A deep eutectic solvent for extracting aescin from seeds of Sauvignon ovata and a method for extracting and enriching aescin from the same

Abstract

The present invention discloses a low eutectic solvent for extracting aescin from saro seeds and a method for extracting and recovering aescin, wherein the low eutectic solvent includes a hydrogen bond donor of butanediol or n-propanol, and a hydrogen bond acceptor of lactic acid or acetamide. The present invention crushes and sieves the saro seeds, adds a DES-water mixed extract, and performs ultrasonic water bath extraction; the extract is filtered, and the filtrate is centrifuged to collect the supernatant, which is the aescin extract; the above extract is subjected to column chromatography recovery to achieve almost complete recovery. The present invention develops a reusable, non-exhaustible, and more efficient low eutectic solvent-assisted ultrasonic extraction method, and obtains an aescin extraction rate that is much higher than the method reported in the pharmacopoeia or literature; further adopts a macroporous resin, uses an environmentally friendly, reusable column chromatography method, and achieves almost complete recovery, solving the problem of difficulty in separating the extract that is common in DES solvent extraction.

Description

Technical Field

[0001] The invention relates to the utilization of medicinal and edible resources, and in particular to a low eutectic solvent for extracting aescin from sauerkraut seeds and a method for extracting and enriching aescin. Background Art

[0002] As a traditional Chinese medicine, the Chinese medicine classic "Supplement to Compendium of Materia Medica" records that Sauerkraut seeds have therapeutic effects on heart and stomach cold pain. In recent years, experts and scholars at home and abroad have conducted a series of studies on its active ingredients, pharmacological activities and clinical applications, confirming that aescin is indeed the active ingredient of Sauerkraut seeds, which has anti-inflammatory, anti-tumor, neuroprotective, gastrointestinal protective, analgesic, antiviral, energy metabolism regulating and vascular tone enhancing effects [Chen Changjun, Xu Weihua, Zhang Mengchen, et al. Research progress on the pharmacological activity and clinical application of aescin preparations [J]. Journal of Wuhan University (Medical Edition), 2020, 41(01): 157-163.]. It is also used clinically for thrombotic diseases such as cerebral thrombosis [Fang Zebo, Hou Yuqing, Xie Jinguo. Clinical study on the treatment of hypertensive cerebral hemorrhage with sodium aescinate injection combined with nicardipine hydrochloride injection [J]. Chinese Journal of Clinical Pharmacology, 2018, 34(06): 618-621.], the regulation of intestinal dysfunction [Zhang Baoshan. Analysis of the effect of octreotide combined with sodium aescinate in the treatment of adhesive intestinal obstruction [J]. Primary Medicine Forum, 2016, 20(12): 1628.] and the treatment of various inflammations. It is a drug with a wide range of pharmacological effects and application prospects. Escin is the main active ingredient in Sauvignon ovata seeds. It is a general name for more than 30 triterpenoid saponin compounds extracted from Sauvignon ovata seeds. Its main forms are escin A (escin Ia), escin B (escin Ib), escin C (isoescin Ia), and escin D (isoescin Ib). Its structural formula is as follows Figure 1As shown. Aescin A and aescin B are also known as β-aescin, which are the two aescins with the highest content; aescin C and aescin D are also known as α-aescin, and their content is second only to aescin A and aescin B. These four aescins account for more than 60% of the total aescins [Gao Jing. Research on fingerprint of sauerkraut seeds and extraction and purification process of active ingredient aescin [D]. Beijing University of Chinese Medicine, 2018: Part I Literature Review 12-42.]. Deep eutectic solvent (DES) is a green solvent formed by the synthesis of intermolecular hydrogen bonds by hydrogen bond donors and hydrogen bond acceptors according to a certain amount of substance ratio. Typical HBA (hydrogen electron acceptors) are substances such as quaternary ammonium salts, and typical HBD (hydrogen electron donors) include substances such as polyols, organic acids and sugars. The solid raw materials are combined to produce a low eutectic mixture. Hydrogen bonds or van der Waals forces will interfere with the crystallization ability of the initial solid raw materials, so the mixture is liquid at ambient temperature and exhibits excellent solvent properties. The main applications of low eutectic solvents are transition metal catalytic reactions and acid-base catalytic reactions. Because of their excellent biocompatibility, they have been widely used in organic synthesis and extraction and separation of natural drug products in recent years. DES can also increase the extraction rate by destroying cell walls, so the application of DES greatly improves the extraction efficiency. However, more important than improving the extraction efficiency is that the obtained extract maintains its biological effect while being harmless to the human body and the environment. DES has low toxicity and is not volatile. In most cases, the extraction rate can be increased in a shorter time.

[0003] The current extraction and preparation methods of aescin include ethanol reflux extraction, methanol ultrasonic extraction, organic solvent extraction, and preparative liquid chromatography:

[0004] 1. Ethanol reflux extraction method: Orthogonal experiments were designed for ethanol concentration, solid-liquid ratio, extraction time, and extraction times. The conclusion is that the best extraction effect can be obtained by using 80% ethanol, 10 times the amount, and extracting twice for 1 hour, that is, the paste rate is 23.6% in the literature, and the contents of aescin A and B in the paste are 12.5% ​​and 5.23%. [Gao Jing, Ding Nan, Chen Danyang, et al. Study on ethanol extraction technology of aescin from Sauerkraut seeds [J]. Modern Chinese Medicine Research and Practice, 2018, 32(02): 34-36.]. Referring to this method, the experimental verification of the extraction rate is about 3%, that is, 30 mg of aescin can be extracted from 1g of Sauerkraut seeds powder.

[0005] 2. Methanol ultrasonic extraction method: 1:10 solid-liquid ratio, 25℃ methanol ultrasonic extraction for 30min (power 250W, frequency 33kHz) is the extraction method for laboratory measurement of aescin content in Sauerkraut seed powder [Nie Fang, Lv Weiqi, Tang Shengling. Simultaneous determination of 4 aescins in Sauerkraut seeds from 14 origins by HPLC [J]. Chinese Traditional Patent Medicine, 2018, 40(12): 2703-2708.]. The literature shows that the content is between 3.1% and 5.6%. With reference to this method, the experimental verification shows that the extraction rate is about 4%, that is, 40mg aescin can be extracted from 1g of Sauerkraut seed powder.

[0006] The above two extraction methods have their own disadvantages. The ethanol reflux extraction method, which is commonly used in industrialization, has the disadvantages of high temperature and long heating time, which destroys aescin, and easily converts β-aescin to α-aescin, and the extraction rate is not high; methanol ultrasonic extraction has the disadvantages of high noise, cannot be mass-produced, and methanol has certain toxicity, so it is only used for content determination in the laboratory.

[0007] 3. Organic solvent extraction: After the intermediate product is extracted with methanol or ethanol, it is usually purified by organic solvent extraction in industry. Chloroform, dichloromethane and ethyl acetate are commonly used for extraction. For example, in the patent application "A method for preparing sodium aescinate" (application publication number CN 108218948 A), the preparation process is as follows: 1kg powder, 1:4 solid-liquid ratio, 50% methanol extraction at room temperature for 12h, extraction three times, the second and third 6h, and the filtrate is combined, and the solution is concentrated under reduced pressure until foam is generated, and the pH is adjusted to about 3 with saturated oxalic acid solution. (1) A large amount of solid is generated by dripping chloroform, centrifugation is performed to obtain a crude solid of aescinate, 100ml of water is added, and the pH is adjusted to about 6 with 1% sodium hydroxide, and then 60g of needle-use activated carbon is added for decolorization for 2 hours, filtered, and the filtrate is concentrated under reduced pressure to about 100ml, and the solution is dripped into 1L of acetone, centrifuged and dried in vacuum to obtain a crude solid of sodium aescinate. The sodium aescinate content is 98.5%; (2) 300ml of dichloromethane is added dropwise, a large amount of solid is produced, and centrifugal filtration is performed to obtain aescin. 100ml of water is added thereto, and the pH is adjusted to about 5 with 1% sodium hydroxide, and then 80g of needle-grade activated carbon is added for decolorization for 6 hours, filtered, and the filtrate is concentrated under reduced pressure to about 100ml, and the solution is dropped into 1L of acetone, and centrifugal filtration and vacuum drying are performed to obtain a crude sodium aescinate. The sodium aescinate content is 98.0%; (3) 300ml of ethyl acetate is added dropwise, a large amount of solid is produced, and centrifugal filtration is performed to obtain aescin. 100ml of water is added thereto, and the pH is adjusted to about 7 with 1% sodium hydroxide, and then 100g of needle-grade activated carbon is added for decolorization for 4 hours, filtered, and the filtrate is concentrated under reduced pressure to about 100ml, and the solution is dropped into acetone, filtered, and vacuum dried to obtain a crude sodium aescinate. The sodium aescinate content is 98.5%. The extraction method requires a large amount of organic solvents, and the recovery of solvents consumes a lot of energy. It is highly polluting to the environment, has many process steps, is time-consuming, and has high costs. According to the EU 2009 / 32 / EC limit on residues in food, the three commonly used organic solvents cannot exceed 10 mg / kg. Chloroform, in particular, is a Class II solvent that is controlled for use in raw materials. It is also a restricted raw material for making drugs and should not be used in large quantities in production. Although the extraction method is relatively simple and the finished product purity is relatively high, it is very harmful to the human body and the environment.

[0008] 4. Preparative liquid chromatography: It can obtain high-purity monomer substances, but it is inefficient and costly, and is not suitable for large-scale industrial production. Summary of the invention

[0009] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a low eutectic solvent for extracting aescin from Sauvignon ovata seeds, and based on the low eutectic solvent, a reusable, non-excreted and more efficient low eutectic solvent-assisted ultrasonic extraction method is developed, and the aescin extraction rate is much higher than the extraction process / method reported in the pharmacopoeia or literature, while still maintaining a high proportion of aescin A and B; further, by optimizing the recovery process and using an environmentally friendly, reusable column chromatography method, a recovery rate of more than 90% (even up to 100%) is obtained, further overcoming the disadvantage that the extract of the low eutectic solvent is difficult to recover and enrich.

[0010] The extraction and recovery method of the present invention can be used as a complete aescin extraction process flow for manufacturing a medicinal raw material of the effective part of aescin, and the process is characterized by no emission, green and efficient extraction, and the extraction rate is much higher than that reported in domestic and foreign literature, and effectively overcomes the disadvantages of the existing aescin extraction process in Salacia chinensis, such as low efficiency, large amount of organic solvent used, and environmental unfriendliness.

[0011] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses a low eutectic solvent for extracting aescin from Sphaerocarpus rotundus, wherein the low eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor is butanediol or n-propanol, and the hydrogen bond acceptor is lactic acid or acetamide, and the molar ratio thereof is 1-2:2-1.

[0012] Wherein, the low eutectic solvent is butanediol and lactic acid, and the molar ratio thereof is equal.

[0013] The method for preparing the deep eutectic solvent for extracting aescin from Sphaerocarpus rotundus according to the present invention comprises the following steps:

[0014] The hydrogen bond donor and the hydrogen bond acceptor are mixed, heated in a water bath and stirred to make the solution uniform, which is a low eutectic solvent.

[0015] Furthermore, the water bath heating temperature is 60-70°C and the time is 0.5h-1h.

[0016] The method for extracting and purifying aescin from seeds of Smilax china is characterized by comprising the following steps:

[0017] (1) preparing a DES-water mixed extract of the deep eutectic solvent;

[0018] (2) crushing and sieving the seeds of Sauvignon ovata, adding DES-water mixed extract, and mixing the two thoroughly for ultrasonic water bath extraction;

[0019] (3) filtering the extract, centrifuging the filtrate and collecting the supernatant, which is the aescin extract;

[0020] (4) The extract is recovered and enriched by chromatography.

[0021] Wherein, the water content in the DES-water mixed extract of the low eutectic solvent in step (1) is 30-70% by volume.

[0022] Wherein, in step (2), after the pulverized and sieved saro seeds, the mass volume ratio of the mixed extract with DES-water is 1:5-30 g / mL.

[0023] The conditions for ultrasonic water bath extraction in step (2) are as follows: ultrasonic power of 10-40 kHz, temperature of 25° C. to 80° C., and extraction time of 10-50 min.

[0024] Preferably, the conditions for the ultrasonic waterbath extraction are: ultrasonic power of 30-40 kHz, temperature of 50° C. to 70° C., and extraction time of 10-30 min.

[0025] Furthermore, the extract is filtered through 3-6 layers of gauze and centrifuged in a centrifuge to collect the supernatant, wherein the centrifugal speed is 2000-5000 rpm and the centrifugal time is 10-40 min.

[0026] The total time for the extraction and chromatography steps does not exceed 17 h.

[0027] Wherein, the chromatography time in step (5) is controlled at 12-16 hours, and the macroporous resin used is AB-8 macroporous resin.

[0028] Furthermore, the chromatography comprises the following steps: (1) column loading: loading the washed resin into the chromatography column to 4 / 5 of the column height (e.g., 50 cm column plus 40 cm resin); (2) adsorption: adding the supernatant after extraction and centrifugation as the loading liquid to the chromatography column for adsorption, with a flow rate of 5 ml / min to 20 ml / min, and stopping loading after reaching the leakage point; (3) washing: washing the resin with purified water until the effluent is clear; (4) elution: eluting with an ethanol solution with a volume percentage of 10-40%, a total of 4-6 times the column volume, and a flow rate of 5 ml / min to 20 ml / min to elute impurities; eluting with an ethanol solution with a volume percentage of 50-90%, a total of 3-7 times the column volume, and a flow rate of 5 ml / min to 20 ml / min to elute the target product. (5) collection: collecting the 50-90% ethanol effluent.

[0029] In the present invention, the preliminarily dried and crushed sauerkraut seeds powder is firstly taken, an appropriate amount of aqueous DES solution is added, ultrasonic water bath extraction is performed, a suitable DES is screened, the extraction conditions are further optimized, and recovery is performed with a macroporous resin, a suitable macroporous resin is screened, and the adsorption and elution conditions are optimized to achieve a recovery rate of 100%. The extraction rate of the aescin recovered by the method of the present invention is far higher than the extraction method for determining the content in the Chinese Pharmacopoeia, and the selection of the macroporous resin enables the recovery rate to reach more than 90%, or even 100%.

[0030] The present invention provides a method and application of extracting aescin from smilax china seed powder with a specific low eutectic solvent. The extraction is carried out using a low eutectic solvent-water mixture, and an extraction rate is obtained that is much higher than the results reported in various literatures. On this basis, the process flow of recovery, enrichment and purification is optimized, which solves the problem of difficulty in recovering the low eutectic solvent extract and obtains a higher recovery rate and higher purity.

[0031] The present invention adopts a low eutectic solvent for the first time to extract aescin from Smilax china seed powder. By using a low eutectic solvent to assist ultrasonic extraction of aescin, the crude extraction rate is higher than the best extraction rate reported in the literature, and after optimization by CCD response surface method, the extraction rate is greatly improved to 61.29-87.95 mg / g, which is much higher than the pharmacopoeia test method (30-40 mg / g extraction rate). At the same time, the proportion of aescin A and B in the four types of aescin A, B, C, and D is still maintained at more than 50%, which is a significant improvement.

[0032] At the same time, the present invention uses a specific AB-8 macroporous resin to recover aescin from the low eutectic solvent extract, optimizes the conditions, and obtains a complete recovery effect, which solves the disadvantages that it is difficult to recover the target substance from the low eutectic solvent and it cannot be completely recovered. The prior art also uses macroporous resin to recover the target substance from the low eutectic solvent extract, but it has not been found that it can be almost completely recovered, and basically only about half can be recovered. The present invention uses AB-8 macroporous resin, which can be completely recovered from the low eutectic solvent after optimization, which is an unexpected effect.

[0033] The present invention can extract aescin from the radix strychnifoliae powder substantially and completely through a single extraction with DES, with high efficiency and fast speed. In addition, DES can be reused at least 3-4 times, and its extraction efficiency is almost the same as that of the new DES, which is truly green, low-consumption and environmentally friendly. The present invention can extract aescin more quickly and completely, thereby achieving the purpose of efficient extraction, which is truly green, low-consumption and environmentally friendly.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] The normal traditional extraction method is methanol ultrasonic extraction or ethanol reflux extraction. With aescin A, B, C, and D as the measurement index of aescin, the general extraction rate is 3-4%, i.e., 30-40 mg / g. However, the present invention adopts a specific extraction method of a specific low eutectic solvent. If the influence of the limit case on aescin A and B is not considered to cause its conversion, the maximum value can reach 8.8%, i.e., 88 mg / g, through response surface design (the increase of 1% extraction rate of aescin in the present invention represents a significant improvement in the effect of the prior art. Specifically, the original 4% extraction rate has reached 8%. Although the extraction rate has only increased by 4%, the efficiency has been improved by 100%), which is much higher than the extraction method of the traditional solvent, and the proportion of aescin A and B can still be kept above 50%. In the case of comprehensively considering the extraction rate and maximizing the proportion of aescin A and B, the extraction rate can also reach 61 mg / g through response surface design, which is still much higher than the traditional extraction method.

[0036] Although low eutectic solvents have high extraction efficiency, many related literatures on the extraction of natural products using low eutectic solvents lack a suitable method for recovering the extracts from the low eutectic solvents. Using macroporous resin to recover the target extract is a common method, but when the target extract in the low eutectic solvent is recovered by macroporous resin, the recovery rate is generally low, which is why low eutectic solvents have high efficiency in extracting natural products but are rarely used in actual production. In the present invention, the recovery rate of four types of aescin by macroporous resin is 95-100%, which almost achieves the effect of complete recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The chemical structures of aescin A, B, C, and D;

[0038] Figure 2 A is a high performance liquid chromatogram of a mixture of four monomers of aescin ABCD; B is a high performance liquid chromatogram of a 97% standard product of aescin produced by Yuanye Company; C is a high performance liquid chromatogram of a 97% standard product of sodium aescinate produced by Yuanye Company; D is a high performance liquid chromatogram of the recovered product of Example 3 of the present invention;

[0039] Figure 3 is the static adsorption recovery rate of different macroporous resins, different letters indicate significant differences (p<0.05), and the same letters indicate insignificant differences;

[0040] Figure 4 It is the adsorption rate of eluent with different column volumes after dynamic adsorption on AB-8 macroporous resin;

[0041] Figure 5 It is the dynamic adsorption gradient elution and single elution recovery rate. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0043] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0044] Experimental materials:

[0045] The raw material of the experiment, the saro seed powder, can be derived from common commercial products, and the present invention collects it from Hanzhong, Shaanxi.

[0046] Among them, the detection method of the four types of aescin ABCD refers to the HPLC method that started after the "Pharmacopoeia of the People's Republic of China (2015 Edition)". Specifically, the system with a mobile phase of acetonitrile-water (0.2% phosphoric acid) ratio of 36:64 was used for measurement. The specific conditions are the mobile phase acetonitrile-water (0.2% phosphoric acid) ratio of 36:64; volume flow rate 1.0mL / min; detection wavelength 220nm; column temperature 25℃; injection volume 10μL.

[0047] Take four kinds of aescin (aescin A, B, C, D) standard powders, dissolve them in methanol, prepare 10mg / ml stocks respectively, and then mix them to prepare four kinds of aescin containing 1mg / ml, 0.5mg / ml, 0.4mg / ml, 0.3mg / ml, 0.2mg / ml, 0.1mg / ml, 0.05mg / ml gradient standards. Draw the standard curve with mass concentration as the horizontal axis and peak area as the vertical axis, and get the regression equation, as shown in Table 1.

[0048] Table 1 shows the regression equations of four types of aescin

[0049]

[0050] Example 1

[0051] (1) Preparation of DES and Shorea seed powder: Butanediol and lactic acid were mixed in a 1:1 molar ratio and stirred in a 60°C water bath for half an hour to make the solution uniform. The DES preparation was completed. Shorea seed powder was a powder of 80-100 mesh that was dried, crushed and sieved. (2) DES was added with water to prepare a DES-water mixed extract with a moisture content of 42.5%. According to the material-liquid ratio of 1:25g / ml, 100ml was taken to extract 4g of powder. After the two were fully mixed, they were put into a glass bottle and placed in an ultrasonic water bath (600W, 40kHz) at 70°C for extraction for 27.5min. The extract was filtered through 6 layers of medical gauze, the filtrate was collected and centrifuged in a centrifuge at 4000rpm for 10min, and the supernatant was collected again as the extract. At this time, the total content of the four types of aescin ABCD was 3.52 mg / ml, totaling 309.57 mg, and the extraction rate was 8.79%, or 87.95 mg / g, far exceeding the efficiency of traditional methanol ultrasonic extraction and ethanol reflux extraction.

[0052] At the same time, aescin A and B account for 53.94% of the four aescins A, B, C, and D. This is an extraction partial scheme that only considers the extraction rate, but compared with the existing ethanol extraction scheme, the result of reference patent CN104402963 is 45-60%, which shows that the extraction method of this embodiment can not only achieve an extremely high extraction rate, but also ensure that aescin A and B have a higher proportion in the four aescins A, B, C, and D.

[0053] Example 2

[0054] (1) Preparation of DES and Shorea seed powder: Butanediol and lactic acid were mixed in a 1:1 molar ratio and stirred in a 70°C water bath for half an hour to make the solution uniform. The DES preparation was completed. Shorea seed powder was a powder of 80-100 mesh that was dried, crushed and sieved. (2) DES was added with water to prepare a DES-water mixed extract with a moisture content of 48.5%. According to the material-liquid ratio of 1:14g / ml, 100ml DES was used to extract 7.14g of powder. After the two were fully mixed, they were put into a glass bottle and extracted in an ultrasonic water bath (600W, 40kHz) at 70°C for 10 minutes. The extract was filtered through 6 layers of medical gauze, the filtrate was collected and centrifuged in a centrifuge at 4000rpm for 10 minutes, and the supernatant was collected again as the extract. At this time, the total content of the four main aescins A, B, C, and D was 4.38 mg / ml, totaling 354.48 mg, and the extraction rate was 6.13%, or 61.292 mg / g, far exceeding the efficiency of traditional methanol ultrasonic extraction and ethanol reflux extraction.

[0055] At the same time, in this embodiment, aescin A and B account for 57.74% of the four aescins A, B, C, and D. This is an extraction partial scheme that comprehensively considers the extraction rate and the AB ratio. Referring to the results of patent CN104402963, the conventional ethanol extraction has an AB ratio of 45-60%. The extraction method of this embodiment can still maintain a very high extraction rate, and at the same time, it can also increase the proportion of aescin A and B in the four aescins A, B, C, and D.

[0056] Example 3

[0057] (1) Preparation of DES and Shorea seed powder: Butanediol and lactic acid were mixed in a 1:1 molar ratio and stirred in a 70°C water bath for half an hour to make the solution uniform, and the DES preparation was completed. Shorea seed powder was a powder of 80-100 mesh that was dried, crushed and sieved. (2) DES was added with water to prepare a DES-water mixed extract with a moisture content of 48.5%. According to the material-liquid ratio of 1:14g / ml, 2800ml was taken to extract 200g of powder. After the two were fully mixed, they were put into a glass bottle and placed in an ultrasonic water bath (600W, 40kHz) at 70°C for extraction for 10 minutes. The extract was filtered through 6 layers of medical gauze, the supernatant was collected and centrifuged in a centrifuge at 4000rpm for 10 minutes, and the supernatant was collected again as the extract. At this time, the total content of the four main aescins A, B, C, and D is 5.001 mg / ml, a total of 11068.24 mg, and the extraction rate is 5.53%, i.e., 55.34 mg / g, far exceeding the traditional ethanol reflux extraction efficiency. At the same time, among the four aescins, A and B account for 58.64%. This embodiment is based on Example 2 and further expands the production and extraction scale, which not only maintains a very high extraction rate, but also ensures the proportion of aescin A and B in the four aescins A, B, C, and D.

[0058] (2) Chromatographic recovery and enrichment: The above extract is set aside. Pre-treat the macroporous resin, soak the AB-8 macroporous resin in ethanol for 4 hours, and wash it with water until there is no alcohol smell. Then use the wet method to fill the column, fill it into a glass column with an inner diameter of 6 cm, and make it 27 cm high, compact it, and spread a layer of quartz sand on the top. Wash the macroporous resin column again with deionized water until the effluent is clear, and then use one column volume of DES-water mixed solution with a DES content of 51.5% to balance the resin column (the actual DES content of the sample solution is also 51.5%). Then add the sample solution (the extract solution in step (1)) to the chromatography column, with a sample volume of 3 column volumes, control the flow rate to 15 ml / min, slowly flow out, and collect the effluent. The effluent can be recovered and used for extraction again. After testing, the extraction efficiency will not decrease, so it can be reused without being discharged into the environment to cause pollution, and it will hardly evaporate. The resin column was washed with two column volumes of deionized water, and the low polar impurities adsorbed on the macroporous resin column were washed with four column volumes of 30% ethanol aqueous solution, with a flow rate of 15 ml / min. The aescin enriched on the resin column was eluted and recovered with five column volumes of 60% ethanol aqueous solution, with a flow rate of 15 ml / min. At this time, the eluted product was basically most of the aescin and pigment, and a small part of impurities with polarity similar to aescin. The entire chromatography time was controlled at 12 hours, and the five column volumes of eluent were mixed, reaching a recovery rate of 101.81%, which was close to 100%, extremely high and excellent. Figure 2 A is the HPLC chromatogram of the mixture of four monomers of aescin, ABCD; B is the HPLC chromatogram of 97% aescin standard product of Yuanye Company; C is the HPLC chromatogram of 97% aescin sodium standard product of Yuanye Company; D is the HPLC chromatogram of the recovered product of Example 3; it proves that the recovered product of the example is consistent with the standard product.

[0059] This example is a larger-scale extraction, and verifies the ability of macroporous resin to recover DES extracts. In fact, the biggest problem with DES extraction in the prior art is that the extracted substances are difficult to separate and recover, and the recovery efficiency is almost only about half. This solution can achieve complete recovery using conventional macroporous resin and ethanol elution.

[0060] Example 4

[0061] Step (1) of this embodiment adopts the same method as that of Embodiment 3.

[0062] (2) Macroporous resin column chromatography recovery: The extract is used as the loading solution for standby use. Pre-treat the macroporous resin, soak the AB-8 macroporous resin in ethanol for 4 hours, and wash it with water until there is no alcohol smell. Then use the wet method to fill the column, fill it into a glass column with an inner diameter of 6 cm, and make it 27 cm high. Compact it, and spread a layer of quartz sand on the top. Wash the macroporous resin column again with deionized water until the effluent is clear, and then balance it with one column volume of DES-water mixed solution with a DES content of 51.5% (the actual DES content of the loading solution is 51.5%). The loading solution is added to the chromatography column, with a loading amount of 3 column volumes, and the flow rate is controlled to 15 ml / min. Slowly flow out for adsorption, and the effluent is collected. The effluent after adsorption by the macroporous resin is used as a recoverable DES extract and extracted again. The resin column was washed with two column volumes of deionized water at a natural flow rate, and the impurities adsorbed on the macroporous resin column were washed with four column volumes of 30% ethanol aqueous solution at a flow rate of 15 ml / min. The aescin enriched on the resin column was eluted with five column volumes of 60% ethanol aqueous solution at a flow rate of 15 ml / min. At this time, the eluted product was basically most of the aescin and pigments, and a small part of impurities with polarity similar to aescin. The whole chromatography time was controlled at 12h each time. After the macroporous resin was repeatedly treated for 5 times, five times the column volume was mixed, and a recovery rate of 88.42% was achieved. The recovery rate was slightly reduced here because the macroporous resin had been reused 5 times in this experiment without extreme conditions of regeneration. Because the macroporous resin was reused 5 times, the recovery rate decreased this time, but by detecting the content of aescin in DES waste liquid, its adsorption effect was still 100%. Although the elution effect decreased, it was still close to 90% overall, and this time it was still significantly better than the existing technology in which the macroporous resin could only recover about 50% of natural drugs from DES, and the highest state was less than 80%. Its AB accounted for 57.54%, which is also a relatively high level in the existing technology.

[0063] Example 5

[0064] Reuse of DES solution: The recovered DES extract is extracted again as new DES, and is recycled and adsorbed again with AB-8 macroporous resin for reuse. The specific extraction and recovery are according to the method of Example 4. The effect of repeated use of DES on the extraction rate is shown in Table 2, and the effect of adsorption and recovery rate of macroporous resin is shown in Table 3. With reference to the two tables, the feasibility of repeated use of macroporous resin and DES is verified, which is feasible for reducing costs and protecting the environment in industrial production. In summary, the extraction efficiency will not decrease, so it can be reused without being discharged into the environment to cause pollution, and it will hardly evaporate.

[0065] Table 2 Multiple use table of DES (n=3)

[0066]

[0067] Table 3 Multiple utilization of macroporous resin (n=3)

[0068]

[0069]

[0070] Test Example 1

[0071] Under the initial screening conditions, the effects of different DES compositions and traditional extraction methods on the extraction rate of aescin were investigated using a more common extraction method. The results are shown in Table 4.

[0072] Table 4 Effect of different DES compositions on extraction effect

[0073]

[0074] The DES used is candidate DES#1, which is a mixture of butanediol and lactic acid in a molar ratio of 1:1.

[0075] (1) Preparation of DES and Shorea seed powder: Butanediol and lactic acid were mixed in a molar ratio of 1:1, and stirred in a 70°C water bath for half an hour to make the solution uniform, which means that the DES preparation was completed. Shorea seed powder was dried, crushed, and sieved to 80-100 mesh powder.

[0076] (2) DES was added with water to prepare a DES-water mixed extract with a water content of 30%. According to the solid-liquid ratio of 1:20g / mL, 10ml was taken to extract 0.5g of powder. After the two were fully mixed, they were put into a glass bottle and placed in an ultrasonic water bath (400W, 40kHz) at a temperature of 50°C for extraction for 30min. The extract was centrifuged at a speed of 4000rpm for 10min in a centrifuge, and the supernatant was collected again as the extract. At this time, the total content of the four saponins, the main saponin aescin ABCD, was 2.1725mg / ml, a total of 21.725mg, and the extraction rate was 4.35%, i.e. 43.45mg / g.

[0077] Among them, comparative example 1 is DES#2 which is a mixture of betaine and lactic acid in a 1:1 molar ratio, and the other steps are the same as the above steps (1) and (2). The total content of the four saponins, the main saponin aescin ABCD, is 1.7733 mg / ml, a total of 17.733 mg, and the extraction rate is 3.55%, i.e. 35.47 mg / g.

[0078] Among them, comparative example 2 is DES#3 which is a mixture of n-propanol and acetamide in a molar ratio of 1:1, and the other steps are the same as the above steps (1) and (2). The total content of the four saponins, the main saponin aescin ABCD, is 1.8315 mg / ml, a total of 18.315 mg, and the extraction rate is 3.66%, i.e. 36.63 mg / g.

[0079] DES#3 is a mixture of n-propanol and acetamide in a 1:1 molar ratio, DES#5 is a mixture of n-propanol and lactic acid in a 1:1 molar ratio, DES#6 is a mixture of choline chloride and ethylene glycol in a 1:1 molar ratio, DES#13 is a mixture of choline chloride and lactic acid in a 1:1 molar ratio, and DES#14 is a mixture of choline chloride and glycerol in a 1:1 molar ratio. The extraction rates of each DES are detailed in Table 4.

[0080] In this example, different DES were used to determine the extraction rate of aescin. Under general conditions, the extraction effect of butanediol and lactic acid in a molar ratio of 1:1 was the best.

[0081] Test Example 2

[0082] The results of DES#1 were further compared with those of the traditional extraction methods of methanol ultrasonic extraction and ethanol reflux extraction. Its structure is shown in Table 5 below.

[0083] Table 5 shows the effects of different extraction methods on the extraction rate

[0084]

[0085] Among them, comparative example 3 is pure methanol extraction:

[0086] (1) Preparation of methanol and smilax china seed powder: Methanol is analytical grade methanol of Tiandi brand. Smilax china seed powder is dried, crushed and sieved to 80-100 mesh powder.

[0087] (2) Methanol extraction Refer to the Chinese Pharmacopoeia, according to the solid-liquid ratio of 1:10g / mL, take 20ml to extract 2g of powder. After the two are fully mixed, put them into a glass bottle and put them into an ultrasonic water bath (400W, 40kHz) at 50℃ for extraction for 30min. The extract is centrifuged at 4000rpm for 10min, and the supernatant is collected again as the extract. At this time, the extraction rate of the four main aescins is 3.15%, that is, 31.55mg / g.

[0088] Among them, comparative example 4 is 80% methanol reflux extraction:

[0089] (1) Preparation of 80% ethanol and smilax china seed powder: ethanol is Tiandi brand analytical pure ethanol, diluted with deionized water to 80% ethanol content. Smilax china seed powder is dried, crushed and sieved 80-100 mesh powder.

[0090] (2) Ethanol reflux extraction, according to the solid-liquid ratio of 1:20g / mL, take 100ml to extract 6g of powder. After fully mixing the two, put them into the reflux extractor to form a reflux extraction for 30min, and repeat once. The extract is centrifuged at 4000rpm for 10min in a centrifuge, and the supernatant is collected again as the extract. At this time, due to the high-temperature evaporation of the organic solvent, 80% ethanol is 84ml. At this time, the extraction rate of the four main aescins is measured to be 3.79%, that is, 37.93mg / g.

[0091] The extraction efficiency of methanol or ethanol varies, but is significantly lower than that of DES#1 (extraction rate 4.54%) under the initial unoptimized general extraction conditions.

[0092] Test Example 3

[0093] The optimal DES was preliminarily screened according to Experimental Example 1, and the effects of different molar ratios of hydrogen bond donors and acceptors of different DES on the extraction rate of aescin were investigated.

[0094] (1) Preparation of DES and Shorea seed powder: Butanediol and lactic acid were mixed in a molar ratio of 1:1, and stirred in a 70°C water bath for half an hour to make the solution uniform, which means that the DES preparation was completed. Shorea seed powder was dried, crushed, and sieved to 80-100 mesh powder.

[0095] (2) DES was added with water to prepare a DES-water mixed extract with a water content of 30%. According to the solid-liquid ratio of 1:20 g / mL, 10 ml was taken to extract 0.5 g of powder. After the two were fully mixed, they were put into a glass bottle and placed in an ultrasonic water bath (400 W, 40 kHz) at a temperature of 50°C for extraction for 30 min. The extract was centrifuged at a speed of 4000 rpm for 10 min in a centrifuge, and the supernatant was collected again as the extract. At this time, the total content of the four saponins, the main saponin aescin ABCD, was 49.26 mg / g (different batches from Test Example 1), and the extraction rate was 4.93%.

[0096] Comparative Example 5 is as follows: the above method is used, except that the molar ratio of butanediol to lactic acid is 1:2.

[0097] The total content of the four saponins, the main saponin aescin ABCD, extracted by the above method, was 48.18 mg / g.

[0098] Comparative Example 6 is as follows: the above method is used, except that the molar ratio of butanediol to lactic acid is 2:1.

[0099] The total content of the four saponins, the main saponin aescin ABCD, extracted by the above method, was 49.20 mg / g.

[0100] At the same time, when the molar ratio of butanediol to lactic acid was 3:1 and the extraction was performed according to the above method, the total content of the four saponins, mainly aescin ABCD, was 49.42 mg / g.

[0101] In general, the extraction efficiency is not much different under different ratios. The extraction efficiency will be slightly improved after the addition of butanediol. Therefore, it is believed that the change in molar ratio has no effect on the extraction rate, and a 1:1 molar ratio of butanediol and lactic acid is still used.

[0102] Test Example 4

[0103] According to the optimal DES formula and ratio screened in Experimental Example 1, the effects of different DES water content ratios on the extraction rate of aescin were investigated.

[0104] (1) Preparation of DES and Shorea seed powder: Butanediol and lactic acid were mixed in a molar ratio of 1:1, and stirred in a 70°C water bath for half an hour to make the solution uniform, which means that the DES preparation was completed. Shorea seed powder was dried, crushed, and sieved to 80-100 mesh powder.

[0105] (2) DES was added with water to prepare a DES-water mixed extract with a water content of 30%. According to the solid-liquid ratio of 1:20 g / mL, 10 ml was taken to extract 0.5 g of powder. After the two were fully mixed, they were put into a glass bottle and placed in an ultrasonic water bath (400 W, 40 kHz) at a temperature of 50°C for extraction for 30 min. The extract was centrifuged at a speed of 4000 rpm for 10 min in a centrifuge, and the supernatant was collected again as the extract. At this time, the total content of the four saponins, the main saponin aescin ABCD, was 45.63 mg / g (different batches from Test Example 1), and the extraction rate was 4.56%.

[0106] Comparative Example 7: According to the above method, a DES-water mixed extract with a moisture content of 20% was prepared, and other conditions remained unchanged, and the total content of the four saponins, the main saponin aescin ABCD, was 42.55 mg / g.

[0107] Comparative Example 8 is prepared according to the above method with a DES-water mixed extract having a moisture content of 40%, and other conditions remain unchanged, to extract four saponins. The total content of the main saponin aescin ABCD is 39.75 mg / g.

[0108] Comparative Example 9 is prepared according to the above method with a DES-water mixed extract having a moisture content of 10%, and other conditions remain unchanged, to extract four saponins, and the total content of the main saponin aescin ABCD is 41.00 mg / g.

[0109] Comparative Example 10 is prepared according to the above method with a DES-water mixed extract having a moisture content of 50%, and other conditions remain unchanged, to extract four saponins. The total content of the main saponin aescin ABCD is 39.53 mg / g.

[0110] From the above experiments, it can be seen that different moisture contents of DES have a certain influence on the extraction rate. However, if the viscosity of DES is too high, it will greatly affect the mass transfer resistance. If the content of DES is too low, the extraction efficiency will also decrease. Under the above conditions, a moisture content of 30% is the best result.

[0111] Test Example 5

[0112] According to the extraction method further optimized in Experimental Example 1, the effect of extraction temperature on the extraction rate of aescin was investigated.

[0113] (1) Preparation of DES and Shorea seed powder: Butanediol and lactic acid were mixed in a molar ratio of 1:1, and stirred in a 70°C water bath for half an hour to make the solution uniform, which means that the DES preparation was completed. Shorea seed powder was dried, crushed, and sieved to 80-100 mesh powder.

[0114] (2) DES was added with water to prepare a DES-water mixed extract with a water content of 30%. According to the solid-liquid ratio of 1:20 g / mL, 10 ml was taken to extract 0.5 g of powder. After the two were fully mixed, they were put into a glass bottle and placed in an ultrasonic water bath (400 W, 40 kHz) at a temperature of 50°C for extraction for 30 minutes. The extract was centrifuged at a speed of 4000 rpm for 10 minutes, and the supernatant was collected again as the extract. The total content of the main saponin aescin ABCD of the four saponins was 40.94 mg / g (different batch from Test Example 1), and the extraction rate was 4.09%.

[0115] Comparative Example 11: According to the above method, the extraction was carried out at a temperature of 40°C for 30 minutes, and other conditions remained unchanged. The total content of the four saponins, the main saponin aescin ABCD, was 27.76 mg / g.

[0116] Comparative Example 12: According to the above method, the extraction was carried out at a temperature of 60°C for 30 minutes, and other conditions remained unchanged. The total content of the four saponins, the main saponin aescin ABCD, was 40.96 mg / g.

[0117] Comparative Example 14: According to the above method, the extraction was carried out at a temperature of 30°C for 30 minutes, and other conditions remained unchanged. The total content of the four saponins, the main saponin aescin ABCD, was 23.93 mg / g.

[0118] Comparative Example 15: According to the above method, the extraction was carried out at a temperature of 20°C for 30 minutes, and other conditions remained unchanged. The total content of the four saponins, the main saponin aescin ABCD, was 18.33 mg / g.

[0119] From the above experiments, it can be seen that the DES extraction of aescin is stable at 50°C and above, and the extraction effect is better, so considering energy saving, it can be set to 50°C.

[0120] Test Example 6

[0121] According to the extraction method further optimized in Experimental Example 1, the effect of the solid-liquid ratio on the extraction rate of aescin was investigated:

[0122] (1) Preparation of DES and Sphaerocarpus chinensis powder: Butanediol and lactic acid were mixed in a molar ratio of 1:1, and stirred in a 50°C water bath for half an hour to make the solution uniform, which means that the DES preparation was completed. Sphaerocarpus chinensis powder was dried, crushed, and sieved to 80-100 mesh powder.

[0123] (2) DES was added with water to prepare a DES-water mixed extract with a water content of 30%. According to the solid-liquid ratio of 1:20 g / mL, 10 ml was taken to extract 0.5 g of powder. After the two were fully mixed, they were put into a glass bottle and placed in an ultrasonic water bath (400 W, 40 kHz) at a temperature of 50°C for extraction for 30 minutes. The extract was centrifuged at a speed of 4000 rpm for 10 minutes in a centrifuge, and the supernatant was collected again as the extract. The total content of the main saponin aescin ABCD of the four saponins was 41.78 mg / g (different batches from Experimental Example 1), and the extraction rate was 4.18%.

[0124] Comparative Example 16: According to the above method, at a solid-liquid ratio of 1:10, 5 ml was taken to extract 0.5 g of powder, and other conditions remained unchanged. The total content of the four saponins, the main saponin aescin ABCD, was 39.86 mg / g.

[0125] Comparative Example 17: According to the above method, at a solid-liquid ratio of 1:15, 7.5 ml was taken to extract 0.5 g of powder, and other conditions remained unchanged. The total content of the four saponins, the main saponin aescin ABCD, was 41.74 mg / g.

[0126] Comparative Example 18: According to the above method, at a solid-liquid ratio of 1:7.5, 3.75 ml was taken to extract 0.5 g of powder, and other conditions remained unchanged. The total content of the four saponins, the main saponin aescin ABCD, was 39.91 mg / g.

[0127] Comparative Example 19: According to the above method, at a solid-liquid ratio of 1:5, 2.5 ml was taken to extract 0.5 g of powder, and the total content of the four saponins, the main saponin aescin ABCD, was 40.67 mg / g.

[0128] The above experiments show that the solid-liquid ratio has a certain influence on the DES extraction rate of aescin, and a solid-liquid ratio of 1:20 is the best.

[0129] Test Example 7

[0130] The extraction method was further optimized according to the above test examples 1-6, and the limitation of extraction time was taken into consideration. A general range was set for response surface design experiment and verification. Because the single factor design has the disadvantage of mutual interference among various conditions, the design is shown in Table 6 below.

[0131] Table 6 Different factor settings

[0132]

[0133]

[0134] Furthermore, on the basis of four factors and five levels, Design Expert 12.0.3 was used to design and the Central composite design (CCD) response surface experimental design method was selected. A total of 30 experimental points were arranged and the experiment was carried out according to this design. The results are shown in Table 7.

[0135] Table 7 Response surface experimental design factors and levels

[0136]

[0137]

[0138] As shown in Table 8, after multivariate nonlinear regression fitting of each experimental point, the quadratic polynomial regression equations of the extraction rate of aescin Y1 and the proportion of aescin A and B Y2 to moisture content (A), extraction temperature (B), liquid-solid ratio (C) and extraction time (D) were obtained:

[0139] y1=62.72+1.45A+1.23B+11.82C+0.4557D-0.0351AB+0.0242AC-

[0140] 0.1427AD+0.0076BC-0.2519BD+0.0534CD-0.5957A 2 -0.1866B 2 -0.0494C 2 -0.2224D 2

[0141] y2=57.81+0.6207A+0.0787B-2.81C-0.096D+0.0457AB-0.0852AC

[0142] -0.0122AD-0.0135BC-0.082BD+0.0686CD-0.1613A 2 -0.1431B 2 +0.0445C 2+0.0067D 2

[0143] The variance regression analysis showed that the model had a very high degree of fit, and finally the results in Table 8 were obtained. The actual experiment verified that the prediction was accurate.

[0144] Table 8 Comparison between the predicted values ​​of the response surface model and the actual values

[0145]

[0146] Compared with all the previous single factor analyses, the extraction rates of the results far exceeded the relevant conditions. In summary, combined with the single factor test and the response surface test, Example 1 of the present invention is the experimental scheme for the optimal extraction rate; Example 2 is the optimal extraction scheme that takes into account both the extraction rate and the proportion of aescin A and B in the four types of aescin A, B, C, and D. Example 3 is to expand the extraction and recovery of aescin on the basis of Example 2. Example 4 is to obtain a large amount of extract according to Example 3, and then pass it through the same macroporous resin column for adsorption and elution many times, and detect the changes in the adsorption rate and recovery rate after multiple uses of the macroporous resin.

[0147] Test Example 8

[0148] The extract was obtained according to Example 3, and the adsorption rates of different macroporous resins such as D101, AB-8, ADS-17, NKA-9 and polyamide on aescin were compared by static adsorption.

[0149] The specific method is as follows: According to polarity, the resins are divided into five types: non-polar, neutral, weakly polar, polar, and strongly polar. According to the instructions of various resins, one commonly used, inexpensive resin suitable for adsorbing natural products with similar structures such as triterpenes or saponins is selected for comparison for each polarity. There are five types, namely AB-8, D101, ADS-17, NKA-9, and polyamide. After each resin is activated according to the instructions, 5g of pretreated macroporous resin is weighed into a conical flask at a mass volume ratio of 1:4, and the DES extract of aescin that has been extracted and divided into 5 parts is added for adsorption. The adsorption is carried out at 150rpm for 6 hours at room temperature. After centrifugation at 4700rpm for 10 minutes, the supernatant is regarded as the effluent after adsorption, and the adsorption rate is calculated based on the aescin concentration in the sample solution and the effluent after adsorption. The resin with the highest adsorption (i.e., the lowest effluent content) is selected. After calculation, the adsorption rate (100% - the percentage of effluent in the extract content) is shown in Table 9 below and Figure 3 As shown, it is shown that AB-8 macroporous resin has the best effect in static adsorption and significantly exceeds other macroporous resins.

[0150] Table 9 Resin static adsorption rate

[0151] Resin Type D101 ADS-17 AB-8 NKA-9 Polyamide Adsorption rate 74.45％ 68.52％ 79.15％ 62.32％ 27.90％

[0152] Furthermore, AB-8 was selected for further experiments on dynamic adsorption.

[0153] ① Optimization of sample volume: Take 220 ml of excess aescin extract, take 33 ml of the optimal resin for static adsorption (ADS-17 resin) for adsorption, control the flow rate, and collect the effluent every 10 ml to determine the leakage point of the macroporous resin column, and use this as the sample volume for subsequent experiments.

[0154] ② Optimization of eluent concentration and volume: 50%, 60%, 70%, 80% ethanol, four concentrations, refer to the static adsorption experiment, use excess volume to gradient elute in sequence, sample each column volume (BV) of the eluent and measure its aescin concentration, and select the appropriate concentration of ethanol as the desorbent for elution according to the amount of aescin eluted by each concentration of ethanol. Then use the excess ethanol of this concentration to elute the resin that has been fully adsorbed once to confirm the eluent volume.

[0155] During the dynamic adsorption process, the saturation point of resin adsorption, also called the leakage point, can be determined by overloading. At a flow rate of 10 ml / min, a sample is collected every 10 ml through a macroporous resin column with a column height of 42 cm and an inner diameter of 2.8 cm. Several points with obvious changes are preliminarily screened out by colorimetry (because the impurities interfering with the colorimetric results are significantly reduced after elution, although it is still not suitable for direct quantitative use, it can be better for horizontal comparison). Through HPLC detection, the leakage point is reached when the loading amount is 3 times the column volume, as shown below Figure 4 As shown. After that, two column volumes of deionized water were used to wash away the residue, and 4 column volumes of 30% ethanol were used to wash away the impurities. Then, gradient elution was performed with 50%, 60%, and 70% ethanol for two column volumes respectively. At the same time, an excess elution with 60% ethanol was also performed. The recovery rates were calculated respectively. It was found that the recovery rate of a single 60% ethanol with 5 volumes was the highest, which could achieve complete recovery, while the gradient elution could only achieve a recovery rate of about 80%, as shown below Figure 5 shown.

Claims

1. A method for extracting and recovering aescin from seeds of Sphaerocarpus rotundus, characterized in that: The steps include: (1) preparing a DES-water mixed extract solution containing a low eutectic solvent; (2) Grind and sieve the seeds of Sauvignon ovata, add DES-water mixed extract, mix the two thoroughly and perform ultrasonic water bath extraction; (3) Filtering the extract, centrifuging the filtrate and collecting the supernatant, which is the aescin extract; (4) recovering and enriching the extract by chromatography; The deep eutectic solvent is butanediol and lactic acid, and the molar ratio thereof is equal; The water content in the DES-water mixed extract of the low eutectic solvent in step (1) is 30-70%; In step (2), after the seeds of Sauerkraut are crushed and sieved, the mass volume ratio of the mixed extract with DES-water is 1:5-30 g / mL; the conditions of ultrasonic water bath extraction in step (2) are: ultrasonic power of 10-40 kHz, temperature of 25°C-80°C, and extraction time of 10-50 min; In step (4), AB-8 macroporous resin is used for chromatography.

2. The method for extracting and recovering aescin from seeds of Sphaerocarpus ovata according to claim 1, characterized in that: The preparation method of a low eutectic solvent for extracting aescin from seeds of Sphaerocarpus rotundus comprises the following steps: The hydrogen bond donor and the hydrogen bond acceptor are mixed, heated in a water bath and stirred to make the solution uniform, which is a low eutectic solvent.

3. The method for extracting and recovering aescin from seeds of Sphaerocarpus ovata according to claim 2, characterized in that: The water bath heating temperature is 60-70°C and the heating time is 0.5h-1h.

4. The method for extracting and recovering aescin from seeds of Sphaerocarpus rotundus according to claim 1, characterized in that: The total time for extraction and chromatography did not exceed 17 h.

5. The method for extracting and recovering aescin from seeds of Sphaerocarpus rotundus according to claim 1, characterized in that: In step (4), the chromatography time is controlled at 12-16 hours.

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