Method for extracting procyanidine from cerasus humilis fruits

Abstract

The invention belongs to the technical field of procyanidine extraction, and particularly relates to a method for extracting procyanidine from cerasus humilis fruits. An alcohol-acid system is introduced to replace a single alcohol solvent, the acid environment of the solution is adjusted, chemical bonds between the proanthocyanidins and other molecules are destroyed, release of the combined-state proanthocyanidins is promoted, dissolution of some water-soluble impurities can be inhibited through the acid environment, and the purity of the proanthocyanidins in the extract is higher. By combining an ultrasonic-assisted extraction technology, cell wall breaking is accelerated through an ultrasonic cavitation effect, the dissolution rate is increased, meanwhile, the extraction time is shortened, the temperature is reduced, and the activity of the procyanidine is protected.

Description

Technical Field

[0001] The invention belongs to the technical field of proanthocyanidin extraction, and particularly relates to a method for extracting proanthocyanidins from 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] Traditional methods mostly use a single alcohol solvent (such as ethanol, methanol) or static extraction for extraction, but the solubility of the bound and highly polymerized forms of proanthocyanidins is insufficient, resulting in low extraction efficiency. In addition, a single alcohol solution has a certain solubility for a variety of plant secondary metabolites (such as sugars, proteins, lipids, pigments, etc.), which may lead to a higher impurity content in the extract and reduce the purity of the target. Summary of the invention

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

[0005] (1) Sample preparation: Select mature, non-moldy Prunus cerasifera fruits, remove soil, impurities and contaminants on the surface of the fruits, remove the core and stalk, and obtain Prunus cerasifera fruit samples;

[0006] (2) Preparation of the extract: Add concentrated hydrochloric acid to the ethanol solution and mix thoroughly to obtain the extract;

[0007] (3) Sample extraction: Add the extract to the European plum sample, grind it thoroughly, and then perform ultrasonic treatment. After centrifugation, anthocyanin sample solution is obtained.

[0008] Furthermore, the volume fraction of the concentrated hydrochloric acid in step (2) is 36% to 38%.

[0009] Furthermore, the ethanol solution in step (2) is a 40% ethanol aqueous solution.

[0010] Furthermore, in step (2), the ratio of concentrated hydrochloric acid to ethanol solution is 1:400.

[0011] Furthermore, the mass volume ratio of the European plum sample to the extract in step (3) is 0.6g:10mL.

[0012] Furthermore, the ultrasonic treatment in step (3) 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.

[0013] Furthermore, the centrifugation in step (3) is performed at 5000 r / min for 10 min.

[0014] The present invention has the following beneficial effects:

[0015] 1. Significant improvement in extraction efficiency

[0016] By using alcohol-acid system and ultrasonic-assisted extraction technology, the extraction time is shortened to 1.5 hours, which is only 1 / 2 of the traditional method. The extraction temperature is reduced to 25-30℃ to avoid high temperature oxidative degradation of proanthocyanidins and protect their activity.

[0017] 2. Enhanced green environmental performance

[0018] The ethanol concentration is reduced to 40% to 50%, and the amount of solvent is reduced by about 30%. The extraction temperature is reduced to 25°C, which has a significant energy-saving effect and a more environmentally friendly process.

[0019] 3. Improved accuracy of results

[0020] The proanthocyanidin standard has the same chemical composition as the sample, and the color reaction efficiency of the standard curve is consistent with that of the sample, which significantly improves the accuracy of the measurement results. The measured value can be directly expressed as the "proanthocyanidin content" in the sample, without the need to convert it into catechin equivalents, which is closer to the actual situation of the sample and can simultaneously reflect the color contribution of low-polymerization and high-polymerization proanthocyanidins, making the results more representative.

[0021] 4. Improvement of detection efficiency and accuracy

[0022] Using an ELISA reader to test 96 samples at a time increases efficiency by about 10 times, making it suitable for rapid screening and analysis of large quantities of samples. The test results are more accurate, and the standard curve correlation coefficient R2 = 0.9998, ensuring the reliability of the results. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] 1. Extraction of proanthocyanidins from Prunus armeniaca fruit

[0029] 1.1 Sample preparation

[0030] 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.

[0031] 1.2 Preparation of extract

[0032] Extraction solution composition: 0.1% hydrochloric acid in 40% ethanol aqueous solution.

[0033] Preparation method: Take 40mL of anhydrous ethanol and add 60mL of distilled water to make a 40% ethanol solution.

[0034] 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, and mix thoroughly to obtain the extract.

[0035] 1.3 Sample extraction

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 1.4 Volume determination and storage

[0042] 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.

[0043] 2. Quantitative analysis of proanthocyanidins content in Prunus armeniaca fruit

[0044] 2.1 Sample measurement operation

[0045] 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.

[0046] 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.

[0047] 2.2 Preparation of standard curve

[0048] 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.

[0049] 2.2.2 Dilution of standard solution

[0050] 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.

[0051] 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

[0052] Pipette 100 μL into each tube, add 600 μL vanillin methanol solution, shake well, add 300 μL concentrated hydrochloric acid, and shake well again.

[0053] 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.

[0054] 2.2.3 Drawing the standard curve

[0055] 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.

[0056] The standard curve conforms to the linear relationship and is fitted by linear regression. The regression equation is: y = kx + b

[0057] Where: y is absorbance; x is proanthocyanidin concentration (mg / mL).

[0058] 2.2.4 Calculation formula of proanthocyanidin content

[0059] The calculation formula of proanthocyanidin content in the experiment is:

[0060] 3. Experimental data and results analysis

[0061] 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

[0062] Linear regression results:

[0063] The standard curve regression equation is: y = 0.5648x + 0.0237

[0064] Regression coefficient R 2 =0.9998, indicating that the linear fitting effect is good.

[0065] Sample measurement data: The sample absorbance value (OD500nm) was measured to be 0.4712.

[0066] 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.

[0067] Calculation of proanthocyanidin content:

[0068] 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 extracting proanthocyanidins from Prunus cerasifera fruit, characterized in that: The following steps are involved: (1) Sample preparation: Select mature, non-moldy Prunus cerasifera fruits, remove soil, impurities and pollutants on the surface of the fruits, remove the core and stalk, and obtain Prunus cerasifera fruit samples; (2) Preparation of the extract: Add concentrated hydrochloric acid to the ethanol solution and mix thoroughly to obtain the extract; (3) Sample extraction: Add the extract to the European plum sample, grind it thoroughly, treat it with ultrasound, and then centrifuge it to obtain the proanthocyanidin sample solution.

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

3. The extraction method according to claim 1, characterized in that The ethanol solution in step (2) is a 40% ethanol aqueous solution.

4. The extraction method according to claim 1, characterized in that The ratio of concentrated hydrochloric acid to ethanol solution in step (2) is 1:

400.

5. The extraction method according to claim 1, characterized in that The mass volume ratio of the European plum sample to the extract in step (3) is 0.6g:10mL.

6. The extraction method according to claim 1, characterized in that The ultrasonic treatment in step (3) 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 extraction method according to claim 1, characterized in that The centrifugation in step (3) is 5000 r / min for 10 min.

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