Pueraria flavone extraction process

Abstract

The invention discloses a pueraria flavone extraction process, and belongs to the technical field of natural product extraction. Aiming at the problems of low extraction efficiency and more impurities of the traditional solvent, a natural deep eutectic solvent (NADES) is combined with a resin purification technology. The method specifically comprises the following steps: pre-treating radix puerariae to obtain radix puerariae powder, extracting by adopting NADES consisting of a hydrogen bond donor (glycerol / ethylene glycol / saccharide) and an acceptor (soluble carbonate), heating at 40-80 DEG C, carrying out 20-50kHz ultrasonic co-treatment, centrifuging, and purifying through macroporous resin. Compared with a traditional ethanol extraction method, the extraction efficiency is improved by 2.3 times, the solvent can be recycled, and the method has the advantages of being environmentally friendly, simple in process and suitable for industrialization.

Description

Technical Field

[0001] The invention belongs to the field of extraction and separation of natural compounds, and particularly relates to a pueraria flavonoids extraction process. Background Art

[0002] Pueraria root is a plant recognized by the National Health Commission as both a medicine and food. It has extremely high edible and health-promoting value and is recorded in many traditional Chinese medicine texts. In traditional medicine, Pueraria root is often used as an adjunct treatment for fever, diarrhea, angina pectoris, etc. Modern pharmacological research has shown that Pueraria root has multiple biological activities, such as estrogen-like activity, anti-inflammatory and antioxidant effects, blood pressure control, blood sugar reduction, and cancer reduction. Numerous studies have shown that isoflavone compounds are the main functional components of Pueraria root. In Western countries, Pueraria root total flavonoids and puerarin extracted from Pueraria root have been widely used in health foods and biopharmaceuticals. Incorporating the active ingredients of Pueraria root total flavonoids into foods or cosmetics to form functional foods or cosmetics to form a Pueraria root specialty product line has practical significance for meeting the consumer needs of different groups.

[0003] my country boasts abundant kudzu root resources. Compared to developed countries, my country boasts significant advantages in natural product resources and traditional Chinese medicine (TCM) culture. However, due to a lack of scientific deep-processing technologies for the extraction, separation, and purification of active ingredients from these natural resources, a significant proportion of kudzu root is lost nationwide each year. This results in the root's nutritional benefits not being fully utilized, significantly limiting its overall economic benefits. In terms of food processing applications, kudzu root is currently primarily used for primary processing into kudzu powder and starch, with most kudzu products confined to laboratory research. The sustainable development of kudzu resources requires addressing how to achieve industrialized production, increase product diversity, and make kudzu products a popular, healthy, and safe food. Summary of the Invention

[0004] The present invention aims to solve the problems of low extraction efficiency and difficult separation and purification of traditional solvents in the existing technology. A new method using natural deep eutectic solvents (NADES) for extraction combined with resin adsorption purification is proposed to achieve the technical effect of improving extraction efficiency and reducing impurities.

[0005] To achieve the above object, the technical solution adopted by the present invention is: a Pueraria lobata flavonoids extraction process, comprising the following steps:

[0006] The kudzu root is washed, dried, crushed, ground and sieved to obtain kudzu root powder;

[0007] Extracting kudzu root powder using natural deep eutectic solvent;

[0008] heating and ultrasonically treating the extract;

[0009] The supernatant was obtained by centrifugation to obtain the total brass of Pueraria root;

[0010] Pueraria isoflavones (puerarin) are obtained by adsorption with exchange resin and elution with eluent.

[0011] Furthermore, the passing rate of the kudzu root powder through a 10-20 mesh sieve after grinding is above 95%.

[0012] Furthermore, the natural deep eutectic solvent consists of a hydrogen bond donor, a hydrogen bond acceptor and water.

[0013] Furthermore, the hydrogen bond donor is selected from one or more of glycerol, ethylene glycol, glucose, and sucrose.

[0014] Furthermore, the hydrogen bond acceptor is selected from soluble carbonates.

[0015] Furthermore, the soluble carbonate is selected from one or more of potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate.

[0016] Furthermore, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is (4-20):1.

[0017] Further, the natural deep eutectic solvent is selected from one of the following combinations: glycerol and potassium carbonate, glycerol and sodium carbonate, glycerol and potassium bicarbonate, glycerol and sodium bicarbonate, ethylene glycol and potassium carbonate, ethylene glycol and sodium carbonate, ethylene glycol and potassium bicarbonate, ethylene glycol and sodium bicarbonate, glucose and potassium carbonate, glucose and sodium carbonate, glucose and potassium bicarbonate, glucose and sodium bicarbonate, sucrose and potassium carbonate, sucrose and sodium carbonate, sucrose and potassium bicarbonate, sucrose and sodium bicarbonate.

[0018] Furthermore, the natural deep eutectic solvent comprises 80-95 wt % of hydrogen bond acceptors and hydrogen bond donors, and the rest is water.

[0019] Furthermore, the heating temperature is 40-80° C.; and the ultrasonic treatment is performed at 20-50 kHz for 20-60 min.

[0020] Furthermore, the centrifugal speed is 5000-15000 rpm, and the centrifugal time is 10-30 min.

[0021] Furthermore, the exchange resin is selected from: macroporous resin or ion exchange resin.

[0022] Furthermore, the macroporous resin is selected from one of AB-8, D101 and HPD100.

[0023] Further, the liquid is eluted into an ethanol solution of water.

[0024] Furthermore, the liquid elution is an ethanol solution with a volume concentration of 50-80%.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The NADES used in the present invention is composed of a hydrogen bond donor (such as glycerol) and an acceptor (such as potassium carbonate), wherein the hydrogen bond donor and the hydrogen bond acceptor form a three-dimensional network structure through intermolecular hydrogen bonds. Potassium carbonate is an alkaline hydrogen bond acceptor, and its CO3 2- The ions form multiple hydrogen bonds with the hydroxyl groups of glycerol, significantly enhancing the polarity of the solvent and promoting the dissolution of cellulose and hemicellulose in the cell walls of Pueraria lobata, thereby extracting more flavonoids.

[0027] 2. The polyhydroxy structure of glycerol can specifically bind to the phenolic hydroxyl groups of isoflavones, improving the solubility of the target ingredients through π-π stacking and hydrophobic interactions. At the same time, the alkaline environment of potassium carbonate (pH>10) inhibits the oxidative degradation of isoflavones. In contrast, the acidic environment of other acidic NADES (such as the L-proline-malic acid system) will lead to the hydrolysis of the isoflavone glycosidic bonds, and acidic groups such as carboxylic acids compete with isoflavones for binding sites, reducing selectivity.

[0028] 3. Potassium carbonate hydrolyzes in the aqueous phase, releasing CO2 gas in a stepwise manner, creating a microbubble effect. This synergistically acts with ultrasound to destroy plant cell walls and enhance solvent permeability. The static permeability of the non-gas-producing NADES system is weaker, requiring ultrasound assistance to achieve a similar effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The purified kudzu root isoflavones according to Example 1 of the present invention are 1 H NMR spectrum. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. 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 creative efforts are within the scope of protection of the present invention.

[0031] The macroporous resin in the present invention is selected from AB-8 macroporous resin (purchased from Beijing Huideyi Technology Co., Ltd., with a particle size range of 0.3-1.25 mm, a water content of 65-75%, and a specific surface area of 480-520 m 2 / g, skeleton density 1.13-1.17g / ml), ethanol, potassium carbonate, sodium carbonate, potassium bicarbonate, L-proline, malic acid, glycerol, ethylene glycol, choline chloride, glucose, sucrose, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd. with a purity of 98%.

[0032] Example 1

[0033] A flavonoid extraction process for kudzu root comprises the following steps: washing 1 kg of collected kudzu root with ultrapure water, drying at 50°C, crushing, grinding, and sieving to obtain kudzu root powder; adding a natural low eutectic solvent (a molar ratio of potassium carbonate to glycerol of 10:1, containing 15% water), heating to 50°C, and ultrasonically treating for 20-60 minutes (ultrasonic treatment frequency of 50 kHz); centrifuging at 13,000 rpm for 20 minutes, filtering, and spinning the filtrate to obtain kudzu root total flavonoids; using a dry loading method, adding the kudzu root total flavonoids to an AB-8 macroporous resin purification column, eluting with 70% ethanol, and spinning to obtain kudzu root isoflavones.

[0034] Example 2

[0035] Referring to the Pueraria lobata flavonoids extraction process involved in Example 1, the molar ratio of glycerol to potassium carbonate was adjusted to 6:1, and the rest remained unchanged.

[0036] Example 3

[0037] Referring to the Pueraria lobata flavonoids extraction process involved in Example 1, the natural deep eutectic solvent system was replaced with glycerol and sodium carbonate, and the rest remained unchanged.

[0038] Example 4

[0039] Referring to the Pueraria lobata flavonoids extraction process involved in Example 1, the natural deep eutectic solvent system was replaced with glycerol and potassium bicarbonate, and the rest remained unchanged.

[0040] Example 5

[0041] Referring to the Pueraria lobata flavonoids extraction process involved in Example 1, the natural deep eutectic solvent system was replaced with ethylene glycol and potassium carbonate, and the rest remained unchanged.

[0042] Example 6

[0043] Referring to the Pueraria lobata flavonoids extraction process involved in Example 1, the natural deep eutectic solvent system was replaced with glucose and potassium bicarbonate, and the rest remained unchanged.

[0044] Example 7

[0045] Referring to the Pueraria lobata flavonoids extraction process involved in Example 1, the natural deep eutectic solvent system was replaced with sucrose and potassium carbonate, and the rest remained unchanged.

[0046] Comparative Example 1

[0047] Referring to the extraction process of kudzu root flavonoids in Example 1, the natural deep eutectic solvent was replaced with choline chloride and glycerol, and the rest was the same as in Example 1.

[0048] Comparative Example 2

[0049] Referring to the extraction process of kudzu root flavonoids in Example 1, the natural deep eutectic solvent was replaced with L-proline and malic acid, and the rest was the same as in Example 1.

[0050] Comparative Example 3

[0051] Referring to the extraction process of kudzu root flavonoids in Example 1, the molar ratio of glycerol to potassium carbonate was adjusted to 2:1, and the rest remained unchanged.

[0052] Comparative Example 4

[0053] Referring to the extraction process of kudzu root flavonoids in Example 1, the molar ratio of glycerol to potassium carbonate was adjusted to 22:1, and the rest remained unchanged.

[0054] Extraction and purification performance test:

[0055] The purity was determined by HPLC using a Poroshell 120 EC-C18 (150 mm × 4.6 mm, 4 μm) column. The mobile phase was 0.1% phosphoric acid solution-acetonitrile with a gradient elution at a flow rate of 1.0 mL / min. The detection wavelength was 250 nm. The column temperature was 25°C, and the injection volume was 10 μL. Cluster analysis and principal component analysis were used for data analysis. The data results are shown in Table 1.

[0056] The puerarin extracted in Example 1 of the present invention 1 HNMR (600MHz, Methanol-d4): δ8.16(s,1H,H-2),8.03(d,J=8.9Hz,1H,H-5),7.42–7.25(m,2H, H-2',H-6'),6.97(d,J=8.8Hz,1H,H-6),6.86–6.76(m,2H,H-3',H-5'),5.09(d,J=9.9Hz,1H,H -1-glu),4.12(m,1H,H-2-glu),3.89(dd,J=12.1,2.2Hz,1H,H-6a-glu),3.75(dd,J=12.4,5.3 Hz,1H,H-6b-glu),3.52(dt,J=8.9,4.4Hz,2H,H-3-glu,H-4-glu),3.49–3.43(m,1H,H-5-glu).

[0057] Table 1. Extraction efficiency and HPLC results of different natural deep eutectic solvents for total flavonoids and puerarin from Pueraria root.

[0058] .

[0059] According to the data in Table 1, Example 1, employing a natural deep eutectic solvent system of glycerol and potassium carbonate (10:1), demonstrated the optimal extraction efficiency for total flavonoids from Pueraria root (10.5 g / kg) and puerarin (8.4 g / kg), significantly outperforming the other systems. Experiments demonstrated that the type of alkaline salt, type of hydrogen bond donor, and their ratio had a key impact on extraction efficiency: potassium carbonate was superior to sodium carbonate / potassium bicarbonate, and glycerol was more effective as a hydrogen bond donor than ethylene glycol, glucose, and sucrose. A glycerol-potassium carbonate molar ratio of 10:1 was the optimal ratio (Comparative Examples 2 / 3 / 4). The remaining natural systems (Comparative Examples 1-2) or those with unbalanced ratios (Comparative Examples 3-4) resulted in efficiency reductions of over 30%.

[0060] The key difference between Comparative Example 1 (Choline chloride: Glycerol) and Example 1 (Glycerol: Potassium carbonate) lies in the synergistic effect of the alkaline strength of the solvent system and the hydrogen bonding. The phenolic hydroxyl groups of kudzu root flavonoids need to be deprotonated (forming -O - ) to enhance hydrophilicity, while the choline chloride-glycerol system lacks a strong alkaline component (such as potassium carbonate), which leads to a decrease in the dissociation degree of flavonoids and a decrease in dissolution efficiency. In addition, the quaternary ammonium structure of choline chloride causes steric hindrance in the hydrogen bond network, hindering the effective binding with flavonoids; while in the glycerol-potassium carbonate system, carbonate ions (CO3 2- ) forms a dynamic hydrogen bond network with flavonoids through electrostatic interaction, significantly improving the solubility.

[0061] The drawbacks of Comparative Example 2 (L-proline:malic acid) are closely related to solvent pH and hydrogen bond selectivity. The neutral or weakly acidic environment formed by L-proline and malic acid inhibits the dissociation of the phenolic hydroxyl groups of the flavonoids, maintaining their hydrophobic state and making them difficult to dissolve. Furthermore, the carboxylic acid group of malic acid preferentially forms intramolecular hydrogen bonds with the amino groups of proline rather than with the flavonoids, resulting in inefficient hydrogen bond donor efficiency. In contrast, the strong alkalinity (pH > 10) of the glycerol-potassium carbonate system not only promotes flavonoid dissociation but also enhances release efficiency by disrupting plant cell walls (such as pectin and cellulose). Acidic or neutral solvents (such as those in Comparative Example 2) lack this synergistic effect.

[0062] The solvent system directly affects the release and dissolution of the target compound by regulating the solvent's alkalinity, hydrogen bond network density, and solubility selectivity. Taking the glycerol-potassium carbonate system as an example, a molar ratio of (4-20):1 achieves a synergistic balance between the alkaline salt (potassium carbonate) and the hydrogen bond donor (glycerol): the high concentration of potassium carbonate ensures that the system pH is > 10, promoting the full deprotonation of the hydroxyl groups of flavonoid phenols (-O -), enhancing hydrophilicity; simultaneously, excess glycerol acts as a hydrogen bond donor, forming a dynamic hydrogen bond network with carbonate ions, effectively encapsulating flavonoid molecules and enhancing solubility. An unbalanced ratio (such as 2:1 in Comparative Example 3 or 22:1 in Comparative Example 4) with excessively high or low potassium carbonate content will cause the system pH to deviate from the optimal range or provide insufficient hydrogen bond donors, respectively, impairing the solvent's alkaline dissociation ability and the strength of intermolecular interactions, ultimately reducing extraction efficiency by over 30%.

[0063] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A process for extracting flavonoids from kudzu root, characterized in that: The extraction method comprises the following steps: Pueraria root powder is added to a natural deep eutectic solvent, and the extraction is completed by heating and ultrasonication. The supernatant is centrifuged to obtain the total flavonoids of Pueraria root, and the Pueraria root isoflavones are obtained by adsorption with an exchange resin and elution with an eluent. The natural deep eutectic solvent comprises: a hydrogen bond acceptor, a hydrogen bond donor, and water; The hydrogen bond acceptor is selected from: soluble carbonate, soluble bicarbonate; The hydrogen bond donor is selected from one or more of glycerol, ethylene glycol, glucose, and sucrose.

2. The process for extracting flavonoids from kudzu root according to claim 1, wherein: The soluble carbonate is selected from one or more of potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate.

3. The process for extracting flavonoids from kudzu root according to claim 1, wherein: The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is (4-20):

1.

4. The process for extracting flavonoids from kudzu root according to claim 2, wherein: The natural deep eutectic solvent is selected from one of the following combinations: glycerol and potassium carbonate, glycerol and sodium carbonate, ethylene glycol and potassium carbonate, ethylene glycol and sodium carbonate, glucose and potassium carbonate, glucose and sodium carbonate, sucrose and potassium carbonate, sucrose and sodium carbonate.

5. The process for extracting flavonoids from kudzu root according to claim 1, wherein: The natural deep eutectic solvent comprises 80-95 wt % of hydrogen bond acceptors and hydrogen bond donors, and the rest is water.

6. The process for extracting flavonoids from kudzu root according to claim 1, wherein: The heating temperature is 40-80° C.; the ultrasonic treatment method is 20-50 kHz for 20-60 min.

7. The process for extracting flavonoids from kudzu root according to claim 1, wherein: The exchange resin is selected from: macroporous resin or ion exchange resin.

8. The process for extracting flavonoids from kudzu root according to claim 7, wherein: The macroporous resin is selected from one of AB-8, D101 and HPD100.

9. The process for extracting flavonoids from kudzu root according to claim 1, wherein: The eluent is an ethanol solution with a volume concentration of 50-80%.

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