Method for extracting and purifying rutin or quercetin in sophora flower-bud by one-pot subcritical water without solvent

Abstract

This application provides a solvent-free subcritical water one-pot extraction and purification method for flavonoids from Sophora japonica buds, belonging to the field of green extraction technology and natural product processing. First, the raw material undergoes enzyme treatment. The Sophora japonica buds are loaded into a sealed stirred reactor; an acetate buffer solution with pH 4.4-5.0 and 0.07-0.15M is added, along with ascorbic acid as an antioxidant; nitrogen gas is purged to establish an inert atmosphere; then the enzyme preparation is added; incubation is performed; followed by subcritical water extraction to obtain crude flavonoids; finally, crystallization purification is carried out to obtain the final flavonoid product. This method eliminates the need for organic solvents, achieving integrated extraction and purification. Selective production of rutin or quercetin is achieved through simple parameter control. The production process is green and environmentally friendly, improving the yield and purity of flavonoids, reducing processing time, and lowering production costs.

Description

Technical Field

[0001] This invention belongs to the field of green extraction technology and natural product processing, specifically involving a method for extracting and purifying rutin or quercetin from Sophora japonica buds using a solvent-free subcritical water one-pot method. Background Technology

[0002] Conventional methods for extracting rutin from plant materials mainly rely on organic solvents (such as ethanol and methanol) and require multi-stage purification processes, including membrane filtration, recrystallization, and column chromatography. These methods suffer from high solvent consumption, lengthy processes, high wastewater treatment costs, and significant safety risks, resulting in poor economic efficiency and low overall product quality. Particularly for rutin-rich raw materials such as Sophora japonica buds, further preparation of quercetin requires additional acid or enzymatic hydrolysis, further increasing process complexity and operating costs.

[0003] Several solvent-free alternatives exist in existing technologies, such as alkaline extraction and acid precipitation (see CN106928290A, CN104592329A), enzymatic fermentation extraction (see WO2004 / 027074 A2), aqueous two-phase systems, and eutectic solvents (DESs). However, these methods can only independently solve local problems in extraction or purification, and have not yet achieved full integration of the entire process from pretreatment, extraction to purification. In particular, there is a lack of integrated systems that can collaboratively complete enzymatic pretreatment, buffer stabilization extraction, antioxidant protection, and solvent-free recrystallization in the same device, resulting in limited overall process efficiency and difficulty in simultaneously improving product yield and purity.

[0004] More importantly, existing technologies cannot achieve selective production of rutin and quercetin by simply adjusting parameters without changing the basic process flow.

[0005] Therefore, there is an urgent need to develop a new green extraction process that can avoid the use of organic solvents, achieve integrated extraction and purification, and selectively produce rutin or quercetin through simple parameter control, so as to overcome the multiple bottlenecks of existing technologies in terms of process complexity, product selectivity and environmental friendliness, and promote the industrial application of plant extraction technology. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides a method for extracting and purifying flavonoids from Sophora japonica buds using a solvent-free subcritical water one-pot method.

[0007] This application provides a method for solvent-free subcritical water one-pot extraction and purification of flavonoids, comprising the following steps:

[0008] S1: Raw material enzyme treatment

[0009] The Sophora japonica flowers were placed in a sealed stirred reactor; an acetate buffer solution of pH 4.4-5.0 and 0.07-0.15M was added, along with ascorbic acid as an antioxidant; nitrogen gas was introduced to establish an inert atmosphere; then the enzyme preparation was added; and then incubation was carried out.

[0010] S2: Subcritical water extraction

[0011] After the raw material enzyme treatment in step S1, the reactor is heated to 110-165℃ and extracted under autogenous pressure; the liquid extract and residue are separated by filtration.

[0012] The liquid extract was cooled, allowed to stand, and the precipitated flavonoid crystals were filtered out, washed, and dried to obtain crude flavonoids.

[0013] S3: Crystallization and purification

[0014] The crude flavonoids obtained in step S2 were dissolved in a hot, weakly alkaline carbonate buffer solution, and an adsorbent was added. The solution was stirred and filtered to remove insoluble impurities and the adsorbent, resulting in a clear solution. The clear solution was adjusted to a weakly acidic state, cooled, and allowed to stand for recrystallization. The crystal product was filtered, washed, and dried to obtain the finished flavonoids.

[0015] Furthermore, in some embodiments, the mass-to-volume ratio of the Sophora japonica flower to the acetic acid buffer in step S1 is 1:(8-12).

[0016] Furthermore, in some embodiments, the amount of ascorbic acid added in step S1 is 0.08-0.12% of the mass of the Sophora japonica flower.

[0017] Furthermore, in some embodiments, the nitrogen gas is introduced in step S1 for 5-10 minutes.

[0018] Furthermore, in some embodiments, the amount of enzyme preparation added in step S1 is 0.4-0.6% of the mass of the Sophora japonica flower.

[0019] Furthermore, in some embodiments, the enzyme preparation described in step S1 is an enzyme preparation containing cellulase or pectinase.

[0020] Furthermore, in some embodiments, the incubation temperature in step S1 is 40-50°C; the incubation time is 40-90 minutes.

[0021] Furthermore, in some embodiments, the flavonoid compound in step S2 is rutin or quercetin.

[0022] Furthermore, in some embodiments, when crude rutin is obtained in step S2, the reactor temperature is raised to 110-125°C.

[0023] Furthermore, in some embodiments, when crude quercetin is obtained in step S2, the reactor temperature is raised to 155-165°C.

[0024] Furthermore, in some embodiments, the extraction time in step S2 is 50-70 minutes.

[0025] Furthermore, in some embodiments, the cooling temperature in step S2 is 5-10°C; the standing time is 2-4 hours; and the washing is done with water at 5-10°C.

[0026] Furthermore, in some embodiments, step S2 further includes a secondary extraction of the residue by adding the same weakly acidic buffer solution as in step S1 to the residue and extracting it again at the same extraction temperature and extraction time.

[0027] Combine the liquid extracts, cool, let stand, filter out the precipitated flavonoid crystals, wash and dry to obtain crude flavonoids.

[0028] Furthermore, in some embodiments, when the product obtained in step S3 is rutin, the temperature of the carbonate buffer solution is 70-85°C and the pH is 8.5-9.2.

[0029] The mass-to-volume ratio of crude rutin to carbonate buffer is 1:(10-15); preferably, the mass-to-volume ratio of crude rutin to carbonate buffer is 1:(10-12).

[0030] The mass ratio of crude rutin to the adsorbent is 1:(1.6-1.8).

[0031] Furthermore, in some embodiments, when the product obtained in step S3 is quercetin, the temperature of the carbonate buffer solution is 70-75°C; the pH is 8.8-9.0; the mass-to-volume ratio of the crude quercetin to the carbonate buffer solution is 1:(6-8); and the mass ratio of the crude quercetin to the adsorbent is 1:(2-4).

[0032] In step S3, the crude flavonoid compound is dissolved in a hot, weakly alkaline carbonate buffer solution. The mass-to-volume ratio of the crude flavonoid compound to the carbonate buffer solution ensures that the crude flavonoid compound has sufficient solubility.

[0033] Furthermore, in some embodiments, the adsorbent in step S3 is a composite adsorbent composed of activated carbon and diatomaceous earth; when the product obtained in step S3 is rutin, the mass ratio of activated carbon to diatomaceous earth in the composite adsorbent is 1:(3-5); when the product obtained in step S3 is quercetin, the mass ratio of activated carbon to diatomaceous earth in the composite adsorbent is 1:(5-7).

[0034] Furthermore, in some embodiments, the clarified solution in step S3 is adjusted to a weakly acidic pH by adjusting the pH of the clarified solution to 4-5 with acetic acid; the cooling temperature in step S3 is 0-10°C; the standing time is 2-3 hours; and the washing is done with water at 0-10°C.

[0035] Furthermore, in some embodiments, the yield of the rutin product in step S3 is above 77%, and the purity is above 92%; the yield of the quercetin product is above 76%, and the purity is above 94%.

[0036] In step S1 of the first aspect of this application, an antioxidant is added to prevent the oxidation of flavonoids such as rutin and quercetin; nitrogen purging is used in the reactor to create an inert atmosphere, minimizing oxidation losses.

[0037] In step S1 of the first aspect of this application, an enzyme preparation is added and then incubated, which can destroy the cell wall, hydrolyze pectin and cellulose, and improve the extraction efficiency of flavonoids such as rutin and quercetin, thus providing raw materials for subcritical water extraction in step S2.

[0038] In step S1 of the first aspect of this application, an acetate buffer with a pH of 4.5-5.0 and a concentration of 0.07-0.15 M is used, providing an optimal ionic strength environment for the enzymatic reaction. Within the pH range of 4.5-5.0, the buffering effect is fully utilized, maintaining high catalytic activity of the enzyme and effectively degrading cell wall polysaccharides. The weakly acidic environment inhibits the alkaline hydrolysis or oxidative degradation of flavonoids rutin and quercetin, protecting the structural integrity of these compounds. The weakly acidic acetate buffer synergizes with the subcritical water extraction in step S2. At subcritical temperatures (110-160℃), the acetate buffer maintains pH stability, preventing excessive hydrolysis of rutin due to excessive acidity or oxidation of quercetin under alkaline conditions. The 0.07-0.15 M concentration enhances osmotic pressure, balancing the promoting effect of ionic strength on extraction with the decrease in solubility caused by excessive ionization. In step S3, the weakly acidic conditions of the acetate buffer inhibit the excessive dissolution of impurities such as pectin and proteins, reducing the purification burden.

[0039] In the first aspect of this application, step S2 employs extraction under autogenous pressure. Autogenous pressure refers to the internal pressure naturally generated in a closed reaction system due to the evaporation of liquid due to heating and the expansion of gases (such as air and water vapor) due to heating. This pressure is not externally injected but spontaneously formed by changes in matter and energy within the system. In the subcritical water extraction in step S2, the reactor is sealed. When the aqueous buffer solution inside the reactor is heated to a temperature far above its atmospheric boiling point (100°C), the water vaporizes. However, because the reactor is sealed, the water vapor cannot escape, causing the density of vapor phase molecules to continuously increase. These trapped high-energy water vapor molecules collide with the container walls and the liquid surface, thus spontaneously generating a high-pressure environment inside the system. This pressure generated by the vaporization of water itself is the "autogenous pressure." In the first aspect of this application, step S2 employs self-generated pressure extraction, ensuring that water does not boil or vaporize at extraction temperatures of 110-125℃ and 155-165℃, thereby maintaining a continuous liquid reaction environment and guaranteeing extraction efficiency. This method of maintaining subcritical liquid water under high temperature and pressure reduces its dielectric constant and polarity, significantly improving its solubility and extraction efficiency for moderately polar compounds (such as rutin and quercetin). The self-generated pressure environment reduces the residual oxygen space at the top of the reactor, working in conjunction with the nitrogen introduced in step S1 to create a low-oxygen, inert atmosphere, effectively inhibiting the oxidative degradation of flavonoids. The required high-temperature and high-pressure environment can be achieved simply by heating and sealing the reactor, eliminating the need for complex external pressurization devices. This greatly simplifies the equipment, reduces investment and operating costs, and facilitates laboratory research and industrial scale-up. Self-generated pressure extraction is a key design that cleverly utilizes physical principles to achieve efficient and green extraction, avoiding complex external pressurization equipment and serving as a crucial guarantee for the economic viability and feasibility of this application.

[0040] When crude rutin is obtained in step S2 of the first aspect of this application, extraction is carried out under autogenous pressure at an extraction temperature of 110-125℃ and a pH of 4.4-5.0, which achieves efficient extraction of rutin with minimal degradation. Water is in a subcritical state within the temperature range of 110-125℃, where its dielectric constant decreases and polarity weakens, similar to organic solvents (such as ethanol), thereby significantly improving the solubility of rutin (a moderately polar compound). High temperature accelerates molecular diffusion and cell wall rupture, promoting mass transfer of rutin from the plant matrix to the solvent and improving extraction efficiency. Strict temperature control below 125℃ avoids excessive hydrolysis or oxidative degradation of rutin glycoside bonds. A weakly acidic environment protects the stability of rutin glycoside bonds; inhibits the ionization of phenolic hydroxyl groups, reducing the rate of oxidative degradation; and it connects seamlessly with the enzymatic reaction stage of step S1, avoiding drastic pH fluctuations. Extraction under autogenous pressure can suppress water vaporization. In a closed system, autogenous pressure can maintain a liquid aqueous phase and avoid concentration polarization or local overheating caused by vaporization at high temperatures. The autogenous pressure environment can reduce oxygen residue and, together with the nitrogen inert atmosphere in step S1, further inhibit the oxidative degradation of rutin.

[0041] In step S2 of the first aspect of this application, when crude quercetin is obtained, extraction is performed under autogenous pressure at an extraction temperature of 155-165℃. This allows rutin to be hydrolyzed to produce quercetin, enabling the production of both rutin and quercetin in the same facility without transferring raw materials or changing the solvent system, significantly reducing equipment investment and production line switching costs. Rutin (quercetin-3-O-rutin glycoside) undergoes specific hydrolysis in subcritical water at 155-165℃, removing the rutinyl group to produce quercetin, while the flavonoid skeleton of quercetin itself remains stable within this temperature range. The autogenous pressure in the closed system ensures that the water remains in a liquid state, avoiding uneven hydrolysis or localized overheating degradation caused by high-temperature vaporization. This technical solution directly utilizes the weak ionization characteristics of subcritical water (H2O). + and OH - (Increased concentration) serves as a hydrolysis catalyst, eliminating the need for additional acid / base or enzyme preparations, thus avoiding the introduction of impurities and purification burden.

[0042] In step S2 of the first aspect of this application, at least one extraction is used, which can make full use of the raw materials and improve the extraction rate.

[0043] In the first aspect of this application, during the preparation of the crude product in step S2 and the finished product in step S3, cold water washing is used to remove trace impurity ions (such as carbonate and acetate ions) adsorbed on the surface, thus avoiding crystal redissolution loss caused by hot water washing.

[0044] In the first aspect of this application, a weakly alkaline carbonate buffer system is used in step S3 to dissolve crude flavonoids such as rutin or quercetin, ensuring complete dissolution of the flavonoids such as rutin or quercetin, while avoiding glycosidic bond hydrolysis or B-ring opening degradation caused by strong alkaline conditions. Rutin and quercetin have different polarities, and the pH range of the carbonate buffer system is different. Heating to 70-85℃ can significantly improve the solubility of flavonoids such as rutin / quercetin, ensuring complete dissolution while inhibiting oxidation caused by high temperature.

[0045] In the first aspect of this application, a composite adsorbent is used in step S3. In this composite adsorbent, activated carbon and diatomaceous earth work together to purify flavonoids such as rutin or quercetin. Activated carbon efficiently adsorbs pigments, tannins, and small organic molecules. Diatomaceous earth traps particulate impurities through its porous structure and aids filtration, preventing activated carbon from clogging the filter membrane. Since quercetin molecules are more polar than rutin, more diatomaceous earth is needed to disperse the adsorbent to avoid over-adsorption of the target product. Therefore, the amount of diatomaceous earth used in quercetin purification is higher than that used in rutin purification. The amount of composite adsorbent used must ensure the removal of impurities and minimize the loss of the target compound.

[0046] In the first aspect of this application, acetic acid is used in step S3 to adjust the clarified solution to a weakly acidic state, which can disrupt the alkaline dissolution environment and trigger crystallization; it can also achieve acid-selective induced precipitation, improve the purity of flavonoids such as rutin or quercetin, promote the optimization of crystal morphology and yield, and possess environmental friendliness and process compatibility. In the carbonate buffer solution, the phenolic hydroxyl groups of flavonoids such as rutin or quercetin ionize to form water-soluble phenoxysalts (-O). - ); after adding acetic acid, H + Neutralizing the charge restores flavonoids to their free state (-OH), drastically reducing their solubility and thus facilitating efficient crystal precipitation. Acetic acid, being a weak acid, allows for a stable reduction in solubility when adjusted to pH 4-5, preventing the decomposition of strong flavonoid skeletons or hydrolysis of glycosidic bonds. At pH 4-5, some acidic impurities (such as organic acids and residual ascorbic acid) remain dissolved, while flavonoids preferentially precipitate, enabling selective separation of flavonoids. Furthermore, the surface charge change of the composite adsorbent under weakly acidic conditions makes it easier to filter and retain, preventing it from entering the crystalline product. The mild acidification conditions of pH 4-5 promote slow crystal formation, reducing lattice defects and impurity entrapment, resulting in high-purity crystals with uniform particle size. Under pH 4-5 conditions, the low solubility of rutin or quercetin ensures high precipitation rates. Acetic acid can be removed by washing with water, eliminating the need to introduce difficult-to-separate ions, aligning with the solvent-free purification principle.

[0047] Rutin hydrolyzes at high temperatures to form quercetin, and quercetin and its glycosyl groups further degrade into various small molecules (such as 3,4-dihydroxybenzoic acid, catechol, and 5-HMF). This application utilizes a multi-parameter synergistic process to effectively regulate the subcritical water reaction pathway, achieving efficient conversion of the target product while minimizing its degradation. In the rutin preparation mode, a stable reaction environment was constructed by using an acetate buffer system with a pH of 4.4-5.0, adding antioxidants, establishing an inert atmosphere, and precisely controlling the extraction temperature at 110-125℃ and the extraction time at 50-70 minutes. This synergistic system effectively inhibits premature hydrolysis of rutin glycoside bonds and prevents oxidative degradation, ensuring a stable crude rutin yield of over 77%. The subsequent purification stage employs an alkali-dissolution, acid-precipitation, and crystallization process, combined with a composite adsorbent of activated carbon and diatomaceous earth to directionally remove impurities, ultimately obtaining a rutin product with a purity ≥92%.

[0048] Similarly, in the quercetin preparation mode, by maintaining the same buffer system and antioxidant environment, and raising the extraction temperature to 155-165℃, the complete conversion of rutin to quercetin can be promoted, while precise temperature control can prevent excessive degradation of quercetin. This synergistic process achieves a crude quercetin yield of over 76%, which is then purified using an optimized alkali-dissolution, acid-precipitation, and crystallization process with a specially formulated composite adsorbent to finally obtain a quercetin product with a purity of ≥95%.

[0049] By precisely matching the composition of the reaction medium, temperature parameters, and purification methods, the selective preparation and efficient purification of rutin and quercetin were achieved in the same device, without the need for organic solvents throughout the process.

[0050] Compared with the prior art, the technical solution of this application has the following beneficial technical effects:

[0051] This application has been approved.

[0052] Raw material enzyme treatment in a weakly acidic buffer solution and antioxidant stabilization under an inert atmosphere, slow down

[0053] This process involves subcritical water extraction in a buffer medium, dissolution of crude flavonoids such as rutin or quercetin in a carbonate buffer solution, and recrystallization purification using a composite adsorbent. Through a multi-step synergistic approach, rutin yields exceeding 77% and quercetin purity exceeds 92%, while quercetin yields exceeding 76% and quercetin purity exceeds 94%, all without the need for organic solvents. This improves yield and purity, reduces processing time, and makes the production process environmentally friendly.

[0054] This application allows for the selective production of flavonoids rutin and quercetin by adjusting the subcritical water extraction temperature in the buffer medium. This eliminates the need to transfer raw materials or change the solvent system, and allows for the production of both rutin and quercetin in the same device, significantly reducing equipment investment and production line switching costs. Attached Figure Description

[0055] The present application will be described in further detail below with reference to the accompanying drawings and preferred embodiments. However, those skilled in the art will understand that these drawings are drawn only for the purpose of explaining the preferred embodiments and therefore should not be construed as limiting the scope of this disclosure. Furthermore, unless specifically indicated, the drawings are only schematic representations of the composition or structure of the described objects and may contain exaggerated depictions, and the drawings are not necessarily drawn to scale.

[0056] Figure 1 This is a schematic diagram of a solvent-free subcritical water one-pot extraction and purification method for flavonoids. Detailed Implementation

[0057] The following is in conjunction with the appendix Figure 1 This disclosure will be explained in detail.

[0058] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0059] Chemicals and reagents

[0060] Sophora japonica buds: Origin: Linyi, Shandong.

[0061] Glacial acetic acid: purity greater than 99.7%; purchased from Aladdin Reagents.

[0062] Anhydrous sodium acetate: ACS grade, purchased from Aladdin Reagents.

[0063] Ascorbic acid: AR grade, purchased from Aladdin Reagents.

[0064] Pectinase preparation: 300,000 u / g, purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.

[0065] Activated carbon: Product number C112241, purchased from Aladdin Reagents.

[0066] Diatomaceous earth: analytical grade, content ≥85%, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0067] Anhydrous sodium carbonate: ACS grade, purchased from Aladdin Reagents.

[0068] Sodium bicarbonate: AR grade, purchased from Aladdin Reagents.

[0069] Unless otherwise specified, all other reagents used in the embodiments of this application are from conventional commercially available products.

[0070] Instruments and equipment

[0071] Sealed stirred reactor: TGYF-C benchtop high-pressure reactor, manufacturer: Henan Yuhua Instrument Technology Co., Ltd.

[0072] Solution preparation:

[0073] Acetic acid buffer: Weigh a certain amount of anhydrous sodium acetate and glacial acetic acid and place them in a beaker; add about 800 mL of deionized water to the beaker and stir until the solids are completely dissolved; immerse the pH meter electrode in the solution and adjust the pH value using concentrated acetic acid or saturated sodium acetate solution, while continuously stirring until the pH value is accurately stable at the target value; transfer the entire solution to a 1 L volumetric flask, dilute to the mark with deionized water, and shake well.

[0074] Carbonate buffer solution: Weigh a certain amount of anhydrous sodium carbonate and sodium bicarbonate and place them in a beaker; add about 800 mL of deionized water preheated to the target temperature to the beaker and stir until the solids are completely dissolved; immerse the calibrated pH meter electrode in the solution and adjust the pH with saturated sodium carbonate until the pH value is accurately stable at the target value; bring the volume to 1 L with deionized water and stir well.

[0075] Specifically, this application provides a solvent-free subcritical water one-pot extraction and purification method for flavonoids, such as... Figure 1 As shown, it includes the following steps:

[0076] S1: Raw material enzyme treatment

[0077] The Sophora japonica flowers were placed in a sealed stirred reactor; an acetate buffer solution of pH 4.4-5.0 and 0.07-0.15M was added, along with ascorbic acid as an antioxidant; nitrogen gas was introduced to establish an inert atmosphere; then the enzyme preparation was added; and then incubation was carried out.

[0078] S2: Subcritical water extraction

[0079] After the raw material enzyme treatment in step S2, the reactor is heated to 110-165℃ and extracted under autogenous pressure; the liquid extract and residue are separated by filtration.

[0080] The liquid extract was cooled, the precipitated flavonoid crystals were filtered out, washed and dried to obtain crude flavonoids.

[0081] S3: Crystallization and purification

[0082] The crude flavonoids obtained in step S2 were dissolved in hot, weakly alkaline carbonate buffer solution, and an adsorbent was added; the mixture was stirred; insoluble impurities and the adsorbent were removed by hot filtration; the clarified solution was adjusted to weakly acidic; the solution was cooled and allowed to stand for recrystallization; the crystalline product was filtered, washed, and dried to obtain the finished flavonoids.

[0083] Furthermore, in step S1, the mass-to-volume ratio of the Sophora japonica flower to the acetic acid buffer is 1:(8-12).

[0084] Furthermore, the amount of ascorbic acid added in step S1 is 0.08-0.12% of the mass of the Sophora japonica flower.

[0085] Furthermore, the inert gas is introduced for 5-10 minutes in step S1.

[0086] Furthermore, the amount of enzyme preparation added in step S1 is 0.4-0.6% of the mass of the Sophora japonica flower.

[0087] Furthermore, the enzyme preparation mentioned in step S1 is an enzyme preparation containing cellulase or pectinase.

[0088] Furthermore, the incubation temperature in step S1 is 40-50℃; the incubation time is 40-90 minutes.

[0089] Furthermore, the flavonoid compound in step S2 is rutin or quercetin;

[0090] Furthermore, when crude rutin is obtained in step S2, the reactor temperature is raised to 110-125°C.

[0091] Furthermore, when crude quercetin is obtained in step S2, the reactor temperature is raised to 155-165°C.

[0092] Furthermore, in some embodiments, the extraction time in step S2 is 50-70 minutes;

[0093] Furthermore, in step S2, the cooling temperature is 5-10℃; the standing time is 2-4 hours; and the washing is done with water at 5-10℃.

[0094] Furthermore, step S3 also includes a second extraction of the residue by adding the same weakly acidic buffer solution as in step S1 to the residue and extracting it again at the same extraction temperature and extraction time.

[0095] Combine the liquid extracts, cool, let stand, filter out the precipitated flavonoid crystals, wash and dry to obtain crude flavonoids.

[0096] Furthermore, when obtaining rutin product in step S3, the temperature of the carbonate buffer solution is 70-85℃; pH is 8.5-9.2; the mass-to-volume ratio of crude rutin to carbonate buffer solution is 1:(10-15); and the mass ratio of crude rutin to adsorbent is 1:(1.6-1.8).

[0097] Furthermore, when the product obtained in step S3 is quercetin, the temperature of the carbonate buffer solution is 70-75℃; the pH is 8.8-9.0; the mass-to-volume ratio of the crude quercetin to the carbonate buffer solution is 1:(6-8); and the mass ratio of the crude quercetin to the adsorbent is 1:(2-4).

[0098] Furthermore, the adsorbent in step S3 is a composite adsorbent composed of activated carbon and diatomaceous earth; when the product obtained in step S3 is rutin, the mass ratio of activated carbon to diatomaceous earth in the composite adsorbent is 1:(3-5); when the product obtained in step S3 is quercetin, the mass ratio of activated carbon to diatomaceous earth in the composite adsorbent is 1:(5-7).

[0099] Furthermore, in step S3, the clarified solution is adjusted to a weakly acidic pH by using acetic acid to adjust the pH to 4-5; the cooling temperature in step S3 is 0-10℃; the standing time is 2-3 hours; and the washing is done with cold water at 0-10℃.

[0100] Furthermore, in step S3, the yield of rutin is above 77% and the purity is above 92%; the yield of quercetin is above 76% and the purity is above 94%.

[0101] Example 1

[0102] S1: Raw material enzyme treatment

[0103] 100.00g of Sophora japonica buds were placed into a sealed stirred reactor; 1.0L of 0.1M acetate buffer solution with pH 4.76 was added; 0.1% ascorbic acid was added as an antioxidant; nitrogen gas was purged into the reactor for 8 minutes to establish an inert atmosphere; then 0.5% of the Sophora japonica buds' mass of pectinase preparation was added; and then the mixture was incubated at 45℃ for 60 minutes.

[0104] S2: Subcritical water extraction

[0105] After the raw material enzyme treatment in step S1, the reactor was heated to 120°C and extracted under autogenous pressure for 60 minutes. The liquid extract and residue were separated by filtration. The residue was then extracted a second time by adding 1.0 L of 0.1 M acetate buffer (pH 4.76) to the residue and extracting again at 120°C for 60 minutes. The extracts were combined, cooled to 8°C, and allowed to stand for 3 hours. The precipitated rutin crystals were filtered, washed with water at 8°C, and dried to obtain crude rutin.

[0106] S3: Crystallization and purification

[0107] 14.40 g of crude rutin obtained in step S2 was dissolved in 150 ml of carbonate buffer solution at 80 °C and pH 9.00. A composite adsorbent consisting of 5 g of activated carbon and 20 g of diatomaceous earth was added. The mixture was stirred for 30 min. Insoluble impurities and the composite adsorbent were removed by hot filtration. The pH of the clarified solution was adjusted to 4.50 with acetic acid. The solution was cooled to 8 °C and allowed to stand for 2 hours for recrystallization. The crystalline product was filtered, washed, and dried to obtain the finished rutin product.

[0108] Example 2

[0109] In Example 2, step S1, the raw material enzyme treatment, is the same as in Example 1. The remaining steps are as follows:

[0110] S2: Subcritical water extraction

[0111] After the raw material enzyme treatment in step S1, the reactor was heated to 160°C and extracted under autogenous pressure for 60 minutes. The liquid extract and residue were separated by filtration. The residue was then extracted a second time by adding 1.0 L of 0.1 M acetate buffer (pH 4.76) to the residue and extracting again at 160°C for 60 minutes. The extracts were combined, cooled to 10°C, and allowed to stand for 2 hours. The precipitated quercetin crystals were filtered, washed with water at 10°C, and dried to obtain crude quercetin.

[0112] S3: Crystallization and purification

[0113] Dissolve 7g of crude quercetin obtained in step S2 in 50ml of carbonate buffer solution at 73℃ and pH 9.00, add a composite adsorbent consisting of 3g of activated carbon and 20g of diatomaceous earth; stir for 30min; remove insoluble impurities and composite adsorbent by hot filtration; adjust the pH of the clarified solution to 4.50 with acetic acid; cool to 10℃, let stand for 2 hours, and recrystallize; filter the crystal product, wash with water at 10℃ and dry to obtain the finished quercetin product.

[0114] The yield and purity of rutin in Example 1 were tested, and the results are shown in Table 1.

[0115] Table 1. Results of rutin yield and purity testing in Example 1

[0116]

[0117] Based on Examples 1 and 2, this application has studied the process parameters, and the specific process parameters are shown in Tables 2-6.

[0118] Table 2. Process parameters for raw material enzyme treatment in step S1.

[0119]

[0120] Table 3. Process parameters for crude rutin prepared by subcritical water extraction in step S2.

[0121]

[0122] Table 4. Process parameters for crude quercetin prepared by subcritical water extraction in step S2.

[0123]

[0124] Table 5. Process parameters for step S3, crude rutin crystallization and purification.

[0125]

[0126] Table 6. Process parameters for step S3, quercetin crude product crystallization and purification.

[0127]

[0128] The yield and purity of the rutin or quercetin products prepared in Examples 3-6 were tested, and the results are shown in Table 7.

[0129] Table 7 shows the yield and purity of the rutin or quercetin products prepared in Examples 3-6.

[0130]

[0131] Table 7 shows that the yield and purity of the rutin or quercetin products prepared in Examples 3-6 were tested. The yield of the rutin products prepared in Examples 3-4 was above 77%, and the purity was above 92%. The yield of the quercetin products prepared in Examples 5-6 was above 76%, and the purity was above 94%.

[0132] Based on Example 1, this application conducted a comparative study on the pH value and concentration of the acetic acid buffer in step S1. The specific parameters are shown in Table 8.

[0133] Table 8. Comparison of pH and concentration of acetate buffer in step S1. (Process parameters)

[0134]

[0135] The yields of the rutin products prepared in Comparative Examples 1-4 were tested, and the results are shown in Table 9.

[0136] Table 9 shows the yield of the rutin products prepared in Comparative Examples 1-4.

[0137]

[0138] As shown in Table 9, the yield of rutin prepared in Comparative Example 1 was 20% lower than that in Example 1; the yield of rutin prepared in Comparative Example 2 was 15% lower than that in Example 1; the yield of rutin prepared in Comparative Example 3 was 18% lower than that in Example 1; and the yield of rutin prepared in Comparative Example 4 was 12% lower than that in Example 1. The reason for these phenomena is that pH < 4.4 inhibits enzyme activity, resulting in insufficient cell wall disruption; pH < 4.4 also exacerbates the hydrolysis of rutin glycoside bonds, leading to a significant decrease in yield. Rutin cannot be fully extracted and may also be decomposed during the extraction process, resulting in a double loss. Similarly, pH > 5.0 is not conducive to optimal enzyme activity; furthermore, the phenolic hydroxyl groups in the rutin molecule are more easily ionized, forming phenolic oxygen free radicals, which can lead to oxidative polymerization or degradation, significantly reducing the yield. When the concentration of acetate buffer is below 0.07M, the buffering capacity is insufficient, failing to provide the optimal working environment for the enzyme, resulting in incomplete cell wall disruption and low rutin dissolution rate. When the concentration of acetate buffer is above 0.15M, the high ionic strength reduces the solubility of rutin, and the high concentration may damage the hydration layer or charge distribution on the surface of the enzyme protein, thus inhibiting its activity and reducing the rutin extraction rate.

[0139] Based on Examples 1 and 2, this application conducted a comparative study on the temperature of the reactor in step S2, and the specific parameters are shown in Table 10.

[0140] Table 10 Comparison of pH and concentration of acetate buffer in step S2 (process parameters)

[0141]

[0142] The yield and purity of the rutin products prepared in Comparative Examples 5-8 were tested, and the results are shown in Table 11.

[0143] Table 11 shows the yield and purity of the rutin or quercetin products prepared in Comparative Examples 5-8.

[0144]

[0145] Table 11 shows that the yield of the rutin product prepared in Comparative Example 5 was 20% lower than that in Example 1, and the purity was 8% lower than that in Example 1; the yield of the rutin product prepared in Comparative Example 6 was 15% lower than that in Example 1, and the purity was 18% lower than that in Example 1. Regarding the extraction temperature of rutin, when the extraction temperature is lowered, impurities with better water solubility are more easily extracted due to insufficient cell wall disruption of the raw material or reduced rutin solubility, thus reducing the yield and purity of the rutin product; when the extraction temperature is increased, rutin degrades, the proportion of impurities increases, and the yield and purity decrease significantly.

[0146] The yield of the quercetin product prepared in Comparative Example 7 was 28% lower than that in Example 2, and the purity was 35% lower than that in Example 2. The yield of the quercetin product prepared in Comparative Example 8 was 17% lower than that in Example 2, and the purity was 20% lower than that in Example 2. Regarding the extraction temperature of quercetin, if the extraction temperature is below 155°C, the hydrolysis rate is too slow, and residual rutin reduces the yield and purity of quercetin. If the extraction temperature is above 165°C, quercetin is oxidized or undergoes ring-opening degradation, increasing impurities and reducing the yield and purity.

[0147] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present disclosure and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for extracting and purifying flavonoids by one-pot subcritical water without solvent, characterized in that, The method comprises the following steps: S1: raw material enzyme treatment The flos sophorae japonicae is loaded into a sealed stirring reactor; a pH 4.4-5.0, 0.07-0.15 M acetic acid buffer is added, ascorbic acid is added as an antioxidant; nitrogen is introduced to establish an inert atmosphere; enzyme preparation is added; and then incubation is performed; S2: subcritical water extraction After the raw material enzyme treatment of step S1, the reactor is heated to 110-125 ℃ or 155-165 ℃, and extraction is performed under autogenous pressure; the liquid extract and residue are separated by filtration; The liquid extract is cooled, allowed to stand, and the separated flavonoid crystals are filtered, washed, and dried to obtain crude flavonoids; S3: crystallization and purification The crude flavonoids obtained in step S2 are dissolved in hot weakly alkaline carbonate buffer, and an adsorbent is added; stirring is performed; insoluble impurities and the adsorbent are removed by filtration to obtain a clear solution; the clear solution is adjusted to weakly acidic; and recrystallization is performed by cooling and allowing to stand; the crystal product is filtered, washed, and dried to obtain finished flavonoids; The enzyme preparation in step S1 is a pectinase-containing enzyme preparation; The flavonoids in steps S2 and S3 are rutin or quercetin; When the crude rutin is obtained in step S2, the reactor temperature is raised to 110-125 ℃; When the crude quercetin is obtained in step S2, the reactor temperature is raised to 155-165 ℃; The extraction time in step S2 is 50-70 minutes.

2. The method according to claim 1, wherein the solvent-free subcritical water one-pot extraction and purification of flavonoids is carried out by the following steps: (1) extracting flavonoids from the raw material with subcritical water; (2) purifying the flavonoids extracted in step (1) with subcritical water; and (3) collecting the purified flavonoids. The mass / volume ratio of the flos sophorae japonicae to the acetic acid buffer in step S1 is 1:(8-12); The amount of ascorbic acid added is 0.08-0.12% of the mass of the flos sophorae japonicae; The amount of enzyme preparation added is 0.4-0.6% of the mass of the flos sophorae japonicae.

3. The method according to claim 1, wherein the solvent-free subcritical water one-pot extraction and purification of flavonoids is carried out by the following steps: (1) extracting flavonoids from the raw material with subcritical water; (2) purifying the flavonoids extracted in step (1) with subcritical water; and (3) collecting the purified flavonoids. The incubation temperature in step S1 is 40-50 ℃; and the incubation time is 40-90 minutes.

4. The method for solvent-free subcritical water one-pot extraction and purification of flavonoids as described in claim 1, characterized in that, The cooling temperature in step S2 is 5-10 ℃; the standing time is 2-4 hours; and the washing is performed with 5-10 ℃ water.

5. The method for solvent-free subcritical water one-pot extraction and purification of flavonoids as described in claim 1, characterized in that, Step S2 further comprises secondary extraction of the residue, wherein the same weakly acidic buffer as in step S1 is added to the residue, and the same extraction temperature and extraction time are used for the secondary extraction; The liquid extracts are combined, cooled, allowed to stand, and the separated flavonoid crystals are filtered, washed, and dried to obtain crude flavonoids.

6. The method for solvent-free subcritical water one-pot extraction and purification of flavonoids as described in claim 1, characterized in that, When the finished rutin is obtained in step S3, the temperature of the carbonate buffer is 70-85 ℃; the pH is 8.5-9.2; the mass / volume ratio of the crude rutin to the carbonate buffer is 1:(10-15); and the mass ratio of the crude rutin to the adsorbent is 1:(1.6-1.8).

7. The method for solvent-free subcritical water one-pot extraction and purification of flavonoids as described in claim 1, characterized in that, When the finished quercetin is obtained in step S3, the temperature of the carbonate buffer is 70-75 ℃; the pH is 8.8-9.0; the mass / volume ratio of the crude quercetin to the carbonate buffer is 1:(6-8); and the mass ratio of the crude quercetin to the adsorbent is 1:(2-4).

8. The method for solvent-free subcritical water one-pot extraction and purification of flavonoids as described in claim 1, characterized in that, The adsorbent in step S3 is a composite adsorbent composed of activated carbon and diatomite; When the finished rutin is obtained in step S3, the mass ratio of activated carbon to diatomite in the composite adsorbent composed of activated carbon and diatomite is 1:(3-5); The mass ratio of the activated carbon and diatomite in the composite adsorbent composed of the activated carbon and diatomite is 1: (5-7) when the quercetin product is obtained in step S3.

9. The method according to claim 1, wherein the solvent-free subcritical water one-pot extraction and purification of flavonoids is carried out by the following steps: (1) extracting flavonoids from the raw material with subcritical water; (2) purifying the flavonoids extracted in step (1) with subcritical water; and (3) collecting the purified flavonoids. The clear solution is adjusted to weak acidity in step S3 by using acetic acid to adjust the clear solution to pH 4-5; the cooling temperature in step S3 is 0-10 DEG C; the standing time is 2-3 hours; and the washing is performed with water at 0-10 DEG C. The mass ratio of the activated carbon and diatomite in the composite adsorbent composed of the activated carbon and diatomite is 1: (5-7) when the quercetin product is obtained in step S3. The clear solution is adjusted to weak acidity in step S3 by using acetic acid to adjust the clear solution to pH 4-5; the cooling temperature in step S3 is 0-10 DEG C; the standing time is 2-3 hours; and the washing is performed with water at 0-10 DEG C.

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