Purslane polysaccharide extraction process based on deep eutectic solvent

Abstract

The invention discloses a purslane polysaccharide extraction process based on a deep eutectic solvent, and relates to the technical field of natural product extraction and food processing. The deep eutectic solvent adopted by the invention has excellent permeating and dissolving capacities on plant cell walls, and can release polysaccharide more efficiently; especially under the conditions that 1, 4-butanediol is used for HBD of the deep eutectic solvent, the molar ratio of choline chloride to 1, 4-butanediol is 1: 4, the water content is 30-50 wt.%, the extraction temperature is 70-80 DEG C, and the extraction time is 30-40 min, the highest extraction rate of the portulaca oleracea polysaccharide can reach about 1.3%, and compared with a traditional hot water extraction process (0.51%), the extraction rate of the portulaca oleracea polysaccharide is remarkably improved. Meanwhile, the deep eutectic solvent disclosed by the invention is composed of natural and low-toxicity components (such as choline salt, urea, glycerol and the like) and is biodegradable, so that the problems of toxicity, volatility and environmental pollution of a traditional organic solvent (such as methanol and acetone) are fundamentally avoided.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and food processing technology, specifically to a process for extracting purslane polysaccharides based on a deep eutectic solvent. Background Technology

[0002] Purslane (Portulaca oleracea L.) is a typical plant with both medicinal and edible properties, rich in polysaccharides, flavonoids, phenolic acids, and other active ingredients. Among these, purslane polysaccharides have been proven to possess various physiological activities, including antioxidant, anti-inflammatory, and immunomodulatory effects, showing promising application prospects in the development of functional foods and natural health products. To obtain purslane polysaccharides with high purity and biological activity, the rational design of extraction and purification processes is crucial.

[0003] In existing technologies, the extraction of purslane polysaccharides typically follows this route: Fresh or dried purslane raw materials are selected, washed, and coarsely pulverized. Extraction is primarily performed using hot water, with a few processes supplemented by ultrasonic, microwave, or enzymatic hydrolysis enhancement methods. The extract is filtered, ethanol is added for precipitation, and centrifugation yields the crude polysaccharide precipitate. This precipitate is then deproteinized using the Sevag method, dialyzed to remove small molecule impurities, and finally freeze-dried to obtain the crude polysaccharide powder. To further improve purity, some processes also employ ion exchange or gel filtration column chromatography for separation and purification.

[0004] However, the above-mentioned traditional extraction and purification processes still have the following shortcomings in practical applications: (1) It is difficult to balance extraction efficiency and polysaccharide structure protection. Since water, dilute acids, or dilute alkalis are often used as solvents, the adjustability of the solvent system and its ability to dissolve polysaccharides are limited. To improve the yield of purslane polysaccharides, repeated extraction at high temperatures (90-100℃) for extended periods (2-3 hours) is often required. This can easily lead to structural changes such as degradation or deacetylation of the polysaccharide molecular chains, affecting the original conformation and biological activity of the purslane polysaccharides and making it difficult to obtain polysaccharide products with intact structures and stable activity.

[0005] (2) Poor extraction selectivity and heavy impurity load Traditional water extraction systems have a single polarity and poor selectivity for dissolving polysaccharides, proteins, pigments, and some small molecules, often resulting in the simultaneous dissolution of a large amount of proteins and pigments. Even with subsequent use of the Sevag method to remove proteins and dialysis to remove small molecules, multiple operations are still required, resulting in a long process and a heavy purification burden.

[0006] In summary, existing purslane polysaccharide extraction processes still have significant shortcomings in improving extraction efficiency, protecting the structural integrity of polysaccharides, and effectively removing impurities and residual components. There is an urgent need to provide a technical solution with a clear process flow and high extraction efficiency. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a purslane polysaccharide extraction process based on a deep eutectic solvent, which addresses the shortcomings of existing purslane polysaccharide extraction processes such as low extraction efficiency, high energy consumption, and heavy environmental burden on the solvent. This process achieves comprehensive optimization of extraction efficiency, solvent greenness, and energy consumption, and significantly improves the yield of purslane polysaccharides while ensuring the green safety and economy of the process.

[0008] The technical solution of this invention is as follows: The extraction process of purslane polysaccharides based on eutectic solvents includes the following steps: S1 Raw material selection and pretreatment: Select fresh purslane, wash and dry it to constant weight, and then cool it for later use; S2 Crushing: The cooled purslane is crushed into a uniform powder or slurry; S3 Preparation of deep eutectic solvent: Mix hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) in water and stir at 60-80℃ until completely dissolved to obtain a transparent and homogeneous solution. After cooling, the deep eutectic solvent (DES) is obtained. Seal and store it in the dark for later use. S4 Extraction and alcohol precipitation: Mix the purslane powder or slurry obtained in step S2 with the deep eutectic solvent and stir evenly. After standing, extract at 70-80℃ for 30-40 min. After cooling, centrifuge and take the supernatant for alcohol precipitation. Centrifuge again to collect the precipitate, redissolve with distilled water to obtain the extract. S5 protein removal: Mix the extract at a volume ratio of (2-4):1 with Sevag reagent, shake, centrifuge to separate the layers, remove the protein layer, and repeat the above protein removal operation on the upper aqueous phase. S6 Dialysis: Dialysis is performed in deionized water using a dialysis bag to remove small molecule impurities from the extract to obtain dialysis solution; S7 Freeze-drying: The dialysate is frozen and then freeze-dried to obtain crude polysaccharide powder; S8 column chromatography separation: The crude polysaccharide powder was passed through a DEAE-52 chromatography column, the main peak fraction was collected by water washing, dialyzed and freeze-dried to obtain purified polysaccharide, thus completing the extraction of purslane polysaccharide.

[0009] Preferably, in step S1, when selecting fresh purslane, choose fresh purslane that is free from rot, insect infestation, and damage, and remove withered yellow leaves and woody stem segments.

[0010] Preferably, in step S1, during cleaning, the purslane is rinsed with tap water to remove mud and sand, then rinsed once with purified water and drained of surface moisture; during drying, the cleaned purslane is spread flat on a drying tray and dried at 50-55℃ to constant weight.

[0011] Preferably, in step S3, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is urea, thiourea, ethylene glycol (EG), 1,4-butanediol (1,4-BDO), citric acid (CA), oxalic acid (OA), lactic acid (LA), glycerol, or levulinic acid (XY).

[0012] Preferably, in step S3, the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:(3-5).

[0013] Preferably, in step S3, the water content of the eutectic solvent is 30-50 wt.%.

[0014] Preferably, in step S4, the mass-to-volume ratio of purslane powder or slurry to eutectic solvent is 1 g: (30-50) mL.

[0015] Preferably, in step S4, the centrifugation speed is 8000-10000 r / min and the centrifugation time is 10-20 min; the alcohol precipitation operation is as follows: add 4 times the volume of anhydrous ethanol to the supernatant and precipitate for 12-24 h.

[0016] Preferably, in step S5, the shaking time is 20-40 minutes, and the protein removal operation in the upper aqueous phase is repeated 3-5 times.

[0017] Preferably, in step S6, during dialysis, a 3500Da dialysis bag is used, and dialysis is performed in deionized water for 48-72 hours.

[0018] Compared with the prior art, the present invention has the following advantages: 1. The purslane polysaccharide extraction process of this invention uses DES composed of food-grade or low-toxicity raw materials such as choline chloride, urea, ethylene glycol, glycerol, and citric acid. It is not a traditional organic solvent and does not generate volatile organic compounds (VOCs) during extraction, significantly reducing environmental pollution and exhibiting good biodegradability and environmental compatibility. Furthermore, DES possesses a unique hydrogen bond network structure, exhibiting stronger swelling capacity with plant cell wall components, thus enhancing polysaccharide leaching efficiency. This invention achieves thorough cell wall disruption of purslane, resulting in a significantly higher polysaccharide dissolution rate than traditional hot water extraction. Compared to traditional hot water extraction requiring high temperatures (90-100℃), long extraction times (2-3 hours), and multiple repetitions, the DES extraction of this invention achieves the same or higher yields at lower temperatures and in shorter extraction times, significantly reducing heating energy consumption, shortening operation time, improving production efficiency, and offering a simple and easy-to-implement process suitable for scale-up production.

[0019] 2. The entire process of this invention requires no complex equipment, the solvent can be reused, and it is suitable for laboratory research and industrial applications. The operation steps are simple, and it can be carried out continuously and on a large scale. The raw materials and solvents are widely available and the cost is low. Traditional heat treatment can easily cause polysaccharide chain segment breakage or dehydration reaction, while the strong swelling ability of DES allows it to dissolve polysaccharides at lower temperatures, which helps to maintain their molecular weight and structural stability, improve the retention rate of active polysaccharides, maintain biological activity (antioxidant, immunomodulatory, etc.), and improve the purity and functionality of polysaccharides.

[0020] 3. In this invention, the combination of DES can achieve "customized dissolution capacity" by changing the HBA / HBD or adjusting the molar ratio. This invention screens nine types of DES, making the method highly versatile and scalable. Multiple DES can be applied to different plants, and can be extended to the green extraction of polysaccharides from Polygonatum, Lycium barbarum, Jerusalem artichoke, etc., demonstrating high adaptability and forming a series of process platforms. Attached Figure Description

[0021] Figure 1 These are test graphs showing the extraction rate of purslane polysaccharides in Examples 1-7 of this invention.

[0022] Figure 2 These are test graphs of the extraction rate of purslane polysaccharides in Examples 3, 8-9 and Comparative Examples 1-2 of the present invention.

[0023] Figure 3 These are test graphs showing the extraction rate of purslane polysaccharides in Examples 3, 10-11 and Comparative Examples 3-4 of this invention.

[0024] Figure 4 These are test graphs showing the extraction rate of purslane polysaccharides in Examples 3, 12-13 and Comparative Examples 5-6 of this invention.

[0025] Figure 5 These are test graphs showing the extraction rate of purslane polysaccharides in Examples 3, 14, and Comparative Examples 7-9 of this invention.

[0026] Figure 6 These are test graphs showing the extraction rate of purslane polysaccharides in Examples 3, 15, and Comparative Examples 10-12 of this invention.

[0027] Figure 7 This is the response surface methodology test diagram in Embodiment 16 of the present invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention.

[0029] The purslane polysaccharide extraction process based on a deep eutectic solvent of the present invention includes the following steps: S1 Raw Material Selection and Pretreatment: Select fresh purslane that is free from rot, insect infestation, and damage, and remove withered yellow leaves and woody stem segments; rinse the purslane with tap water to remove mud and sand, then rinse it once with purified water and drain the surface water; spread the washed purslane flat on a drying tray and place it in a 55℃ hot air circulating oven to dry to constant weight, in order to reduce the moisture content of the raw material and inhibit enzymatic browning and polysaccharide degradation, and cool it for later use after drying.

[0030] S2 Crushing: The cooled purslane is crushed using a pulverizer to make it into a uniform powder for subsequent DES extraction.

[0031] S3 Preparation of deep eutectic solvent: Mix HBA and HBD in water and stir until completely dissolved in a 70°C water bath to obtain a transparent and homogeneous solution. After cooling, the resulting solution is transparent and free of crystals, which is DES. Seal and store it in the dark for later use.

[0032] S4 Extraction and Alcohol Precipitation: Place the purslane powder obtained in step S2 into different conical flasks, add the DES prepared in step S3, stir evenly, let stand for 5 min, and then extract at 70-80℃ for 30-40 min; after cooling, centrifuge (8000 r / min, 10 min), take the supernatant, add 4 times the volume of anhydrous ethanol, precipitate for 24 h, centrifuge again to collect the precipitate, and redissolve with distilled water to obtain the extract.

[0033] S5 Protein Removal: Mix the extract (volume ratio 3:1) with Sevag reagent (an organic phase composed of chloroform and n-butanol in a volume ratio of 4:1), shake for 30 min, centrifuge to separate the layers, remove the protein layer, and repeat the above protein removal operation 4 times with the upper aqueous phase.

[0034] S6 Dialysis: Using a 3500Da dialysis bag, dialyze in deionized water for 72 hours to remove small molecule impurities from the extract and obtain the dialysis solution.

[0035] S7 Freeze-drying: The dialysate was frozen at -80℃ and then freeze-dried to obtain crude polysaccharide powder.

[0036] S8 column chromatography separation: The crude polysaccharide powder was passed through a DEAE-52 chromatography column, the main peak fraction was collected by water washing, dialyzed and freeze-dried to obtain purified polysaccharide, thus completing the extraction of purslane polysaccharide.

[0037] Example 1 According to the process of this invention, HBD is set as urea, and DES solvent of ChCl-U is prepared. The molar ratio of HBA to HBD is 1:4, the water content of DES solvent is 30%, the extraction temperature is 70℃, the extraction time is 40min, and the mass-volume ratio of purslane powder to DES solvent is 1g:30mL.

[0038] Example 2 The difference from Example 1 is that HBD is lactic acid, and a DES solvent of ChCl-LA is prepared.

[0039] Example 3 The difference from Example 1 is that HBD is 1,4-butanediol, and the DES solvent for preparing ChCl-B is obtained.

[0040] Example 4 The difference from Example 1 is that HBD is ethylene glycol, and the DES solvent for preparing ChCl-EG is obtained.

[0041] Example 5 The difference from Example 1 is that HBD is glycerol, and the DES solvent for preparing ChCl-G is obtained.

[0042] Example 6 The difference from Example 1 is that HBD is levulinic acid, and the DES solvent ChCl-XY is prepared.

[0043] Example 7 The difference from Example 1 is that HBD is citric acid, and a DES solvent of ChCl-CA is prepared.

[0044] The extraction rates of purslane polysaccharides obtained in Examples 1-7 were tested, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that when the HBD of the DES solvent is 1,4-butanediol, the extraction rate of purslane polysaccharide reaches the highest level (0.8%).

[0045] Example 8 The difference from Example 3 is that the molar ratio of HBA to HBD is set to 1:3.

[0046] Example 9 The difference from Example 3 is that the molar ratio of HBA to HBD is set to 1:5.

[0047] Comparative Example 1 The difference from Example 3 is that the molar ratio of HBA to HBD is set to 1:1.

[0048] Comparative Example 2 The difference from Example 3 is that the molar ratio of HBA to HBD is set to 1:2.

[0049] The extraction of purslane polysaccharides was repeated according to the extraction process of Example 3. The extraction rates of purslane polysaccharides obtained from Examples 3, 8-9, and Comparative Examples 1-2 are as follows: Figure 2 As shown. By Figure 2It can be seen that as the HBD content increases, the extraction rate also increases. When the molar ratio of HBA to HBD is 1:3, 1:4, and 1:5, the extraction rate is much higher than that when the molar ratio is 1:1 and 1:2. When the molar ratio is 1:5, the extraction rate is the highest, reaching 0.92%.

[0050] Example 10 The difference from Example 3 is that the water content of the DES solvent is 40%.

[0051] Example 11 The difference from Example 3 is that the water content of the DES solvent is 50%.

[0052] Comparative Example 3 The difference from Example 3 is that the water content of the DES solvent is 10%.

[0053] Comparative Example 4 The difference from Example 3 is that the water content of the DES solvent is 20%.

[0054] The extraction of purslane polysaccharides was repeated according to the extraction process of Example 3. The extraction rates of purslane polysaccharides obtained from Examples 3, 10-11, and Comparative Examples 3-4 are as follows: Figure 3 As shown. By Figure 3 It can be seen that when the water content of the DES solvent is 30-50%, the extraction rate of purslane polysaccharide remains at a high level and reaches its highest level (0.97%) when the water content of the DES solvent is 40%. When the water content of the DES solvent is too low, it is not conducive to the extraction of purslane polysaccharide, resulting in a decrease in the extraction rate.

[0055] Example 12 The difference from Example 3 is that the mass-to-volume ratio of purslane powder to DES solvent is 1g:40mL.

[0056] Example 13 The difference from Example 3 is that the mass-to-volume ratio of purslane powder to DES solvent is 1g:50mL.

[0057] Comparative Example 5 The difference from Example 3 is that the mass-to-volume ratio of purslane powder to DES solvent is 1g:10mL.

[0058] Comparative Example 6 The difference from Example 3 is that the mass-to-volume ratio of purslane powder to DES solvent is 1g:20mL.

[0059] The extraction of purslane polysaccharides was repeated according to the extraction process of Example 3. The extraction rates of purslane polysaccharides obtained from Examples 3, 12-13, and Comparative Examples 5-6 are as follows: Figure 4 As shown. By Figure 4 It can be seen that when the mass-volume ratio of purslane powder to DES solvent is 1g:(30-50)mL, the extraction rate of purslane polysaccharide remains at a high level, much higher than that of comparative examples 5-6, and reaches the highest (1.04%) when the mass-volume ratio of purslane powder to DES solvent is 1g:30mL.

[0060] Example 14 The difference from Example 3 is that the extraction time is 30 minutes.

[0061] Comparative Example 7 The difference from Example 3 is that the extraction time is 10 minutes.

[0062] Comparative Example 8 The difference from Example 3 is that the extraction time is 20 minutes.

[0063] Comparative Example 9 The difference from Example 3 is that the extraction time is 50 minutes.

[0064] The extraction of purslane polysaccharides was repeated according to the extraction process of Example 3. The extraction rates of purslane polysaccharides obtained from Examples 3, 14, and Comparative Examples 7-9 are as follows: Figure 5 As shown. By Figure 5 It can be seen that the extraction rate gradually increased with the extension of extraction time in the early stage, but when the extraction time was extended to 50 min, the extraction rate decreased instead; when the extraction time was 40 min, the extraction rate of purslane polysaccharide reached the highest (1.1%).

[0065] Example 15 The difference from Example 3 is that the extraction temperature is 80°C.

[0066] Comparative Example 10 The difference from Example 3 is that the extraction temperature is 90°C.

[0067] Comparative Example 11 The difference from Example 3 is that the extraction temperature is 50°C.

[0068] Comparative Example 12 The difference from Example 3 is that the extraction temperature is 60°C.

[0069] The extraction of purslane polysaccharides was repeated according to the extraction process of Example 3. The extraction rates of purslane polysaccharides obtained from Examples 3, 15, and Comparative Examples 10-12 are as follows: Figure 6 As shown. By Figure 6It can be seen that the extraction rate gradually increases with the increase of extraction temperature in the early stage. When the extraction temperature reaches 70℃, the extraction rate reaches a relatively high level. When the extraction temperature is 80℃, the extraction rate of purslane polysaccharide reaches the highest (1.3%). However, when the extraction temperature continues to rise to 90℃, the extraction rate decreases instead. Moreover, the temperature is too high at this time, which can easily lead to structural changes such as degradation or deacetylation of polysaccharide molecular chains, affecting the original conformation and biological activity of purslane polysaccharide, and making it difficult to obtain polysaccharide products with intact structure and stable activity.

[0070] Example 16 Response surface methodology experiments were conducted to evaluate the extraction temperature, extraction time, and water content of the DES solvent in this invention (during the experiments, HBD was 1,4-butanediol, the molar ratio of HBA to HBD was 1:4, and the mass-to-volume ratio of purslane powder to DES solvent was 1 g:30 mL), as shown in Tables 1-2: Table 1

[0071] Table 2

[0072] Test graph as shown Figure 7 As shown. Based on the aforementioned examples and comparative examples, using Design-Expert 13.0 software and employing a Box-Behnken design, with extraction temperature (A), extraction time (B), and DES solvent water content (C) as independent variables, and purslane polysaccharide extraction rate (Y) as the response value, response surface analysis and quadratic regression fitting were performed. The regression equations for purslane polysaccharide yield and each factor are as follows: .

[0073] The results of the analysis of variance are shown in Table 2. The significance level of the model is P < 0.0001, and the lack-of-fit term P = 0.4134 (P > 0.05) is not significant, indicating that the regression model has good fit and reliability with small experimental error. The coefficient of determination of the model is R0. 2 =0.9944, adjusted coefficient of determination R 2 The adj=0.9871 indicates that the model can explain the changes in the extraction rate of purslane polysaccharides well, and the goodness of fit is extremely high.

[0074] Significance analysis showed that extraction temperature (A) and moisture content (C) had significant effects on polysaccharide yield (P<0.01), with moisture content having the most significant effect, followed by extraction temperature; while extraction time (B) had a relatively weak effect (P>0.05). The interaction terms AB, AC, and BC all significantly affected the yield, with the BC interaction being the most significant (P<0.01), indicating that under higher moisture content conditions, extending the extraction time can further improve the yield. The quadratic term A... 2 B 2 C 2 All results were highly significant (P<0.01), indicating that the polysaccharide yield exhibited a significant nonlinear trend with changes in factors.

[0075] Response surface and contour plot as follows Figure 7 As shown in the figure, under the interaction of extraction temperature and extraction time (A×B), the response surface exhibits a distinctly curved convex shape, with contour lines approaching elliptical shapes, indicating a significant interaction between the two factors. The polysaccharide yield initially increases and then decreases with increasing extraction temperature and time, reaching its highest point at approximately 80℃ and 40 min. The response surface for extraction temperature and moisture content (A×C) also shows a distinctly curved shape, indicating a significant interaction between the two factors; the polysaccharide yield reaches a relatively high level when the temperature is 75-85℃ and the moisture content is 40-45%. Under the interaction of extraction time and moisture content (B×C), the response surface shows significant curvature and the contour lines are elliptical, indicating that this interaction significantly affects the yield; excessively high or low moisture contents are detrimental to polysaccharide dissolution.

[0076] In summary, the yield of purslane polysaccharides is significantly affected by extraction temperature, water content, and their interaction. The model shows high fitting accuracy and can be used to predict and optimize extraction conditions. According to the software optimization results, the theoretically optimal extraction conditions are: extraction temperature of approximately 80.365℃, extraction time of approximately 41.142 min, and DES solvent water content of approximately 43.154%, with a predicted yield of 1.975%. Based on actual conditions, the optimal conditions were adjusted to an extraction temperature of 80℃, an extraction time of 41 min, and a DES solvent water content of 43%. To verify the reliability of the model, three parallel verification experiments were conducted. The actual extraction rates were 1.93%, 2.05%, and 1.97%, with an average value of (1.99±0.06)%, differing from the predicted value by 0.76%. This deviation is less than 3%, indicating that the model has good predictive accuracy and stability and can be used in actual extraction processes.

[0077] Comparative Example 13 Comparative Example 13 uses a traditional hot water extraction process, including the following steps: S1 selects fresh or dried purslane raw materials, removes mud and impurities, and washes them repeatedly with clean water; S2 fresh raw materials are dried to constant weight, cooled and pulverized into uniform powder; S3 was added to purslane powder with distilled water at a solid-liquid ratio of 1g:30mL. After pre-soaking, it was extracted by heating in a water bath at 100℃ for 2h. The extraction was repeated 3 times. The extracts were combined and filtered through a filter cloth or centrifuged to remove insoluble matter, thus obtaining an aqueous extract. S4 concentrated the aqueous extract under vacuum by rotary evaporation, and slowly added 4 times the volume of anhydrous ethanol under stirring to precipitate the precipitate. After standing for 24 hours, the precipitate was collected by centrifugation, the supernatant was discarded, and the precipitate was dissolved in ultrapure water to obtain a crude polysaccharide solution. S5 crude polysaccharide solution was mixed with Sevag reagent at a volume ratio of 3:1. The protein layer was removed by shaking and centrifugation multiple times. The upper aqueous phase was placed in a 3500 Da dialysis bag and dialyzed in ultrapure water for 72 h to remove small molecule impurities such as inorganic salts, small molecule sugars and organic solvents.

[0078] S6 freezes the dialysate at -80°C and then freezes it to obtain crude polysaccharide powder.

[0079] S7 passed the crude polysaccharide powder through a DEAE-52 chromatography column, washed with water to collect the main peak fraction, dialyzed, and freeze-dried to obtain purified polysaccharide, thus completing the extraction of purslane polysaccharide.

[0080] The extraction rate of purslane polysaccharides in test comparison 13 was 0.51%.

[0081] The purslane polysaccharide extraction process of this invention utilizes a deep eutectic solvent with excellent penetration and dissolution capabilities to plant cell walls, enabling more efficient polysaccharide release. Simultaneously, the deep eutectic solvent of this invention is composed of natural, low-toxicity components (such as choline salts, urea, and glycerol), which are biodegradable, fundamentally avoiding the toxicity, volatility, and environmental pollution problems of traditional organic solvents (such as methanol and acetone). The entire production process is cleaner and safer, aligning with the development direction of green chemistry. The process flow of this invention is simple, requiring no complex equipment or harsh reaction conditions (such as high temperature and high pressure), and is easy to control and implement. Furthermore, the extraction time is relatively shortened, and it can be carried out at lower temperatures, significantly reducing energy consumption and making the production process more economical. The raw materials for the deep eutectic solvent of this invention are inexpensive and readily available, and most of the deep eutectic solvent can be recovered and reused through simple methods, greatly reducing solvent consumption costs and wastewater discharge. This makes the technology both economical and sustainable, highly suitable for large-scale production. The polysaccharide extracted by the process of this invention has fewer impurities, the subsequent purification steps are relatively simple, the resulting product has good quality, and it is easier to meet the strict requirements of raw materials in the fields of functional food, medicine and high-end cosmetics, with broad application prospects.

Claims

1. A deep eutectic solvent-based extraction process for polysaccharides from Portulaca oleracea L., characterized in that, Comprising the following steps: S1 raw material selection and pretreatment: select fresh purslane, wash and dry to constant weight, then cool and reserve; S2 crushing: crush the cooled purslane into uniform powder or slurry; S3 preparation of deep eutectic solvent: mix the hydrogen bond acceptor and the hydrogen bond donor in water, stir at 60-80°C until completely dissolved, obtain a transparent uniform solution, cool and obtain the deep eutectic solvent, seal and store in the dark; S4 extraction and alcohol precipitation: mix the purslane powder or slurry obtained in step S2 with the deep eutectic solvent and stir uniformly, stand for 30-40 min at 70-80°C; centrifuge after cooling, take the supernatant for alcohol precipitation, and centrifuge to collect the precipitate, then redissolve with distilled water to obtain the extract; S5 protein removal: mix the extract with Sevag reagent in a volume ratio of (2-4):1, shake and centrifuge to separate the layers, remove the protein layer, and repeat the protein removal operation for the upper aqueous phase 3-5 times; S6 dialysis: dialyze in deionized water using a dialysis bag to remove small molecular impurities in the extract to obtain a dialysate; S7 freeze-drying: freeze the dialysate and then freeze-dry to obtain a crude polysaccharide powder; S8 column chromatography separation: pass the crude polysaccharide powder through a DEAE-52 chromatography column, collect the main peak fraction eluted with water, dialyze and freeze-dry to obtain purified polysaccharide, and the extraction of purslane polysaccharide is completed.

2. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, characterized in that, In step S1, when selecting fresh purslane, select fresh purslane without rot, insect damage, and damage, and remove dry and yellow leaves and lignified stems.

3. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, characterized in that, In step S1, when washing, wash the purslane with tap water to remove dirt, and then rinse once with pure water, and drain the surface water; when drying, lay the washed purslane on the baking tray, and dry at 50-55°C to constant weight.

4. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, characterized in that, In step S3, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is urea, thiourea, ethylene glycol, 1,4-butanediol, citric acid, oxalic acid, lactic acid, glycerol or levulinic acid.

5. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, wherein, In step S3, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(3-5).

6. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, wherein, In step S3, the water content of the deep eutectic solvent is 30-50wt.%.

7. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, wherein, In step S4, the mass-volume ratio of the purslane powder or slurry to the deep eutectic solvent is 1g:(30-50)mL.

8. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, wherein, In step S4, the centrifugal speed is 8000-10000r / min, and the centrifugal time is 10-20 min; the alcohol precipitation operation is: add 4 times the volume of anhydrous ethanol to the supernatant, and precipitate for 12-24 h.

9. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, wherein, In step S5, the shaking time is 20-40 min, and the upper aqueous phase is repeated 3-5 times.

10. The deep eutectic solvent-based extraction process of purslane polysaccharides according to claim 1, wherein, In step S6, when dialysis, use a 3500Da dialysis bag and dialyze in deionized water for 48-72 h.

Images