample solution, place it in a colorimetric tube, add phenol solution, mix well on a rotary mixer, carefully add concentrated sulfuric acid, mix well again, boil in a boiling water bath, cool, and obtain the test solution.

[0100] Example 4

[0101] Different from Example 3, in step S2, the mixed solvent includes choline chloride, amyl alcohol and chloroform in a volume ratio of 1:1:3.

[0102] In step S2, the mixed solvent contains 15 wt% of water.

[0103] Example 5

[0104] Different from Example 3, in step S2, the mixed solvent includes choline chloride, amyl alcohol and chloroform in a volume ratio of 1:1:3.

[0105] In step S2, the mixed solvent contains 20 wt% of water.

[0106] Comparative Example 1

[0107] The difference from Example 3 is that the mixed solvent is anhydrous ethanol.

[0108] Comparative Example 2

[0109] Different from Example 3, in step S1, no enzymatic hydrolysis step is performed.

[0110] Comparative Example 3

[0111] The difference from Example 3 is that there is no pre-treatment in step S1.

[0112] The performance tests of the above embodiments and comparative examples are shown in Table 1.

[0113] Table 1 Determination results of polysaccharides

[0114]

[0115] As can be seen from the above table, the performance of the embodiment is better than that of the comparative example. The main reason may be that in the embodiment, the sample is first pretreated by water dissolution, then frozen, and then combined with ultrasound, so that the sample particles are loose and expanded, which is more conducive to the subsequent dissolution in the solvent. In addition, before the determination, starch and dextrin are enzymatically hydrolyzed into glucose in sequence, and then precipitated with anhydrous ethanol, which reduces the influence of starch and dextrin auxiliary materials on polysaccharides and greatly improves the detection accuracy of crude polysaccharides. At the same time, the present invention uses a mixed solvent to extract the sample, which can effectively remove the interfering components of monosaccharides, oligosaccharides, glycosides and alkaloids, making the measurement of polysaccharide content more accurate.

[0116] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting crude polysaccharides in an immunity enhancing product, characterized in that: The following steps are involved: S1: Pretreatment of immunity-boosting products; Grind the immunity enhancing product, add water and mix, then freeze at -20 to -10°C for 20 to 30 minutes to obtain ice particles, then heat the ice particles to 20 to 40°C and dry them, while starting ultrasound to obtain pre-sample powder; Add water to the sample powder and boil it, then cool it down and add amylase to perform enzymolysis and inactivate the enzyme, then continue to add maltase to perform enzymolysis and inactivate the enzyme, and dry it to obtain the sample powder; S2: taking sample powder, extracting with a mixed solvent to remove interfering components such as monosaccharides, oligosaccharides, glycosides and alkaloids, and obtaining a first sample solution; S3: taking the first sample solution, using a basic divalent copper reagent to precipitate dextran, and then reacting with phenol-sulfuric acid to obtain a test solution; S4: Use the standard solution to draw a standard curve, measure the absorbance value of the test solution at a wavelength of 400-600nm, and calculate the crude polysaccharide content of the test solution based on the standard curve.

2. The method for detecting crude polysaccharides in an immunity enhancing product according to claim 1, characterized in that: In step S1, the frequency of ultrasound is 20-30 kHz.

3. The method for detecting crude polysaccharides in an immunity enhancing product according to claim 1, characterized in that: In step S1, the enzymolysis temperature is 30-60°C, and the enzymolysis time is 5-20 minutes.

4. The method for detecting crude polysaccharides in an immunity enhancing product according to claim 1, characterized in that: In step S2, sample powder is taken, mixed solvent is added, mixed and centrifuged at 2800-3500r / min for 3-6min, and the supernatant is discarded; the residue is washed with ethanol, centrifuged and the supernatant is discarded, the operation is repeated 3-4 times, the residue is dissolved with water and fixed to volume, and mixed to make the first sample solution.

5. The method for detecting crude polysaccharides in an immunity enhancing product according to claim 4, characterized in that: In step S2, the mixed solvent comprises choline chloride, amyl alcohol and chloroform in a volume ratio of 1:1:3-5.

6. The method for detecting crude polysaccharides in an immunity enhancing product according to claim 4, characterized in that: In step S2, the mixed solvent contains 10-20 wt% of water.

7. The method for detecting crude polysaccharides in an immunity enhancing product according to claim 1, characterized in that: In step S3, the extraction is carried out by ultrasonic extraction, specifically, ultrasonic extraction is carried out 3-5 times at 100-150W and 30-60°C, each time for 10-20 minutes.

8. The method for detecting crude polysaccharides in an immunity enhancing product according to claim 1, characterized in that: Take the first sample solution, add sodium hydroxide solution and divalent copper reagent solution, boil in a boiling water bath, centrifuge after cooling, discard the supernatant, wash the residue with washing solution and centrifuge, discard the supernatant, repeat 3 times, dissolve the residue with sulfuric acid solution and transfer to a 50mL volumetric flask, add water to dilute to the scale, mix well, and obtain the second sample solution; accurately pipette the second sample solution, place it in a colorimetric tube, add phenol solution, mix well on a rotary mixer, carefully add concentrated sulfuric acid, mix well again, boil in a boiling water bath, cool, and obtain the test solution.

9. The method for detecting crude polysaccharides in an immunity enhancing product according to claim 1, characterized in that: In step S4, the method of the standard curve is as follows: Measure 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of glucose standard application solution and place them in colorimetric tubes respectively, add 2 ml, add 1 ml of phenol solution, and 10 ml of concentrated sulfuric acid, mix well, boil in water bath for 2 min, cool to room temperature, measure the absorbance at 485 nm with a 1 cm colorimetric dish, and draw a standard curve.

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