Method for determining procyanidine in cerasus humilis fruits

Abstract

The invention belongs to the technical field of procyanidine determination, and particularly relates to a method for determining procyanidine in cerasus humilis fruits. A standard curve is made by using a procyanidine standard substance and is closer to the chemical composition of a sample, so that the actual content of procyanidine in the sample is more accurately reflected, the determination deviation caused by structural difference is avoided, a microplate reader high-throughput detection method is introduced, a vanillin developing method is combined, a plurality of samples are detected at one time, and the detection accuracy is high. The sample detection speed and flux are greatly improved, and a rapid and accurate analysis tool is provided for sample content determination.

Description

Technical Field

[0001] The invention belongs to the technical field of proanthocyanidin determination, and particularly relates to a method for determining proanthocyanidins in European plum fruits. Background Art

[0002] Proanthocyanidins are a general term for a large class of polyphenolic compounds that are widely found in plants. They have strong antioxidant and free radical scavenging effects, can effectively eliminate superoxide anion free radicals and hydroxyl free radicals, and are also involved in the metabolism of phosphate and arachidonic acid and protein phosphorylation, protecting lipids from peroxidation damage; they are powerful metal chelators that can chelate metal ions to form inert compounds in the body; they protect and stabilize vitamin C, and help absorb and utilize vitamin C. Proanthocyanidins are widely distributed and exist in the skins, shells, seeds, cores, flowers, and leaves of many plants. Grape seeds have the highest content of proanthocyanidins and are rich in variety.

[0003] The prior art expresses the content of proanthocyanidins in terms of catechin equivalents. Catechins are the basic monomers of proanthocyanidins, and proanthocyanidins are usually a mixture of oligomers and polymers formed by polymerization of catechins and epicatechins. Using catechins as a standard cannot fully represent the actual structure and properties of polymer proanthocyanidins. The results may underestimate the content of high-polymerization proanthocyanidins in the sample. In addition, the existing detection of proanthocyanidin content mostly relies on a single spectrophotometer for sample-by-sample analysis and detection, which has a 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

[0004] In order to solve the above problems, the present invention provides a method for determining proanthocyanidins in Prunus cerasifera fruit, comprising the following steps:

[0005] (1) Sample solution preparation: Select mature, non-moldy Prunus cerasus fruit, remove soil, impurities and pollutants on the surface of the fruit, remove the core and stalk, and obtain a Prunus cerasus fruit sample; add concentrated hydrochloric acid to the ethanol solution and mix thoroughly to obtain an extract; add the extract to the Prunus cerasus sample, grind thoroughly, perform ultrasonic treatment, and centrifuge to obtain an anthocyanin sample solution;

[0006] (2) draw a standard curve;

[0007] (3) Sample determination: Take 100 μL of the extracted proanthocyanidin sample solution, add it to a test tube, add 600 μL of 0.04 g / mL vanillin methanol solution, shake well, add 300 μL of concentrated hydrochloric acid, shake well again, and place the sample determination tube in a 30°C water bath for 20 minutes to ensure sufficient color development. Use the extract instead of the sample as a blank tube; take 200 μL of the reaction solution in a 96-well plate, and use an enzyme reader to detect the absorbance of the sample tube and the blank tube at a wavelength of 500 nm;

[0008] (4) Data calculation: According to the formula Calculate the content of proanthocyanidins in the samples.

[0009] Furthermore, the volume fraction of the concentrated hydrochloric acid in step (1) is 36% to 38%.

[0010] Furthermore, the ethanol solution in step (1) is a 40% ethanol aqueous solution.

[0011] Furthermore, in step (1), the ratio of concentrated hydrochloric acid to ethanol solution is 1:400.

[0012] Furthermore, the mass volume ratio of the European plum sample to the extract in step (1) is 0.6g:10mL.

[0013] Furthermore, the ultrasonic treatment in step (1) is as follows: ultrasonic extraction at 25° C. for 30 min; the frequency of the ultrasonic wave is set to 40 kHz and the power is 200 W.

[0014] Furthermore, the centrifugation in step (1) is performed at 5000 r / min for 10 min.

[0015] Furthermore, the concentration of concentrated hydrochloric acid in step (3) is 36% to 38%.

[0016] The present invention has the following beneficial effects:

[0017] The present invention uses proanthocyanidin standards to prepare a standard curve, which is closer to the chemical composition of the sample, thereby more accurately reflecting the actual content of proanthocyanidins in the sample and avoiding measurement deviations caused by structural differences. In addition, a high-throughput detection method of an ELISA reader is introduced, combined with a vanillin colorimetric method, to detect multiple samples at one time, greatly improving the sample detection speed and throughput, and providing a fast and accurate analysis tool for sample content determination. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] 1. Extraction of proanthocyanidins from Prunus armeniaca fruit

[0024] 1.1 Sample preparation

[0025] Fruit treatment: Select ripe, mildew-free European plum fruits, remove dirt, impurities and contaminants from the surface of the fruit, and remove the core and stalk. Accurately weigh 0.6g of cored fruit pulp sample and put it into a mortar for later use.

[0026] 1.2 Preparation of extract

[0027] Extraction solution composition: 0.1% hydrochloric acid in 40% ethanol aqueous solution.

[0028] Preparation method: Take 40mL of anhydrous ethanol and add 60mL of distilled water to make a 40% ethanol solution.

[0029] Take 0.1 mL of concentrated hydrochloric acid (volume fraction of concentrated hydrochloric acid is 36% to 38%), add it to the above 40% ethanol solution, mix thoroughly, and the extract is obtained.

[0030] 1.3 Sample extraction

[0031] Preliminary grinding: Add 2 mL of extract into a mortar and grind the pulp sample thoroughly with a pestle until the sample becomes a uniform slurry.

[0032] Mortar cleaning: Use 2 mL of extraction solution to clean the mortar and pestle, and flush the remaining pulp slurry into a 10 mL centrifuge tube.

[0033] Ultrasonic assisted extraction: The centrifuge tube was placed in an ultrasonic chamber and subjected to ultrasonic extraction for 30 min at 25°C. The frequency of the ultrasonic wave was set to 40 kHz and the power was 200 W.

[0034] Centrifugation: After extraction, place the sample in a centrifuge and centrifuge at 5000r / min for 10 minutes. Collect the supernatant after centrifugation and transfer it to a clean 10mL centrifuge tube.

[0035] Repeat extraction: Repeat the above ultrasonic extraction and centrifugal separation process on the centrifuged sediment, and use fresh extraction solution to repeat the extraction 3 times. Collect the supernatant each time and combine the supernatants of the 3 times.

[0036] 1.4 Volume determination and storage

[0037] Transfer the combined supernatant to a 10 mL volumetric flask and dilute to 10 mL with an extract (0.1% hydrochloric acid in 40% ethanol). Transfer the dilute extract to a sealed container and store it in a freezer at -20°C to avoid degradation of proanthocyanidins caused by light and high temperature. The proanthocyanidin content will be determined later.

[0038] 2. Quantitative analysis of proanthocyanidins content in Prunus armeniaca fruit

[0039] 2.1 Sample measurement operation

[0040] Take 100 μL of the extracted proanthocyanidin sample solution, add it to a test tube, add 600 μL of 0.04 g / mL vanillin methanol solution, shake well, add 300 μL of concentrated hydrochloric acid (36% to 38%), shake well again, and place the sample measurement tube in a 30°C water bath for 20 minutes to ensure sufficient color development. Use the extract instead of the sample as a blank tube.

[0041] Take 200 μL of the reaction solution into a 96-well plate and use an ELISA reader to detect the absorbance of the sample tube and the blank tube at a wavelength of 500 nm.

[0042] 2.2 Preparation of standard curve

[0043] 2.2.1 Preparation of standard solution: Accurately weigh 20 mg of proanthocyanidins and add 2 mL of extract to prepare a 10 mg / mL proanthocyanidin standard solution.

[0044] 2.2.2 Dilution of standard solution

[0045] Prepare 8 1.5 mL centrifuge tubes and dilute the 10 mg / mL standard solution with the extract to produce 0, 0.5, 1, 1.5, 2, 2.5, 3, and 4 mg / mL standard solutions.

[0046] Tube No. Proanthocyanidins standard solution (μL) Extraction solution (μL) Proanthocyanidin concentration (mg / mL) 1 0 1000 0 2 50 950 0.5 3 100 900 1 4 150 850 1.5 5 200 800 2 6 250 750 2.5 7 300 700 3 8 400 600 4

[0047] Pipette 100 μL into each tube, add 600 μL vanillin methanol solution, shake well, add 300 μL concentrated hydrochloric acid, and shake well again.

[0048] Place all test tubes in a 30°C water bath for 20 minutes. Pipette 200 μL of the reaction solution from each tube into a 96-well plate and measure the absorbance of each tube at a wavelength of 500 nm using an ELISA reader. Use the extract instead of the sample as a blank tube.

[0049] 2.2.3 Drawing the standard curve

[0050] The measured absorbance value (OD500nm) was used as the ordinate and the proanthocyanidin concentration (mg / mL) was used as the abscissa to draw a standard curve.

[0051] The standard curve conforms to the linear relationship and is fitted by linear regression. The regression equation is: y = kx + b

[0052] Where: y is absorbance; x is proanthocyanidin concentration (mg / mL).

[0053] 2.2.4 Calculation formula of proanthocyanidin content

[0054] The calculation formula of proanthocyanidin content in the experiment is:

[0055] 3. Experimental data and results analysis

[0056] Tube No. Proanthocyanidin concentration (mg / mL) Absorbance (OD500nm) 1 0 0 2 0.5 0.3118 3 1 0.5929 4 1.5 0.8621 5 2 1.1452 6 2.5 1.4264 7 3 1.7375 8 4 2.2798

[0057] Linear regression results:

[0058] The standard curve regression equation is: y = 0.5648x + 0.0237

[0059] Regression coefficient R 2 =0.9998, indicating that the linear fitting effect is good.

[0060] Sample measurement data: The sample absorbance value (OD500nm) was measured to be 0.4712.

[0061] 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.

[0062] Calculation of proanthocyanidin content:

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for determining proanthocyanidins in Prunus armeniaca fruit, characterized in that: The following steps are involved: (1) Sample solution preparation: Select mature, non-moldy Prunus cerasus fruit, remove soil, impurities and pollutants on the surface of the fruit, remove the core and stalk, and obtain a Prunus cerasus fruit sample; add concentrated hydrochloric acid to the ethanol solution and mix thoroughly to obtain an extract; add the extract to the Prunus cerasus sample, grind thoroughly, perform ultrasonic treatment, and centrifuge to obtain an anthocyanin sample solution; (2) draw a standard curve; (3) Sample determination: Take 100 μL of the extracted proanthocyanidin sample solution, add it to a test tube, add 600 μL of 0.04 g / mL vanillin methanol solution, shake well, add 300 μL of concentrated hydrochloric acid, shake well again, and place the sample determination tube in a 30°C water bath for 20 minutes to ensure sufficient color development. Use the extract instead of the sample as a blank tube; take 200 μL of the reaction solution in a 96-well plate, and use an enzyme reader to detect the absorbance of the sample tube and the blank tube at a wavelength of 500 nm; (4) Data calculation: Proanthocyanidin content according to the formula Calculate the content of proanthocyanidins in the samples.

2. The measuring method according to claim 1, characterized in that The volume fraction of concentrated hydrochloric acid in step (1) is 36% to 38%.

3. The measuring method according to claim 1, characterized in that The ethanol solution in step (1) is a 40% ethanol aqueous solution.

4. The measuring method according to claim 1, characterized in that The ratio of concentrated hydrochloric acid to ethanol solution in step (1) is 1:

400.

5. The measuring method according to claim 1, characterized in that The mass volume ratio of the European plum sample to the extract in step (1) is 0.6g:10mL.

6. The measuring method according to claim 1, characterized in that The ultrasonic treatment in step (1) is as follows: ultrasonic extraction at 25° C. for 30 min; the frequency of the ultrasonic wave is set to 40 kHz and the power is 200 W.

7. The measuring method according to claim 1, characterized in that The centrifugation in step (1) is 5000 r / min for 10 min.

8. The measuring method according to claim 1, characterized in that The concentration of concentrated hydrochloric acid in step (3) is 36% to 38%.

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