Method for determining total flavonoids in cerasus humilis fruits

Abstract

The invention belongs to the field of biological agents, and particularly relates to a method for determining total flavonoids in cerasus humilis fruits. The microplate reader is used for detecting 96 samples at a time, the efficiency is improved by about 10 times, and the method is suitable for rapid screening and analysis of large-batch samples. The detection result is more accurate, the correlation coefficient R2 of a standard curve is equal to 0.9972, and the result reliability is ensured. Background interference is corrected through the sample control tube, and data analysis is simpler, more convenient and more accurate. The absorbance difference (delta A) between the sample and the control tube directly reflects the content of flavone, and the content of flavone can be compared with a standard curve more accurately, so that the accurate flavone concentration can be quickly obtained.

Description

Technical Field

[0001] The invention belongs to the field of biological preparations, and particularly relates to a method for determining total flavonoids in Prunus cerasifera fruits. Background Art

[0002] Flavonoids are widely found in plants in nature and are secondary metabolites of plants. Flavonoids are a type of yellow pigment derived from flavonoids (2-phenylchromone) as the parent nucleus, including isomers of flavonoids and their hydrogenation and reduction products, that is, a series of compounds with C6-C3-C6 as the basic carbon frame. Flavonoids are widely distributed in the plant kingdom. Most of them exist in the form of glycosides or carbon sugars combined with sugars in plants, and some exist in free form.

[0003] There are many compounds with medicinal value in flavonoids. These compounds are used to prevent and treat cardiovascular and cerebrovascular diseases, such as reducing the fragility of blood vessels, improving vascular permeability, reducing blood lipids and cholesterol, preventing and treating hypertension in the elderly, cerebral hemorrhage, coronary heart disease, angina pectoris, dilating coronary blood vessels, and increasing coronary flow. Many flavonoid components have antitussive, expectorant, antiasthmatic and antibacterial activities. They also have liver protection, liver detoxification, antifungal, treatment of acute and chronic hepatitis, cirrhosis, and anti-free radical and antioxidant effects. In addition, flavonoids have the same effects as phytoestrogens. In animal production in animal husbandry, the application of flavonoids can significantly improve animal production performance, improve animal disease resistance, and improve animal immune function.

[0004] In traditional methods, the determination of the total content of flavonoids often relies on relatively simple color reaction methods, but these methods may be affected by background interference. It is not considered that the red or purple sample background may interfere with the color reaction, affect the accurate determination of absorbance, and lead to inaccurate results. The existing detection of flavonoid content mostly relies on a single spectrophotometer for sample-by-sample analysis, which has low sample throughput and cumbersome operation, low efficiency, and long time consumption, making it difficult to meet the needs of efficient analysis. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for determining total flavonoids in Prunus cerasifera fruit, comprising the following steps:

[0006] (1) Assay tube setup: Take 30 μL of the extracted total flavonoid sample solution and add it to a 2 mL centrifuge tube, add 30 μL of 5% NaNO2 solution, vortex thoroughly to mix, let stand at room temperature for 5 min, add 30 μL of 10% Al(NO3)3 solution, vortex thoroughly to mix again, let stand at room temperature for 5 min, immediately add 400 μL of 1 mol / L NaOH solution, then add 510 μL of 40% ethanol, and place the sample assay tube in a 37°C water bath for 30 min to ensure sufficient color development;

[0007] (2) Control tube setting: replace Al(NO3)3 solution with distilled water; take 30 μL of the extracted total flavonoid sample solution, add it to a 2 mL centrifuge tube, add 30 μL of 5% NaNO2 solution, vortex thoroughly, let it stand at room temperature for 5 min, add 30 μL of distilled water, let it stand at room temperature for 5 min, immediately add 400 μL of 1 mol / L NaOH solution, then add 510 μL of 40% ethanol, and place the sample control tube in a 37°C water bath for 30 min to ensure sufficient color development;

[0008] (3) Absorbance determination: Take 200 μL of the reaction solution in the test tube and the control tube and place it in a 96-well plate. Use an ELISA reader to measure the absorbance of the sample tube and the control tube at a wavelength of 510 nm.

[0009] (4) Preparation of standard curve: Prepare a standard solution, dilute the standard solution and measure the absorbance value, use the measured absorbance value as the ordinate and the total flavonoids concentration as the abscissa to draw a standard curve.

[0010] Furthermore, the preparation method of the standard solution in step (4) is: accurately weigh 20 mg of rutin, add 2 mL of extract, and prepare a 10 mg / mL rutin standard solution.

[0011] The present invention has the following beneficial effects:

[0012] By adding a sample control tube, the experiment can more accurately reflect the actual content of flavonoids. The use of the control tube helps to eliminate the influence of other substances or background color, so that the measured absorbance change directly reflects the change of flavonoids. Compared with the prior art, the introduction of the sample control tube significantly improves the accuracy and reliability of the determination.

[0013] The present invention uses an ELISA instrument to detect 96 samples at a time, which increases the efficiency by about 10 times and is suitable for rapid screening and analysis of large quantities of samples. The test results are more accurate, and the correlation coefficient of the standard curve R 2 =0.9972, ensuring the reliability of the results. The background interference is corrected by the sample control tube, making data analysis simpler and more accurate. The absorbance difference (ΔA) between the sample and control tube directly reflects the content of flavonoids, which can be more accurately compared with the standard curve to quickly obtain the accurate flavonoid concentration. DETAILED DESCRIPTION

[0014] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0015] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0016] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0017] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0018] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0019] Example 1 Sample measurement operation

[0020] Determination principle: The experiment uses the NaNO2-Al(NO3)3-NaOH colorimetric method to determine the total flavonoid content, that is, in an alkaline nitrite solution, flavonoid compounds and aluminum ions form a red complex with a characteristic absorption peak at 510nm. The absorbance value of the reaction product at 510nm can be measured to calculate the total flavonoid content in the sample. Since the fruit of European plum is red, in order to remove the influence of the background color, a determination tube and a control tube are used during the determination.

[0021] Assay tube: Take 30 μL of the extracted total flavonoid sample solution, add it to a 2mL centrifuge tube, add 30 μL of 5% mass concentration NaNO2 solution, vortex mix thoroughly, let stand at room temperature for 5 minutes, add 30 μL of 10% mass concentration Al(NO3)3 solution, vortex mix thoroughly again, let stand at room temperature for 5 minutes, immediately add 400 μL 1mol / LNaOH solution, then add 510 μL40% ethanol, and place the sample assay tube in a 37°C water bath to react for 30 minutes to ensure sufficient color development.

[0022] Control tube: Replace Al(NO3)3 solution with distilled water. Take 30μL of the extracted total flavonoid sample solution, add it to a 2mL centrifuge tube, add 30μL of 5% NaNO2 solution, vortex thoroughly, let stand at room temperature for 5min, add 30μL of distilled water, let stand at room temperature for 5min, immediately add 400μL of 1mol / LNaOH solution, then add 510μL of 40% ethanol, and place the sample control tube in a 37℃ water bath for 30min to ensure full color development.

[0023] Take 200 μL of the reaction solution in the test tube and the control tube and put it in a 96-well plate, and use an enzyme-labeled instrument to detect the absorbance of the sample tube and the control tube at a wavelength of 510 nm.

[0024] Example 2 Standard Curve Preparation

[0025] 2.1 Preparation of standard solution: Accurately weigh 20 mg of rutin and add 2 mL of extract to prepare a 10 mg / mL rutin standard solution.

[0026] 2.2 Dilution of standard solution

[0027] Prepare 8 1.5 mL centrifuge tubes and dilute the 10 mg / mL standard solution with the extract to produce 0, 0.25, 0.5, 1, 1.5, 2, 2.5, and 3 mg / mL standard solutions.

[0028]

[0029]

[0030] Pipette 30 μL from each tube and add it to a 2 mL centrifuge tube. Add 30 μL of 5% NaNO2 solution, vortex thoroughly to mix, let stand at room temperature for 5 min, add 30 μL of 10% Al(NO3)3 solution, vortex thoroughly to mix again, let stand at room temperature for 5 min, immediately add 400 μL of 1 mol / L NaOH solution, then add 510 μL of 40% ethanol, and place in a 37°C water bath to react for 30 min to ensure adequate color development.

[0031] Take 200 μL of the above reaction solution into a 96-well plate and use an ELISA reader to detect the absorbance value of the standard tube at a wavelength of 510 nm.

[0032] 2.3 Drawing the standard curve

[0033] The measured absorbance value (OD510nm) was used as the ordinate, and the total flavonoids concentration (mg / mL) expressed as rutin concentration was used as the abscissa to draw a standard curve.

[0034] The standard curve conforms to the linear relationship and is fitted by linear regression. The regression equation is: y = kx + b

[0035] Where: y is absorbance; x is total flavonoid concentration (mg / mL).

[0036] 2.4 Calculation formula of total flavonoid content

[0037] The calculation formula of total flavonoid content in the experiment is:

[0038]

[0039] Example 3. Experimental data and result analysis

[0040]

[0041]

[0042] Linear regression results:

[0043] The standard curve regression equation is: y = 0.2225x-0.0047

[0044] Regression coefficient R 2 =0.9972, indicating that the linear fitting effect is good.

[0045] Sample measurement data: The absorbance value (OD510nm) of the sample measurement tube was measured to be 0.156.

[0046] The absorbance value (OD510nm) of the sample control tube was measured to be 0.038.

[0047] ΔAsample = Aassay - Acontrol = 0.156 - 0.038 = 0.118

[0048] Substitute into the standard curve equation: The total volume of the extract was 10 mL, and the fresh weight of the sample was 0.6 g.

[0049] Calculation of total flavonoid content: Total flavonoid content

Claims

1. A method for determining total flavonoids in Prunus cerasifera fruit, characterized in that: The following steps are involved: (1) Assay tube setup: Take 30 μL of the extracted total flavonoid sample solution and add it to a 2 mL centrifuge tube, add 30 μL of 5% NaNO2 solution, vortex thoroughly to mix, let stand at room temperature for 5 min, add 30 μL of 10% Al(NO3)3 solution, vortex thoroughly to mix again, let stand at room temperature for 5 min, immediately add 400 μL of 1 mol / L NaOH solution, then add 510 μL of 40% ethanol, and place the sample assay tube in a 37°C water bath for 30 min to ensure sufficient color development; (2) Control tube setting: replace Al(NO3)3 solution with distilled water; take 30 μL of the extracted total flavonoid sample solution, add it to a 2 mL centrifuge tube, add 30 μL of 5% NaNO2 solution, vortex thoroughly, let it stand at room temperature for 5 min, add 30 μL of distilled water, let it stand at room temperature for 5 min, immediately add 400 μL of 1 mol / L NaOH solution, then add 510 μL of 40% ethanol, and place the sample control tube in a 37°C water bath for 30 min to ensure sufficient color development; (3) Absorbance determination: Take 200 μL of the reaction solution in the test tube and the control tube and place it in a 96-well plate. Use an ELISA reader to measure the absorbance of the sample tube and the control tube at a wavelength of 510 nm. (4) Preparation of standard curve: Prepare a standard solution, dilute the standard solution and measure the absorbance value, use the measured absorbance value as the ordinate and the total flavonoids concentration as the abscissa to draw a standard curve.

2. The method according to claim 1, characterized in that: The preparation method of the standard solution in step (4) is: accurately weigh 20 mg of rutin, add 2 mL of extract, and prepare a 10 mg / mL rutin standard solution.

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