Green flavonoid compound extraction method based on deep eutectic solvent

Abstract

The invention discloses a flavonoid compound green extraction method based on a deep eutectic solvent, which comprises the following steps: crushing and sieving a qi-tonifying and mass-eliminating formula to obtain compound powder; adding the compound powder and a deep eutectic solvent (DES) into a grinder for MCE grinding extraction to obtain a compound extract; adding the compound extract into pure water, carrying out vortex and centrifugation, and taking supernate, namely a compound extracting solution; according to the method, the DES solvent is used for assisting MCE ball milling to extract the flavonoid components (proanthocyanidin B1, hesperidin, quercetin, aloe-emodin 8-glucoside and neohesperidin) in the formula for tonifying qi and eliminating diseases for the first time, the extraction efficiency is remarkably improved, the extraction process is more environmentally friendly, and the method is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the extraction of active ingredients from traditional Chinese medicine, specifically to a novel, green, and efficient method for extracting flavonoids from traditional Chinese medicine compound formulas based on deep eutectic solvents. Background Technology

[0002] Mechanical cell disruption (MCE) is an innovative and efficient pretreatment technology widely recognized for its environmentally friendly and pollution-free characteristics. This technology shows broad application prospects in the extraction of natural products and traditional Chinese medicine. By inducing chemical and physical changes in materials through mechanical force, MCE can effectively reduce particle size at room temperature, offering significant advantages such as high extraction rate, short processing time, and minimal environmental impact. Therefore, based on its advantages of low cost, high efficiency, room temperature operation, and the use of pure water rather than organic solvents as the extraction medium, MCE is widely used for the extraction of flavonoids, alkaloids, and other bioactive compounds. However, traditional MCE is mainly used as a simple extraction process, lacking integrated enrichment steps, which limits its detection efficiency for low-abundance components. Furthermore, this method typically relies on solid alkaline reagents (including inorganic and organic bases), posing potential environmental risks. Therefore, developing novel environmentally friendly reagents for the extraction of target compounds is of great significance. Such reagents are particularly crucial for microextraction applications and the detection of low-abundance components in complex matrices.

[0003] With increasing global emphasis on environmental protection and sustainable development, traditional organic solvents face increasingly stringent restrictions due to their high toxicity and poor biodegradability. Therefore, the development of green solvents has become a major focus of green chemistry research. Deep eutectic solvents (DESs) are mixtures of two or more components (typically hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD)) that form a eutectic system through strong intermolecular forces at a specific molar ratio. During DES formation, HBA and HBD form hydrogen bonds at a specific molar ratio, resulting in a mixture with a melting point lower than the pure components. This unique composition endows DESs with significant physicochemical properties, including low volatility, low toxicity, ease of preparation, biodegradability, and environmental compatibility, all of which have been confirmed by scientific research. In recent years, DESs have increasingly replaced traditional organic solvents as a green alternative for extracting and enriching active ingredients from various natural products. However, this extraction method is rarely used for the extraction and separation of complex compound mixtures because most current methods still rely on toxic organic solvents and rarely utilize green solvents such as DESs. Therefore, combining MCEs with DESs instead of traditional reagents is a promising research direction.

[0004] Yiqi Xiaozheng Formula (YQXZF) is a modified version of a formula for treating chronic kidney disease (CKD) developed by the Nephrology Department of Ningbo Municipal Hospital of Traditional Chinese Medicine, based on a formula by Zhang Peiqiu, a nationally renowned TCM physician. This formula consists of Astragalus membranaceus, Codonopsis pilosula, Angelica sinensis, Ligusticum chuanxiong, Bombyx batryticatus, Rheum palmatum, Curcuma zedoaria, and Citrus reticulata peel. It can reduce urinary protein levels, improve renal function, and inhibit high glucose-induced renal tubular epithelial cell fibrosis, epithelial-mesenchymal transition, and inflammatory responses. Hesperidin, quercetin, and neohesperidin are natural flavonoids. These three compounds are designated as quantitative markers in the Chinese Pharmacopoeia and exhibit a range of pharmacological activities, including antioxidant, anti-inflammatory, cardiovascular protective, and hypoglycemic effects. Furthermore, YQXZF also contains various flavonoids, including proanthocyanidin B1 and aloe-emodin 8-glucoside, both of which possess significant anti-inflammatory and anti-fibrotic activities. It is widely used in clinical practice to treat mild cases of kidney blood stasis caused by spleen and kidney qi deficiency, weak blood circulation, and qi stagnation and blood stasis.

[0005] This invention provides a green, practical, and fundamental DES-assisted MCE method for extracting proanthocyanidin B1, hesperidin, quercetin, aloe-emodin 8-glucoside, and neohesperidin from YQXZF. Quantitative analysis of the five target compounds was performed by HPLC. Key parameters—including DES type, DES dosage, grinding time, solid-liquid ratio, and vortexing time—were optimized through a single-factor experimental system. Response surface methodology (RSM) was further employed to investigate the interaction among the three main influencing factors (DES dosage, grinding time, and vortexing time) to determine the optimal extraction conditions. The feasibility of the method was verified through comprehensive method validation and sample analysis. Finally, the contents of the five key components were determined under the optimized extraction scheme. Summary of the Invention

[0006] To address the problem of insufficient extraction efficiency of components in traditional Chinese medicine compound preparations using conventional extraction methods, this invention provides a green extraction method for flavonoids based on deep eutectic solvents.

[0007] This invention is the first to propose using DES solvent-assisted MCE ball milling to extract flavonoid components (proanthocyanidin B1, hesperidin, quercetin, aloe-emodin 8-glucoside and neohesperidin) from Yiqi Xiaozheng formula, which significantly improves extraction efficiency and makes the extraction process more green and environmentally friendly.

[0008] The technical solution of the present invention is as follows:

[0009] A green extraction method for flavonoids based on deep eutectic solvents, comprising:

[0010] A. Crush the Yiqi Xiaozheng formula into powder, sieve it (50 mesh) to obtain the compound powder;

[0011] The Qi-Boosting and Mass-Eliminating Formula consists of the following ingredients in the indicated weight ratios: Astragalus membranaceus 30 parts, Codonopsis pilosula 10 parts, Angelica sinensis 10 parts, Ligusticum chuanxiong 15 parts, Bombyx mori 10 parts, Rheum palmatum 6 parts, Curcuma zedoaria 15 parts, and Citrus reticulata 5 parts.

[0012] B. Add the compound powder obtained in step A and deep eutectic solvent (DES) to a grinder for MCE grinding and extraction to obtain the compound extract;

[0013] The preferred material-to-liquid ratio of compound powder and deep eutectic solvent is 10:1, mg / μL;

[0014] The preferred method is to grind and extract at 540 rpm for 240 s;

[0015] The composition of the deep eutectic solvent is selected from any of the following:

[0016] The molar ratio of choline chloride (ChCl) to urea is 1:2.

[0017] The molar ratio of choline chloride to ethylene glycol is 1:2.

[0018] Choline chloride / lactic acid molar ratio 1:2 (preferred);

[0019] The molar ratio of choline chloride / ethylene glycol / lactic acid is 1:1:2.

[0020] The preparation method of the deep eutectic solvent is as follows:

[0021] Mix the components according to the formula, heat to 80°C under nitrogen atmosphere, and stir magnetically at 300 rpm (1~4 h) until a uniform and transparent liquid is obtained.

[0022] C. Add the compound extract obtained in step B to pure water, vortex and centrifuge, and take the supernatant as the compound extract.

[0023] The preferred liquid-to-solid ratio of pure water to the compound powder obtained in step A is 1:50, mL / mg;

[0024] The preferred method is to vortex for 3 minutes and centrifuge at 15,000 rpm for 3 minutes.

[0025] The obtained compound extract was filtered through a syringe filter and then analyzed by HPLC.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention provides a highly efficient, environmentally friendly, and simple method that can simultaneously and accurately extract multiple flavonoid components from traditional Chinese medicine formulas. Furthermore, this invention does not use organic solvents, ensuring its environmental friendliness, safety, and reliability, making it suitable for industrial production. Attached Figure Description

[0028] Figure 1Comparison of five flavonoid components in various extraction methods: ultrasonic extraction (UE), reflux extraction (RE), DES-assisted ball milling extraction (MCE+DES), and ball milling extraction (MCE).

[0029] Figure 2 Comparison of different DES-assisted MCE ball milling extraction methods.

[0030] Figure 3 Comparison of different DES solvent amounts.

[0031] Figure 4 Comparison of extraction efficiency under different ball milling times.

[0032] Figure 5 Comparison of different feed-to-liquid ratios.

[0033] Figure 6 Comparison of extraction efficiency under different vortex times. Detailed Implementation

[0034] The present invention is further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0035] In the following embodiments,

[0036] The fully automatic sample rapid grinding instrument (JXFSTPRP-32L) is equipped with 5 grinding balls (stainless steel grinding balls, 10mm in diameter) as grinding tools;

[0037] vortex generator (VORTEX-5, Killin-Bell);

[0038] The compositions of the four deep eutectic solvents are as follows:

[0039] DES1 ChCl: urea, molar ratio 1:2;

[0040] DES2 ChCl: Ethylene glycol, molar ratio 1:2;

[0041] DES3 ChCl: lactic acid, molar ratio 1:2;

[0042] DES4 ChCl: Ethylene glycol: Lactic acid, molar ratio 1:1:2.

[0043] Example 1: Method for extracting flavonoids from Yiqi Xiaozheng Formula using different DES-assisted MCE ball milling techniques

[0044] The Qi-Boosting and Zheng-Eliminating Formula was ground into powder according to the compound ratio. A high-speed, multi-functional pulverizer was used to pulverize the eight medicinal materials into a 50-mesh compound powder according to the compound ratio. The compound ratio is as follows: Astragalus membranaceus 30g, Codonopsis pilosula 10g, Angelica sinensis 10g, Ligusticum chuanxiong 15g, Bombyx mori 10g, Rheum palmatum 6g, Curcuma zedoaria 15g, and Citrus reticulata 5g.

[0045] Four DES compounds (DES1, DES2, DES3, and DES4) were synthesized from different component compounds for the extraction of flavonoids. Due to their structural differences, these DES compounds interact with the target analytes through different mechanisms. To evaluate and compare the extraction efficiencies of the four DES compounds, 50 μL of DES was added to 500 mg of sample powder, mixed, and milled at 540 rpm for 320 seconds. Then, 10 mL of water was added, the mixture was transferred to a 15 mL centrifuge tube, vortexed for 2 minutes, and then centrifuged at 15,000 rpm for 3 minutes. The supernatant was collected to obtain the compound extract.

[0046] The compound extract was filtered through a needle filter (Φ13 mm × 0.22 μm) to obtain the final compound sample, which was used for subsequent analysis and research.

[0047] The samples were analyzed using a Shimadzu LC-20A HPLC system purchased from Shimadzu Corporation, Kyoto, Japan. Chromatographic separation and analysis were performed using an Agilent Infinity LabPoroshell 120 EC-C18 column (4.6 × 150 mm, 2.7 μm). Chromatographic conditions: flow rate 1.0 mL / min, column temperature 25℃, detection wavelength set to full wavelength scan (260 nm, 276 nm), injection volume 10 μL. Mobile phase A was water containing 0.1% (v / v) acetic acid, and mobile phase B was acetonitrile. Gradient elution: 0–1 min, 5% B; 1–10 min, 10% B; 10–15 min, 17% B; 15–30 min, 30% B; 30–40 min, 40% B; 40–60 min, 100% B.

[0048] like Figure 2 As shown, DES2 and DES3 exhibited the highest extraction efficiencies for proanthocyanidins B1 and hesperidin. This is likely due to the ability of these dehydrated esters to stabilize multiple phenolic hydroxyl groups while maintaining a hydrophobic structure. DES3 showed the best extraction performance. The peak area of ​​DES3 for extracting proanthocyanidins B1 was approximately twice that of DES1. The superior performance of DES3 may stem from its acidity—inhibiting the ionization of the target analyte while promoting the breakdown of the plant matrix. Therefore, DES3, composed of choline chloride (ChCl) and lactic acid (LAC), is considered the optimal extractant.

[0049] Example 2: A method for extracting flavonoids from Yiqi Xiaozheng formula by ball milling with different DES solvent amounts

[0050] The Qi-Boosting and Zheng-Eliminating Formula was ground into powder according to the compound ratio. A high-speed multi-functional pulverizer was used to pulverize the eight medicinal materials into a 50-mesh compound powder according to the compound ratio. During the ball milling process of the compound powder, 20, 50, 80, and 110 μL of DES3 were added respectively to determine the optimal conditions. Other experimental conditions remained unchanged: grinding time 320 seconds, 10 mL of water added as the extraction solvent, and vortexing for 2 minutes.

[0051] like Figure 3 As shown, the DES volume significantly affected the extraction efficiency of the five analytes. When the DES volume increased from 20 μL to 50 μL, the peak area of ​​the target compounds gradually increased, reaching its peak at 50 μL. This indicates that higher DES volumes enhance the interaction with the analytes until saturation is achieved. When the concentration exceeded 50 μL, the peak areas of naringin and neohesperidin decreased significantly. This phenomenon may be due to the increased system viscosity at higher DES volumes, leading to reduced mass transfer efficiency. Furthermore, excess DES alters the polarity of the solvent environment, thereby reducing the partition coefficient of the target compounds. Based on these results, 50 μL was determined to be the optimal DES volume for extracting the five target analytes.

[0052] Example 3: A method for extracting flavonoids from Yiqi Xiaozheng formula by DES-assisted MCE ball milling at different ball milling times.

[0053] The Yiqi Xiaozheng formula was ground into powder according to the compound ratio. A high-speed multi-functional pulverizer was used to pulverize the eight medicinal materials into a 50-mesh compound powder according to the compound ratio. Under fixed extraction conditions (DES3 dosage of 50 μL, extraction solvent of 10 mL water, vortex time of 2 min), the grinding times were 120, 240, 320 and 480 s, respectively.

[0054] like Figure 4 As shown, a grinding time of 120 seconds resulted in larger particle sizes and relatively lower peak areas, indicating insufficient sample fragmentation. When the grinding time was extended to 240 seconds, the sample was thoroughly ground, resulting in smaller particle sizes and increased specific surface area. Under these conditions, the extraction efficiency of all target compounds reached its maximum. This effectively disrupted the cell structure, promoted the complete release of active ingredients, and enhanced their interaction with DES. However, after grinding for 320 seconds, the peak area decreased significantly. This reduction may be due to the generation of excessively fine particles with high specific surface areas, which adsorb the target compounds and re-adsorb them onto the solid matrix. Furthermore, prolonged grinding converts mechanical energy into heat, potentially leading to degradation of heat-sensitive components. Therefore, 240 seconds was ultimately selected as the optimal grinding time.

[0055] Example 4: A method for extracting flavonoids from Yiqi Xiaozheng formula by DES-assisted MCE ball milling with different material-to-liquid ratios.

[0056] The Yiqi Xiaozheng formula was ground into powder according to the compound ratio, and the eight medicinal materials were pulverized into 50-mesh compound powder using a high-speed multi-functional pulverizer. Under certain conditions (DES3 dosage: 50 μL, grinding time: 240 s, extraction solvent: water, vortex time: 2 min), YQXZF was extracted using different material-to-liquid ratios to determine the optimal extraction conditions. In this experiment, the mass of the YQXZF sample powder was fixed at 500 mg, and the material-to-liquid ratio was changed by adjusting the volume of pure water (8, 10, 12, 14 mL). As the volume of pure water increased from 8 mL to 14 mL, the peak area of ​​the target compound gradually decreased, indicating that peak area alone cannot reliably reflect the extraction efficiency. Therefore, the absolute extraction yield of each analyte was calculated using the corresponding quantitative formula.

[0057] The results are as follows Figure 5 As shown, the absolute extraction rate was highest when the water volume was 10 mL. 10 mL was ultimately chosen as the optimal condition because it ensures both high extraction efficiency and a balance between ease of operation and cost-effectiveness. Based on these results, 0.05 g / mL (equivalent to 10 mL of pure water) was selected as the optimal material-to-liquid ratio.

[0058] Example 5: A method for extracting flavonoids from Yiqi Xiaozheng formula by DES-assisted MCE ball milling at different vortex times.

[0059] The Yiqi Xiaozheng formula was ground into powder according to the compound ratio, and the eight medicinal materials were pulverized into 50-mesh compound powder using a high-speed multi-functional pulverizer. Under fixed extraction conditions (DES3 volume 50 μL, grinding time 240 s, extraction solvent water, extraction volume 10 mL), the effect of vortexing time (1, 2, 3, 4 min) on the extraction efficiency of the target compound was evaluated.

[0060] like Figure 6 As shown, extending the vortexing time from 1 minute to 3 minutes resulted in an increase in the peak area of ​​all analytes. This enhancement stems from the rapid and thorough wetting of the herbal powder during vortexing, which disrupts surface hydrophobic barriers and facilitates solvent penetration into the interparticle voids. The vigorous mixing also enhances mass transfer between the solid and liquid phases, promoting the dissolution and elution of readily available target compounds from the surface and shallow pores of the herbal matrix. The extraction efficiency was highest at a vortexing time of 3 minutes, indicating that the target analytes were almost completely dissolved at this point. Beyond 3 minutes, the peak area decreased slightly, possibly due to the conversion of mechanical energy into heat, leading to an increase in system temperature and triggering degradation or structural changes in flavonoids. Therefore, 3 minutes was chosen as the optimal vortexing time.

Claims

1. A green extraction method for flavonoids based on deep eutectic solvents, characterized in that, include: A. Pulverize the Qi-Boosting and Zheng-Eliminating Formula, sieve it, and obtain the compound powder; B. Add the compound powder obtained in step A and the deep eutectic solvent to a grinder for MCE grinding and extraction to obtain the compound extract; The composition of the deep eutectic solvent is selected from any of the following: The molar ratio of choline chloride to urea is 1:

2. The molar ratio of choline chloride to ethylene glycol is 1:

2. The molar ratio of choline chloride to lactate is 1:

2. The molar ratio of choline chloride / ethylene glycol / lactic acid is 1:1:2; C. Add the compound extract obtained in step B to pure water, vortex and centrifuge, and take the supernatant as the compound extract.

2. The green extraction method for flavonoids based on deep eutectic solvent as described in claim 1, characterized in that, In step A, the Qi-Boosting and Zheng-Eliminating Formula consists of the following medicinal materials in the following weight ratios: Astragalus membranaceus 30 parts, Codonopsis pilosula 10 parts, Angelica sinensis 10 parts, Ligusticum chuanxiong 15 parts, Bombyx mori 10 parts, Rheum palmatum 6 parts, Curcuma zedoaria 15 parts, and Citrus reticulata 5 parts.

3. The green extraction method for flavonoids based on deep eutectic solvent as described in claim 1, characterized in that, In step B, the ratio of compound powder to deep eutectic solvent is 10:1, mg / μL.

4. The green extraction method for flavonoids based on deep eutectic solvent as described in claim 1, characterized in that, In step B, the extraction was performed by grinding at 540 rpm for 240 s.

5. The green extraction method for flavonoids based on deep eutectic solvent as described in claim 1, characterized in that, In step B, the composition of the deep eutectic solvent is choline chloride / lactic acid molar ratio of 1:

2.

6. The green extraction method for flavonoids based on deep eutectic solvent as described in claim 1, characterized in that, In step B, the deep eutectic solvent is prepared as follows: the components are mixed according to the ratio, heated to 80 °C under nitrogen atmosphere, and magnetically stirred at 300 rpm until a uniform and transparent liquid is obtained.

7. The green extraction method for flavonoids based on deep eutectic solvent as described in claim 1, characterized in that, In step C, the liquid-to-solid ratio of pure water to the compound powder obtained in step A is 1:50, mL / mg.

8. The green extraction method for flavonoids based on deep eutectic solvent as described in claim 1, characterized in that, In step C, vortex for 3 minutes and centrifuge at 15,000 rpm for 3 minutes.

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