Method for extracting high-purity cyanidin galactoside from Aronia nigra

Abstract

The present invention discloses a method for efficiently extracting cyanidin galactoside from Aronia nigra, which is characterized by comprising the following steps: 1) raw material pretreatment; 2) ethanol extraction; 3) solvent recovery concentration; 4) macroporous adsorption resin adsorption and analysis; 5) solvent recovery concentration; 6) microsphere reverse phase chromatography filler purification; 7) low temperature concentration crystallization and recrystallization; 8) freeze drying. The method of the present invention can prepare cyanidin galactoside with a purity greater than 95% through secondary macroporous resin adsorption and combined with microsphere reverse phase chromatography filler purification. The process of the present invention is simple, the extraction process is safe and mild, and it is conducive to the promotion and application of cyanidin galactoside in large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of plant active ingredient extraction, and particularly relates to a method for extracting high-purity cyanidin galactoside from Sorbus melanocarpa. Background Art

[0002] Aronia nigra, also known as immortal berry and wild aronia, is native to the northeastern United States and is a perennial deciduous shrub of the genus Aronia in the Rosaceae family. In the 1990s, the Liaoning Arid Area Research Institute introduced a cultivar of Aronia nigra to Northeast my country, and since then, research on Aronia nigra in my country has begun. Since the introduction of the species, my country has mastered the widespread application of sexual reproduction and asexual propagation technology after years of research, and has successively completed research on cultivation and regional trials. The current planting areas are mainly concentrated in the eastern and central parts of my country. Aronia nigra has three main values: medicinal, edible, and ornamental. Aronia nigra is rich in anthocyanins, proanthocyanidins, flavonoids, chlorogenic acid, dietary fiber and other substances. In 2018, the National Health Commission issued the "Announcement on New Food Ingredients of Aronia nigra Fruit", stating that Aronia nigra and its products have the potential to promote health and have potential market prospects in the health care products, natural food additives, cosmetics and other industries.

[0003] Anthocyanin is the glycoside form of anthocyanidin, which is widely present in the cell sap of flowers, fruits, stems, leaves and root organs of plants, making plants appear in different colors from red, purple to blue. Anthocyanin mainly includes 6 non-glycosides: pelargonidin, cyanidin, delphinidin, peony glycoside, petunidin and malvadin. Studies have shown that anthocyanin has antioxidant, anti-fatigue, anti-inflammatory, anti-tumor and vision-enhancing effects. It has been widely used in food, biology, medicine and other fields. The fruit of Aralia nigra is rich in polyphenols, and the polyphenol content is the highest in the known fruit, of which anthocyanin content can account for a quarter of the total phenol. The anthocyanins of Aralia nigra are mainly composed of four types of anthocyanins, including cyanidin-3-O-galactoside, cyanidin-3-arabinoside, cyanidin-3-glucoside, and cyanidin-3-xyloside, among which cyanidin galactoside accounts for the largest proportion of 68.68%.

[0004] Cyanidin 3-galactoside, also known as cyanidin galactoside, has a variety of physiological and biochemical effects such as protecting blood vessels and anticoagulation. Therefore, using the fruit of Aralia oleracea as raw material to extract, separate and purify cyanidin galactoside to prepare a high-purity cyanidin galactoside product will have broad market prospects. At present, most of the existing studies use conventional methods such as solvent extraction and ultrasonic-assisted extraction to extract anthocyanins from Aralia oleracea, but there are few reports on the extraction, separation and purification of cyanidin galactoside. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for extracting high-purity cyanidin galactoside from Aronia melanocarpa. The method can prepare cyanidin galactoside with a purity greater than 95% through secondary macroporous resin adsorption combined with microsphere reverse phase chromatography filler purification.

[0006] The present invention is achieved through the following technical solutions.

[0007] A method for efficiently extracting cyanidin galactoside from Aronia melanocarpa, characterized by comprising the following steps:

[0008] 1) Raw material pretreatment

[0009] Pick plump, disease-free and insect-free Aronia nigra fruits as raw materials, wash and drain the surface water, then quickly freeze them, and then store them at a low temperature below -18 for later use;

[0010] 2) Ethanol extraction

[0011] The raw material of step (1) is subjected to microwave thawing-slow freezing cycle treatment for 2 to 3 times, and then ethanol is added for ultrasonic-assisted extraction, and then the extract is collected by centrifugation and filtration for later use;

[0012] 3) Concentrate and recover the solvent

[0013] 4) Macroporous adsorption resin adsorption and analysis

[0014] The concentrated solution of step (3) is adsorbed by macroporous adsorption resin type I, and then desorbed by ethanol step by step, and then adsorbed by macroporous adsorption resin type II, and then desorbed by ethanol step by step, and the desorbed solution finally obtained is collected for later use;

[0015] 5) Concentrate and recover the solvent

[0016] 6) Purification by microsphere reverse phase chromatography filler

[0017] Add the microsphere reverse phase chromatography filler to the concentrated solution of step (5), stir for 10 to 30 minutes, then let it stand to take out the precipitated microsphere reverse phase chromatography filler, then elute the microsphere reverse phase chromatography filler, collect the eluate, and obtain a purified solution;

[0018] 7) Low temperature concentration crystallization and recrystallization

[0019] The purified solution obtained in step (6) is sequentially subjected to low temperature concentration crystallization and recrystallization, and the precipitated crystals are collected for later use;

[0020] 8) Freeze drying

[0021] The crystals obtained in step (7) are freeze-dried to obtain cyanidin galactoside.

[0022] Preferably, in step (2), the solid-liquid ratio of Aronia melanocarpa fruit to ethanol is 1:6-12 kg / L.

[0023] Preferably, in step (2), the power of the ultrasound is 150-200W, and the ultrasonic extraction time is 15-30min.

[0024] Preferably, in the step (2), the specific parameters of microwave thawing-slow freezing are: firstly placing the frozen Aronia nigra fruits in a container, then thawing them at a microwave power of 0.2 to 2.5 kW / kg for 2 to 5 min, and then placing the thawed Aronia nigra fruits at -12 to -8°C for slow freezing for 4 to 10 h.

[0025] Preferably, in step (4), the macroporous adsorption resin type I is D101 type, and the macroporous adsorption resin type II is AB-8 type.

[0026] Preferably, in step (6), the microsphere reverse phase chromatography filler is porous silica gel with a particle size of 18 to 30 μm.

[0027] Preferably, in step (6), the microsphere reverse phase chromatography filler is pretreated before use, that is, the microsphere reverse phase chromatography filler is mixed with sewage ethanol at a solid-liquid ratio of 1:3-5 kg / L, and then placed in ultrasonic oscillation for 10-15 minutes, and then filtered and separated to obtain the microsphere reverse phase chromatography filler, and then rinsed with distilled water and dried at low temperature.

[0028] Preferably, in step (8), the freeze-drying parameters are as follows: pre-freezing temperature of -55 to -45°C, time of 4 to 6 hours; sublimation drying temperature of -35 to -30°C, time of 22 to 24 hours; decomposition temperature of 30°C, time of 16 to 20 hours; freeze dryer vacuum degree of 1.0 Pa, cold trap temperature of -55 to -45°C.

[0029] Beneficial effects of the present invention:

[0030] 1) The present invention can retain the anthocyanin components in the black fruit aronia fruits to the maximum extent without being destroyed or lost by quick freezing and low temperature storage of the harvested black fruit aronia fruits, thereby facilitating the acquisition of high-purity cyanidin galactoside; and, the present application uses a microwave thawing-slow freezing cycle process in ethanol extraction, which can destroy the cell structure of the black fruit aronia fruits through repeated thawing and freezing, thereby facilitating the dissolution of effective ingredients in the cells, thereby improving the yield of ethanol extraction, and further enhancing the extraction effect by means of ultrasound assistance.

[0031] 2) The method of the present invention can prepare cyanidin galactoside with a purity greater than 95% through secondary macroporous resin adsorption, combined with microsphere reverse phase chromatography filler purification, low temperature concentration crystallization and recrystallization.

[0032] 3) The process of the present invention is simple, and the extraction process is safe and mild, which is conducive to the promotion and application in the large-scale production of cyanidin galactoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the liquid chromatogram of the solvent blank;

[0034] Figure 2 This is the liquid chromatogram of cyanidin galactoside sample. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific implementation methods. It should be pointed out that the following implementation methods are only illustrative descriptions of the present invention in the form of examples, but the protection scope of the present invention is not limited thereto. All equivalent replacements made by those skilled in the art to the present invention in the spirit of the present invention fall within the protection scope of the present invention.

[0036] Example 1

[0037] A method for efficiently extracting cyanidin galactoside from Aronia melanocarpa comprises the following steps:

[0038] 1) Raw material pretreatment

[0039] Pick plump, disease-free and insect-free Aronia nigra fruits as raw materials, wash and drain the surface water, then quickly freeze them, and then store them at a low temperature below -18 for later use;

[0040] 2) Ethanol extraction

[0041] The raw material of step (1) is subjected to microwave thawing-slow freezing cycle treatment for 2 to 3 times, and then ethanol is added for ultrasonic-assisted extraction, and then the extract is collected by centrifugation and filtration for later use;

[0042] 3) Concentrate and recover the solvent

[0043] 4) Macroporous adsorption resin adsorption and analysis

[0044] The concentrated solution of step (3) is adsorbed by macroporous adsorption resin type I, and then ethanol is desorbed step by step, and then adsorbed by macroporous adsorption resin type II, and then ethanol is desorbed step by step, and the desorbed solution finally obtained is collected for later use;

[0045] 5) Concentrate and recover the solvent

[0046] 6) Purification by microsphere reverse phase chromatography filler

[0047] Add the microsphere reverse phase chromatography filler to the concentrated solution of step (5), stir for 10 to 30 minutes, then let it stand to take out the precipitated microsphere reverse phase chromatography filler, then elute the microsphere reverse phase chromatography filler, collect the eluate, and obtain a purified solution;

[0048] 7) Low temperature concentration crystallization and recrystallization

[0049] The purified solution obtained in step (6) is sequentially subjected to low temperature concentration crystallization and recrystallization, and the precipitated crystals are collected for later use;

[0050] 8) Freeze drying

[0051] The crystals obtained in step (7) are freeze-dried to obtain cyanidin galactoside.

[0052] Example 2

[0053] A method for efficiently extracting cyanidin galactoside from Aronia melanocarpa comprises the following steps:

[0054] 1) Raw material pretreatment

[0055] Pick plump, disease-free and insect-free Aronia nigra fruits as raw materials, wash and drain the surface water, then quickly freeze them, and then store them at a low temperature below -18 for later use;

[0056] 2) Ethanol extraction

[0057] The raw material of step (1) is subjected to microwave thawing-slow freezing cycle treatment for 2 to 3 times, wherein the specific parameters of microwave thawing-slow freezing are as follows: firstly, the frozen Aronia nigra fruits are placed in a container, and then thawed at a microwave power of 0.2 to 2.5 kw / kg for 2 to 5 minutes, and then the thawed Aronia nigra fruits are placed at -12 to -8°C for slow freezing for 4 to 10 hours;

[0058] After the raw material is subjected to microwave thawing-slow freezing cycle treatment for 2-3 times, ethanol is added according to the solid-liquid ratio of Aronia nigromaculata fruit to ethanol of 1:6-12 kg / L, ultrasonic-assisted extraction is performed under the conditions of ultrasonic power of 150-200 W and ultrasonic extraction time of 15-30 min, and then the extract is collected by centrifugal filtration for later use;

[0059] 3) Concentrate and recover the solvent

[0060] 4) Macroporous adsorption resin adsorption and analysis

[0061] The concentrated solution of step (3) is adsorbed by D101 macroporous adsorption resin, and then desorbed by ethanol step by step, and then adsorbed by AB-8 macroporous adsorption resin, and then desorbed by ethanol step by step, and the desorbed solution finally obtained is collected for later use;

[0062] 5) Concentrate and recover the solvent

[0063] 6) Purification by microsphere reverse phase chromatography filler

[0064] Porous silica with a particle size of 18 to 30 μm is selected as a microsphere reverse phase chromatography filler. The microsphere reverse phase chromatography filler is first mixed with sewage ethanol at a material-liquid ratio of 1:3 to 5, and then placed in ultrasonic oscillation for 10 to 15 minutes, and then filtered and separated to obtain the microsphere reverse phase chromatography filler, and then rinsed with distilled water and dried at low temperature;

[0065] Add the microsphere reverse phase chromatography filler to the concentrated solution of step (5), stir for 10 to 30 minutes, then let it stand to take out the precipitated microsphere reverse phase chromatography filler, then elute the microsphere reverse phase chromatography filler, collect the eluate, and obtain a purified solution;

[0066] 7) Low temperature concentration crystallization and recrystallization

[0067] The purified solution obtained in step (6) is sequentially subjected to low temperature concentration crystallization and recrystallization, and the precipitated crystals are collected for later use;

[0068] 8) Freeze drying

[0069] The crystals obtained in step (7) are freeze-dried to obtain cyanidin galactoside; the freeze-drying parameters are as follows: pre-freezing temperature of -55 to -45°C, time of 4 to 6 hours; sublimation drying temperature of -35 to -30°C, time of 22 to 24 hours; decomposition temperature of 30°C, time of 16 to 20 hours; freeze dryer vacuum degree of 1.0 Pa, cold trap temperature of -55 to -45°C.

[0070] Example 3

[0071] A method for efficiently extracting cyanidin galactoside from Aronia melanocarpa comprises the following steps:

[0072] 1) Raw material pretreatment

[0073] Pick plump, disease-free and insect-free Aronia nigra fruits as raw materials, wash and drain the surface water, then quickly freeze them, and then store them at a low temperature below -18 for later use;

[0074] 2) Ethanol extraction

[0075] The raw material of step (1) is subjected to microwave thawing-slow freezing cycle treatment twice, wherein the specific parameters of microwave thawing-slow freezing are as follows: firstly, the frozen Aronia nigra fruits are placed in a container, and then thawed at a microwave power of 0.5 kW / kg for 5 min, and then the thawed Aronia nigra fruits are placed at -10°C for slow freezing for 10 h;

[0076] After the raw material is subjected to microwave thawing-slow freezing cycle treatment twice, ethanol is added at a solid-liquid ratio of 1:10 kg / L of Aronia nigromaculata fruit and ethanol, and ultrasonic-assisted extraction is performed under the conditions of ultrasonic power of 150-200 w and ultrasonic extraction time of 15 min, and then the extract is collected by centrifugal filtration for later use;

[0077] 3) Concentrate and recover the solvent

[0078] 4) Macroporous adsorption resin adsorption and analysis

[0079] The concentrated solution of step (3) is adsorbed by D101 macroporous adsorption resin, and then desorbed by ethanol step by step, and then adsorbed by AB-8 macroporous adsorption resin, and then desorbed by ethanol step by step, and the desorbed solution finally obtained is collected for later use;

[0080] 5) Concentrate and recover the solvent

[0081] 6) Purification by microsphere reverse phase chromatography filler

[0082] Porous silica with a particle size of 20 μm was selected as the microsphere reverse phase chromatography filler. The microsphere reverse phase chromatography filler was first mixed with sewage ethanol at a material-liquid ratio of 1:5, and then placed in ultrasonic oscillation for 10 minutes, and then filtered and separated to obtain the microsphere reverse phase chromatography filler, and then continued to be eluted with distilled water and dried at low temperature;

[0083] Add the microsphere reverse phase chromatography filler to the concentrated solution of step (5), stir for 30 minutes, then let it stand to remove the precipitated microsphere reverse phase chromatography filler, then elute the microsphere reverse phase chromatography filler, collect the eluate, and obtain a purified solution;

[0084] 7) Low temperature concentration crystallization and recrystallization

[0085] The purified solution obtained in step (6) is sequentially subjected to low temperature concentration crystallization and recrystallization, and the precipitated crystals are collected for later use;

[0086] 8) Freeze drying

[0087] The crystals obtained in step (7) were freeze-dried. The freeze-drying parameters were as follows: pre-freezing temperature of -55 to -45°C for 4 to 6 hours; sublimation drying temperature of -35 to -30°C for 22 to 24 hours; desorption temperature of 30°C for 16 to 20 hours; freeze dryer vacuum degree of 1.0 Pa, cold trap temperature of -55 to -45°C, and finally 6.09 g of cyanidin galactoside was obtained. The purity of the obtained cyanidin galactoside was tested by liquid chromatography. The testing conditions were as follows:

[0088] Chromatographic column: C18 ME, 5um, 100A, 4.6*250mm;

[0089] Detector: UV 200~600nm;

[0090] Detection wavelength: 520nm;

[0091] The mobile phase is as follows:

[0092] Time (min) A Acetonitrile B 0.2% phosphoric acid water 0 13 87 15 13 87

[0093] The liquid chromatogram of the obtained cyanidin galactoside is shown in Figure 2 , it was calculated that the purity of cyanidin galactoside reached 95.62%.

Claims

1. A method for extracting high-purity cyanidin galactoside from Aronia nigra, characterized in that The steps include: 1) Raw material pretreatment Pick plump, disease-free and insect-free Aronia nigra fruits as raw materials, wash and drain the surface water, then quickly freeze them, and then store them at a low temperature below -18°C for later use; 2) Ethanol extraction The raw material of step (1) is subjected to microwave thawing-slow freezing cycle treatment for 2 to 3 times, and then ethanol is added for ultrasonic-assisted extraction, and then the extract is collected by centrifugal filtration for later use; wherein the specific parameters of microwave thawing-slow freezing are as follows: firstly, the frozen Aronia nigra fruits are placed in a container, and then thawed at a microwave power of 0.2 to 2.5 kw / kg for 2 to 5 min, and then the thawed Aronia nigra fruits are placed at -12 to -8°C for slow freezing for 4 to 10 h; 3) Concentrated recovery of solvent 4) Macroporous adsorption resin adsorption and analysis The concentrated solution of step (3) is adsorbed by macroporous adsorption resin type I, and then desorbed by ethanol step by step, and then adsorbed by macroporous adsorption resin type II, and then desorbed by ethanol step by step, and the desorbed solution finally obtained is collected for use; wherein the macroporous adsorption resin type I is D101 type, and the macroporous adsorption resin type II is AB-8 type; 5) Concentration and recovery of solvent 6) Microsphere reverse phase chromatography packing purification Add the microsphere reverse phase chromatography filler to the concentrated solution of step (5), stir for 10 to 30 minutes, then let it stand to take out the precipitated microsphere reverse phase chromatography filler, then elute the microsphere reverse phase chromatography filler, collect the eluate, and obtain the purified solution; 7) Low temperature concentration crystallization and recrystallization The purified solution obtained in step (6) is sequentially subjected to low temperature concentration crystallization and recrystallization, and the precipitated crystals are collected for later use; 8) Freeze drying The crystals obtained in step (7) are freeze-dried to obtain cyanidin galactoside.

2. The method for extracting high-purity cyanidin galactoside from Aronia melanocarpa as claimed in claim 1, characterized in that: In the step (2), the solid-liquid ratio of Aronia melanocarpa fruit to ethanol is 1:6-12 kg / L.

3. The method for extracting high-purity cyanidin galactoside from Aronia melanocarpa as claimed in claim 1, characterized in that: In the step (2), the power of the ultrasound is 150-200 W, and the ultrasonic extraction time is 15-30 min.

4. The method for extracting high-purity cyanidin galactoside from Aronia melanocarpa as claimed in claim 1, characterized in that: In the step (6), the microsphere reverse phase chromatography filler is porous silica gel with a particle size of 18 to 30 μm.

5. The method for extracting high-purity cyanidin galactoside from Aronia melanocarpa as claimed in claim 1, characterized in that: In the step (6), the microsphere reverse phase chromatography filler is pretreated before use, that is, the microsphere reverse phase chromatography filler is mixed with sewage ethanol at a solid-liquid ratio of 1:3-5 kg / L, and then placed in ultrasonic oscillation for 10-15 min, and then filtered and separated to obtain the microsphere reverse phase chromatography filler, and then rinsed with distilled water and dried at low temperature.

6. The method for extracting high-purity cyanidin galactoside from Aronia melanocarpa as claimed in claim 1, characterized in that: In the step (8), the freeze-drying parameters are as follows: pre-freezing temperature of -55 to -45°C, time of 4 to 6 h; sublimation drying temperature of -35 to -30°C, time of 22 to 24 h; desorption temperature of 30°C, time of 16 to 20 h; freeze-drying machine vacuum degree of 1.0 Pa, cold trap temperature of -55 to -45°C.

Images