A method for ultrasonic-assisted extraction of agarwood tetraols using deep eutectic solvents

Abstract

The present invention discloses a method for ultrasonically assisted extraction of agarwood tetraol from agarwood using a deep eutectic solvent. The method uses agarwood powder as a raw material and a deep eutectic solvent as an extraction solvent, and ultrasonic heating-assisted extraction is performed to obtain an extract. Through extensive experiments, the present invention screens out an optimal deep eutectic solvent formed by quaternary ammonium salt choline chloride and polyol ethylene glycol, which is of the same type or has similar material components as agarwood tetraol. The deep eutectic solvent is used in conjunction with ultrasonic extraction of agarwood tetraol, eliminating the need for further removal. Furthermore, the extraction process is environmentally friendly and safe, without damaging the structure of agarwood tetraol. This can better simplify the process, save costs, improve extraction efficiency, and protect the environment.

Description

Technical Field

[0001] The present invention relates to the technical field of natural substance extraction, and in particular to a method for extracting agarwood tetraols with the assistance of ultrasound using a deep eutectic solvent. Background Art

[0002] Agarwood is a commonly used herb in Traditional Chinese Medicine (TCM), with a history of over a thousand years. The earliest record of agarwood as a medicine can be found in the Liang Dynasty book "Ming Yi Bie Lu" by Tao Hongjing: "Agarwood, Xunluxiang, Jishexiang, Patchouli, Zhantangxiang, Fengxiang, and are slightly warm." Agarwood is the resinous wood of Aquilaria sinensis (Lour.) Gilg, a member of the Thymelaeaceae family, and can be harvested year-round. Agarwood is a precious traditional Chinese medicinal material with high medicinal value. It contains a variety of bioactive components, such as volatile oils, resins, and lignin, which exhibit various pharmacological effects, including anti-inflammatory, antioxidant, and anti-tumor properties.

[0003] Existing techniques often use organic solvents such as methanol, ethanol, and water mixtures to extract agarwood tetraols. However, these solvents require large amounts of volatile and toxic reagents, posing significant safety and health risks and incompatible with the principles of green chemistry. Therefore, developing an efficient, rapid, and low-cost agarwood extraction method is of great practical significance.

[0004] Deep eutectic solvents (DES) are composed of hydrogen bond acceptors and hydrogen bond donors in a specific molar ratio. Through intermolecular hydrogen bonding, they form a homogeneous mixture that is liquid at room temperature. Deep eutectic solvents have rapidly developed as a green solvent in recent years. They offer advantages such as simple preparation, high extraction efficiency, and environmental friendliness. They have garnered increasing attention and have shown promising applications in extraction and separation, functional materials, and electrochemistry. Due to their strong solubility for both polar and non-polar compounds, deep eutectic solvents offer high extraction efficiency for a variety of natural active ingredients, holding great potential for development in the field of natural substance extraction. Currently, there are no reports on the extraction of tetraols from agarwood using this technology, suggesting promising applications and development prospects. Summary of the Invention

[0005] Technical Problem Solved: To address the problems of the existing technology, the present invention provides a method for the ultrasonic-assisted extraction of tetraols from agarwood using a deep eutectic solvent. This method, which uses a deep eutectic solvent as the extraction solvent and ultrasonic-assisted heating, offers advantages such as environmental friendliness, a simple preparation process, high extraction efficiency, low cost, and a safe and non-toxic product.

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

[0007] A method for ultrasonically assisted extraction of tetraols from agarwood using a deep eutectic solvent, the operating steps are as follows:

[0008] A. Pre-treating agarwood to obtain agarwood powder;

[0009] B. adding the agarwood powder into an ultrasonic extraction container, adding a deep eutectic solvent and water to mix, and performing ultrasonic treatment to obtain a mixed treatment liquid;

[0010] C. Centrifuge the mixed treated liquid to obtain the supernatant to obtain the agarwood tetraol extract.

[0011] As a preferred embodiment, the deep eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor (HBA) is a quaternary ammonium salt, and the hydrogen bond acceptor (HBD) is a polyol;

[0012] As a particularly preferred solution, the hydrogen bond acceptor is choline chloride; and the hydrogen bond donor is ethylene glycol, 1,2-propylene glycol or glycerol.

[0013] As a preferred embodiment, the molar ratio of HBA and HBD in the above deep eutectic solvent is 1:2 to 1:6, preferably 1:3 of choline chloride and ethylene glycol, or the molar ratio is 1:2 to 1:6, preferably 1:3 of choline chloride and 1,2-propylene glycol, or the molar ratio is 1:2 to 1:6, preferably 1:3 of choline chloride and glycerol.

[0014] The preparation method of the deep eutectic solvent of the present invention is as follows: HBA and HBD are fully mixed in a certain molar ratio, and heated in a water bath at a temperature of 80-90° C. to form a uniform, stable and transparent liquid.

[0015] As a preferred embodiment, the mixed system of the deep eutectic solvent and water described above has a water content of 30% to 60% (mass ratio).

[0016] As a preferred embodiment, the material-to-liquid ratio between the agarwood powder and the aqueous solution of the deep eutectic solvent is 1:10 to 1:25, in g / mL.

[0017] The temperature of the ultrasonic extraction is 40-60° C., the extraction power is 100-150W, and the extraction time is 30-60 minutes.

[0018] For the solid-liquid separation, the centrifugal speed is 6000-8000 rpm and the centrifugal time is 5-10 minutes.

[0019] Beneficial effects: Agarwood is a precious traditional Chinese medicine, and the structural stability of agarwood tetraol is generally poor. Therefore, a method is needed that has high extraction efficiency and does not damage the agarwood tetraol structure.

[0020] The deep eutectic solvent used in the present invention can efficiently extract agarwood tetraol from agarwood. Through a large number of experiments, the best deep eutectic solvent formed by quaternary ammonium salt choline chloride and polyol ethylene glycol was screened out. It is of the same type as agarwood tetraol or an analogue with similar material components. After using it in combination with ultrasound to extract agarwood tetraol, the subsequent extract does not need to undergo a removal and purification step, and will not damage the structure of agarwood tetraol. The entire extraction process is green and safe, with a simple preparation process, low cost, simple extraction equipment operation, and no special requirements for samples. The extraction efficiency is higher than that of conventional methods, and the obtained product is natural and non-toxic. It can avoid the disadvantages of using organic solvents, a long extraction process, and easy deterioration of the extract. It has good application value and development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the extraction yield of agarwood tetraols using deep eutectic solvents obtained by mixing choline chloride and ethylene glycol in different molar ratios.

[0022] Figure 2 This is the HPLC chromatogram of agarwood tetraol standard.

[0023] Figure 3 is the HPLC chromatogram of DES-1 extract.

[0024] Figure 4 is the HPLC chromatogram of DES-2 extract.

[0025] Figure 5 is the HPLC chromatogram of DES-3 extract.

[0026] Figure 6 HPLC chromatogram of the water extract.

[0027] Figure 7 HPLC chromatogram of methanol extract. DETAILED DESCRIPTION

[0028] The present invention provides a method for ultrasonically assisted extraction of agarwood tetraols using a deep eutectic solvent. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. The specific examples described in the embodiments of the invention are merely illustrative of the specific embodiments of the present invention and are not intended to limit the present invention.

[0029] Example 1

[0030] A method for extracting agarwood tetraols with the aid of ultrasound using a deep eutectic solvent, the method comprising the following steps:

[0031] Step A: Prepare a deep eutectic solvent containing choline chloride-ethylene glycol: specifically, mix choline chloride and ethylene glycol in molar ratios of 1:2, 1:3, 1:4, 1:5, and 1:6, respectively, heat in a water bath at 80° C. to form a uniform, stable, transparent liquid, and seal for storage.

[0032] Step B: The deep eutectic solvents of each molar ratio in step (1) were mixed with water to form a mixture system (water content of 40%), and then mixed with agarwood powder at a material-liquid ratio of 1:20, unit g / mL, and subjected to ultrasonic heating assisted extraction (temperature 50°C, time 40min, extraction power 120W) to obtain each extract, which was allowed to stand and cooled;

[0033] Step C: The extracts obtained in step (2) were centrifuged at 6000-8000 rpm for 10 min, and the supernatant was collected to obtain the agarwood tetraol extract. The sample solution was subjected to HPLC analysis using an Elite HPLC 1260 system (Agilent, USA) and a C18 column (4.6×250 mm, 5 μm S.No.1). The flow rate was 0.7 ml per minute; the column temperature was 30°C; the detection wavelength was 252 nm; and the mobile phase ratio was shown in Table 1. The agarwood tetraol content results are shown in Table 1. Figure 1 shown.

[0034] Table 1 HPLC separation method of agarwood tetraols

[0035]

[0036]

[0037] Depend on Figure 1 As shown in the figure, the content of agarwood tetraols extracted using deep eutectic solvents with different molar ratios of choline chloride and ethylene glycol varies. As the molar ratio of choline chloride to ethylene glycol increases, the extraction efficiency first increases and then decreases, reaching the highest extraction efficiency when the molar ratio of choline chloride to ethylene glycol is 1:3.

[0038] Example 2

[0039] The present invention provides a method for ultrasonically assisted extraction of agarwood tetraols using a deep eutectic solvent, the method comprising the following steps:

[0040] Step A: Preparation of a deep eutectic solvent (DES-1) containing choline chloride and ethylene glycol: Specifically, choline chloride and ethylene glycol were mixed at a molar ratio of 1:3, heated in a water bath at 80° C. to form a homogeneous, stable, and transparent liquid, which was then sealed and stored for later use.

[0041] Step B: The deep eutectic solvent (DES-1) in step (1) is mixed with water to form a mixture system (water content is 40%), and then mixed with agarwood powder at a material-liquid ratio of 1:20, unit g / mL, and subjected to ultrasonic heating assisted extraction (temperature 50°C, time 40min, extraction power 120W) to obtain an extract, which is then allowed to stand and cooled;

[0042] Step C: Centrifuge the extract obtained in step (2) at 6000-8000 rpm for 10 min, and collect the supernatant to obtain the agarwood tetraol extract.

[0043] The HPLC determination method is the same as in Example 1. The results of the content of agarwood tetraol are shown in Table 2 and Figure 3 shown.

[0044] Example 3

[0045] The same as Example 1, except that: a deep eutectic solvent (DES-2) containing choline chloride-1,2-propylene glycol was prepared: the specific method was as follows: choline chloride and 1,2-propylene glycol were mixed in a molar ratio of 1:3, heated in a water bath at 80°C to form a uniform, stable and transparent liquid, and sealed for storage.

[0046] The same as in Example 1, the content of agarwood tetraol was detected by high performance liquid chromatography (HPLC). The content of agarwood tetraol was shown in Table 2 and Figure 4 shown.

[0047] Example 4

[0048] The same as Example 1, except that: a deep eutectic solvent (DES-3) containing choline chloride and glycerol was prepared: the specific method was as follows: choline chloride and glycerol were mixed in a molar ratio of 1:3, heated in a water bath at 80°C to form a uniform, stable and transparent liquid, and sealed for storage.

[0049] The same as in Example 1, the content of agarwood tetraol was detected by high performance liquid chromatography (HPLC). The content of agarwood tetraol was shown in Table 2 and Figure 5 shown.

[0050] Comparative Example 1

[0051] The same as Example 1, except that: a deep eutectic solvent (DES-4) containing choline chloride-lactic acid was prepared: the specific method was as follows: choline chloride and lactic acid were mixed in a molar ratio of 1:3, heated in a water bath at 80°C to form a uniform, stable and transparent liquid, and sealed for storage.

[0052] Similar to Example 1, the content of agarwood tetraol was determined by high performance liquid chromatography (HPLC) and was 3.16 mg / g, as shown in Table 2.

[0053] Comparative Example 2

[0054] The same as Example 1, except that: a deep eutectic solvent (DES-5) containing choline chloride-glycolic acid was prepared: the specific method was as follows: choline chloride and glycolic acid were mixed in a molar ratio of 1:3, heated in a water bath at 80°C to form a homogeneous, stable, transparent liquid, and sealed for storage.

[0055] Similar to Example 1, the content of agarwood tetraol was determined by high performance liquid chromatography (HPLC) and was 2.89 mg / g, as shown in Table 2.

[0056] Comparative Example 3

[0057] Pure water ultrasonic extraction of agarwood tetraols is carried out as follows:

[0058] Step A: Add agarwood powder into a round-bottom flask, add ultrapure water at a material-liquid ratio of 1:20 (g / mL), and perform ultrasonic heating-assisted extraction (temperature 50°C, time 40 min, extraction power 120 W) to obtain an extract, which is then allowed to stand and cooled;

[0059] Step B: Centrifuge the extract obtained in step (2) and take the supernatant to obtain the agarwood tetraol extract. The content of tetraol in the extract was detected by high performance liquid chromatography (HPLC). The results are shown in Table 2 and Figure 6 shown.

[0060] Comparative Example 4

[0061] Agarwood tetraols were extracted using 80% methanol solution by ultrasonic method as follows:

[0062] Step A: Add agarwood powder into a round-bottom flask, add 80% methanol solution at a solid-liquid ratio of 1:20 (g / mL), and perform ultrasonic heating-assisted extraction (temperature 50°C, time 40 min, extraction power 120W) to obtain an extract, which is then allowed to stand and cooled;

[0063] Step B: Centrifuge the extract obtained in step (2) and take the supernatant to obtain the agarwood tetraol extract. The content of tetraol in the extract was detected by high performance liquid chromatography (HPLC). The results are shown in Table 2 and Figure 7 shown.

[0064] The extracts of Examples 2 to 4 and Comparative Examples 1 to 4 were tested and analyzed. The specific results are shown in Table 2:

[0065] Table 2 Extraction rates of agarwood tetraols in different solvents

[0066]

[0067] The HPLC chromatogram shows that water-extracted agarwood extracts contain very few components and have a low extraction rate of agarwood tetraols, while DES-extracted agarwood extracts can extract more active ingredients. The data show that the extraction rates of agarwood tetraols by all five DESs are higher than those of pure water and methanol, with DES1 achieving the highest extraction rate of 4.83 mg / g, demonstrating significant technological progress.

[0068] It should be noted that the parameters of the embodiments of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the conditions under which the present invention can be implemented. Any changes in the proportion relationship or adjustments in the components should still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

Claims

1. A method for ultrasonically assisted extraction of agarwood tetraols using a deep eutectic solvent, characterized in that: The method comprises the following preparation steps: A. Pre-treating agarwood to obtain agarwood powder; B. adding the agarwood powder into an ultrasonic extraction container, adding a deep eutectic solvent and water to mix, and performing ultrasonic treatment to obtain a mixed treatment liquid; C. Centrifuging the mixed treated liquid to obtain the supernatant to obtain an agarwood tetraol extract; The deep eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is choline chloride; the hydrogen bond acceptor is selected from one or more of ethylene glycol, 1,2-propylene glycol, and glycerol; The preparation method of the deep eutectic solvent is as follows: a hydrogen bond donor and a hydrogen bond acceptor are fully mixed in a molar ratio of 1:2 to 1:6, and heated in a water bath at a temperature of 80 to 90° C. to form a uniform, stable and transparent liquid.

2. The method for ultrasonically assisted extraction of agarwood tetraols using a deep eutectic solvent according to claim 1, characterized in that The preparation method of the deep eutectic solvent is as follows: choline chloride and ethylene glycol are fully mixed in a molar ratio of 1:

3.

3. The method for ultrasonically assisted extraction of agarwood tetraols using a deep eutectic solvent according to claim 1, characterized in that: In step B, the mixed system of the deep eutectic solvent and water has a water content of 30% to 60% by mass.

4. The method for ultrasonically assisted extraction of agarwood tetraols using a deep eutectic solvent according to claim 1, characterized in that In step B, the material-to-liquid ratio between the agarwood powder and the aqueous solution of the deep eutectic solvent is 1:10 to 1:25, unit: g / mL.

5. The method for ultrasonically assisted extraction of agarwood tetraols using a deep eutectic solvent according to claim 1, characterized in that: In step B, the temperature of the ultrasonic treatment is 40-60° C., the extraction power is 100-150 W, and the time of the ultrasonic treatment is 30-60 min.

6. The method for ultrasonically assisted extraction of agarwood tetraols using a deep eutectic solvent according to claim 1, characterized in that: In step C, the centrifugal separation is performed at a speed of 6000 to 8000 rpm, and the centrifugal separation time is 5 to 10 minutes.

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