Method for extracting total flavonoids from cerasus humilis fruits

Abstract

The invention belongs to the field of biological agents, and particularly relates to a method for extracting total flavonoids from cerasus humilis fruits. According to the method, an alcohol-acid system is introduced to replace a single alcohol solvent, and an ultrasonic-assisted extraction technology is adopted, so that extraction can be performed at a relatively low temperature, and the risk of thermal degradation of flavonoid substances caused by high temperature is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biological preparations, and particularly relates to a method for extracting total flavonoids from European plum 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] Traditional extraction methods usually use conventional grinding, soaking and high-temperature extraction. Although these methods are widely used, they have low extraction efficiency, take a long time, and may not be able to effectively extract all flavonoid components. In particular, for the extraction of flavonoid glycosides, they are often unable to be fully hydrolyzed, resulting in low extraction efficiency.

[0005] Although commonly used solvents such as water or ethanol can extract certain flavonoids, their solubility and stability for certain flavonoid compounds are still limited. In addition, some methods do not add acidic solutions or other auxiliary reagents to promote the hydrolysis of flavonoid glycosides, thus affecting the extraction effect.

[0006] In the existing method, only one extraction is performed, the extracted flavonoid content is not high, and the maximum extraction potential of the extraction solution is not fully utilized, resulting in insufficient flavonoid concentration in the extract, affecting the accuracy of subsequent analysis. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for extracting total flavonoids from Prunus cerasifera fruit, comprising the following steps:

[0008] (1) Sample preparation: Select mature, non-moldy Prunus cerasifera fruits, remove dirt, impurities and contaminants on the surface of the fruits, and remove the core and stalk to obtain the samples to be extracted;

[0009] (2) Preparation of extract: Take anhydrous ethanol and add distilled water to prepare an ethanol solution; then take concentrated hydrochloric acid and add it to the ethanol solution, mix thoroughly, and obtain an extract;

[0010] (3) Total flavonoids extraction: The sample to be extracted is mixed with the extract and ground to obtain a homogenate, the homogenate is subjected to ultrasonic treatment, and the homogenate after ultrasonic treatment is centrifuged to obtain the supernatant to obtain the total flavonoids in the fruit of European plum.

[0011] Furthermore, the concentration of the ethanol solution in step (2) is 40%.

[0012] Furthermore, the concentration of concentrated hydrochloric acid in step (2) is 36% to 38% by volume.

[0013] Furthermore, the volume ratio of the ethanol solution to concentrated hydrochloric acid in step (2) is 400:1.

[0014] Furthermore, in step (3), the ratio of the sample to be extracted to the extract is 0.6 g:10 mL.

[0015] Furthermore, the ultrasonic treatment conditions in step (3) are ultrasonic extraction at 25° C. for 30 min, with an ultrasonic frequency of 40 kHz and a power of 200 W.

[0016] Furthermore, the centrifugation condition in step (3) is 5000 r / min for 10 minutes.

[0017] The present invention has the following beneficial effects:

[0018] The present invention introduces an alcohol acidification system (40% ethanol + 0.1% hydrochloric acid) to replace a single alcohol solvent and adjusts the acidic environment of the solution to promote the hydrolysis of flavonoid glycosides and increase the solubility of flavonoid compounds, thereby effectively increasing the extraction amount of flavonoids.

[0019] By adopting ultrasound-assisted extraction technology, extraction can be performed at a lower temperature, reducing the risk of thermal degradation of flavonoids by high temperature. At the same time, the use of low-temperature storage (-20°C) after extraction can maintain the stability of flavonoids and avoid degradation or oxidation during storage.

[0020] Repeated extraction was performed multiple times, and the supernatant of each extraction was combined to increase the extraction amount and concentration of flavonoids and improve the overall extraction effect of the sample. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] 1. Extraction of total flavonoids from Prunus armeniaca fruit

[0027] 1.1 Sample preparation

[0028] 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.

[0029] 1.2 Preparation of extract

[0030] Extraction solution composition: 0.1% hydrochloric acid in 40% ethanol aqueous solution.

[0031] Preparation method: Take 40mL of anhydrous ethanol and add 60mL of distilled water to make a 40% ethanol solution.

[0032] 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.

[0033] 1.3 Sample extraction

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 1.4 Volume determination and storage

[0040] The combined supernatant was transferred to a 10 mL volumetric flask and diluted to 10 mL mark with an extract (0.1% hydrochloric acid in 40% ethanol). The diluted extract was transferred to a sealed container and stored at -20°C to avoid degradation of flavonoids and maintain their stability. The total flavonoid content was determined later.

[0041] 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 total flavonoids from Prunus cerasifera fruit, characterized in that: The following steps are involved: (1) Sample preparation: Select mature, non-moldy Prunus cerasifera fruits, remove dirt, impurities and contaminants on the surface of the fruits, and remove the core and stalk to obtain the samples to be extracted; (2) Preparation of extract: Take anhydrous ethanol and add distilled water to prepare an ethanol solution; then take concentrated hydrochloric acid and add it to the ethanol solution, mix thoroughly, and obtain an extract; (3) Total flavonoids extraction: The sample to be extracted is mixed with the extract and ground to obtain a homogenate, the homogenate is subjected to ultrasonic treatment, and the homogenate after ultrasonic treatment is centrifuged to obtain the supernatant to obtain the total flavonoids in the fruit of European plum.

2. The method according to claim 1, characterized in that: The concentration of the ethanol solution in step (2) is 40%.

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

4. The method according to claim 1, characterized in that: The volume ratio of the ethanol solution to concentrated hydrochloric acid in step (2) is 400:

1.

5. The method according to claim 1, characterized in that The ratio of the sample to be extracted to the extract in step (3) is 0.6 g:10 mL.

6. The method according to claim 1, characterized in that The ultrasonic treatment conditions in step (3) are ultrasonic extraction at 25° C. for 30 min, with an ultrasonic frequency of 40 kHz and a power of 200 W.

7. The method according to claim 1, characterized in that The centrifugation condition in step (3) is 5000r / min for 10 minutes.