Bai-muxiang leaf flavonoid compound, and extraction method and application thereof

Abstract

This invention discloses a method for extracting flavonoids from Aquilaria sinensis leaves and its application. Ultrasonic-assisted aqueous two-phase extraction (APEA) is used to extract flavonoids from Aquilaria sinensis leaves. The optimal extraction process was determined through single-factor experiments and response surface methodology: 31% ethanol (v / v), 34% ammonium sulfate (w / v), a solid-liquid ratio of 60:1 (mL / g), ultrasonic time of 50 min, and ultrasonic power of 100 W. This method combines the high-efficiency mass transfer advantages of ultrasonic disruption with the selective enrichment characteristics of an APEA system, achieving a total flavonoid extraction rate of 34.65 ± 0.14%, which is 87.6% higher than the traditional ethanol extraction method and 31.1% higher than the non-ultrasonic APEA method. This method is highly efficient, simple to operate, environmentally friendly, and effectively maintains the molecular structural integrity of flavonoids.

Description

Technical Field

[0001] This invention relates to the field of flavonoid extraction technology, and more specifically to a flavonoid compound from Aquilaria sinensis leaves, its extraction method, and its application. Background Technology

[0002] Aquilaria sinensis is a unique and precious medicinal plant resource in my country. Its leaves, a major byproduct of the agarwood industry, are abundant but often discarded or burned, failing to be effectively utilized. Research indicates that Aquilaria sinensis leaves are rich in structurally diverse and broadly active flavonoids, possessing high development potential and application prospects. However, there is currently a lack of systematic extraction process optimization for flavonoids in Aquilaria sinensis leaves, resulting in low utilization rates of its active ingredients and hindering efficient extraction and functional applications.

[0003] Currently, there are various extraction technologies for flavonoids, among which ultrasound-assisted aqueous two-phase extraction (UA-ATPE) is widely used due to its high extraction efficiency, mild reaction conditions, and environmental friendliness. The ultrasonic field can disrupt the structure of plant powder through mechanical oscillation and cavitation, accelerating the dissolution of active substances. The aqueous two-phase system relies on the two-phase partitioning environment formed by organic solvents and salt solutions in the aqueous phase, utilizing the difference in partition coefficients of flavonoids in the two phases to achieve efficient separation. The combined use of these two technologies can significantly improve the extraction efficiency of flavonoids while maximally preserving their molecular structure and biological activity.

[0004] However, existing technologies lack optimization of ultrasound-assisted aqueous two-phase extraction processes for flavonoids from Aquilaria sinensis leaves, particularly comparative studies on the effects of different extraction methods on the microstructure of the material. Therefore, providing an efficient, stable, and suitable extraction method for flavonoids from Aquilaria sinensis leaves, and clarifying its differences from traditional methods, is a technical problem urgently needing to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a flavonoid compound from Aquilaria sinensis leaves, its extraction method, and its application. Using Aquilaria sinensis leaves as raw material, the present invention significantly improves the flavonoid yield through an optimized ultrasound-assisted aqueous two-phase extraction process, providing technical support for the resource utilization of Aquilaria sinensis leaves.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for extracting flavonoids from Aquilaria sinensis leaves, comprising the following steps:

[0008] S1. Raw material pretreatment: Take the leaves of Aquilaria sinensis, dry and pulverize them, and then perform Soxhlet extraction with petroleum ether as solvent to remove chlorophyll and lipid components. Then dry them to obtain the pretreated Aquilaria sinensis leaf powder.

[0009] S2. Ultrasonic-assisted aqueous two-phase extraction: The Aquilaria sinensis leaf powder pretreated in S1 was mixed with an ammonium sulfate / ethanol aqueous two-phase system and subjected to ultrasonic-assisted extraction; the volume fraction of ethanol in the aqueous two-phase system was 22%~32%, the mass fraction of ammonium sulfate was 26%~36%, the solid-liquid ratio was 20:1~70:1 (mL / g), and the ultrasonic time was 20~70 min;

[0010] S3. Collection and separation: After extraction, centrifuge and collect the supernatant to obtain the extract containing flavonoids.

[0011] As a preferred technical solution, the optimal process parameters for the ultrasound-assisted aqueous two-phase extraction in S2 are: ethanol volume fraction 31% (v / v), ammonium sulfate mass fraction 34% (w / v), material-liquid ratio 60:1 (mL / g), ultrasound time 50 min, and ultrasound power 100 W.

[0012] As a preferred technical solution, the aqueous two-phase system in S2 is an ammonium sulfate / ethanol aqueous two-phase system, which can promote the precipitation of the ethanol phase through salting out, forming two phases with significant volume differences, providing an efficient enrichment environment for flavonoids.

[0013] As a preferred technical solution, the drying in S1 is natural air drying, and the pulverization is passing through a 100-mesh sieve.

[0014] As a preferred technical solution, the centrifugation conditions in S3 are 8000 rpm for 5 min.

[0015] Another object of the present invention is to provide: the flavonoid compounds of Aquilaria sinensis leaves prepared by the above method.

[0016] Another object of the present invention is to provide the use of the above-mentioned flavonoid compounds from Aquilaria sinensis leaves in the preparation of functional foods or medicines with antioxidant, hypoglycemic, or anti-Alzheimer's disease properties.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: The present invention systematically establishes an ultrasound-assisted aqueous two-phase method for efficiently extracting flavonoids from Aquilaria sinensis leaves. Using Aquilaria sinensis leaves as raw material, this method optimizes the ultrasound-assisted aqueous two-phase extraction process through single-factor experiments and response surface methodology. The total flavonoid extraction rate can reach 34.65±0.14%, which is 87.6% higher than the traditional ethanol extraction method (18.47%) and 31.1% higher than the method without ultrasound-assisted aqueous two-phase extraction (26.43%). The ultrasound-assisted aqueous two-phase extraction process provided by the present invention combines the efficient mass transfer advantages of ultrasonic disruption with the selective enrichment characteristics of the aqueous two-phase system, significantly improving the flavonoid yield. It is also simple to operate, environmentally friendly, and fully utilizes Aquilaria sinensis leaf resources, realizing their high-value utilization. It provides high-quality natural active raw materials for the development of pharmaceuticals or functional foods and has broad application prospects. Attached Figure Description

[0018] Figure 1 For example: the ultraviolet spectral scanning curve of rutin;

[0019] Figure 2 For: Standard working curve for rutin content determination;

[0020] Figure 3 The effects of different aqueous two-phase systems on the phase ratio, partition coefficient and recovery rate of total flavonoids;

[0021] Figure 4 The following parameters were used to determine the recovery rate, yield, and partition coefficient of total flavonoids from Aquilaria sinensis leaves at different ethanol volume fractions.

[0022] Figure 5 The following parameters were determined: recovery rate, yield, and partition coefficient of total flavonoids from Aquilaria sinensis leaves under different ammonium sulfate mass fractions.

[0023] Figure 6 The following parameters were used to determine the recovery rate, yield, and partition coefficient of total flavonoids from Aquilaria sinensis leaves under different liquid-to-solid ratios.

[0024] Figure 7 The following parameters were used to determine the recovery rate, yield, and partition coefficient of total flavonoids from Aquilaria sinensis leaves under different ultrasonic times:

[0025] Figure 8 Here is a three-dimensional surface plot of the response surface to the interaction between the volume fraction of ethanol and the mass fraction of ammonium sulfate in the extraction efficiency of total flavonoids.

[0026] Figure 9 Here is a three-dimensional surface plot of the response surface to the interaction between the total flavonoid extraction efficiency and the liquid-solid ratio.

[0027] Figure 10 Here is a three-dimensional surface plot of the response surface of the total flavonoid extraction efficiency as a function of the interaction between ethanol volume fraction and ultrasonic time.

[0028] Figure 11 The following is a three-dimensional surface plot of the response surface to the interaction between the mass fraction of ammonium sulfate and the liquid-solid ratio in the extraction efficiency of total flavonoids.

[0029] Figure 12 Here is a three-dimensional surface plot of the response surface to the interaction between ammonium sulfate mass fraction and ultrasonic time in the extraction efficiency of total flavonoids.

[0030] Figure 13 The figure shows the three-dimensional surface plot of the response surface of the total flavonoid extraction efficiency affected by the interaction between the liquid-solid ratio and the ultrasonic time. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] Establishment of maximum absorption wavelength and standard curve

[0034] The total flavonoid content in Aucklandia lappa leaves was quantitatively determined using the sodium nitrite-aluminum nitrate colorimetric method. A certain volume of rutin standard working solution was measured, and 0.5 mL of 5% NaNO₂ solution and 0.5 mL of 10% Al(NO₃)₃ solution were added sequentially. The mixture was stirred and allowed to stand for 5 min. Then, 4 mL of 4% NaOH solution was added, and the solution was diluted to volume with 50% ethanol. The mixture was shaken well and allowed to stand for 15 min for color development. A microplate reader was used to scan the entire wavelength range of 230–1000 nm.

[0035] Results analysis: such as Figure 1 As shown, the rutin colorimetric reaction system exhibits a characteristic maximum absorption peak at a wavelength of 510 nm, and therefore 510 nm was selected as the detection wavelength. Under optimized conditions, a standard curve was plotted with the mass concentration of rutin in the range of 0–0.1 mg / mL as the independent variable and the absorbance value as the dependent variable. The regression equation was Y = 5.5106X + 0.0005, and the coefficient of determination R² = 0.9998. Figure 2 The linear relationship is good, which can meet the requirements for accurate quantitative analysis of total flavonoids in subsequent analysis.

[0036] Example 2

[0037] Screening of the optimal aqueous two-phase extraction system

[0038] To optimize the aqueous two-phase extraction process for total flavonoids from Aquilaria sinensis leaves, the effects of different salt / alcohol combinations (ammonium sulfate / ethanol, sodium citrate / ethanol, dipotassium hydrogen phosphate / ethanol, ammonium sulfate / n-propanol, sodium citrate / n-propanol, dipotassium hydrogen phosphate / n-propanol, ammonium sulfate / isopropanol, sodium citrate / isopropanol, dipotassium hydrogen phosphate / isopropanol) on the phase ratio, partition coefficient, and total flavonoid recovery rate were investigated.

[0039] Results analysis: such as Figure 3 As shown, the partition characteristics of different aqueous two-phase systems differ significantly. The ammonium sulfate / ethanol system exhibits the best performance, with a phase ratio of 2.01, a partition coefficient of 0.64, and a total flavonoid recovery rate of 79.27%. This is because ammonium sulfate has a strong salting-out effect, effectively promoting the precipitation of the ethanol phase and forming two phases with significant volume differences, providing an efficient enrichment environment for flavonoids. With increasing alcohol carbon chain length, the phase ratio, partition coefficient, and recovery rate generally show a decreasing trend, indicating that ethanol is more suitable as the phase-forming solvent for short-chain alcohols in this study. Considering both extraction efficiency and cost, the ammonium sulfate / ethanol aqueous two-phase system was selected as the optimal extraction system.

[0040] Example 3

[0041] Single-factor experiment on ultrasound-assisted aqueous two-phase extraction process

[0042] The effects of four factors—ethanol volume fraction, ammonium sulfate mass fraction, liquid-solid ratio, and ultrasonic time—were investigated using the total flavonoid recovery rate, ethanol phase yield, and partition coefficient of Aquilaria sinensis leaves as indicators.

[0043] (1) Ethanol volume fraction: With a fixed ammonium sulfate concentration of 30%, a liquid-to-solid ratio of 50:1, and an ultrasonic time of 30 min, the concentration was investigated within the range of 22% to 32%. The results are as follows: Figure 4 As shown, all indicators initially increased and then stabilized with increasing ethanol volume fraction, reaching a peak at 30%. Too low a concentration resulted in insufficient phase separation, while too high a concentration led to excessive changes in polarity, weakening the hydrophobic and hydrogen bonding interactions between flavonoids and the ethanol phase. Therefore, 30% was determined to be the optimal ethanol volume fraction.

[0044] (2) Ammonium sulfate mass fraction: With a fixed ethanol volume fraction of 26%, a liquid-to-solid ratio of 50:1, and an ultrasonic time of 30 min, the concentration was investigated within the range of 26% to 36%. The results are as follows: Figure 5 As shown, the total flavonoid recovery, ethanol phase yield, and partition coefficient all increased first and then decreased with increasing ammonium sulfate mass fraction, reaching a peak at 34%. Too low a concentration resulted in insufficient ionic strength, while too high a concentration increased system viscosity and decreased mass transfer efficiency. Therefore, 34% was determined to be the optimal ammonium sulfate mass fraction.

[0045] (3) Liquid-to-solid ratio: With a fixed ethanol volume fraction of 26%, ammonium sulfate mass fraction of 30%, and ultrasonic time of 30 min, the ratio was investigated within the range of 20:1 to 70:1. The results are as follows: Figure 6 As shown, within the liquid-to-solid ratio range of 20:1 to 60:1, all indicators increase with the increase of the liquid-to-solid ratio, reaching a peak at 60:1; after exceeding 60:1, they tend to stabilize. Too low a liquid-to-solid ratio results in insufficient contact between the solvent and the raw materials, while too high a ratio dilutes the system and increases subsequent concentration costs. Therefore, 60:1 was determined to be the optimal liquid-to-solid ratio.

[0046] (4) Ultrasonic time: With a fixed ethanol volume fraction of 26%, ammonium sulfate mass fraction of 30%, and liquid-to-solid ratio of 50:1, the time was observed within the range of 20-70 min. The results are as follows: Figure 7 As shown, the total flavonoid recovery rate, ethanol phase yield, and partition coefficient all increased and then decreased with increasing ultrasonic time, reaching a peak at 50 min. Cavitation effect promoted flavonoid dissolution within 20–50 min; after 50 min, the system temperature increased, leading to partial flavonoid oxidation and degradation, and ultrasonication may disrupt the phase equilibrium of the aqueous two-phase system. Therefore, 50 min was determined to be the optimal ultrasonic time.

[0047] Example 4

[0048] Response surface methodology for optimizing extraction process

[0049] Based on single-factor experiments, ethanol volume fraction (A), ammonium sulfate mass fraction (B), liquid-to-solid ratio (C), and ultrasonic time (D) were selected as independent variables, and total flavonoid extraction rate (Y) was selected as the response value. A Box-Behnken design was used for four-factor, three-level response surface optimization. Experimental design and ANOVA results showed that the model differences were extremely significant (P<0.0001), the lack-of-fit term was not significant (P=0.0514), Adj R²=0.9007, and the signal-to-noise ratio=13.8412, indicating a good model fit. Among these, A, the interaction term AB, and the quadratic terms A², B², C², and D² had extremely significant effects on the extraction rate (P<0.01). From the response surface plot and contour plot (…),… Figures 8-13 It can be clearly seen that the interaction between the volume fraction of ethanol and the mass fraction of ammonium sulfate is extremely significant, the interaction between the volume fraction of ethanol and the ultrasonic time is significant, and the other interactions are not significant.

[0050] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0051] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting flavonoids from Aquilaria sinensis leaves, characterized in that, Includes the following steps: S1. After drying and pulverizing the leaves of Aquilaria sinensis, Soxhlet extraction was performed using petroleum ether as a solvent to remove chlorophyll and lipid components. The powder was then dried to obtain pretreated Aquilaria sinensis leaf powder. S2. Mix the pretreated Aquilaria sinensis leaf powder with an ammonium sulfate / ethanol aqueous two-phase system and perform ultrasonic-assisted extraction. The extraction conditions are: ethanol volume fraction 22%~32%, ammonium sulfate mass fraction 26%~36%, material-liquid ratio 20:1~70:1 (mL / g), and ultrasonic time 20~70 min. S3. After extraction, centrifuge and collect the supernatant to obtain the extract containing flavonoids.

2. The method according to claim 1, characterized in that... The optimal extraction conditions in step S2 are: ethanol volume fraction 31% (v / v), ammonium sulfate mass fraction 34% (w / v), solid-liquid ratio 60:1 (mL / g), ultrasonic time 50 min, and ultrasonic power 100W.

3. The method according to claim 1, characterized in that... The aqueous two-phase system mentioned in step S2 is an ammonium sulfate / ethanol aqueous two-phase system.

4. The method according to claim 1, characterized in that... The drying in step S1 is natural air drying, and the pulverization is passing through a 100-mesh sieve.

5. The method according to claim 1, characterized in that... The centrifugation conditions described in step S3 are 8000 rpm for 5 min.

6. The method according to claim 1, characterized in that... The leaves of the Aquilaria sinensis mentioned are healthy leaves free from pests and diseases, which are cleaned and then naturally air-dried.

7. Flavonoids from Aquilaria sinensis leaves extracted by the method according to any one of claims 1 to 6.

8. The use of the flavonoid compounds from Aucklandia lappa leaves according to claim 7 in the preparation of functional foods or medicines with antioxidant, hypoglycemic, or anti-Alzheimer's disease related functions.

9. The application according to claim 8, characterized in that... The total flavonoid extraction rate of the flavonoids from the leaves of the plant can reach 34.65 ± 0.14%.

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