A lannea coromelli leaf flavonoid compound, and an extraction method and application thereof

Abstract

The application discloses a kind of chess nan linaloe leaf flavonoids compound extraction purification method and application.It belongs to flavonoids extraction technical field.The application extracts flavonoids compound in chess nan linaloe leaf by ultrasonic wave auxiliary ethanol extraction method, and is determined by single factor test and response surface method optimization optimum extraction process;Further, macroporous adsorption resin is used to purify crude extract, and obtain purified flavonoids compound.Flavonoids compound contains many active ingredients such as acacia leaf glycoside, quercetin, sakura flower element by identification.The method of the application is high in extraction efficiency, simple in operation, environmentally friendly and safe, and the obtained flavonoids compound has significant antioxidant activity and inhibitory activity of acetylcholinesterase and alpha-glucosidase, which provides technical support for the development and utilization of chess nan linaloe leaf.

Description

Technical Field

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

[0002] Agarwood, known as Qinan agarwood, is formed from the resin of the artificially selected high-quality Aquilaria sinensis tree. It boasts excellent resin quality, a unique aroma, and advantages such as easy resin formation, high yield, and short growth cycle. Qinan agarwood leaves, a byproduct of agarwood cultivation, are rich in resources, but currently are mostly discarded or burned, failing to be effectively utilized. Research shows that flavonoids in Qinan agarwood leaves possess various biological activities, including antioxidant, hypoglycemic, anti-Alzheimer's disease, and anti-inflammatory effects, exhibiting high medicinal and health-promoting value. Therefore, research on the extraction and utilization of flavonoids from Qinan agarwood leaves is of significant importance for resource development.

[0003] Currently, extraction methods for flavonoids mainly include organic solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, and enzyme-assisted extraction. Among these, ultrasound-assisted extraction is widely used in plant flavonoid extraction research due to its advantages such as low cost, simple operation, high extraction efficiency, and minimal damage to active ingredients. However, existing technologies mostly focus on the preliminary extraction of agarwood leaves, lacking systematic extraction process optimization, purification methods, and activity evaluation for flavonoids from *Aquilaria sinensis* leaves. This results in low utilization of active ingredients, making it difficult to achieve efficient extraction and functional applications. Furthermore, the flavonoids obtained from *Aquilaria sinensis* leaves through ultrasound extraction have low purity and contain various impurities, affecting their efficacy.

[0004] Therefore, how to provide an efficient, stable method for extracting flavonoids from Aquilaria sinensis leaves, and further clarify their composition, structure and bioactivity, is a technical problem that urgently needs 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 obtains high-purity, high-activity flavonoid compounds through an optimized ultrasound-assisted extraction process and macroporous resin purification 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 the leaves of Aquilaria sinensis includes the following steps:

[0008] S1. Raw material pretreatment: Take healthy, disease-free agarwood leaves, clean them, air dry them naturally, crush them to a 100-mesh sieve, and seal them for later use.

[0009] S2, Flavonoid Extraction: The powdered agarwood leaves pretreated by S1 were mixed with ethanol solution at a certain material-liquid ratio, and ultrasonic-assisted extraction was performed. After extraction, the mixture was centrifuged and the supernatant was collected to obtain the extract.

[0010] S3. Preparation of crude extract: The extract of S2 was concentrated under reduced pressure in a rotary evaporator to remove ethanol and obtain crude flavonoid extract;

[0011] S4. Purification: The crude extract was dissolved in water and purified by passing it through a D3520 macroporous adsorption resin column. The extract was eluted with water and 70% ethanol in sequence. The 70% ethanol eluent was collected and concentrated under reduced pressure to obtain the purified flavonoids.

[0012] As a preferred technical solution, the concentration of the ethanol solution in S2 is 60%; the material-to-liquid ratio is 1g:50mL; the ultrasonic power of the ultrasonic-assisted extraction is 100W, and the ultrasonic time is 30min; the centrifugation speed is 5000rpm, and the time is 5min.

[0013] As a preferred technical solution, the specific conditions for the purification process in S4 are as follows: the pH value of the loading solution is 3; the adsorption rate is 1.5 mL / min, the elution rate is 1.0 mL / min, and the eluent volume is 50 mL.

[0014] Another object of the present invention is to provide: flavonoid compounds prepared by the above method.

[0015] Another object of the present invention is to provide the application of the above-mentioned flavonoids, including in any of the following directions:

[0016] A: Applications in the preparation of foods or pharmaceuticals with antioxidant activity;

[0017] B: Application in the preparation of pharmaceuticals or health products that inhibit acetylcholinesterase activity;

[0018] C: Application in the preparation of pharmaceuticals or health products that inhibit α-glucosidase activity.

[0019] 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 a method for the efficient extraction, separation and purification of flavonoids from Aquilaria sinensis leaves. Using Aquilaria sinensis leaves as raw material, this method optimizes the ultrasound-assisted ethanol extraction process using response surface methodology, achieving a flavonoid yield of 6.68±0.05%, significantly higher than traditional methods. Further combining macroporous resin purification technology, D3520 resin was selected as the optimal purification medium, and by optimizing parameters such as loading pH and adsorption / elution flow rate, the flavonoid purity was increased by 1.49 times. The extraction and purification process provided by the present invention is efficient and stable, fully utilizing Aquilaria sinensis leaf resources and realizing their high-value utilization. Activity experiments confirmed that the obtained flavonoids have excellent antioxidant activity and significant inhibitory effects on acetylcholinesterase and α-glucosidase, providing high-quality natural active raw materials for the development of pharmaceuticals or functional foods, and has broad application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 The effect of different ethanol concentrations on the extraction rate of total flavonoids (FQP);

[0022] Figure 2 To: the DPPH free radical scavenging activity of different groups of products;

[0023] Figure 3 For: the ABTS free radical scavenging activity of different group products;

[0024] Figure 4 For: Total reducing antioxidant capacity of products in different groups; Detailed Implementation

[0025] 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.

[0026] Example 1

[0027] Ultrasonic-assisted extraction ethanol concentration screening

[0028] Under the fixed conditions of a material-to-liquid ratio of 1:50 (g / mL), ultrasonic extraction time of 30 min, and ultrasonic power of 100 W, the effects of ethanol concentrations of 50%, 60%, 70%, 80%, and 90% on the extraction rate of total flavonoids (FQP) from Aquilaria sinensis leaves were investigated. Figure 1 ).

[0029] Results Analysis: The total flavonoid extraction rate from Aquilaria sinensis leaves showed a trend of first increasing and then decreasing. Within the ethanol concentration range of 50%–60%, the flavonoid extraction yield rapidly increased and reached its maximum value; within the range of 60%–80%, the yield slowly decreased with increasing ethanol concentration; and within the range of 80%–90%, the yield rapidly decreased with increasing ethanol concentration. Further optimization experiments were conducted using the ethanol concentrations (50%, 60%, and 70%) that resulted in the maximum extraction rate.

[0030] Example 2

[0031] Screening of optimal conditions for ultrasound-assisted extraction

[0032] Based on the optimal ethanol concentration in Example 1, the extraction parameters of FQP were optimized, and the extraction parameters were determined to be: liquid-to-solid ratio (40-60%), ethanol concentration (50-70%), ultrasonic time (20-40 min), and ultrasonic power of 100 W. Based on the single-direction experiment, a three-factor, three-level Box-Behnken design (BBD) was adopted, where the liquid-to-solid ratio (A, %), ethanol concentration (B, mL / g), and ultrasonic time (C, min) were independent variables, and the total flavonoid extraction rate (Y) was the response value. The results are shown in Table 1.

[0033] Table 1

[0034]

[0035] Results analysis: As shown in Table 1, based on the significance test, the optimal liquid-to-solid ratio is 1:50, the optimal ethanol concentration is 60%, and the optimal ultrasonic time is 30 min.

[0036] Example 3

[0037] A method for extracting flavonoids from the leaves of Aquilaria sinensis includes the following steps:

[0038] S1. Raw material pretreatment: Collect healthy, disease-free agarwood leaves, clean them thoroughly, air dry them naturally, crush them, pass them through a 100-mesh sieve, and seal them for later use.

[0039] S2. Ultrasonic-assisted extraction: Weigh the pretreated Aquilaria sinensis leaf powder from S1, add 60% ethanol solution at a material-to-liquid ratio of 1:50 (g / mL), and extract under ultrasonic power of 100 W for 30 min. After extraction, centrifuge at 5000 rpm for 5 min and collect the supernatant. Repeat the extraction three times and combine the extracts. The total flavonoid content was determined by the NaNO-Al(NO)-NaOH colorimetric method. After three repeated experiments, the average extraction rate of total flavonoids was found to be 6.68 ± 0.05%.

[0040] S3. Preparation of crude extract: The combined extract obtained in S2 was concentrated under reduced pressure in a rotary evaporator, and impurities such as chlorophyll and oil were removed by petroleum ether extraction. The extract was freeze-dried into powder for later use.

[0041] S4. Macroporous Resin Purification: The crude extract obtained in S3 was reconstituted with deionized water, and the pH was adjusted to 3.0. The sample solution was passed through a D3520 macroporous adsorption resin column at a flow rate of 1.0 mL / min for dynamic adsorption. After adsorption, the sample was washed twice with distilled water to remove impurities. Then, elution was performed with 70% ethanol at a flow rate of 1.0 mL / min. 50 mL of the eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain the purified flavonoids.

[0042] S5. Purity Determination: The flavonoid content was determined using the NaNO-Al(NO)-NaOH colorimetric method. The total flavonoid purity of the crude extract before purification was 12.56±0.12 mg / g, and the total flavonoid purity after purification was 18.83±0.42 mg / g, an increase of 1.49 times compared to before purification. The results indicate that D3520 macroporous resin has a good purification effect on flavonoid compounds from Aquilaria sinensis leaves.

[0043] To verify the efficacy of the flavonoids extracted in this invention, the vitamin C group was used as a positive control group. The DPPH radical scavenging activity, ABTS radical scavenging activity, and total reducing power of the products in different groups were measured. The experimental results are as follows: Figures 2-4 As shown.

[0044] Results Analysis: Figure 2 It can be seen that when the concentration reaches 50 μg / mL, the scavenging rates of crude extract, alcohol extract and VC are 91.39±0.16%, 97.63±1.2% and 98.7±0.09%, respectively. At this time, the DPPH scavenging ability of the purified product is comparable to that of VC.

[0045] Depend on Figure 3 It can be seen that when the concentration of the purified product is 200 μg / mL, the ABTS free radical scavenging rates of the crude extract, purified product and VC are comparable, all reaching close to 100.

[0046] Depend on Figure 4 It was found that within the concentration range of 12.5–200 μg / mL, the total reducing power of the crude extract, purified extract, and vitamin C all increased with increasing concentration. The difference in total reducing power between the crude extract, purified extract, and vitamin C of *Aquilaria sinensis* leaves increased with increasing concentration, but this difference was not significant at lower concentrations. This indicates that both the crude extract and purified extract of *Aquilaria sinensis* leaves possess a certain total reducing power, with the purified extract having a higher total reducing power than the crude extract.

[0047] 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.

[0048] 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 and purifying flavonoids from Aquilaria sinensis leaves, comprising the following steps: S1. After drying and pulverizing the leaves of Aquilaria sinensis, extract flavonoids using ultrasonic-assisted ethanol extraction. The extraction conditions are: ethanol concentration 50%~70%, material-liquid ratio 1:40~1:60 (g / mL), and ultrasonic time 20~40 min. S2. After centrifugation, filtration, and concentration, the extract was subjected to static adsorption and desorption using D3520 macroporous adsorption resin. The static adsorption conditions were: pH of the loading solution 3-5, and elution ethanol concentration 60%-80%. S3. The saturated resin is dynamically eluted at a flow rate of 0.5-1.5 mL / min and an eluent volume of 40-60 mL. The eluent is collected, concentrated, and dried to obtain purified flavonoids.

2. The method according to claim 1, characterized in that... The optimal extraction conditions in step (1) are: ethanol concentration of 60%, material-to-liquid ratio of 1:50 (g / mL), and ultrasonic time of 30 min.

3. The method according to claim 1, characterized in that... The optimal pH for static adsorption in step (2) is 3, and the optimal elution ethanol concentration is 70%.

4. The method according to claim 1, characterized in that... The optimal flow rate for dynamic elution in step (3) is 1.0 mL / min, and the amount of eluent used is 50 mL.

5. The method according to claim 1, characterized in that... The D3520 macroporous resin is a polar or non-polar resin with a specific surface area ≥480 m² / g and an average pore size of 85~90 nm.

6. The flavonoids extracted and purified from Aquilaria sinensis leaves according to the method described in claim 1, characterized in that... It contains at least one of the following flavonoid components: luteolin, quercetin, safflowerin, zeaxanthin, genistein, physalisin, veselin-2, quercetin 3-O-glucoside, genistein, and luteolin.

7. The use of the flavonoids extracted and purified from Aquilaria sinensis leaves by the method of claim 1 in the preparation of pharmaceuticals or functional foods that have antioxidant properties, inhibit acetylcholinesterase, or inhibit α-glucosidase.

8. The application according to claim 7, characterized in that... The antioxidant activity includes scavenging DPPH free radicals and ABTS free radicals and enhancing reducing power.

9. The application according to claim 7, characterized in that... The IC50 value for inhibiting acetylcholinesterase was 60.48 ± 0.08 μg / mL, and the IC50 value for inhibiting α-glucosidase was 22.07 ± 0.15 μg / mL.

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