The carbon nutritional status and quality traits of plants are often assessed using sugar content as an important indicator. Monosaccharides and certain oligosaccharides (e.g., maltose) contain free aldehyde or ketone groups and possess reducing properties, thus they are classified as reducing sugars. Polysaccharides and sucrose, among others, are non-reducing sugars. Taking advantage of the fact that polysaccharides can be hydrolyzed by acid into monosaccharides, the total sugar content can be determined by measuring the monosaccharide content after hydrolysis.
The detection principle of the DNS Reagent (NY/T Method) is that reducing sugars are oxidized to sugar acids under alkaline conditions, while 3,5-dinitrosalicylic acid is reduced to a brownish-red amino compound. Within a certain range, the amount of reducing sugars is proportional to the color intensity of the brownish-red product. The absorbance of the brownish-red substance is measured at 540 nm, and this absorbance value exhibits a linear relationship with the reducing sugar content. The content of reducing sugars and total sugars in samples can be determined using colorimetry and a standard curve.
This reagent is intended for research use only and is not suitable for clinical diagnosis or other applications.
Materials Required (User-Supplied):
1.Distilled water, 6 M hydrochloric acid solution, 6 M sodium hydroxide solution
2.50 mL centrifuge tubes
3.Centrifuge
4.Water bath or constant temperature incubator
5.Spectrophotometer and cuvettes
6.Homogenizer or mortar and pestle
7.I₂-KI solution: Dissolve 5 g of iodine and 10 g of potassium iodide in 100 mL of distilled water.
8.Glucose (Glu) standard solution: 1 mg/mL
Operating Procedure (For Reference Only):
1. Extraction of Reducing Sugars:
① Weigh 0.5–3 g of plant sample, cut into small pieces, add approximately 3 mL of distilled water, and homogenize. Transfer the homogenate to a beaker or Erlenmeyer flask. Rinse the homogenizer 2–3 times with 12 mL of distilled water and combine the washings into the same container.
② Incubate in a 50°C water bath for 30 min with intermittent stirring to ensure complete extraction of reducing sugars.
③ Transfer the precipitate and extract to a 50 mL centrifuge tube and centrifuge at 4000 × g for 5 min.
④ Retain the supernatant. Add 20 mL of distilled water to the precipitate, mix well, and centrifuge again at 4000 × g for 5 min.
⑤ Retain the supernatant. Combine the two supernatants, bring the volume to 100 mL with distilled water (extraction solution), and mix well. This serves as the reducing sugar test solution.
2. Hydrolysis and Extraction of Total Sugars:
① Weigh 0.5–3 g of plant sample, cut into small pieces, add approximately 3 mL of distilled water, and homogenize. Transfer the homogenate to a beaker or Erlenmeyer flask. Rinse the homogenizer 2–3 times with 12 mL of distilled water and combine the washings into the same container.
② Add 10 mL of 6 M hydrochloric acid solution to the container, mix well, and boil for 30 min with intermittent stirring.
③ Spot 2 drops of the hydrolysate onto a glass slide, add 1 drop of indicator solution (I₂-KI solution is recommended; see concentration details under "Materials Required" on the first page of the manual), and check whether hydrolysis is complete. Complete hydrolysis is indicated by the absence of a blue color.
④ After hydrolysis is complete, cool to room temperature. Add 6 M sodium hydroxide solution to adjust the pH to 7.4. Bring the volume to 100 mL with distilled water, mix well, and centrifuge at 4000 × g for 5 min or filter.
⑤ Take 10 mL of the supernatant or filtrate and bring the volume to 100 mL with distilled water to obtain a 10-fold diluted total sugar hydrolysate (extraction solution). Take 1 mL of this total sugar hydrolysate for determination of reducing sugar content.
3. Preparation of Glucose Standard Curve:
Take clean centrifuge tubes or test tubes and proceed according to the table below. Use tube No. 0 as the blank to zero the instrument. Measure the absorbance at 540 nm. Plot the absorbance values on the y-axis against the standard concentrations (mg/mL) on the x-axis to generate the standard curve.
Reagent Added
| 0 | 1 | 2 | 3 | 4 | 5 | | Glu standard (1 mg/mL) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 | | Distilled water (mL) | 1 | 0.8 | 0.6 | 0.4 | 0.2 | 0 | | DNS Reagent (mL) | 2 | 2 | 2 | 2 | 2.5 | 2 | | Distilled water (mL) | 9 | 9 | 9 | 9 | 9.5 | 9 | | Equivalent glucose amount (mg) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 |
Note: After adding the DNS reagent, heat in a boiling water bath for exactly 5 minutes, then remove and cool to room temperature under running tap water. |
4. Determination of Total Sugars and Reducing Sugars:
Take 1 mL of the prepared reducing sugar extract or total sugar hydrolysate, add 2 mL of DNS Reagent, and proceed with the same operations as for the standard curve. Measure the absorbance of each tube at 540 nm.
Calculation:
Reducing sugar content (%):
Reducing sugar content (%) = (C × VT) / (m × VS ) × 100
Total sugar content (%):
Total sugar content (%) = (C × VT) / (m × VS) × 100 × 10 × 0.9
C = Sugar amount determined from the standard curve (mg/mL)
VT = Volume of the extraction solution (mL)
m = Mass of the plant sample (mg)
VS= Sample volume used for the assay (mL)
Precautions:
1.Avoid repeated freeze-thaw cycles of the above low-temperature reagents and store them protected from light to prevent loss of activity or efficiency.
2.If samples cannot be assayed immediately, store at 2–8°C; they are stable for 3 days.
3.If the reducing sugar concentration of the sample is too high, dilute with distilled water and re-measure, then multiply the result by the dilution factor.
4.The total sugar calculation formula is applicable when interfering impurities are minimal and the reducing sugar content is relatively low compared to the total sugar content. The factor × 0.9 is used to account for the water consumed during hydrolysis when converting total sugars into monosaccharides.
5.Commercially available hydrochloric acid is typically around 12 M and should be diluted to 6 M for use (the exact dilution factor depends on the concentration of the customer's hydrochloric acid. Note that 6 M hydrochloric acid is volatile and should be prepared fresh immediately before use).
6.The reagent should be used as soon as possible after opening to avoid affecting subsequent experimental results.
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