Method for extracting anthocyanin in red beans and bilberries by combining ternary eutectic solvent with microwave extraction method
By using a ternary eutectic solvent and microwave extraction method, the environmental pollution problem of extracting anthocyanins from red bean and blueberry was solved, achieving efficient and stable anthocyanin extraction and reuse. The extract also has antioxidant properties.
Patent Information
- Application Number
- CN202511403096.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2026-02-24
AI Technical Summary
In existing technologies, the extraction of anthocyanins from red beans and blueberries using organic solvents presents problems of environmental pollution and high wastewater treatment costs, and there is a lack of environmentally friendly alternatives.
Anthocyanin components in red bean and blueberry were extracted using a ternary eutectic solvent combined with microwave extraction. The extraction efficiency was optimized by adjusting the solvent composition and extraction conditions. Extraction was carried out using a ternary eutectic solvent composed of choline chloride, citric acid and 1,4-butanediol under microwave assistance, and the results were quantitatively analyzed by ultra-high performance liquid chromatography.
It has achieved efficient extraction of anthocyanins from red beans and blueberries. The extract is stable and can be stored for 2-3 months, and the solvent can be reused 2-5 times. The extract has strong antioxidant activity and can be applied in the field of antioxidants.
Abstract
Description
Technical Field
[0001] This invention pertains to a novel method for extracting green solvents. Background Technology
[0002] Red bilberry is a highly nutritious natural wild berry containing various active ingredients such as phenols, anthocyanins, flavonoids, and terpenes. Due to its excellent biological activity, it is used for purposes such as scavenging free radicals, anti-inflammation, lowering blood sugar, liver protection, prevention and treatment of cardiovascular diseases, anti-tumor activity, vision protection, and prevention of Alzheimer's disease. Therefore, in-depth research and development of red bilberry resources is of great significance. Different types of extraction solvents are an important factor affecting the anthocyanin content of red bilberry.
[0003] Currently, the extraction of anthocyanins from plants still mainly relies on organic solvent extraction, with solvents such as methanol, ethanol, water, or mixed solvents being commonly chosen. However, organic solvents are toxic and harmful to the environment, and wastewater treatment is costly. Replacing organic solvents with more environmentally friendly extraction technologies will be a further development trend. Deep eutectic solvents (DESs) are green solvents that can be degraded by soil microorganisms into low-toxicity or non-toxic substances, making them environmentally safe and friendly. They also offer mild operating conditions and can be used in the pharmaceutical, cosmetic, and food industries, making them an effective alternative to traditional extraction technologies. DESs are a eutectic mixture composed of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). Due to their lower freezing point, they can remain liquid at room temperature. Furthermore, the interaction between HBDs and HBAs in DESs can inhibit anion and cation exchange under sensitive conditions, reduce the degradation rate of unstable substances, and improve the stability of the extract.
[0004] To our knowledge, there is currently no method for extracting anthocyanins from red bean and blueberry using a ternary eutectic solvent combined with microwave extraction. This invention reports a method for extracting anthocyanins from red bean and blueberry using a ternary eutectic solvent combined with microwave extraction, and uses ultra-high performance liquid chromatography (UHPLC) to quantitatively study the two most abundant anthocyanins in red bean and blueberry. Summary of the Invention
[0005] This invention utilizes a ternary eutectic solvent combined with microwave extraction to extract anthocyanin components from red beans and blueberries. The specific extraction method is as follows:
[0006] 1. A eutectic solvent was prepared using choline chloride as a hydrogen bond acceptor. Lactic acid, DL-malic acid, citric acid, ethylene glycol, 1,2-propanediol, 1,3-butanediol, and 1,4-butanediol were mixed in molar ratios of 1:1, 1:2, and 2:1, and 20% water was added. The mixture was stirred at 80°C to synthesize a binary eutectic solvent. The cyanidin-3-O-glucoside and delphinidin-3-O-glucoside were quantitatively studied using ultra-high performance liquid chromatography (UHPLC).
[0007] 2. Using the binary eutectic solvent synthesized in step 1, add red bean and blueberry pulp at a material-to-liquid ratio of 1:10. Microwave-assisted extraction at 50℃ for 15 min was performed. The highest contents of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside were extracted when the molar ratio of synthesized choline chloride-citric acid (ChCl-Citric acid) and choline chloride-1,4-butanediol (ChCl-1,4Bu) was 1:2. The contents of cyanidin-3-O-glucoside were between 1.78 and 1.88 mg / g, and the contents of delphinidin-3-O-glucoside were between 4.35 and 4.5 mg / g.
[0008] 3. Choline chloride, citric acid, and 1,4-butanediol, which yielded the highest extraction yields, were combined with 20% water in molar ratios of 1:1:1, 1:2:1, 1:2:2, and 2:1:1 to synthesize choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu). Microwave extraction at 50℃ for 15 min yielded the highest extraction contents of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside at a molar ratio of 1:2:2. The contents of cyanidin-3-O-glucoside ranged from 2.0 to 2.2 mg / g, and the contents of delphinidin-3-O-glucoside ranged from 5.9 to 6.1 mg / g.
[0009] 4. Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) in a molar ratio of 1:2:2 was extracted with 10%, 15%, 20%, 25%, and 30% water, respectively, at 50℃ using microwave-assisted extraction for 15 min. The highest contents of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside were obtained when the water content was 15%. The contents of cyanidin-3-O-glucoside were between 2.13 and 2.3 mg / g, and the contents of delphinidin-3-O-glucoside were between 6.17 and 6.3 mg / g.
[0010] 5. The synthesized choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was added to red bean and blueberry pulp at material-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50. Microwave-assisted extraction was performed at 50℃ for 15 min. The highest contents of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside were obtained at a material-liquid ratio of 1:30. The contents of cyanidin-3-O-glucoside were between 2.4 and 2.5 mg / g, and the contents of delphinidin-3-O-glucoside were between 6.3 and 6.5 mg / g.
[0011] 6. The synthesized choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was added to red bean and blueberry pulp at a material-to-liquid ratio of 1:30. Microwave-assisted extraction was performed at 40℃, 45℃, 50℃, 55℃, and 60℃ for 15 min. The highest contents of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside were obtained at 50℃. The contents of cyanidin-3-O-glucoside were between 2.58 and 2.7 mg / g, and the contents of delphinidin-3-O-glucoside were between 6.52 and 6.6 mg / g.
[0012] 7. The synthesized choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was added to red bean and blueberry pulp at a material-to-liquid ratio of 1:30. Microwave-assisted extraction was performed at 50℃ for 10 min, 15 min, 20 min, 25 min, 30 min, and 25 min, at which the contents of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside were highest. The contents of cyanidin-3-O-glucoside were between 2.71 and 2.8 mg / g, and the contents of delphinidin-3-O-glucoside were between 6.6 and 6.8 mg / g.
[0013] 8. An orthogonal experiment with four factors and three levels was designed using orthogonal design software. The highest contents of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside were obtained when the water content was 15%, the liquid-solid ratio was 1:30, the extraction temperature was 50℃, and the extraction time was 25 min. The highest contents of cyanidin-3-O-glucoside were between 1.97 and 2.8 mg / g, and the highest contents of delphinidin-3-O-glucoside were between 5.93 and 6.8 mg / g.
[0014] 9. The anthocyanin extract of red bean bilberry obtained by choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) has strong antioxidant activity and can be applied in the field of antioxidants.
[0015] The anthocyanin extract from red bean bilberry obtained using the method of this invention is very stable and can be stored in a refrigerator at 0-7°C for 2-3 months. The choline chloride-citric acid-1,4-bu solution can be recycled and reused 2-5 times. The extracted red bean bilberry cyanidin-3-O-glucoside content is between 2.0-2.8 mg / g, and the delphinidin-3-O-glucoside content is between 5.9-6.8 mg / g, making it suitable for applications in the antioxidant field.
[0016] The advantages of this invention are:
[0017] 1. The ternary eutectic solvent used in this method has the characteristics of low vapor pressure, stable properties, easy biodegradability, environmental friendliness, low price, non-toxicity or low toxicity, and transparency at room temperature. Compared with the currently widely used binary eutectic solvent, it has lower viscosity, resulting in better extraction and separation. The choline chloride-citric acid-1,4-butanediol (ChCl-Citricacid-1,4Bu) solution used in this invention can be recycled and reused 2-5 times.
[0018] 2. The extracts of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside from red bean and blueberry are very stable and can be stored in a refrigerator at 0-7℃ for 2-3 months.
[0019] 3. The extracts of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside from red bean and blueberry have strong antioxidant activity and can be used as natural antioxidants in the field of antioxidants.
[0020] Example 1
[0021] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 10% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:20. The mixture was extracted at 45°C for 20 minutes. The content of cyanidin-3-O-glucoside was between 1.97 and 2.1 mg / g, and the content of delphinidin-3-O-glucoside was between 6.0 and 6.16 mg / g.
[0022] Example 2
[0023] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 10% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:30. The mixture was extracted at 50°C for 25 minutes. The content of cyanidin-3-O-glucoside was between 2.5 and 2.6 mg / g, and the content of delphinidin-3-O-glucoside was between 6.4 and 6.5 mg / g.
[0024] Example 3
[0025] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 10% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:40. The mixture was extracted at 55°C for 30 minutes. The content of cyanidin-3-O-glucoside was between 2.0 and 2.17 mg / g, and the content of delphinidin-3-O-glucoside was between 5.96 and 6.16 mg / g.
[0026] Example 4
[0027] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 10% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:20, and the mixture was extracted at 50°C for 30 min. The content of cyanidin-3-O-glucoside was between 1.97 and 2.07 mg / g, and the content of delphinidin-3-O-glucoside was between 6.0 and 6.16 mg / g.
[0028] Example 5
[0029] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 15% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:30. The mixture was extracted at 55°C for 20 min. The content of cyanidin-3-O-glucoside was between 2.2 and 2.3 mg / g, and the content of delphinidin-3-O-glucoside was between 6.3 and 6.47 mg / g.
[0030] Example 6
[0031] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 15% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:40. The mixture was extracted at 45°C for 25 minutes. The content of cyanidin-3-O-glucoside was between 2.15 and 2.2 mg / g, and the content of delphinidin-3-O-glucoside was between 6.2 and 6.32 mg / g.
[0032] Example 7
[0033] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 20% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:20. The mixture was extracted at 55°C for 25 minutes. The content of cyanidin-3-O-glucoside was between 2.21 and 2.3 mg / g, and the content of delphinidin-3-O-glucoside was between 6.4 and 6.5 mg / g.
[0034] Example 8
[0035] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 20% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:30. The mixture was extracted at 45°C for 30 minutes. The content of cyanidin-3-O-glucoside was between 2.3 and 2.38 mg / g, and the content of delphinidin-3-O-glucoside was between 6.5 and 6.6 mg / g.
[0036] Example 9
[0037] Choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) was mixed in a molar ratio of 1:2:2, and 20% water was added. The mixture was stirred at 80°C to synthesize a eutectic solvent. After cooling at room temperature, red bean and blueberry pulp was added at a material-to-liquid ratio of 1:40. The mixture was extracted at 50°C for 20 min. The content of cyanidin-3-O-glucoside was between 2.21 and 2.3 mg / g, and the content of delphinidin-3-O-glucoside was between 6.47 and 6.5 mg / g.
Claims
1. This invention utilizes a ternary eutectic solvent combined with microwave extraction to extract anthocyanins from red bean and blueberry, and uses an Agilent ultra-high performance liquid chromatograph to quantify the high content of cyanidin-3-O-glucoside and delphinidin-3-O-glucoside. A ternary eutectic solvent is synthesized using choline chloride as a hydrogen bond acceptor and different types of hydrogen bond donors, with the addition of an appropriate amount of water under heating and stirring conditions at 80°C. Red bean and blueberry pulp is added according to a specific material-to-liquid ratio. The mixture is transferred to a microwave extractor and extracted at a selected temperature for a specific time. The extract is then centrifuged at 10000 rpm for 15 minutes.
2. The method for extracting anthocyanin components from red bean and blueberry pulp using a ternary eutectic solvent combined with microwave extraction according to claim 1, wherein choline chloride is used as a hydrogen bond acceptor, and seven different hydrogen bond donors (lactic acid, DL-malic acid, anhydrous citric acid, ethylene glycol, 1,2-propanediol, 1,3-butanediol, and 1,4-butanediol) are mixed in molar ratios of 1:1, 1:2, and 2:1, respectively, and 20% water is added to synthesize a binary eutectic solvent. Red bean and blueberry pulp is added at a material-to-liquid ratio of 1:10, and microwave extraction is performed at 50°C for 15 min. The content of cyanidin-3-O-glucoside is between 0.9 and 1.9 mg / g, and the content of delphinidin-3-O-glucoside is between 3.5 and 4.5 mg / g. The highest content of cyanidin-3-O-glucoside (1.85 mg / g) and delphinidin-3-O-glucoside (4.3 mg / g) was obtained when the molar ratio of choline chloride to citrate was 1:
2. Similarly, the highest content of cyanidin-3-O-glucoside (1.78 mg / g) and delphinidin-3-O-glucoside (4.5 mg / g) was obtained when the molar ratio of choline chloride to 1,4-butanediol was 1:
2.
3. The method for extracting anthocyanin components from red bean and blueberry pulp using a ternary eutectic solvent combined with microwave extraction according to claim 1, wherein the two binary eutectic solvents with the highest extraction rates according to claim 2 are used, with choline chloride as a hydrogen bond acceptor and citric acid and 1,4-butanediol as hydrogen bond donors, mixed in molar ratios of 1:1:1, 1:2:1, 1:2:2, and 2:1:1 respectively, and 20% water is added to synthesize the ternary eutectic solvent. Red bean and blueberry pulp is added at a material-to-liquid ratio of 1:10, and microwave-assisted extraction is performed at 50°C for 15 min. The content of cyanidin-3-O-glucoside is between 1.9 and 2.2 mg / g, and the content of delphinidin-3-O-glucoside is between 5.2 and 6.1 mg / g.
4. The method for extracting anthocyanin components from red bean and blueberry pulp using a ternary eutectic solvent combined with microwave extraction according to claim 1, wherein 10%, 15%, 20%, 25%, and 30% water are added respectively according to the 1:2:2 molar ratio described in claim 3 to synthesize choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu), and red bean and blueberry pulp is added at a material-liquid ratio of 1:
10. Microwave extraction is performed at 50°C for 15 min, and the content of cyanidin-3-O-glucoside is between 1.8 and 2.3 mg / g, and the content of delphinidin-3-O-glucoside is between 4.9 and 6.3 mg / g.
5. The method for extracting anthocyanin components from red bean and blueberry pulp using a eutectic solvent combined with microwave extraction according to claim 1, wherein red bean and blueberry pulp is added to the pulp at material-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50 respectively, using a microwave-assisted extraction method at 50°C for 15 min. The content of cyanidin-3-O-glucoside is between 1.9 and 2.5 mg / g, and the content of delphinidin-3-O-glucoside is between 5.5 and 6.5 mg / g.
6. The method for extracting total anthocyanin components from red bean and blueberry using a eutectic solvent combined with microwave extraction according to claim 1, wherein choline chloride-citric acid-1,4-butanediol (ChCl-Citric acid-1,4Bu) is synthesized according to step 3, and red bean and blueberry pulp is added at a material-to-liquid ratio of 1:
30. Microwave-assisted extraction is performed at 40℃, 45℃, 50℃, 55℃, and 60℃ for 15 min, respectively. The content of cyanidin-3-O-glucoside is between 1.9 and 2.7 mg / g, and the content of delphinidin-3-O-glucoside is between 5.5 and 6.6 mg / g.
7. The method for extracting total anthocyanin components from red beans and blueberries using a eutectic solvent combined with microwave extraction according to claim 1, wherein the mixture is microwave-assisted extracted at 55°C for 10 min; 15 min; 20 min; 25 min; 30 min according to step 6, and the content of cyanidin-3-O-glucoside is between 2.2 and 2.8 mg / g, and the content of delphinidin-3-O-glucoside is between 5.6 and 6.8 mg / g.
8. According to the method for extracting anthocyanin components from red beans and blueberries using a ternary eutectic solvent combined with microwave extraction as described in claim 1, three optimal levels from 4.5.6.7 were selected. An orthogonal experiment with four factors and three levels was designed using orthogonal design software. When the water content was 15%, the material-to-liquid ratio was 1:30, the extraction temperature was 50℃, and the extraction time was 25 min, the content of cyanidin-3-O-glucoside was highest between 1.97 and 2.8 mg / g, and the content of delphinidin-3-O-glucoside was highest between 5.93 and 6.8 mg / g.