Method for extracting polyphenol and flavone in plant by using deep eutectic solvent

Abstract

The invention discloses a method for extracting polyphenol and flavone in plants by using a deep eutectic solvent, and belongs to the technical field of natural product extraction. The invention discloses a method for extracting polyphenol and flavone in plants by using a deep eutectic solvent, which is characterized in that the deep eutectic solvent compounded by an L-proline-1, 3 propylene glycol-n-butyl alcohol ternary system is used as a polyphenol and flavone extracting agent for extracting polyphenol and flavone. The invention discloses a method for extracting polyphenol and flavone in plants by using a deep eutectic solvent. The extraction efficiency of the method is higher than that of a deep eutectic solvent with an organic solvent (ethanol) and choline chloride as main components and a binary deep eutectic solvent. Meanwhile, compared with a traditional organic solvent, the method has the advantages of being environmentally friendly, low in volatility, high in biocompatibility and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and more particularly to a method for extracting polyphenols and flavonoids from plants by utilizing a deep eutectic solvent. Background Art

[0002] Polyphenols and flavonoids are natural compounds widely found in plants. They have attracted widespread attention due to their diverse biological activities, such as antioxidant, anti-inflammatory, antibacterial, and antiviral properties. Traditional solvent extraction methods, based on the principle of like dissolves like, utilize organic solvents such as methanol, ethanol, and ethyl acetate. However, the efficiency of these methods is limited by the choice of solvent and the characteristics of the plant material. Harmful solvent residues may also remain in the final product, impacting its safety and purity.

[0003] Existing methods for extracting polyphenols and flavonoids using organic solvents have problems such as low biocompatibility, poor environmental friendliness, and low extraction efficiency.

[0004] Therefore, providing a method for extracting polyphenols and flavonoids from plants using deep eutectic solvents is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent.

[0006] The present invention uses a deep eutectic solvent composed of a ternary system of L-proline, 1,3-propylene glycol, and n-butanol as a polyphenol and flavonoid extractant. This solvent achieves higher extraction efficiency than deep eutectic solvents based on organic solvents (ethanol), choline chloride, and binary deep eutectic solvents. It also offers advantages over traditional organic solvents, such as being environmentally friendly, having low volatility, and high biocompatibility.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent comprises the following steps:

[0009] (1) Preparing a deep eutectic solvent: L-proline, 1,3-propylene glycol, and n-butanol are mixed in a molar ratio of 1:0.5 to 2:1 to 2; and after mixing, water is added to account for 10 to 50% of the total mass of the deep eutectic solvent.

[0010] (2) Pre-treatment of plant materials: Manually pick the flowers of the tiger lily in full bloom. After harvesting, place the tiger lily flowers in a cool, dry environment with good ventilation conditions for natural drying. When the water content of the flowers drops below 15%, the flowers appear crisp and the petals are easy to break, the drying process is stopped. Subsequently, the dried tiger lily flowers are transferred to a laboratory-grade crushing equipment for crushing. The crushed product is sieved using a 60-mesh standard test sieve, and the fine powder that passes through the sieve is collected. The part that does not pass through the sieve is returned to the crusher for secondary crushing, and the above process is repeated until all materials meet the specified particle size requirements. This step ensures the consistency of the particle size of the obtained tiger lily flower powder, which is conducive to the subsequent extraction of active ingredients.

[0011] (3) The pretreated plant material was mixed with a deep eutectic solvent at a solid-liquid mass ratio of 1:20, and ultrasonicated for 1 to 60 minutes at an ultrasonic power of 100 to 500 W and an ultrasonic temperature of 20 to 60°C. The supernatant was centrifuged at 4000 rpm for 10 minutes in a high-speed centrifuge and the polyphenol and flavonoid content was determined.

[0012] The pretreatment step is not necessary and can be omitted for plant materials that are not easy to pretreat. The inventors recommend the following pretreatment steps, and other steps known to those skilled in the art that can achieve the purpose of pretreatment can also be applied.

[0013] The crushing step can be performed using crushing equipment known in the art, such as a cutter, a wall breaker, a crusher, etc. The crushing step can be completed in one go or in steps from coarse to fine.

[0014] The "plant material" referred to in the present invention is not limited to a specific species and can be a single plant material or a combination of multiple plant materials. The "plant material" referred to in the present invention refers to all parts of a plant, including flowers, fruits, seeds, roots, bark, stems, leaves, etc.

[0015] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent. The natural deep eutectic solvent used has excellent solubility and selectivity, and can significantly improve the extraction efficiency of polyphenols and flavonoids. Experimental results show that compared with traditional organic solvent extraction methods, the extraction method of the present invention can obtain a higher extraction rate in a shorter time. The preparation method of the natural deep eutectic solvent of the present invention is simple and easy, and can be achieved by simple mixing and heating. With the assistance of ultrasound, the extraction conditions are mild and do not require extreme conditions such as high temperature or high pressure. DETAILED DESCRIPTION

[0016] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0017] The raw materials used in the specific embodiments of the present invention are shown in Table 1, and the equipment purchase manufacturers are shown in Table 2.

[0018] Table 1 Source of raw materials

[0019] name Purchase manufacturer Specification Tiger Lily Self-produced (Origin: Jiamusi City, Heilongjiang Province) Moisture content below 15% L-Proline Shanghai Titan Technology Co., Ltd. 99.0％ 1,3-Propanediol Shanghai Titan Technology Co., Ltd. 98.0％ Anhydrous ethanol Liaoning Quanrui Reagent Co., Ltd. 99.7％ Folinol Shanghai MacLean Biochemical Technology Co., Ltd. BR sodium carbonate Liaoning Quanrui Reagent Co., Ltd. 99.8％ <![CDATA[NaNO2]]> Tianjin Kaitong Chemical Reagent Co., Ltd. 99.0％ <![CDATA[Al(NO3)3]]> Liaoning Quanrui Reagent Co., Ltd. 99.0％ NaOH Tianjin Guangfu Technology Development Co., Ltd. 96.0％ n-Butanol Tianjin Kaitong Chemical Reagent Co., Ltd. 99.7％ urea Shanghai Titan Technology Co., Ltd. 99.0％ Ethylene glycol Shanghai Titan Technology Co., Ltd. 99.5％ DL-malic acid Shanghai Titan Technology Co., Ltd. 98.0％ Choline chloride Shanghai Titan Technology Co., Ltd. 99.0％

[0020] Table 2 Equipment sources

[0021]

[0022]

[0023] Example 1

[0024] A method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent comprises the following steps:

[0025] (1) Preparation of deep eutectic solvent:

[0026] L-proline, 1,3-propylene glycol, and n-butanol were mixed in a molar ratio of 1:0.5:1. After mixing, water was added to a concentration of 10% of the total mass of the deep eutectic solvent. The mixture was stirred until completely dissolved to form a transparent and uniform deep eutectic solvent.

[0027] (2) Pre-treatment of plant materials: Manually pick the flowers of the tiger lily in full bloom. After harvesting, place the tiger lily flowers in a cool, dry environment with good ventilation conditions for natural drying. When the water content of the flowers drops below 15%, the flowers appear crisp and the petals are easy to break, the drying process is stopped. Subsequently, the dried tiger lily flowers are transferred to a laboratory-grade crushing equipment for crushing. The crushed product is sieved using a 60-mesh standard test sieve, and the fine powder that passes through the sieve is collected. The part that does not pass through the sieve is returned to the crusher for secondary crushing, and the above process is repeated until all materials meet the specified particle size requirements. This step ensures the consistency of the particle size of the obtained tiger lily flower powder, which is conducive to the subsequent extraction of active ingredients.

[0028] (3) Extraction of polyphenols and flavonoids:

[0029] The pretreated plant material was mixed with a deep eutectic solvent at a solid-to-liquid ratio of 1:20. Ultrasonication was performed for 1 minute at 500W and 40°C. The mixture was centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected for polyphenol and flavonoid analysis. A comparative study was conducted using anhydrous ethanol instead of the deep eutectic solvent under the same conditions (Comparative Example 1).

[0030] Example 2

[0031] A method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent comprises the following steps:

[0032] (1) Preparation of deep eutectic solvent:

[0033] L-proline, 1,3-propylene glycol, and n-butanol were mixed in a molar ratio of 1:1:1.5. After mixing, water was added to account for 30% of the total mass of the deep eutectic solvent. Stir until completely dissolved to form a transparent and uniform deep eutectic solvent.

[0034] (2) Pretreatment of plant raw materials: same as in Example 1.

[0035] (3) Extraction of polyphenols and flavonoids:

[0036] The pretreated plant material was mixed with a deep eutectic solvent at a solid-to-liquid ratio of 1:20. Ultrasonication was performed for 30 minutes at a power of 250 W and a temperature of 20°C. The mixture was centrifuged at 4000 rpm for 10 minutes. The supernatant was then collected and analyzed for polyphenols and flavonoids. A comparative study was conducted using anhydrous ethanol instead of the deep eutectic solvent under the same conditions (Comparative Example 2).

[0037] Example 3

[0038] A method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent comprises the following steps:

[0039] (1) Preparation of deep eutectic solvent:

[0040] Mix L-proline, 1,3-propylene glycol, and n-butanol in a molar ratio of 1:2:2; then add water (50% of the total mass of the deep eutectic solvent) and stir until completely dissolved to form a transparent and uniform deep eutectic solvent.

[0041] (2) Pretreatment of plant raw materials: same as in Example 1.

[0042] (3) Extraction of polyphenols and flavonoids:

[0043] The pretreated plant material was mixed with a deep eutectic solvent at a solid-to-liquid ratio of 1:20. Ultrasonication was performed for 60 minutes at 100W of power and 60°C of temperature. The supernatant was centrifuged at 4000 rpm for 10 minutes and then analyzed for polyphenols and flavonoids. A comparative study was conducted using anhydrous ethanol instead of the deep eutectic solvent under the same conditions (Comparative Example 3).

[0044] Example 4

[0045] A method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent comprises the following steps:

[0046] (1) Preparation of deep eutectic solvent:

[0047] L-proline, 1,3-propylene glycol, and n-butanol were mixed in a molar ratio of 1:1:1. After mixing, water was added to account for 30% of the total mass of the deep eutectic solvent. Stir until completely dissolved to form a transparent and uniform deep eutectic solvent.

[0048] (2) Pretreatment of plant raw materials: same as in Example 1.

[0049] (3) Extraction of polyphenols and flavonoids:

[0050] The pretreated plant material was mixed with a deep eutectic solvent at a solid-liquid mass ratio of 1:20, ultrasonicated for 20 min at an ultrasonic power of 500 W and an ultrasonic temperature of 30°C. The supernatant was centrifuged at 4000 rpm for 10 min in a high-speed centrifuge and then the polyphenols and flavonoids were determined.

[0051] Comparative Example 4

[0052] A method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent comprises the following steps:

[0053] (1) Preparation of binary deep eutectic solvent:

[0054] L-proline and 1,3-propylene glycol were mixed in a molar ratio of 1:1. After mixing, water was added to account for 30% of the total mass of the deep eutectic solvent. The mixture was stirred until completely dissolved to form a transparent and uniform deep eutectic solvent.

[0055] (2) Pretreatment of plant raw materials: same as in Example 1.

[0056] (3) Extraction of polyphenols and flavonoids:

[0057] The pretreated plant material was mixed with a deep eutectic solvent at a solid-liquid mass ratio of 1:20, ultrasonicated for 20 min at an ultrasonic power of 500 W and an ultrasonic temperature of 30°C. The supernatant was centrifuged at 4000 rpm for 10 min in a high-speed centrifuge and then the polyphenols and flavonoids were determined.

[0058] Comparative Example 5

[0059] A method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent comprises the following steps:

[0060] (1) Preparation of deep eutectic solvent with choline chloride as main component:

[0061] Mix choline chloride and 1,3-propylene glycol in a molar ratio of 1:1; add water to a concentration of 30% of the total mass of the deep eutectic solvent; and stir until completely dissolved to form a transparent and uniform deep eutectic solvent.

[0062] (2) Pretreatment of plant raw materials: same as in Example 1.

[0063] (3) Extraction of polyphenols and flavonoids:

[0064] The pretreated plant material was mixed with a deep eutectic solvent at a solid-liquid mass ratio of 1:20, ultrasonicated for 20 min at an ultrasonic power of 500 W and an ultrasonic temperature of 30°C. The supernatant was centrifuged at 4000 rpm for 10 min in a high-speed centrifuge and then the polyphenols and flavonoids were determined.

[0065] Comparative Example 6

[0066] The 1,3-propylene glycol in Comparative Example 5 was replaced by urea, and the remaining operations were the same as those in Comparative Example 5.

[0067] Comparative Example 7

[0068] The 1,3-propylene glycol in Comparative Example 5 was replaced by ethylene glycol, and the remaining operations were the same as those in Comparative Example 5.

[0069] Comparative Example 8

[0070] The 1,3-propylene glycol in Comparative Example 5 was replaced with DL-malic acid, and the remaining operations were the same as those in Comparative Example 5.

[0071] Test example

[0072] 1) Polyphenol measurement method:

[0073] 0.05 mL of supernatant samples from Examples 1-3 and Controls 1-3 were each drawn into a volumetric flask, 0.5 mL of forin phenol was added, and the mixture was allowed to stand in the dark for 3 min. 2 mL of 0.1 g / mL Na₂CO₃ was then added, the volume was made up to 10 mL with triple-distilled water, and the mixture was allowed to stand in the dark for 1 h. The absorbance was measured at 760 nm using a UV spectrophotometer, and the polyphenol content of the sample was calculated by comparison with the standard curve.

[0074] 2) Flavonoids measurement method

[0075] 0.5 mL of supernatant samples of Examples 1-3 and Controls 1-3 were respectively taken into 10 mL volumetric flasks, 0.3 mL of 50 mg / mL NaNO2 was added, the mixture was shaken and allowed to stand for 6 min, 0.3 mL of 100 mg / mL Al(NO3)3 was added, the mixture was shaken and allowed to stand for 6 min, 4 mL of 4 mg / mL NaOH was added, and the volume was finally made up to 10 mL with 70% ethanol, shaken, and allowed to stand at room temperature for 15 min. The absorbance was measured at 510 nm using a UV spectrophotometer and compared with the standard curve to calculate the flavonoid content of the sample.

[0076] The results of flavonoid and polyphenol contents are shown in Table 3.

[0077] Table 3

[0078]

[0079] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent, characterized in that: The following steps are involved: (1) preparing a deep eutectic solvent: mixing L-proline, 1,3-propylene glycol, and n-butanol in a molar ratio of 1:0.5 to 2:1 to 2; and adding water to a concentration of 10 to 50% of the total mass of the deep eutectic solvent after mixing; (2) Pre-treating the plant raw materials: drying and then crushing the plant raw materials; (3) Extraction of polyphenols and flavonoids: The plant material pretreated in step (2) was mixed with the deep eutectic solvent prepared in step (1) at a solid-liquid mass ratio of 1:20, and ultrasonicated for 1 to 60 minutes at an ultrasonic power of 100 to 500 W and an ultrasonic temperature of 20 to 60°C. After centrifugation, the supernatant was collected and the polyphenols and flavonoids were determined.

2. The method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent according to claim 1, characterized in that: When the plant is a tiger lily flower, it is first dried until the water content is below 15%; then the dried tiger lily flower is pulverized, and the pulverized product is sieved using a 60-mesh standard test sieve.

3. The method for extracting polyphenols and flavonoids from plants using a deep eutectic solvent according to claim 1, characterized in that: The centrifugation in step (3) is performed at 4000 rpm for 10 min.

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