Deep eutectic solvent, preparation method thereof and application of deep eutectic solvent in extraction of lophatherum gracile flavone

Abstract

The invention provides a deep-eutectic solvent, a preparation method thereof and application of the deep-eutectic solvent in extraction of lophatherum gracile flavone. According to the deep-eutectic solvent, choline chloride is used as a hydrogen bond acceptor, and any one of an amide compound, an alcohol compound, a carboxylic acid compound and a glycosyl compound is used as a hydrogen bond donor; in the deep eutectic solvent, the molar ratio of the choline chloride to the amide compound is 1: 2, the molar ratio of the choline chloride to the alcohol compound is 1: 2, the molar ratio of the choline chloride to the carboxylic acid compound is 1: 1, and the molar ratio of the choline chloride to the glycosyl compound is (1-3): 1; wherein the amide compound is prepared from urea, N-methylurea and acetamide; the alcohol compounds comprise glycerol, 1, 2-propylene glycol, 1, 4-butanediol and ethylene glycol; the carboxylic acid compounds comprise lactic acid, oxalic acid, malonic acid and citric acid; the glycosyl compound comprises glucose, maltose, fructose and cane sugar. The method has the advantages of greenness, environmental friendliness, improved extraction efficiency and the like.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a low eutectic solvent and a preparation method thereof, and application thereof in extracting flavonoids from bamboo leaves. Background Art

[0002] Lophatherum gracile Brongn is the stem and leaf of the perennial plant Lophatherum gracile Brongn of the Poaceae family. It grows on hillsides, woodlands or forest edges, and in the shade of roads. It is mainly produced in southern my country. It has a very high use value and is a traditional Chinese medicine in my country. Lophatherum gracile Brongn is rich in a variety of effective ingredients such as flavonoids and bioactive polysaccharides. It also contains characteristic ingredients such as phenolic acid compounds, anthraquinone compounds, terpenoid lactones, and trace elements such as manganese, zinc, and selenium. These ingredients together give Lophatherum gracile Brongn unique medicinal and nutritional value. It is recorded in the ancient pharmacopoeia "Cao Mu Bian Fang" that Lophatherum gracile Brongn has the effects of "eliminating phlegm, quenching thirst, treating irritability, cough and asthma, vomiting blood, vomiting, and epilepsy in children". Among them, the content of flavonoids is the richest, which has significant benefits to the human body. Modern research shows that total flavonoids in Lophatherum gracile Brongn have multiple effects such as antioxidant, anti-aging, anti-inflammatory, anti-tumor, antiviral and antibacterial. In addition, it can significantly reduce blood lipids, constrict blood vessels, protect the liver, and relieve fever and diuresis. Therefore, total flavonoids from light bamboo leaves have broad application prospects in the fields of medicine, health products and food.

[0003] Flavonoids from the leaves of Psoralea corylifolia have a wide range of pharmacological activities. How to extract flavonoids from Psoralea corylifolia and optimize its extraction process is of great significance to the development and utilization of flavonoids from the leaves of Psoralea corylifolia. With the development of extraction technology, in addition to the traditional reflux method and water decoction method, some new extraction processes have gradually emerged, but these extraction processes all require a large amount of organic solvents and are less environmentally friendly.

[0004] Therefore, it is very necessary to provide an environmentally friendly solvent for extracting flavonoids from bamboo leaves. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a low eutectic solvent and a preparation method thereof and application thereof in extracting flavonoids from bamboo leaves.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The invention provides a low eutectic solvent. The low eutectic solvent uses choline chloride as a hydrogen bond acceptor, and any one of an amide compound, an alcohol compound, a carboxylic acid compound and a glycosyl compound as a hydrogen bond donor. In the low eutectic solvent, the molar ratio of choline chloride to the amide compound is 1:2, the molar ratio of choline chloride to the alcohol compound is 1:2, the molar ratio of choline chloride to the carboxylic acid compound is 1:1, and the molar ratio of choline chloride to the glycosyl compound is (1-3):1; wherein the amide compound includes urea, N-methyl urea and acetamide; the alcohol compound includes glycerol, 1,2-propylene glycol, 1,4-butanediol and ethylene glycol; the carboxylic acid compound includes lactic acid, oxalic acid, malonic acid and citric acid; and the glycosyl compound includes glucose, maltose, fructose and sucrose.

[0008] Further, the low eutectic solvent includes choline chloride-urea, choline chloride-N-methylurea, choline chloride-acetamide, choline chloride-glycerol, choline chloride-1,2-propylene glycol, choline chloride-1,4-butanediol, choline chloride-ethylene glycol, choline chloride-lactic acid, choline chloride-oxalic acid, choline chloride-malonic acid, choline chloride-citric acid, choline chloride-glucose, choline chloride-maltose, choline chloride-fructose and choline chloride-sucrose; wherein the molar ratio of choline chloride to glucose in the choline chloride-glucose is 2:1, the molar ratio of choline chloride to maltose in the choline chloride-maltose is 3:1, the molar ratio of choline chloride to fructose in the choline chloride-fructose is 1:1, and the molar ratio of choline chloride to sucrose in the choline chloride-sucrose is 2:1.

[0009] The present invention also provides a method for preparing the low eutectic solvent as described above, the preparation method comprising: using choline chloride as a hydrogen bond acceptor, mixing any one of an amide compound, an alcohol compound, a carboxylic acid compound and a glycosyl compound in a corresponding molar ratio to obtain a mixture; adding 20% ​​w / w deionized water to the mixture, heating to 80° C., and stirring until transparent, uniform and stable to obtain the low eutectic solvent.

[0010] The present invention also provides the use of the deep eutectic solvent as described above in the field of traditional Chinese medicine extraction.

[0011] The present invention further provides the use of the above-mentioned low eutectic solvent in extracting flavonoids from Pleistocene chinensis leaves.

[0012] Furthermore, the extraction method includes: drying, crushing and screening the leaves of Bambusa chinensis to obtain coarse powder of Bambusa chinensis; extracting the coarse powder of Bambusa chinensis with the low eutectic solvent by thermal extraction, filtering and combining the obtained extract to obtain a Bambusa chinensis leaf extract.

[0013] Furthermore, the drying temperature is 40°C to 60°C, and the drying time is 18h to 24h; and the crushing and screening process is specifically a 40-60 mesh sieve.

[0014] Furthermore, in the thermal extraction method, the mass volume ratio (solid-liquid ratio) of the crude powder of light bamboo leaves to the low eutectic solvent is 1g:10mL; the extraction temperature is 60°C; and the extraction time is 30min.

[0015] Furthermore, the extraction method also includes: detecting the content of flavonoids in the light bamboo leaf extract extracted by the low eutectic solvent by sodium nitrite-aluminum nitrate colorimetry; the sodium nitrite-aluminum nitrate colorimetric method specifically includes: taking 1 mL of the light bamboo leaf extract, then mixing it with 0.3 mL of 5% sodium nitrite, and then adding 0.3 mL of 10% aluminum nitrate, 4.0 mL of 4% sodium hydroxide and 4.4 mL of distilled water; after incubating at room temperature for 15 minutes, recording the absorbance at 510 nm.

[0016] Furthermore, the low eutectic solvent is choline chloride-N-methylurea.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0018] The present invention provides a deep eutectic solvent and a preparation method thereof and application thereof in extracting flavonoids from bamboo leaves.

[0019] (1) Green and environmentally friendly: The deep eutectic solvent provided by the present invention is usually composed of renewable resources, has good biodegradability, and has little impact on the environment. This is compared with many traditional organic solvents, which may cause long-term pollution to the environment.

[0020] (2) Improved extraction efficiency: The present invention achieves high-efficiency extraction of flavonoids from bamboo leaves by screening and optimizing the composition of the deep eutectic solvent. Compared with traditional extraction methods, such as hot water extraction or organic solvent extraction, the deep eutectic solvent provides a higher extraction rate.

[0021] (3) Easy operation: The preparation process of the low eutectic solvent provided by the present invention is simple, and only the hydrogen bond donor and the hydrogen bond acceptor need to be mixed in a certain proportion and heated until transparent. This preparation method is easy to scale up and has relatively low cost.

[0022] (4) Improved safety: The low eutectic solvent provided by the present invention generally has lower toxicity and irritation. Compared with some toxic organic solvents, extraction using a low eutectic solvent is safer.

[0023] (5) Cost-effectiveness: Since the raw materials for preparing the low eutectic solvent provided by the present invention are relatively cheap and easy to obtain, and the preparation process is simple, the overall cost is low.

[0024] (6) Mild extraction conditions: The extraction conditions (such as temperature and time) used in the present invention are relatively mild, which helps to protect the structure of the flavonoids in the leaves of the bamboo from being destroyed and maintain the biological activity of the extract.

[0025] (7) The extraction process is controllable: By optimizing the extraction conditions through single-factor experiments, the extraction process can be precisely controlled to ensure the quality and consistency of the extract.

[0026] (8) Wide application scope: The low eutectic solvent provided by the present invention is not only suitable for the extraction of flavonoids from bamboo leaves, but can also be extended to the extraction of effective ingredients from other natural plants, and has a wide range of application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results are the results of the flavonoid content test in the extract of Bambusa longituba in each deep eutectic solvent prepared in Example 1;

[0028] Figure 2 This is the result of the effect of the water content of the deep eutectic solvent on the efficiency of extraction of flavonoids from bamboo leaves by ultrasound-assisted deep eutectic solvent in Example 4;

[0029] Figure 3 This is the result of the effect of the molar ratio of the deep eutectic solvent composition on the efficiency of extraction of flavonoids from Psoralea corylifolia by ultrasound-assisted deep eutectic solvent in Example 5;

[0030] Figure 4 This is the result of the effect of the solid-liquid ratio on the efficiency of extraction of flavonoids from Psoralea corylifolia by ultrasound-assisted deep eutectic solvent in Example 6;

[0031] Figure 5 This is the result of the effect of extraction temperature on the efficiency of extraction of flavonoids from Psoralea corylifolia by ultrasound-assisted deep eutectic solvent in Example 7;

[0032] Figure 6 This is the result of the effect of ultrasound time on the efficiency of ultrasound-assisted deep eutectic solvent extraction of flavonoids from lophatherum gracile in Example 8. DETAILED DESCRIPTION

[0033] Low eutectic solvents, also known as deep eutectic solvents, refer to eutectics formed by hydrogen bonding in a certain proportion between two or more solid substances. One of the components is a quaternary ammonium salt, which serves as a hydrogen bond acceptor, and the other component is a metal ion or other form, which serves as a hydrogen bond donor. Similar to the principle of low eutectic mixtures, the melting point of low eutectic solvents is lower than that of their components. The melting point of low eutectic solvents is mostly within 150°C, and some are below 70°C. Some are at room temperature, that is, they are liquid at room temperature, and are also called room temperature low eutectic solvents. Traditional solvents such as methanol, ethanol, and petroleum ether, also known as petrochemical solvents, have the disadvantages of polluting the environment, being highly toxic, and being non-renewable. Scholars have also been seeking alternative solvents, such as ionic solutions. Although ionic solutions are pollution-free, they have a high viscosity and are inconvenient to recycle. Low eutectic solvents overcome the above disadvantages and have the advantages of simple preparation, good stability, high chemical purity, low cost, degradability, and low viscosity. In addition, the polarity of the solvent can be adjusted by adjusting the molar ratio of the components of the low eutectic solvent.

[0034] The inventors have found that low eutectic solvents have been widely used in the extraction of effective ingredients of traditional Chinese medicine, extraction of effective substances in food, preparation of test solutions for quality inspection, electrochemistry and materials science. Low eutectic solvents have advantages in the extraction of natural products, and show good extraction effects on flavonoids, polyphenols, proanthocyanidins, lignins, saponins and other components, especially in-depth research on the extraction of flavonoid compounds. As a new type of environmentally friendly green solvent that can enhance the extraction effect of microwaves and ultrasound, low eutectic solvents have great advantages in the extraction of effective ingredients of traditional Chinese medicine, and have great potential in replacing traditional solvents and toxic solvents.

[0035] As can be seen from the background technology, in the field of natural product extraction, especially in the process of extracting flavonoids from bamboo leaves, traditional methods such as water extraction, alcohol extraction, enzyme extraction, etc. have the problems of long time consumption, low extraction efficiency, high energy consumption, high cost, and great environmental impact. These methods not only extract a small amount, but are also unfriendly to the environment, and the equipment cost is high, which limits their application in large-scale production.

[0036] In view of these shortcomings, the present invention proposes a new method for extracting flavonoids from bamboo leaves by combining a low eutectic solvent with ultrasound. This method aims to improve extraction efficiency, reduce energy consumption and cost, and achieve an environmentally friendly extraction process. By optimizing extraction conditions and using an environmentally friendly low eutectic solvent, the present invention not only improves the extraction efficiency of flavonoids from bamboo leaves, but also enhances the application value of the extract, providing a new efficient, low-cost, and environmentally friendly way for natural product extraction.

[0037] The present invention is based on the characteristics of a low eutectic solvent, that is, a solvent with specific properties is formed through the interaction of a hydrogen bond acceptor and a hydrogen bond donor. Choline chloride, as a hydrogen bond acceptor, combines with hydrogen bond donors such as N-methylurea to form a choline chloride-N-methylurea type low eutectic solvent. This solvent has unique physicochemical properties, such as low toxicity, high solubility and good biodegradability, making it an ideal medium for extracting natural products. In the present invention, by screening different hydrogen bond donors, it is determined that the combination of choline chloride and N-methylurea has the highest efficiency in extracting flavonoids from light bamboo leaves. This optimized low eutectic solvent can more effectively extract flavonoid compounds from light bamboo leaves through ultrasound-assisted extraction technology. Ultrasound-assisted extraction technology utilizes the mechanical effect and cavitation effect of ultrasound to enhance the penetration of the solvent and improve the extraction efficiency.

[0038] The present invention is described in detail below in conjunction with specific implementation modes.

[0039] In the embodiments, the raw material of loblolly leaves was purchased from Guangzhou Qingping Chinese medicinal materials market, and the choline chloride, urea, N-methyl urea, acetamide, glycerol, 1,2-propylene glycol, 1,4-butanediol, ethylene glycol, lactic acid, oxalic acid, malonic acid, citric acid, glucose, maltose, fructose and sucrose used in the preparation of the low eutectic solvent were all purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0040] Example 1 Preparation of deep eutectic solvent

[0041] (1) As shown in Table 1, choline chloride is used as a hydrogen bond acceptor, and the corresponding amides (urea, N-methylurea, acetamide), alcohols (glycerol, 1,2-propylene glycol, 1,4-butanediol, ethylene glycol), carboxylic acids (lactic acid, oxalic acid, malonic acid, citric acid), and glycosyls (glucose, maltose, fructose, sucrose) are respectively used as hydrogen bond donors, mixed in corresponding molar ratios, and 20% (w / w) deionized water is added to the mixture, heated to 80° C., and stirred until a transparent, uniform, and stable low eutectic solvent liquid is obtained, thereby obtaining the corresponding various low eutectic solvents.

[0042] Table 1 Preparation and shape of deep eutectic solvents

[0043]

[0044] (2) In order to measure the pH value of a low eutectic solvent, you first need to prepare a calibrated pH meter and standard buffer. Use standard buffers of pH 4.01, 7.00, and 10.01 to perform a multi-point calibration on the pH meter to ensure the accuracy of the instrument. Then, place the low eutectic solvent sample to be tested in a clean container and ensure that the sample temperature is close to room temperature. Using a glass rod or pH meter probe, gently immerse the probe into the sample, avoiding contact with the container wall. Wait for the reading to stabilize and record the pH value. After each measurement, clean the probe with distilled or deionized water and shake it gently to remove excess water. Record the pH value of each sample, and take care to thoroughly clean the probe between measuring different samples to prevent cross contamination. In this way, the pH value of the low eutectic solvent sample can be accurately determined, providing important data for evaluating its acid-base properties.

[0045] Example 2 Preparation of Bamboo Leaves Extract in Deep Eutectic Solvent

[0046] Accurately weigh 5 g of Lophatherum gracile powder with a solid-liquid ratio of 1:10, mix evenly with the corresponding low eutectic solvent, extract Lophatherum gracile flavonoids by thermal extraction, filter and combine the extracts to obtain Lophatherum gracile extracts of each low eutectic solvent as sample solution.

[0047] Example 3 Determination of the total flavonoids content of the bamboo leaf extract of each deep eutectic solvent by sodium nitrite-aluminum nitrate colorimetric method

[0048] (1) Drawing of standard curve:

[0049] Accurately weigh 5 mg of rutin reference substance, place it in a 10 mL volumetric flask, add a portion of 95% ethanol solution by volume, place it in a water bath and dissolve it with slight heat, after cooling, add 95% ethanol by volume, and dilute to the scale to obtain rutin reference substance solution (0.5 mg / mL). Add the corresponding 95% ethanol to 1 mL of 0, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of rutin standard solution, and then mix with 0.3 mL of 5% sodium nitrite, and then add 0.3 mL of 10% aluminum nitrate, 4.0 mL of 4% sodium hydroxide and 4.4 mL of distilled water. After incubation at room temperature for 15 minutes, record the absorbance at 510 nm.

[0050] (2) Sample testing:

[0051] 0.5 mL of sample solution was taken, diluted to 1 mL with anhydrous ethanol, and 1 mL of sample solution was precisely taken into a 10 mL stoppered test tube. According to the method of drawing the standard curve, it was mixed with 0.3 mL of 5% sodium nitrite, and then 0.3 mL of 10% aluminum nitrate, 4.0 mL of 4% sodium hydroxide and 4.4 mL of distilled water were added. After incubation at room temperature for 15 minutes, the absorbance was recorded at 510 nm, and the content of flavonoids in light bamboo leaves under different extraction conditions was calculated according to the standard curve.

[0052] Flavonoid content in sample (%) = flavonoid content calculated from standard curve (mg) × dilution factor / sampling amount × 100%

[0053] Result analysis:

[0054] The results of the flavonoid content test in the extracts of Bambusa longituba extracted with the low eutectic solvents prepared in Example 2 are shown in Table 2.

[0055] Table 2 Detection results of flavonoids in the extract of Bambusa longituba under various deep eutectic solvents

[0056]

[0057]

[0058] As can be seen from Table 2, when the composition of the low eutectic solvent is choline chloride-N-methylurea, the total flavonoids concentration in the bamboo leaf extract reaches 1.3529±0.0043 mg / mL. Compared with the easy-to-obtain choline chloride-N-methylurea low eutectic solvent, the bamboo leaf flavonoids extracted have the highest content and yield, and the extraction efficiency is higher than that of the traditional distilled water extraction method. Therefore, the choline chloride-N-methylurea low eutectic solvent is identified as the best low eutectic solvent for extracting bamboo leaf flavonoids. Figure 1 shown.

[0059] Example 4 Effect of water content of deep eutectic solvent on the efficiency of extraction of flavonoids from bamboo leaves by ultrasound-assisted deep eutectic solvent

[0060] The choline chloride-N-methylurea low eutectic solvent prepared in Example 1 was added to a centrifuge tube containing 0.5 g of light bamboo leaf powder. Distilled water was added at a water content (0%, 5%, 10%, 20% and 30%), and the low eutectic solvent composition molar ratio was 1:1, and the solid-liquid ratio was 1:10. An ultrasonicator was used, the temperature was 60°C, and the extraction was 30 minutes. Then, the extract was centrifuged at 6000 rpm at 4°C for 10 minutes. The supernatant was collected for further analysis. The total flavonoid content in each low eutectic solvent extract was detected according to the above-mentioned sodium nitrite-aluminum nitrate colorimetric method.

[0061] from Figure 2 It can be observed that the change trend of the total flavonoids content in the leaves of Psoralea corylifolia increases with the increase of water content. From 0 to 20% water content, the total flavonoids content gradually increases, reaches a peak, and then decreases slightly at 30% water content. This shows that there is an optimal water content that makes the total flavonoids content reach the maximum value. It can be seen from the data that this optimal water content is 20%, because at this water content, the average value of the total flavonoids content is the highest, which is 1.1817 mg / mL. This shows that under this experimental condition, the water content of 20% is the best.

[0062] Example 5 Effect of the molar ratio of deep eutectic solvent composition on the efficiency of ultrasound-assisted deep eutectic solvent extraction of flavonoids from bamboo leaves

[0063] The choline chloride-N-methylurea low eutectic solvent prepared in Example 1 was added to a centrifuge tube containing 0.5 g of light bamboo leaf powder. The low eutectic solvent composition molar ratio was (1:1, 1:2, 1:3, 2:1 and 3:1), the water content was 20%, and the solid-liquid ratio was 1:10. An ultrasonicator was used, the temperature was 60°C, and the extraction was 30 minutes. Then, the extract was centrifuged at 6000 rpm at 4°C for 10 minutes. The supernatant was collected for further analysis. The total flavonoid content in each low eutectic solvent extract was detected according to the above-mentioned sodium nitrite-aluminum nitrate colorimetric method.

[0064] from Figure 3 It can be observed that the concentration of total flavonoids in Psoralea corylifolia changes with the increase of the molar ratio. From 1:1 to 1:2, the concentration increases significantly, and then decreases slightly at 1:03. When the molar ratio increases to 2:1 and 3:1, the concentration decreases further. This indicates that there is an optimal molar ratio that allows the concentration to reach a maximum value. It can be seen from the data that this optimal molar ratio is 1:2, because at this molar ratio, the average concentration is the highest, at 1.1820 mg / mL. This shows that under this experimental condition, a composition ratio of 1:2 is optimal.

[0065] Example 6 Effect of solid-liquid ratio on the efficiency of extraction of flavonoids from bamboo leaves by ultrasound-assisted deep eutectic solvent

[0066] The choline chloride-N-methylurea low eutectic solvent prepared in Example 1 was added to a centrifuge tube containing 0.5 g of light bamboo leaf powder. The solid-liquid ratio (in g / mL) was 1:5, 1:10, 1:15, 1:25, 1:35, 1:45, the molar ratio was 1:2, the water content was 20%, and the solid-liquid ratio was 1:10. Use an ultrasonicator, the temperature is 60°C, and the extraction is 30 minutes. Then, the extract was centrifuged at 6000 rpm at 4°C for 10 minutes. The supernatant was collected for further analysis. The total flavonoid content in each low eutectic solvent extract was detected according to the above-mentioned sodium nitrite-aluminum nitrate colorimetric method.

[0067] from Figure 4 It can be observed that with the increase of solid-liquid ratio, the concentration of total flavonoids from Bambusa chinensis showed an upward trend. However, in fact, when the solid-liquid ratio increased from 1:10 to 1:45, the total flavonoids from Bambusa chinensis only increased from 1.0845 mg / mL to 1.1997 mg / mL, proving that the solid-liquid ratio has little effect on the efficiency of extraction of flavonoids from Bambusa chinensis by ultrasound-assisted deep eutectic solvent.

[0068] Example 7 Effect of extraction temperature on the efficiency of extraction of flavonoids from bamboo leaves by ultrasound-assisted deep eutectic solvent

[0069] The choline chloride-N-methylurea low eutectic solvent prepared in Example 1 was added to a centrifuge tube containing 0.5 g of light bamboo leaf powder. The variables were extraction temperatures of 50°C, 55°C, 60°C, 65°C, 70°C and 75°C, a molar ratio of 1:2, a water content of 20%, and a solid-liquid ratio of 1:10. Using an ultrasonicator, the extraction was performed for 30 min. The extract was then centrifuged at 6000 rpm for 10 minutes at 4°C. The supernatant was collected for further analysis. The total flavonoid content in each low eutectic solvent extract was detected according to the above-mentioned sodium nitrite-aluminum nitrate colorimetric method.

[0070] from Figure 5 It can be observed that the concentration changes with increasing temperature. From 50°C to 70°C, the concentration generally shows an increasing trend, but it decreases at 75°C and increases at 80°C. This indicates that there is an optimal temperature range where the concentration reaches its maximum value. From the data, it can be seen that the average concentration at 70°C is the highest, which is 1.7181 mg / mL, followed by 65°C and 80°C. It can be concluded that temperature has a significant effect on concentration, and there is an optimal temperature that maximizes the concentration. In this experiment, the optimal temperature is 70°C.

[0071] Example 8 Effect of Ultrasonic Time on Ultrasonic-Assisted Deep Eutectic Solvent Extraction Efficiency of Flavonoids from Bambusa chinensis Leaves

[0072] The choline chloride-N-methylurea low eutectic solvent prepared in Example 1 was added to a centrifuge tube containing 0.5 g of light bamboo leaf powder. The variables were extraction time of 10 min, 20 min, 30 min, 40 min, 50 min and 60 min, a molar ratio of 1:2, a water content of 20%, and a solid-liquid ratio of 1:10. Using an ultrasonicator, the extraction temperature was 70°C and the extraction was performed for 30 min. The extract was then centrifuged at 6000 rpm at 4°C for 10 minutes. The supernatant was collected for further analysis. The total flavonoid content in each low eutectic solvent extract was detected according to the above-mentioned sodium nitrite-aluminum nitrate colorimetric method.

[0073] from Figure 6 It can be observed that the concentration of flavonoids in the leaves of Bamboo Shoots changes with the increase of extraction time. From 10 minutes to 50 minutes, the concentration generally shows an increasing trend, but it decreases slightly at 60 minutes, and then decreases again at 70 minutes. This indicates that there is an optimal extraction time that allows the concentration of flavonoids in the leaves of Bamboo Shoots to reach its maximum value. From the data, it can be seen that the average concentration at 50 minutes is the highest, which is 1.2415 mg / mL, followed by 60 minutes. It is concluded that the extraction time has a significant effect on the concentration of flavonoids in the leaves of Bamboo Shoots, and there is an optimal extraction time that maximizes the concentration. In this experiment, the optimal extraction time is 50 minutes.

[0074] Based on Examples 4 to 8, it can be concluded that the best method for extracting flavonoids from bamboo leaves with ultrasound-assisted deep eutectic solvent is as follows: the deep eutectic solvent comprises choline chloride: N-methylurea in a molar ratio of 1:2, a water content of 20%, and a solid-liquid ratio of 1:10. An ultrasonic instrument is used, the extraction temperature is 70°C, and the extraction time is 30 minutes.

[0075] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A deep eutectic solvent, characterized in that The deep eutectic solvent uses choline chloride as a hydrogen bond acceptor and any one of an amide compound, an alcohol compound, a carboxylic acid compound and a saccharide compound as a hydrogen bond donor; In the deep eutectic solvent, the molar ratio of choline chloride to amide compounds is 1:2, the molar ratio of choline chloride to alcohol compounds is 1:2, the molar ratio of choline chloride to carboxylic acid compounds is 1:1, and the molar ratio of choline chloride to glycosyl compounds is (1-3):1; Wherein, the amide compounds include urea, N-methylurea and acetamide; The alcohol compounds include glycerol, 1,2-propylene glycol, 1,4-butylene glycol and ethylene glycol; The carboxylic acid compounds include lactic acid, oxalic acid, malonic acid and citric acid; The saccharide compounds include glucose, maltose, fructose and sucrose.

2. The deep eutectic solvent according to claim 1, characterized in that The deep eutectic solvent includes choline chloride-urea, choline chloride-N-methylurea, choline chloride-acetamide, choline chloride-glycerol, choline chloride-1,2-propylene glycol, choline chloride-1,4-butanediol, choline chloride-ethylene glycol, choline chloride-lactic acid, choline chloride-oxalic acid, choline chloride-malonic acid, choline chloride-citric acid, choline chloride-glucose, choline chloride-maltose, choline chloride-fructose and choline chloride-sucrose; The molar ratio of choline chloride to glucose in the choline chloride-glucose is 2:1, the molar ratio of choline chloride to maltose in the choline chloride-maltose is 3:1, the molar ratio of choline chloride to fructose in the choline chloride-fructose is 1:1, and the molar ratio of choline chloride to sucrose in the choline chloride-sucrose is 2:

1.

3. The method for preparing a deep eutectic solvent according to claim 1 or 2, characterized in that: The preparation method comprises: Choline chloride is used as a hydrogen bond acceptor, and any one of an amide compound, an alcohol compound, a carboxylic acid compound and a glycosyl compound is mixed in a corresponding molar ratio to obtain a mixture; 20% w / w deionized water is added to the mixture, heated to 80° C., and stirred until transparent, uniform and stable to obtain the low eutectic solvent.

4. Use of the deep eutectic solvent as claimed in claim 1 or 2 in the field of traditional Chinese medicine extraction.

5. Use of the deep eutectic solvent as claimed in claim 1 or 2 in extracting flavonoids from Pleistocene chinensis leaves.

6. The use according to claim 5, characterized in that: The extraction method comprises: Drying, crushing and screening the leaves of the bamboo to obtain coarse powder of the leaves of the bamboo; The crude powder of the leaves of the bamboo is extracted with the low eutectic solvent by a heat extraction method, and the obtained extract is filtered and combined to obtain the leaves of the bamboo extract.

7. The use according to claim 6, characterized in that: The temperature of the drying treatment is 40°C to 60°C, and the time is 18h to 24h; the crushing and screening treatment is specifically 40 to 60 mesh sieve.

8. The use according to claim 6, characterized in that: In the thermal extraction method, the mass volume ratio (solid-liquid ratio) of the crude powder of light bamboo leaves to the low eutectic solvent is 1g:10mL; the extraction temperature is 60°C; and the extraction time is 30min.

9. The use according to claim 6, characterized in that: The extraction method further comprises: detecting the content of flavonoids in the Bambusa truncatula extract extracted by the deep eutectic solvent by sodium nitrite-aluminum nitrate colorimetry; The sodium nitrite-aluminum nitrate colorimetric method specifically includes: taking 1 mL of the light bamboo leaf extract, then mixing it with 0.3 mL of 5% sodium nitrite, and then adding 0.3 mL of 10% aluminum nitrate, 4.0 mL of 4% sodium hydroxide and 4.4 mL of distilled water; after incubating at room temperature for 15 minutes, recording the absorbance at 510 nm.

10. The use according to claim 6, characterized in that: The deep eutectic solvent is choline chloride-N-methylurea.

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