Method for extracting quercitrin by using natural eutectic solvent and application thereof

Abstract

The present application belongs to the field of biochemistry and health food, and particularly relates to a method for extracting astilbin by using a natural eutectic solvent and application. The present application uses a novel environment-friendly solvent, a natural eutectic solvent (choline chloride-lactic acid), to extract astilbin, and the extraction rate of astilbin is twice as high as that of ethanol extraction. The mechanism of extracting astilbin by using the natural eutectic solvent is obtained by using a molecular dynamics simulation technology. The natural eutectic solvent extract of astilbin is obtained by using a simulated digestion system, and the bioavailability of astilbin in the natural eutectic solvent extract is 84.1% higher than that of ethanol extract, and the antioxidant capacity is 5 times higher than that of ethanol extract. The protection of the natural eutectic solvent in the simulated digestion solves the problem of low bioavailability of active ingredients in each stage of digestion.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a method and application for extracting astilbin using a natural eutectic solvent. Background Technology

[0002] Existing research indicates that α-glucosidase, a digestive enzyme, has high activity and promotes the digestion and absorption of carbohydrates in the intestines, leading to elevated plasma glucose levels. Inhibiting α-glucosidase activity can delay carbohydrate absorption and increase insulin secretion, thus maintaining lower postprandial plasma glucose levels in diabetic patients. Therefore, inhibiting α-glucosidase activity can alleviate diabetes and has a therapeutic effect; most commercially available diabetes medications are inhibitors of this enzyme. Alternatively, they can inhibit α-amylase to block the conversion of starch into sugars, thereby lowering plasma glucose levels and controlling blood sugar.

[0003] Astilbin is a natural dietary flavonoid that exhibits a variety of significant biological activities, including selective immunosuppression, anti-inflammation, antioxidant effects, and regulation of lipids and blood sugar. Due to its hydrophobic nature, astilbin is typically obtained through methanol or ethanol extraction, resulting in low extraction rates that do not meet food safety requirements and pollute the environment. Natural eutectic solvents (NaDES), prepared from food-grade raw materials, have been proven in recent years to be effective solvents for extracting active ingredients such as polyphenols and flavonoids, and can yield ready-made extracts for direct use in food. However, they have not yet been applied to the extraction of astilbin.

[0004] CN116270772A discloses a method for ultrasonic extraction of flavonoid components—isorhamnoside, kaempferol, and quercetin—from sea buckthorn using a natural eutectic solvent (choline chloride-lactic acid). CN115925602A discloses a method for extracting lutein from marigold using a eutectic solvent (choline chloride-lactic acid). CN106565804A discloses an extraction process for astilbin from *Hippophae rhamnoides* leaves, using ethanol extraction. Astilbin is characterized by low solubility, low stability, and low bioavailability. Summary of the Invention

[0005] In order to obtain an astilbin extract with high bioavailability and strong antioxidant properties, this invention uses natural raw materials to prepare a green and environmentally friendly natural eutectic solvent (choline chloride-lactic acid) for extracting astilbin from Smilax glabra. This resulted in a natural eutectic solvent extract with an astilbin content twice that of the 70% ethanol extract.

[0006] This invention analyzes the changes in bioavailability and antioxidant capacity of the natural eutectic solvent extract of astilbin at different stages of digestion by simulating the digestive system in vitro. It was found that the bioavailability and antioxidant capacity of astilbin in the natural eutectic solvent extract of astilbin are significantly higher than those in the 70% ethanol extract.

[0007] The raw materials (choline chloride and lactic acid) used to prepare natural eutectic solvents are present in the body's cells and tissues. Choline chloride is widely used as a vitamin in pharmaceuticals, health products, and food additives; lactic acid is commonly used as a food ingredient and additive.

[0008] The beneficial effects of this invention are:

[0009] NaDES based on choline chloride-lactic acid (CHCl-LAC) is not only an environmentally friendly solvent for extracting phytochemicals, but also serves as an edible carrier to improve the bioavailability of bioactive compounds such as flavonoids. Therefore, the selection of this novel green solvent solves the environmental pollution problem during extraction, while the edibility of the components addresses the issue of subsequent separation. Furthermore, the protective effect of natural eutectic solvents in simulated digestion overcomes the problem of low bioavailability of active ingredients at various stages of digestion.

[0010] This invention utilizes a novel environmentally friendly natural eutectic solvent (choline chloride-lactic acid) to extract astilbin, achieving an astilbin extraction rate twice that of ethanol extraction. Molecular dynamics simulations were employed to elucidate the mechanism of astilbin extraction using the natural eutectic solvent. A simulated digestion system was used to obtain an astilbin natural eutectic solvent extract with 84.1% higher bioavailability and 5 times higher antioxidant capacity than the ethanol extract. Attached Figure Description

[0011] Figure 1 NaDES solvent component screening;

[0012] Figure 2 CHCL-LAC molar ratio screening;

[0013] Figure 3 The effect of the interaction between the three factors of material-liquid ratio (A), moisture (B), and time (c) on the yield of astilbin;

[0014] Figure 4 Validation of optimal conditions for CHCL-LAC extraction of astilbin;

[0015] Figure 5Molecular dynamics simulation analysis: Distribution of astilbene in different solvent systems (70% ethanol (v / v) and CHCL-LAC) at 0 ns and 20 ns (A); Average non-covalent interaction (ANI) analysis and thermal fluctuation index (TFI) analysis of astilbene in different solvent systems (B); Accessible surface area (SASA) (C), interaction energy (D), number of hydrogen bonds and hydrogen bond lifetime (E) between different solvents and astilbene at 0 ns and 20 ns;

[0016] Figure 6 Changes in the bioavailability (A), DPPH antioxidant capacity (B), and ORAC antioxidant capacity (C) of different extracts during in vitro simulated gastrointestinal digestion; Correlation analysis of astilbin and antioxidant capacity in CHCL-LAC extract (D) and 70% ethanol extract (E).

[0017] Figure 7 Figure showing the inhibitory effects of astilbin CHCL-LAC extract on α-amylase and α-glucosidase. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] Example 1

[0020] 1. Preparation of natural eutectic solvent

[0021] A mixture of two components, hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs), was placed in a reaction flask with a magnetic stirrer and heated to 85°C for 60 to 120 minutes, with constant stirring to form a viscous homogeneous liquid. NaDES consisted of choline chloride (CHCl) mixed with eight different HBDs, including lactic acid (LAC), citric acid (CA), glucose (GLU), sucrose (SUC), 1,6-hexanediol (1,6-HEX), 1,4-butanediol (1,4-BUT), ethylene glycol (EG), and glycerol (GLY), with a water content of 20%.

[0022] 2. Screening of natural eutectic solvents

[0023] (1) Solvent selection

[0024] Choline chloride was used as the hydrogen bond acceptor (HBA), and lactic acid (LAC), citric acid (CA), sucrose (SO), glucose (G), 1,6-hexanediol (HDO), 1,4-butanediol (BDO), ethylene glycol (EG), and glycerol (GLY) were used as hydrogen donors (HBD) (Table 1). The system was kept at 20% water content. NADES was prepared with different molar ratios of HBD and HBA. Smilax glabra (SGR) powder was added to different NaDES solvents at a solid-liquid ratio of 1:20 (w / v), and extraction was performed at 50℃ in an ultrasonic water bath for 40 minutes. The commonly used 70% ethanol astilbin extraction method (extraction time 90 min, solid-liquid ratio 10 mL / g) was also compared. Finally, the astilbin content in the extract was determined by HPLC. Figure 1 The yields of astilbin obtained from different types of NaDES extraction are shown. Clearly, under the designed parameters, the astilbin yield obtained from CHCL-LAC extraction (2.44 mg / g) was the highest, significantly higher than that obtained from 70% ethanol (1.27 mg / g).

[0025] Table 1. Different NaDES systems (20% water content)

[0026]

[0027] (2) Molar ratio screening

[0028] The optimal solvent (choline chloride-lactic acid) was optimized by adjusting the molar ratio. The HBA:HBD molar ratios were 1:1, 1:2, 1:3, and 1:4. NaDES solvents were prepared by heating and stirring. 0.25 g of 60-mesh SGR sieve powder was accurately weighed into a 10 mL test tube. The four NADES solvent ratios were added to the test tubes and mixed thoroughly. Extraction was performed in an ultrasonic instrument at 50℃, 40 min, and 100 W, with each group repeated three times. The extracts were precipitated and centrifuged at 12000 rpm for 10 min. The astilbin content extracted by NaDES under different ratios was determined by high-performance liquid chromatography (HPLC), and the results were compared.

[0029] Screening results for the molar ratio of natural eutectic solvents are as follows: Figure 2 As shown, the yields of astilbene at molar ratios of 1:1 and 1:2 were 2.25 mg / g and 2.44 mg / g, respectively, which were significantly higher than the yields at molar ratios of 1:3 (0.12 mg / g) and 1:4 (0.11 mg / g).

[0030] 3. Optimization of extraction conditions for astilbin using natural eutectic solvents

[0031] (1) Response surface optimization

[0032] A three-order Box-Behnke response surface methodology was used to investigate the effects of the material-to-liquid ratio (A), ultrasonic-assisted time (B), and moisture content (C) on the extraction of astilbin content from SGR using the NaDES method (Table 2). Based on the Box-Behnke design, variables were optimized by changing the operating parameters, and regression analysis was performed based on the experimental data. Subsequently, additional confirmatory experiments were conducted to confirm the effectiveness of the statistical experimental strategy. DesignExpert Ver. 8.0.6 software was used for data analysis, and three-dimensional response plots were used to analyze the extraction effect.

[0033] Table 2. Box-Benhnken Test Levels and Factors

[0034]

[0035] The effect of extraction conditions on the yield was further studied using the RSM method. With astilbin yield (Y) as the dependent variable and the solid-liquid ratio (A), moisture content (B), and extraction time (C) as independent variables, the fitted equation for the extraction was: Y = 2.33 - 0.022a + 0.0421B + 0.0217C - 0.0486AB - 0.0037AC - 0.0085BC + 0.1138A² - 0.1154B² + 0.0484C². In the model, the p-values ​​of the linear and quadratic terms (A², B², C²) were all less than 0.05, indicating that they had a significant impact on the extraction of astilbin. (Three-dimensional response surface plot) Figure 3 The results showed the interactive effects of the solid-liquid ratio and water content (AB), water content and time (BC), and solid-liquid ratio and time (AC) on the astilbin yield (Y). Increasing the solid-liquid ratio (15 mL / g) and water content (30%) increased the astilbin yield, while further increases in these parameters moderately decreased the astilbin yield. Furthermore, the astilbin yield was significantly affected by the linear terms of the solid-liquid ratio (A) and water content (B), as well as the quadratic terms of the solid-liquid ratio (A2), water content (B2), and extraction time (C2). Under the conditions of a solid-liquid ratio of 22.87 mL / g, water content of 28.72%, and extraction time of 45.07 min, the highest astilbin yield from *Smilax glabra* reached 2.45 mg / g. To verify the reliability of this process optimization experiment, considering operational feasibility, the actual extraction results were very close to the expected results of the process optimization, significantly higher than the astilbin yield of 70% ethanol (1.27 mg / g) by more than 2 times (Tables 3 and 4). Figure 4 ).

[0036] Table 3. Response Surface Experimental Design and Results

[0037]

[0038]

[0039] Table 4. Response Surface Experimental Regression Simulation (ANOVA)

[0040]

[0041] Note: * indicates a significant difference (p < 0.05)

[0042] To reveal the mechanism behind the high yield of CHCL-LAC relative to other solvents, molecular dynamics simulations were performed using a solvent system (CHCL-LAC-70% ethanol (v / v)) and astilbin. Figure 5 As shown in Figure A, in 70% ethanol (v / v), clusters form from 0 ns to 20 ns, and the molecules remain uniformly distributed in CHCL-LAC, indicating that astilbene is more soluble in CHCL-LAC than in ethanol. Visualizations of ANI and the corresponding TFIs between solvent molecules are listed in... Figure 5 B. Compared to the ethanol system, the structure of the chloride anion in CHCL-LAC allows for the connection of the H atom of the hydroxyl group on astilbene with the H atoms of other hydroxyl groups on the solvent molecule, facilitating the formation of more hydrogen bonds and avoiding collisions. Based on ANI analysis, the stability of the interaction is represented by TFI. The results show that the red region of astilbene in CHCL-LAC is much smaller than in ethanol (red indicates a weaker interaction). This indicates that the interaction between CHCL-LAC and astilbene is more stable, forming a stable astilbene-CHCL-LAC hydrogen-bonded supramolecular group.

[0043] Solvent-accessible surface area (SASA) is a measure of the contact area between molecules and solvent. In CHCL-LAC solvent, the SASA value remains at 260 nm. 2 Around 10 ns, while in 70% ethanol, SASA fluctuates significantly in the first 10 ns. Figure 5 C), which is related to Figure 5 Astilbene aggregates formed in solvent A under 70% ethanol conditions are consistent with the astilbene clusters uniformly distributed in CHCL-LAC. Relatively stable solubility in both solvents was observed within a time range of 10–20 ns. The average SASA value of CHCL-LAC was 255.68 nm. 2 Higher than ethanol's 227.96 nm2 ( Figure 5 C) indicates that astilbin has a larger contact area and a wider distribution in CHCL-LAC. Compared with 70% ethanol, CHCL-LAC forms a much higher number of hydrogen bonds with astilbin molecules. Figure 5E). This can be partly explained by the fact that the yield and solubility of astilbin extracted using CHCL-LAC are higher than those using other solvents. Meanwhile, the non-covalent interaction energy of CHCL-LAC is -9023.37 kJ / mol, lower than the -8685.01 kJ / mol of 70% ethanol solvent. Figure 5 D). This is consistent with the above results, namely that astilbene molecules dissolved in CHCL-LAC are more stable and more evenly distributed than in other solvents.

[0044] The number of hydrogen bonds and average lifetime between astilbene and solvent molecules are as follows: Figure 5 As shown in E, the average number of hydrogen bonds with CHCL-LAC is 660.84, which is much higher than that of ethanol (543.78 hydrogen bonds). Figure 5 E). In the CHCL-LAC system, the average hydrogen bond lifetime of astilbin is 12 ps, which is much higher than the 8.5 ps in the ethanol system. Figure 5 E) indicates that the hydrogen bonds of astilbene formed in the CHCL-LAC system are more stable than those formed in the 70% ethanol system, which also leads to a higher yield of astilbene extracted using the CHCL-LAC system compared to ethanol. Molecular dynamics simulations show that astilbene and CHCL-LAC may form stable hydrogen-bonded supramolecular structures with wider distribution, higher SASA (superoxide dismutase), tighter interactions, lower interaction energies, and longer average hydrogen bond lifetimes.

[0045] (2) Determination of astilbin content by HPLC

[0046] A standard solution was prepared using 95% pure astilbin as the standard, a standard curve was plotted, and the astilbin content in the extract was determined under the following chromatographic conditions:

[0047] Chromatographic column: WAT054275 Symmetry C18 (4.6 mm × 250 mm, 5 μm), flow rate 1.0 mL / min, column temperature 30 ℃, detection wavelength 291 nm, injection volume 10 μL, mobile phase: methanol:water (30:70, v / v)

[0048] 4. Extraction Mechanism Research

[0049] Molecular dynamics (MD) simulations were performed using the GROMACS2019.6 package with the Amber (99SB-ildn) force field. MD simulations were performed within the NPT with a step size of 1 ns and 2 fs, saving trajectory coordinates every 500 frames for a total of 1000 frames. The average non-covalent interaction (ANI) and thermal fluctuation index (TFI) were calculated using Multiwfn3.8 (dev). Partial charges of astilbene, ethanol, choline chloride, and lactate molecules were calculated using Gaussian 16 encoding and 6-31+g(d,p) basis functions. All atoms were parameterized using the OPLSS-AA force field and MKTOP, including bond parameters, angular parameters, and dihedral angles. The distribution of astilbene in ethanol and DES was investigated using molecular dynamics (MD) simulations. The water molecule model was TIP3P. The number of hydrogen bonds, hydrogen bond lifetime, interaction energy, and solvent reachable surface area (SSA) between astilbene and 70% ethanol / NaDES were analyzed using a 20 ns MD simulation.

[0050] 5. In vitro digestion of NaDES extract

[0051] (1) Bioavailability evaluation

[0052] 5 mL of CHCL-LAC extract, 70% ethanol extract (redissolved in PBS), and pure astilbene (dissolved in PBS) were mixed into simulated gastric juice (SGF). Porcine pepsin (0.8 mL, 12500 U / mL) and calcium chloride (2.5 μL, 0.3 M) were then added, and the pH was adjusted to 2 ± 0.2. The final weight of each sample was increased to 10 g with water. The mixture was then incubated at 37 °C and 150 rpm for 30, 60, 90, and 120 min. After gastric digestion, the pH of the whole gastric chyme was adjusted to 6.8 with 1 M NaOH for subsequent intestinal digestion, and then mixed with 4.4 mL of simulated intestinal fluid, 1 mL of pancreatin (800 U / mL), 16 μL of calcium chloride (0.3 M), and 1 mL of bile salts (10 mM). Finally, the pH was adjusted to 7 ± 0.2, and the mixture was incubated at 37 °C for 60, 90, 120, and 180 min. At each digestion point, the mass of the digestate was measured, and the digestate was analyzed by HPLC. The bioavailability (%) of roxithromycin was calculated according to (1).

[0053] Bioavailability of astilbin (%) = (Astilbin content at a specific digestion stage / initial astilbin content) × 100% (1)

[0054] (2) Antioxidant capacity determination

[0055] DPPH assay: Accurately weigh Trolox (98%) standard and dissolve it in anhydrous ethanol to obtain a 1000 μM standard stock solution. Dilute with anhydrous ethanol serially to prepare 0, 50, 100, 200, 300, 400, and 500 μM standard solutions. Prepare a 0.50 mmol / L DPPH solution using anhydrous ethanol. Take 50 μL of each of the above different stages of sample digestion solutions and add 150 μL of DPPH solution. Mix thoroughly and shake well. Incubate at room temperature in the dark for 30 min. Measure the absorbance at 517 nm using a microplate reader and record the value as Asample. Use anhydrous ethanol instead of DPPH solution and mix it with the sample solution in a controlled ratio to remove color interference from the sample solution. Measure the absorbance and record the value as Acontrol. Use anhydrous ethanol instead of the sample solution and mix it with the DPPH solution in a controlled ratio. Measure the absorbance and record the value as Ablank. Use Trolox (water-soluble vitamin E) solution as a positive control. Three parallel measurements were set up for each test, and the DPPH radical scavenging rate was calculated using the average value. The calculation formula (2) is as follows:

[0056] DPPH free radical scavenging rate (%) = [1 - {(A sample -A control ) / A blank}】×100% (2)

[0057] The ORAC reaction system should be carried out at 37°C in PBS solution. Before the assay, preheat the microplate reader, ensuring it is at 37°C before reading the plate. Add different concentrations of the sample solution (dissolved in PBS), PBS (blank group), and 25 μL of standard solution to each well of the black 96-well microplate, along with 150 μL of sodium fluorescein solution. Cover the microplate and incubate for 30 min. Then, quickly add freshly prepared AAPH solution to each well using a multichannel pipette to initiate the reaction. Place the microplate in the microplate reader and continuously measure the fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 518 nm. Acquire the fluorescence intensity signal of each well every 2 min, and continue measuring until the fluorescence decays to baseline. Perform three parallel measurements for each sample. The antioxidant capacity of the sample solution is expressed as Trolox equivalent.

[0058] The bioavailability and antioxidant capacity of astilbin in CHCL-LAC extract and 70% ethanol extract during in vitro simulated gastrointestinal digestion are shown in Tables 5-7. Figure 5 .

[0059] The residual amount of astilbin in the fluids during the gastric and intestinal digestive stages is as follows: Figure 6As shown. After 2 hours of gastric digestion, a small amount of astilbin was lost from the liquid, with the residual amount exceeding 90%, indicating that astilbin is stable during gastric digestion. During intestinal digestion, astilbin in both extracts underwent significant degradation, with the 70% ethanol extract showing the greatest degradation, including isomerization and decomposition. This is related to the instability of astilbin in alkaline solutions. In the final digestive fluid, the recovery rate of astilbin from the CHCL-LAC extract was 84.1%, significantly higher than that from the 70% ethanol extract (…). Figure 6 (A and Table 5)

[0060] The DPPH free radical scavenging capacity and ORAC antioxidant capacity of CHCL-LAC extract and 70% ethanol extract are as follows: Figure 6 B and Figure 6 As shown in Figure C, during gastric digestion, the antioxidant capacity of DPPH significantly decreased, while the antioxidant activity of ORAC remained relatively stable. After 3 hours of intestinal digestion, the antioxidant capacities of DPPH and ORAC remained at 75.7% and 57.7%, respectively, significantly higher than those of the 70% ethanol extract (13.6% DPPH, 21.4% ORAC). Figure 6 Tables B, 6C, 6, and 7 show that CHCL-LAC retains a higher antioxidant capacity than 70% ethanol. Correlation analysis revealed that the antioxidant capacity of ORAC is particularly correlated with changes in astilbene content during digestion. Figure 6 The results (D and 6E) indicate that astilbin is the most effective contributor to the antioxidant capacity of ORAC.

[0061] The inhibitory effects of astilbene CHCL-LAC extract on α-amylase and α-glucosidase are shown in [reference needed]. Figure 7 The inhibition rate of the enzyme increased with increasing CHCL-LAC extract concentration. The higher the extract concentration (0.99 g / L), the higher the IC50 value for inhibiting α-amylase (0.67 g / L). Figure 7 A), and the extract showed a stronger inhibitory effect on α-glucosidase, with an IC50 value of 0.64 g / L. Figure 7 B).

[0062] Table 5. Bioavailability of astilbin during in vitro simulated gastrointestinal digestion.

[0063]

[0064] Table 6. Changes in the DPPH antioxidant capacity of different extracts during simulated gastrointestinal digestion in vitro.

[0065]

[0066]

[0067] Table 7. Antioxidant capacity of ORAC in different extracts during in vitro simulated gastrointestinal digestion.

[0068]

Claims

1. A method for extracting astilbin using a natural eutectic solvent, characterized in that, The method is as follows: Smilax glabra powder is mixed with NaDES solvent, the water content of the system is kept at 10-30%, and the mixture is extracted under ultrasonic water bath for 20-60 min. After centrifugation, the supernatant is collected to obtain a natural eutectic solvent extract containing astilbin. The ratio of Smilax glabra powder to NaDES solvent is 1:1-30. The NaDES solvent is choline chloride-lactic acid, wherein the molar ratio of choline chloride to lactic acid is 1:1-2. The method for preparing the NaDES solvent is as follows: choline chloride and lactic acid are mixed and reacted at 85°C for 60-120 minutes, with continuous stirring to form a viscous homogeneous liquid to obtain the NaDES solvent. The temperature of the ultrasonic water bath is 50℃.

2. The method according to claim 1, characterized in that, The centrifugation time was 10 minutes.

3. The method according to claim 2, characterized in that, The molar ratio of choline chloride to lactic acid is 1:

2.

4. A natural eutectic solvent extract containing astilbin, characterized in that, The astilbin natural eutectic solvent extract is prepared by the method described in any one of claims 1-3.

5. The use of the natural eutectic solvent extract containing astilbene prepared by the method of any one of claims 1-3 or the natural eutectic solvent extract containing astilbene as described in claim 4 in the preparation of antioxidant products.

6. The use of the natural eutectic solvent extract containing astilbin prepared by the method of any one of claims 1-3 or the natural eutectic solvent extract containing astilbin as described in claim 4 in the preparation of products that inhibit the activity of α-amylase and α-glucosidase.

7. The use of the natural eutectic solvent extract containing astilbene prepared by the method of any one of claims 1-3 or the natural eutectic solvent extract containing astilbene as described in claim 4 in the preparation of hypoglycemic products.

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