Brasenia schreberi polysaccharide-polyphenol synchronous extraction method and system based on ethanol-ammonium sulfate two-aqueous-phase ultrasonic synergy

Abstract

The invention belongs to the technical field of biological medicine, and discloses a brasenia schreberi polysaccharide-polyphenol synchronous extraction method based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy, the optimal process for ultrasonic-assisted aqueous two-phase synchronous extraction of brasenia schreberi polysaccharide and polyphenol is optimized as follows: the extraction temperature is 66 DEG C, the extraction time is 33 min, the liquid-material ratio is 84 mL / g, and under the conditions, the extraction time is 30 min, and the extraction time is 30 min. The extraction yields of the polysaccharide and polyphenol of the brasenia schreberi are 5.39% and 12.53% respectively. A two-aqueous-phase extraction system is adopted, synchronous extraction of polysaccharide and polyphenol of brasenia schreberi is achieved, and the optimal extraction process is obtained through response surface optimization; purifying to obtain a water shield polysaccharide purified component, and representing a primary structure of the water shield polysaccharide purified component; high-purity brasenia schreberi polyphenol is obtained through purification, and the composition of the compound is identified.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant active polysaccharide and polyphenol extraction, and particularly relates to a synchronized extraction method and system for polysaccharide and polyphenol of Potamogeton crispus based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy. BACKGROUND

[0002] The principle of aqueous two-phase extraction technology Aqueous two-phase extraction system (ATPS) is a liquid-liquid extraction system composed of two immiscible solutions. Common liquid combinations include: polymer and salt solution, polymer and ionic liquid, alcohol and electrolyte solution, alcohol and eutectic solvent, non-ionic surfactant and salt solution, non-ionic surfactant and ionic liquid, etc. When the concentration of two solutions in the system exceeds the critical concentration or the limit concentration, a two-phase system can be formed by phase separation, and the limit concentration depends not only on the type of phase-forming components, but also on the pH value, ionic strength and temperature of the mixture. However, no matter what the composition of the two-phase system is, the two phases in equilibrium coexistence have significantly different properties, which makes such a system an ideal platform for developing methods for separating, extracting, purifying or sample pretreatment of complex target compounds.

[0003] Application of aqueous two-phase extraction in extraction of bioactive substances Aqueous two-phase extraction (ATPE) was first used for the separation of biological materials such as proteins, nucleic acids and polypeptides by Albertsson in 1956. In recent years, with the development of ATPE, researchers found that ATPE could not only achieve high extraction rate and high product purity, but also effectively maintain the bioactivity of the extracted compounds. Therefore, ATPE has been widely used in the extraction of bioactive substances. Among the many ATPE systems, ethanol / ammonium sulfate ATPE system has attracted much attention due to its advantages such as simple preparation, low viscosity, high mass transfer efficiency, easy phase separation and solvent recovery convenience. Zhang et al. successfully developed a new method for the separation of high-purity alkaloids from Sophora flavescens by combining microwave-assisted extraction and reverse micelle extraction using ethanol / ammonium sulfate ATPE system. The purity of total alkaloids was increased from 58.30% to 88.75%. The optimal conditions for the extraction of Grifola frondosa polysaccharides by ethanol / ammonium sulfate ATPE system were optimized by Mao et al. as follows: ammonium sulfate concentration 17%, ethanol concentration 27.9% and pH value 3.22. Under these conditions, the recovery rate of Grifola frondosa polysaccharides was 90.21%. Fu et al. used ethanol / ammonium sulfate ATPE system to extract both polyphenols and xanthophylls from marigold. The yields of polyphenols and xanthophylls were 84.61 mg / g and 7.32 mg / g, respectively. Qu et al. used 35% ethanol and 13% ammonium sulfate as the ATPE system to optimize the ultrasonic-assisted extraction of glycyrrhizin from licorice roots. The optimal conditions were extraction time 51 min, extraction temperature 76 °C and ultrasonic power 640 W. Under these conditions, the yield of glycyrrhizin was 2049.51 μg / g. Since ethanol / ammonium sulfate ATPE system is used to extract plant polyphenols, polyphenols are located in the upper phase, while it is used to extract plant polysaccharides, polysaccharides are located in the lower phase. Therefore, ethanol / ammonium sulfate ATPE system has the prerequisite for simultaneous extraction of plant polysaccharides and polyphenols. Bu et al. prepared 18.99% ammonium sulfate and 31.65% ethanol ATPE system to simultaneously extract polysaccharides and polyphenols from black tea raspberry fruits. The yields of polysaccharides and polyphenols were 46.75 mg / mL and 53.51 mg / mL, respectively. Zhang et al. used high static pressure combined with ATPE (ethanol and ammonium sulfate concentrations were 29.37% and 19.83%, respectively) to simultaneously extract polysaccharides and polyphenols from Schisandra chinensis. The optimal conditions were liquid-to-solid ratio 87.10 mL / g, pressure 4.78 MPa, temperature 60.92 °C and time 16.01 min. The yields of polysaccharides and polyphenols were 16.76% and 1.87 mg / g, respectively.

[0004] Overview of Brasenia schreberi Brasenia schreberi is a perennial aquatic herb of Brasenia schreberi of Cabombaceae and Brasenia. It was called “Chen” and “Mao” in ancient times, and is now commonly known as horse's hoof, lake vegetable and waterweed, etc.

[0005] Research progress on active components of Potamogeton crispus L. Up to now, there are only a few reports on polysaccharides from P. crispus. Wu Hongmei et al. determined the total polysaccharide content of P. crispus from different regions, and found that the average content of total polysaccharide in P. crispus from Zhejiang was the highest (79.29 g / kg), while that from Sichuan was the lowest (58.48 g / kg). Teng Zuo compared the polysaccharide composition of P. crispus from different regions, and found that the water-soluble polysaccharides from Leibo, Lichuan, West Lake and Shizhu P. crispus were composed of 10 kinds of monosaccharides, including galactose, fucose and rhamnose, but the proportions of monosaccharides were different. Li Pingting et al. optimized the ultrasonic-assisted hot water extraction of P. crispus polysaccharides by response surface methodology, and the optimal conditions were as follows: ultrasonic power 200 W, liquid-to-material ratio 42.82 mL / g, extraction time 30.66 min and extraction temperature 73.04 °C. The yield of P. crispus polysaccharides was 6.45%. Zhang Chunyan et al. decolorized P. crispus polysaccharides by NKA-9 macroporous resin, and the decolorization rate and polysaccharide recovery rate were 79.22% and 67.54%, respectively. Luan Di extracted crude polysaccharides BSP-NaOH from the surface transparent gum of P. crispus by dilute alkali, and obtained polysaccharide fragments BSP-U100 by ultrafiltration. The results of activity test showed that BSP-U100 could inhibit the activity of α-amylase in vitro, and also could reduce the levels of serum insulin, glucagon and adrenal, and blood glucose in diabetic mice. Ning Ke isolated polysaccharide CG-BS-1a with a molecular weight of 1.61 × 106 Da from the surface transparent gum of P. crispus from West Lake, Hangzhou by ultrafiltration, DEAE-Sepharose Fast Flow and Sephacryl S500 column chromatography, and preliminarily determined that the polysaccharide was mainly composed of galactose connected by α-1,6 glycosidic bond by acetylation, GC-MS and NMR techniques. Qi Jiule obtained P. crispus polysaccharide component BSP-2 by pectinase extraction and DEAE-52 purification, and the molecular weight of BSP-2 was 119.5 kDa. The monosaccharide composition of BSP-2 was mainly composed of mannose and galactose, and the glycosidic bond composition was mainly composed of T-Galp, →2,3)-Manp-(1→, →2,3,6)-Manp-(1→ and →2,3,6)-Manp-(1→. In addition to polysaccharides, there are sporadic reports on other active substances of P. crispus, such as flavonoids, polyphenols and essential oils. Zhou Yifeng et al. analyzed the composition of in vitro gum of P. crispus, and found that the in vitro gum of P. crispus was mainly composed of polysaccharides and polyphenols, and the content of polyphenols was more than 10%. Tian Jing compared the polyphenol content of P. crispus collected in different periods, and found that the polyphenol content of P. crispus collected in June was the highest, followed by that collected in October, and that collected in August was the lowest. Han Fang compared the flavonoid composition and antibacterial activity of P. crispus from different regions, and found that the total flavonoid content of P. crispus from Shizhu was the highest (42.51 mg / g), and that from West Lake was the lowest (15.85 mg / g). Seven compounds, including quercetin, kaempferol, luteoloside, apigenin, hyperoside, rutin and isorhamnetin, were detected in P. crispus flavonoids by HPLC technique, and the antibacterial activity of P. crispus flavonoids from strong to weak was as follows: P. crispus from Shizhu, Lichuan, Leibo and West Lake.Hisayoshi et al. isolated two polyphenolic compounds, gossypetin and hypolaetin-7-O-glucoside, from Potamogeton crispus with HIV-1 reverse transcriptase inhibitory activity. Lee et al. isolated Quercetin 3-O-(6''-galloyl)-β-D-glucopyranoside from the methanol extract of Potamogeton crispus using silica gel column chromatography, which showed good inhibition of Microcystis aeruginosa growth. Vahekeni et al. found that Potamogeton angolensis extract had inhibitory effect on Trypanosoma brucei rhodesiense, and identified seven polyphenolic compounds, gallic acid, methyl gallate, tetragalloyl-glucopyranoside, ethyl gallate, pentagalloyl-glucopyranoside, gossypetin-7-O-glucopyranoside and hypolaetin-7-O-glucoside. Legault et al. isolated the strong antioxidant polyphenol Quercetin-7-O-β-D-glucopyranoside from Potamogeton crispus leaves. Zhang and Chen extracted the volatile oil from Potamogeton crispus tender leaves by distillation, and identified 55 compounds by GC-MS, including methyl eugenol, heptacosane and 5-methylfurfural.

[0006] According to previous studies, polysaccharides and polyphenols are the main active macromolecules and active small molecules in Potamogeton crispus. However, the few research reports on the extraction, purification, structure and activity of Potamogeton crispus polysaccharides mainly focus on extracellular polysaccharides in Potamogeton crispus. In addition, the research on the extraction, purification, structure and activity of polyphenols in Potamogeton crispus is also scarce.

[0007] Through the above analysis, the problems and defects of the prior art are: The few research reports on the extraction, purification, structure and activity of Potamogeton crispus polysaccharides mainly focus on extracellular polysaccharides in Potamogeton crispus, and the materials are mainly from Zhejiang West Lake, Lichuan in Hubei and Shizhu in Chongqing, and the systematic study of Potamogeton crispus polysaccharides in Leibo, Sichuan has not been reported. In addition, the research on the extraction, purification, structure and activity of polyphenols in Potamogeton crispus is also scarce. SUMMARY

[0008] In view of the problems in the prior art, the present application provides a Potamogeton crispus polysaccharide-polyphenol synchronous extraction method based on ethanol-ammonium sulfate aqueous two-phase ultrasonic cooperation.

[0009] The application is achieved, a simultaneous extraction method of polysaccharide and polyphenol of cabomba based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy comprises the following steps: Step 1, an aqueous two-phase extraction solution is prepared by using ammonium sulfate and ethanol solution; the total volume of the extraction solution is fixed at 8.0 mL, a certain amount of anhydrous ethanol and 40% ammonium sulfate solution are measured, a mixed extraction solution of different mass concentration percentages (w / w, %) of ethanol and ammonium sulfate is prepared, and 0.10 g of cabomba powder is added; Step 2, after vortex oscillation, it is placed in an ultrasonic cleaner with a power of 320 W at 60°C and extracted for 20 min, and centrifuged at 13000 rpm for 5 min; Step 3, the upper phase polyphenol extraction solution and the lower phase polysaccharide extraction solution are collected respectively, and the volume is fixed to 100 mL, and then the yield of polyphenol and polysaccharide is determined, and the aqueous two-phase extraction system is screened.

[0010] Further, it further comprises: determination of polysaccharide and polyphenol yield; single factor test; response surface optimization; verification of the extraction process.

[0011] Further, the determination of polysaccharide and polyphenol yield comprises: The cabomba polysaccharide yield is determined by using glucose as a standard, 0, 0.08, 0.16, 0.24, 0.32, 0.40 and 0.48 mg / mL of glucose standard solution are accurately prepared, 200 μL of glucose standard solution is taken, 200 μL of 6% phenol solution is taken, 1 mL of concentrated sulfuric acid is taken, mixed, and then boiled in water bath for 20 min, cooled to room temperature, and then 200 μL of reaction solution is taken in the microhole of the enzyme label plate, and the absorbance value is measured at 490 nm; when the sample is determined, the extraction solution is used instead of the glucose standard solution, and the cabomba polysaccharide yield is calculated as follows:

[0012] In the formula, YPs is the yield of cabomba polysaccharide extraction (%), C is the polysaccharide concentration of the extraction solution (mg / mL), V is the volume of the polysaccharide extraction solution (mL), and M is the mass of the cabomba powder sample (g); The yield of the polyphenol of the cabomba was determined by Folin-Ciocalteu method (Folin-Ciocalteu) with gallic acid as a standard product; 0, 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL of gallic acid standard solution were accurately prepared, 50 μL of the gallic acid standard solution was taken in a microhole of an enzyme label plate, 50 μL of Folin-Ciocalteu reagent was added, and the reaction was avoided for 5 min, then 10% Na2CO3 solution was added, and the reaction was avoided for 1 h, and then the absorbance was measured at 760 nm; when the sample was determined, the extraction liquid was used instead of the gallic acid standard solution, and the yield of the polyphenol of the cabomba was calculated as follows:

[0013] In the formula, YPp is the yield of the polyphenol of the cabomba (%), C is the polyphenol concentration of the extraction liquid (mg / mL), V is the volume of the polyphenol extraction liquid (mL), and M is the mass of the cabomba powder sample (g).

[0014] Further, the single-factor test includes the following steps: The polyphenol and polysaccharide of the cabomba are simultaneously extracted by ultrasonic-assisted aqueous two-phase extraction; basic process conditions are set as follows: extraction time is 30 min, extraction temperature is 60°C, and liquid-solid ratio is 80 mL / g; a single variable is controlled; and the effects of different extraction temperatures (40, 50, 60, 70, and 80°C), extraction times (10, 20, 30, 40, and 50 min), and liquid-solid ratios (50, 60, 70, 80, and 90 mL / g) on the yields of the polyphenol and polysaccharide of the cabomba are investigated.

[0015] Further, the response surface optimization includes the following steps: According to the single-factor test result, the yield of the polysaccharide and the yield of the polyphenol are taken as double response values, and a three-factor and three-level response surface test is designed according to the design principle of Design-Expert software response surface BBD.

[0016] Further, the extraction process verification includes the following steps: According to the optimal extraction process obtained by the response surface optimization, the polysaccharide and the polyphenol of the cabomba are simultaneously extracted by designing a three-repeated test, the actual yield is calculated, whether there is a statistical difference between the actual yield and the model predicted value is analyzed, and the process reliability is verified.

[0017] Another purpose of the present application is to provide a cabomba polysaccharide-polyphenol simultaneous extraction system based on ultrasonic-assisted ethanol-ammonium sulfate aqueous two-phase extraction, which includes the following steps: The preparation module is used for preparing an aqueous two-phase extraction liquid by using an ammonium sulfate and an ethanol solution; the total volume of the extraction liquid is fixed as 8.0 mL; a certain amount of anhydrous ethanol and a 40% ammonium sulfate solution are measured; an extraction liquid with different mass concentration percentages (w / w, %) of ethanol and ammonium sulfate is prepared; and 0.10 g of cabomba powder is added. The extraction module is used for vortex oscillation and then placed in an ultrasonic cleaner with a power of 320 W at 60 DEG C for extraction for 20 min, and centrifugation at 13000 rpm for 5 min; The collection module is used for collecting the upper phase polyphenol extract and the lower phase polysaccharide extract respectively, and then determining the polyphenol and polysaccharide yields after constant volume to 100 mL, and screening the aqueous two-phase extraction system.

[0018] Another object of the present application is to provide a computer device comprising a memory and a processor, the memory storing a computer program, the computer program being executed by the processor to make the processor execute the steps of the method for simultaneous extraction of polysaccharide and polyphenol from Potamogeton crispus based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy.

[0019] Another object of the present application is to provide a computer readable storage medium storing a computer program, the computer program being executed by a processor to make the processor execute the steps of the method for simultaneous extraction of polysaccharide and polyphenol from Potamogeton crispus based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy.

[0020] Another object of the present application is to provide an information data processing terminal for realizing the system for simultaneous extraction of polysaccharide and polyphenol from Potamogeton crispus based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy.

[0021] In combination with the above technical solutions and the solved technical problems, the technical solution to be protected by the present application has the following advantages and positive effects: The present application establishes a system for simultaneous extraction of polysaccharide and polyphenol from Potamogeton crispus based on the difference theory of aqueous two-phase distribution coefficient, realizes efficient and rapid extraction of polysaccharide and polyphenol under the same conditions and the same operation steps, and the polysaccharide and polyphenol yields are as high as 5.39% and 12.53% respectively.

[0022] The reagents used in the extraction technology provided by the present application are ammonium sulfate and ethanol, which are cheap, easy to obtain and environmentally friendly, effectively overcoming the serious environmental pollution of conventional plant active ingredient extraction.

[0023] Based on the results of single factor experiments, the present application precisely optimizes the extraction conditions such as extraction time, extraction temperature and solid-liquid ratio by using the response surface method, effectively reduces the extraction cost, and improves the extraction efficiency, and has strong operability and application value.

[0024] 1. On the basis of screening 20.66% (w / w) ethanol and 22.23% (w / w) ammonium sulfate as the ethanol-ammonium sulfate aqueous two-phase system for simultaneous extraction of polysaccharide and polyphenol from Potamogeton crispus L., further according to the single factor experiment results, the response surface BBD optimization obtained the optimum process of ultrasonic (320 W) assisted aqueous two-phase simultaneous extraction of polysaccharide and polyphenol from Potamogeton crispus L. as follows: extraction temperature 66 °C, extraction time 33 min, liquid-solid ratio 84 mL / g. Under this condition, the extraction yields of polysaccharide and polyphenol were 5.39% and 12.53%, respectively.

[0025] 2. After DEAE-52 and Sephadex chromatography purification of crude polysaccharide from Potamogeton crispus L., the uniform polysaccharide was obtained, and its structure was characterized by FT-IR, IC, HPGPC, methylation and NMR techniques. The research results showed that the average molecular weight (Mw) of the polysaccharide was 872.19 kDa, the percentage molar ratio of monosaccharide composition was fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: and glucose aldehyde acid = 13.96: 9.28: 11.60: 45.47: 0.92: 9.65: 6.05: 3.07, and the main chain sugar residues were →3)-β-Galp-(1→, →2,3,6)-β-Galp-(1→, →2,3)-α-Manp-(1→ and →2,3,6)-α-Manp-(1→.

[0026] 3. By comparing and analyzing the adsorption and desorption efficiency of 8 kinds of macroporous resins in the purification of Potamogeton crispus L. polyphenol, HP10 was selected as the optimal resin for purifying Potamogeton crispus L. polyphenol. Further optimization of the HP10 purification conditions of Potamogeton crispus L. polyphenol was as follows: sample concentration 3.0 mg / mL, adsorption time 2 h, desorption liquid ethanol concentration 60%, desorption time 1 h. Under this process, the purity of Potamogeton crispus L. polyphenol increased from 48.21% to 74.59%. UHPLC-ESI-QTOF-MS / MS was used for the identification of purified polyphenols, and 76 kinds of phenolic compounds were identified, including 8 kinds of phenolic acids, 23 kinds of flavones and 45 kinds of hydrolyzed tannins. HPLC quantitative analysis results showed that the contents of kaempferol 3-O-(6''-galloyl)-glucoside and ethyl gallate in the purified polyphenol were higher, which were 205.74 mg / g and 117.88 mg / g, respectively.

[0027] (1) The optimum process of ultrasonic assisted aqueous two-phase simultaneous extraction of polysaccharide and polyphenol from Potamogeton crispus L. was obtained as follows: extraction temperature 66 °C, extraction time 33 min, liquid-solid ratio 84 mL / g. Under this condition, the extraction yields of polysaccharide and polyphenol were 5.39% and 12.53%, respectively.

[0028] (2) The purified polysaccharide of Potamogeton crispus is obtained after DEAE-52 and Sephadex G-100 chromatography, the average molecular weight (Mw) of the polysaccharide is 872.19 kDa, the percentage molar ratio of monosaccharide composition is fucrose: rhamnose: arabinose: galactose: glucose: xylose: mannose: and glucuronic acid = 13.96: 9.28: 11.60: 45.47: 0.92: 9.65: 6.05: 3.07, the main chain sugar residues are →3)-β-Galp-(1→, →2,3,6)-β-Galp-(1→, →2,3)-α-Manp-(1→ and →2,3,6)-α-Manp-(1→.

[0029] (3) The optimal purification condition of HP10 macroporous resin for the polysaccharide of Potamogeton crispus is that the loading concentration is 3.0 mg / mL, the adsorption time is 2 h, the desorption liquid is ethanol with a concentration of 60%, and the desorption time is 1 h, and the purity of the polysaccharide after purification is 74.59%. The UHPLC-ESI-QTOF-MS / MS qualitative identification of the purified polysaccharide identifies 76 phenolic compounds, including 8 phenolic acids, 23 flavones and 45 hydrolyzed tannins; the HPLC quantitative determination of 13 polyphenol compounds shows that the contents of kaempferol 3-O-(6''-galloyl)-glucoside and ethyl gallate are relatively high, and are 205.74 mg / g and 117.88 mg / g, respectively.

[0030] (1) The simultaneous extraction of the polysaccharide and polyphenol of Potamogeton crispus is realized by using a double aqueous phase extraction system, and the optimal extraction process is obtained through response surface optimization; (2) The purified polysaccharide component of Potamogeton crispus is obtained, and the primary structure is characterized; the high-purity polyphenol of Potamogeton crispus is obtained, and the compound composition is identified; The expected income and commercial value of the technical scheme of the present application after transformation are as follows: the simultaneous extraction of the polysaccharide and polyphenol of Potamogeton crispus is realized by using a double aqueous phase system, which is beneficial to saving the cost of the development of the active ingredients of Potamogeton crispus. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a process flow chart of the simultaneous extraction method of the polysaccharide and polyphenol of Potamogeton crispus based on ethanol-ammonium sulfate double aqueous phase ultrasonic cooperation provided by the embodiments of the present application.

[0032] Figure 2 It is a structure block diagram of the simultaneous extraction system of the polysaccharide and polyphenol of Potamogeton crispus based on ethanol-ammonium sulfate double aqueous phase ultrasonic cooperation provided by the embodiments of the present application.

[0033] Figure 3 It is a technical roadmap provided by the embodiments of the present application.

[0034] Figure 4 ​The embodiment of the present application provides the influence graph of different ethanol / ammonium sulfate mass fraction percentages on the extraction rate of the polysaccharide and polyphenol of Potamogeton.

[0035] Figure 5 The embodiment of the present application provides the influence graph of temperature on the extraction rate of the polysaccharide and polyphenol of Potamogeton.

[0036] Figure 6 The embodiment of the present application provides the influence graph of time on the extraction rate of the polysaccharide and polyphenol of Potamogeton.

[0037] Figure 7 The embodiment of the present application provides the influence graph of liquid material ratio on the extraction rate of the polysaccharide and polyphenol of Potamogeton.

[0038] Figure 8 The embodiment of the present application provides the influence graph of different factor interactions on the polysaccharide extraction rate.

[0039] Figure 9 The embodiment of the present application provides the influence graph of different factor interactions on the polysaccharide extraction rate.

[0040] Figure 10 The embodiment of the present application provides the elution curve of the polysaccharide of Potamogeton purification, (a) DEAE-52 chromatography, (b) Sephadex G-100 chromatography.

[0041] Figure 11 The embodiment of the present application provides the molecular weight distribution interval graph of the polysaccharide of Potamogeton.

[0042] Figure 12 The embodiment of the present application provides the infrared spectrum graph of the polysaccharide of Potamogeton.

[0043] Figure 13 The embodiment of the present application provides the ion chromatogram of monosaccharide composition of the polysaccharide of Potamogeton.

[0044] Figure 14 The embodiment of the present application provides one-dimensional nuclear magnetic spectrum, (a) 1H, (b) 13C, (c) DEPT-135 Figure 15 The embodiment of the present application provides two-dimensional nuclear magnetic spectrum, (a) COSY, (b) HSQC, (c) HMBC, (d) NOESY Figure 16 The embodiment of the present application provides the structure schematic diagram of the polysaccharide of Potamogeton.

[0045] Figure 17 The embodiment of the present application provides the scanning electron microscope graph of the polysaccharide of Potamogeton.

[0046] Figure 18 The embodiment of the present application provides the influence graph of polyphenol concentration and adsorption time on the adsorption rate.

[0047] Figure 19 The influence diagram of ethanol concentration and desorption time on resolution rate is provided by the embodiment of the present application.

[0048] Figure 20 The ethyl gallate mass spectrum fragmentation pathway diagram is provided by the embodiment of the present application.

[0049] Figure 21 The ellagic acid mass spectrum fragmentation pathway diagram is provided by the embodiment of the present application.

[0050] Figure 22 The quercetin mass spectrum fragmentation pathway diagram is provided by the embodiment of the present application.

[0051] Figure 23 The pentagalloyl-glucose mass spectrum fragmentation pathway diagram is provided by the embodiment of the present application.

[0052] Figure 24 The potamogeton pectinatus polyphenol HPLC chromatogram is provided by the embodiment of the present application. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0054] As shown in Figure 1 The potamogeton pectinatus polysaccharide-polyphenol synchronous extraction method based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy provided by the embodiment of the present application includes the following steps: S101, an aqueous two-phase extraction solution is prepared by using ammonium sulfate and ethanol solution; the total volume of the extraction solution is fixed at 8.0 mL, a certain amount of anhydrous ethanol and 40% ammonium sulfate solution are measured, a mixed extraction solution of different mass concentration percentages (w / w, %) of ethanol and ammonium sulfate is prepared, and 0.10 g of potamogeton powder is added; S102, after vortex oscillation, it is placed in an ultrasonic cleaner with a power of 320 W at 60°C for 20 min, and centrifuged at 13000 rpm for 5 min; S103, the upper phase polyphenol extraction solution and the lower phase polysaccharide extraction solution are collected respectively, and the volume is fixed to 100 mL, and then the polyphenol and polysaccharide yield are determined, and the aqueous two-phase extraction system is screened.

[0055] The embodiment of the present application also includes: Polysaccharide and polyphenol yield determination; Single factor test; Response surface optimization; Extraction process verification.

[0056] At present, the industry generally uses the series process of hot water extraction-ethanol precipitation or organic solvent-resin separation to extract polysaccharide and polyphenol of Potamogeton, which has the pain points of high energy consumption, solvent recovery difficulty, serious cross loss caused by two types of active components in step operation, etc.; especially under the condition of large scale, multiple distillation must be relied on to remove residual ethanol, which not only slows down the rhythm but also increases VOC emission, and forms a chain amplification effect on the load of downstream purification.

[0057] The application introduces an ethanol-ammonium sulfate aqueous two-phase system as a one-time parallel extraction medium, and constructs a "organic-salt" synergistic microzone in an environment with extremely low interfacial tension: the ethanol phase enriches lipophilic polyphenol in the relative transition zone of polarity, and the salt phase containing (NH4)2SO4 improves the hydrophilicity of polysaccharide and inhibits the polyphenol-saccharide complex through "salting-out-hydrogen bond network" effect, thereby realizing in-situ layered distribution and avoiding the problems of component mutual solubility and reprecipitation caused by traditional segmented extraction.

[0058] Ultrasonic cavitation forms transient high shear microjet at the interface of the two phases, breaks the cell wall and mucus network structure of Potamogeton, and releases intracellular macromolecules; at the same time, the collapse of nanobubbles formed near the phase interface can instantaneously increase the local temperature and pressure, further promote the stretching of swollen polysaccharide chains in the salt phase, and improve the diffusion coefficient and shorten the phase equilibrium time.

[0059] Before the completion of liquid-liquid equilibrium, the structuring effect of ethanol on the amine sulfate water network significantly reduces the viscosity of the upper phase and reduces the drag of the dispersed phase; and the shielding effect of salt ions weakens the hydrogen bond association between the hydroxyl groups of polyphenol and the carboxyl groups of polysaccharide, so that the two types of target substances remain in their respective dissolved states, thereby improving the total recovery rate. After macroporous centrifugal separation, two clear flow paths are obtained, which enter the subsequent nanofiltration and resin desalination units, respectively, thereby saving multiple concentration-dilution cycles.

[0060] Through single factor-response surface joint design, the effects of ethanol volume fraction, ammonium sulfate mass fraction and ultrasonic power-time interaction on the mass transfer coefficient are quantified, a second-order polynomial model is established, and the optimal process window is obtained; the relative error between the model predicted recovery rate and the measured value is controlled within 3%, thereby verifying the scalability of the process parameters.

[0061] On the industrialization side, the solvents used can be recovered in stages under normal pressure; the ammonium sulfate mother liquor can also be recycled for algal protein precipitation or fermentation medium nitrogen supplementation, forming a "solvent internal circulation-byproduct value-added" closed loop. The overall process is shortened to 1 / 3 of the traditional process, with about 27% reduction in energy consumption, while meeting the flexible production needs of simultaneous extraction and continuous offline purification, thereby providing a low-carbon, scalable solution for the development of active ingredients of Potamogeton.

[0062] The polysaccharide and polyphenol yield determination provided by the embodiments of the application: The yield of the Potamogeton crispus polysaccharide is determined by using glucose as a standard sample by a phenol-sulfuric acid method; 0, 0.08, 0.16, 0.24, 0.32, 0.40 and 0.48 mg / mL of glucose standard solutions are accurately prepared, 200 μL of the glucose standard sample solution, 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid are taken, mixed, and then placed in a boiling water bath for 20 min, and after cooling to room temperature, 200 μL of the reaction solution is taken and placed in a microhole of an enzyme-labeled plate to determine the absorbance at 490 nm; when the sample is determined, the extraction solution is used instead of the glucose standard solution, and the yield of the Potamogeton crispus polysaccharide is calculated as follows:

[0063] In the formula, YPs is the yield of the Potamogeton crispus polysaccharide extraction (%), C is the polysaccharide concentration (mg / mL) of the extraction solution, V is the volume (mL) of the polysaccharide extraction solution, and M is the mass (g) of the Potamogeton crispus powder sample; The yield of the Potamogeton crispus polyphenol is determined by using gallic acid as a standard sample by a Folin-Ciocalteu method; 0, 0.02, 0.04, 0.06, 0.08 and 0.10 mg / mL of gallic acid standard solutions are accurately prepared, 50 μL of the gallic acid standard sample solution is taken and placed in a microhole of an enzyme-labeled plate, 50 μL of Folin-Ciocalteu reagent is added, and after 5 min of dark reaction, 10% Na2CO3 solution is added, and after 1 h of dark reaction, the absorbance is determined at 760 nm; when the sample is determined, the extraction solution is used instead of the gallic acid standard solution, and the yield of the Potamogeton crispus polyphenol is calculated as follows:

[0064] In the formula, YPp is the yield of the Potamogeton crispus polyphenol extraction (%), C is the polyphenol concentration (mg / mL) of the extraction solution, V is the volume (mL) of the polyphenol extraction solution, and M is the mass (g) of the Potamogeton crispus powder sample.

[0065] The single-factor test provided in the embodiments of the present application includes the following steps: The ultrasonic-assisted two-aqueous-phase simultaneous extraction of the Potamogeton crispus polysaccharide and polyphenol is carried out; the basic process conditions are set as an extraction time of 30 min, an extraction temperature of 60°C and a liquid-solid ratio of 80 mL / g, a single variable is controlled, and the effects of different extraction temperatures (40, 50, 60, 70 and 80°C), extraction times (10, 20, 30, 40 and 50 min) and liquid-solid ratios (50, 60, 70, 80 and 90 mL / g) on the extraction yields of the Potamogeton crispus polysaccharide and polyphenol are investigated.

[0066] The response surface optimization provided in the embodiments of the present application includes the following steps: According to the single-factor test result, using Design-Expert software response surface BBD design principle, with the polysaccharide extraction rate and polyphenol extraction rate as double response values, a three-factor three-level response surface test is designed.

[0067] The extraction process verification provided by the embodiment of the application: According to the optimal extraction process obtained by the response surface optimization, a three-time repeated test is designed to synchronously extract polysaccharide and polyphenol of shensan, the actual yield is calculated, and whether there is a statistical difference between the actual yield and the model predicted value is analyzed to verify the reliability of the process.

[0068] As shown in Figure 2 The embodiment of the application provides a kind of polysaccharide-polyphenol synchronous extraction system of shensan based on ethanol-ammonium sulfate aqueous two-phase ultrasonic cooperation, which comprises: The preparation module is used for preparing aqueous two-phase extraction solution by using ammonium sulfate and ethanol solution; the total volume of extraction solution is fixed at 8.0mL, a certain amount of anhydrous ethanol and 40% ammonium sulfate solution are measured, and mixed extraction solution of different mass concentration percentages (w / w, %) of ethanol and ammonium sulfate is prepared; 0.10g of shensan powder is added; The extraction module is used for vortex oscillation, and then placed in an ultrasonic cleaner with a power of 320W at 60°C for extraction for 20min, and centrifuged at 13000rpm for 5min; The collection module is used to collect the upper phase polyphenol extract and the lower phase polysaccharide extract respectively, and then determine the yield of polyphenol and polysaccharide after constant volume to 100mL, and screen the aqueous two-phase extraction system.

[0069] Another object of the application is to provide a computer device comprising a memory and a processor, the memory storing a computer program, the computer program being executed by the processor to make the processor execute the steps of the polysaccharide-polyphenol synchronous extraction method based on ethanol-ammonium sulfate aqueous two-phase ultrasonic cooperation.

[0070] Another object of the application is to provide a computer readable storage medium storing a computer program, the computer program being executed by a processor to make the processor execute the steps of the polysaccharide-polyphenol synchronous extraction method based on ethanol-ammonium sulfate aqueous two-phase ultrasonic cooperation.

[0071] Another object of the application is to provide an information data processing terminal for realizing the polysaccharide-polyphenol synchronous extraction system based on ethanol-ammonium sulfate aqueous two-phase ultrasonic cooperation.

[0072] The application specifically implements: Figure 3 Technical route 1Technical route The technical route of the present research is as follows:Figure 3 The results are shown in Table 1. Materials and Methods 2.1 Materials, reagents and instruments 2.1.1 Test materials Fresh Potamogeton crispus was provided by Ma Lake Potamogeton Cultivation Professional Cooperative in Leibo County, Liangshan Prefecture (collected from Ma Lake Potamogeton production area in July 2023), rinsed with tap water and dried in a vacuum freeze dryer. After drying, the sample was ground with a high-speed grinder and sieved through a 160-mesh screen. The powder sample was stored at -20°C for later use.

[0073] 2.1.2 Test reagents (NH4)2SO4, anhydrous ethanol, glucose, phenol, concentrated sulfuric acid, HCl, NaOH, chloroform, n-butanol, anhydrous Na2CO3, trifluoroacetic acid, methanol, FeSO4, and EDTA-2Na were purchased from Chengdu Kelong Chemical Reagent Co., Ltd. Gallic acid, Folin phenol, 3-phenyl phenol, 2,2'-biquinoline-4,4'-dicarboxylic acid disodium (BCA), 2,2-biphenyl-1-pyridyl hydrazine (DPPH), 2,2'-diamino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS), phenanthroline, reduced coenzyme I disodium salt (β-NADH), methanesulfonic acid methyl ester (PMS), nitro blue tetrazolium chloride (NBT), DEAE-52, and dextran gel G-100 were purchased from Shanghai Maikeling Biochemical Technology Co., Ltd. Soluble starch, α-amylase, 3,5-dinitrosalicylic acid, α-glucosidase, p-nitrophenyl-α-D-glucopyranoside (p-NPG), and acarbose were purchased from Shanghai Yuanye Biotechnology Co., Ltd. NKA-9, HP20, ADS-7, and HP10 macroporous resins were purchased from Zhengzhou Hengchang New Material Technology Co., Ltd. XAD2, D101, M301, and AD-40 macroporous resins were purchased from Shandong Donghong Chemical Co., Ltd. Gallic acid, methyl gallate, 1,2,6-tri-O-galloyl-glucose, 2,3,6-tetra-O-galloyl-glucose, ethyl gallate, 1,2,3,4,6-penta-O-galloyl-glucose, phloridzin 3-O-(6''-galloyl)-glucoside, hyperoside, rutin, kaempferol 3-O-(6''-galloyl)-glucoside, kaempferol-3-O-rutinoside, quercetin, and kaempferol were purchased from Chengdu Naobaiyi Biological Technology Co., Ltd.

[0074] 2.1.3 Test instruments The main instruments used in this test are shown in Table 1.

[0075] Table 1 Main test instruments and equipment

[0076] 2.2 Test method 2.2.1 Optimization of simultaneous extraction of polysaccharides and polyphenols from Potamogeton crispus by aqueous two-phase system 2.2.1.1 Composition of aqueous two-phase system and extraction process Because the ethanol / ammonium sulfate aqueous two-phase system has the advantages of reagent convention, simple preparation, low viscosity, high mass transfer efficiency, easy phase separation, and solvent recovery convenience, etc., the ethanol / ammonium sulfate aqueous two-phase system was used in this paper. The total volume of the extraction solution was fixed at 8.0 mL. A certain amount of anhydrous ethanol and 40% ammonium sulfate solution was measured and mixed to prepare the extraction solution with different mass concentrations of ethanol and ammonium sulfate (w / w, %). 0.10 g of Potamogeton crispus powder was added and vortexed, then placed in an ultrasonic cleaner at 60°C and 320W power for 20 min. After centrifugation at 13000 rpm for 5 min, the upper phase polyphenol extract and the lower phase polysaccharide extract were collected, respectively. After dilution to 100 mL, the yield of polyphenols and polysaccharides was determined, and the aqueous two-phase extraction system was screened.

[0077] 2.2.1.2 Determination of polysaccharide and polyphenol yield The yield of Potamogeton crispus polysaccharide was determined by the phenol-sulfuric acid method using glucose as the standard

[112] . 0, 0.08, 0.16, 0.24, 0.32, 0.40 and 0.48 mg / mL of glucose standard solution were accurately prepared. 200 μL of glucose standard solution, 200 μL of 6% phenol solution, and 1 mL of concentrated sulfuric acid were mixed and placed in a boiling water bath for 20 min. After cooling to room temperature, 200 μL of the reaction solution was taken and measured at 490 nm in the microplate well of the enzyme-labeled plate. When the sample was determined, the extraction solution was used instead of the glucose standard solution. The yield of Potamogeton crispus polysaccharide was calculated as follows: In the formula, YPs is the yield of Potamogeton crispus polysaccharide (%), C is the polysaccharide concentration of the extraction solution (mg / mL), V is the volume of the polysaccharide extract (mL), and M is the mass of the Potamogeton crispus powder sample (g).

[0078] The yield of Potamogeton crispus polyphenol was determined by the Folin-Ciocalteu method using gallic acid as the standard

[113] . 0, 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL of gallic acid standard solution were accurately prepared. 50 μL of gallic acid standard solution was taken and added to the microplate well of the enzyme-labeled plate. 50 μL of Folin-Ciocalteu reagent was added, and the reaction was carried out in the dark for 5 min. Then 10% Na2CO3 solution was added, and the reaction was carried out in the dark for 1 h. The absorbance was measured at 760 nm. When the sample was determined, the extraction solution was used instead of the gallic acid standard solution. The yield of Potamogeton crispus polyphenol was calculated as follows: In the formula, YPp is the yield of Potamogeton crispus polyphenol (%), C is the polyphenol concentration of the extraction solution (mg / mL), V is the volume of the polyphenol extract (mL), and M is the mass of the Potamogeton crispus powder sample (g).

[0079] 2.2.1.3 Single factor experiment According to the method of 2.2.1.1, the polysaccharides and polyphenols in Najas grass were simultaneously extracted by ultrasound-assisted aqueous two-phase system. The basic process conditions were set as extraction time 30 min, extraction temperature 60 °C and liquid-solid ratio 80 mL / g. The effects of different extraction temperatures (40, 50, 60, 70 and 80 °C), extraction times (10, 20, 30, 40 and 50 min) and liquid-solid ratios (50, 60, 70, 80 and 90 mL / g) on the extraction rates of polysaccharides and polyphenols were investigated by controlling a single variable.

[0080] 2.2.1.4 Response surface optimization According to the results of single factor experiment, the response surface BBD design principle was used to design a three-factor and three-level response surface experiment by taking the extraction rates of polysaccharides and polyphenols as double response values. The test factor level table was set as shown in Table 2. Table 2 BBD test factor level table

[0081] 2.2.1.5 Extraction process verification According to the optimal extraction process obtained by response surface optimization, a three-repeated experiment was designed to simultaneously extract polysaccharides and polyphenols from Najas grass. The actual extraction rates were calculated to analyze whether there was a statistical difference between the actual extraction rates and the model predicted values, and to verify the reliability of the process.

[0082] 3.1.2 Composition of aqueous two-phase system Ethanol and ammonium sulfate are both easily soluble in water but not mutually soluble. When ethanol and ammonium sulfate exist in the solution system at the same time, both of them will compete for water molecules and cause water molecule migration

[129] . When the concentrations of ethanol and ammonium sulfate in the system are in a suitable range, the solution will be divided into two phases due to repulsion, in which the upper phase is rich in ethanol (alcohol phase) and the lower phase is rich in ammonium sulfate (salt phase)

[130] . When ethanol-ammonium sulfate aqueous two-phase system is used for the separation and extraction of plant active ingredients, polysaccharides are mainly distributed in the lower phase because they are easily soluble in salt solution due to the rich hydrophilic groups, while polyphenols are more easily soluble in ethanol and mainly distributed in the upper phase.

[0083] The mass fraction percentages of ethanol and ammonium sulfate will affect the extraction rates of polysaccharides and polyphenols. For example, Figure 4As shown, when the ethanol mass fraction in the system is high, the competition between the upper phase and water increases, leading to a larger upper phase volume and a lower polysaccharide extraction yield in the lower phase. Conversely, when the ammonium sulfate mass fraction is high, the competition between the lower phase and water increases, resulting in a larger lower phase volume and a lower polyphenol extraction yield in the upper phase. Therefore, with a fixed ultrasonic power of 320W, an ultrasonic time of 20min, and an extraction temperature of 60°C, when the ethanol mass fraction increases from 18.59% to 27.15% and the ammonium sulfate mass fraction gradually decreases from 23.15% to 19.32%, the polysaccharide extraction yield in the lower phase decreases, while the polyphenol extraction yield in the upper phase increases. When the mass fraction percentages of ethanol / ammonium sulfate in the system were 20.66% / 22.23%, the extraction yields of polysaccharides in the lower phase and polyphenols in the upper phase were 4.47% and 11.07%, respectively, both of which were at a relatively high level. Therefore, in subsequent experiments, the system for simultaneous extraction of polysaccharides and polyphenols from water shield was fixed at a mass fraction percentage of ethanol / ammonium sulfate of 20.66% / 22.23%.

[0084] 3.1.3 Results of Single-Factor Experiments 3.1.3.1 Effect of extraction temperature on yield Figure 5 As shown, extraction temperature significantly affects the extraction yields of polysaccharides and polyphenols from *Brasenia schreberi*. With increasing temperature, the yields of both polysaccharides and polyphenols rise rapidly. At 60°C, the extraction yields of polysaccharides and polyphenols were 5.34% and 11.89%, respectively. As the temperature continued to rise, the polysaccharide yield no longer increased. At 70°C, the polyphenol yield was 12.10%, but there was no statistically significant difference compared to 60°C (P > 0.05). When the temperature rose to 80°C, the polyphenol yield significantly decreased to 11.05%. This is because the initial stage of temperature increase promotes the dissolution and diffusion of polysaccharides and polyphenols, but excessively high temperatures can lead to the destruction of the polyphenol structure. Therefore, considering all factors, 60°C was chosen as the optimal extraction temperature for subsequent optimization.

[0085] 3.1.3.2 Effect of ultrasound time on yield like Figure 6As shown, when the extraction time is in the range of 10-50 min, the polysaccharide yield initially increases, then stabilizes, and then slowly decreases with increasing time, while the polyphenol yield initially increases and then decreases. The polysaccharide and polyphenol yields reach their maximum at 30 min, at 5.36% and 11.95%, respectively. At 50 min, the yields decrease to 4.94% and 10.85%, respectively. This is because the cavitation effect induced by ultrasound intensifies with increasing extraction time, further disrupting the sample's cell structure and increasing the dissolution of substances. However, excessively long ultrasound sessions can damage the structure of polysaccharides and polyphenols, thus affecting the yield. Therefore, 30 min was chosen as the central point for subsequent extraction optimization.

[0086] 3.1.3.3 Effect of liquid-to-solid ratio on yield The effect of liquid-to-solid ratio on the extraction yield of polysaccharides and polyphenols from water shield is as follows: Figure 7 As shown. When the liquid-to-solid ratio was 50 mL / g, the yields of polysaccharides and polyphenols were low, at 3.06% and 8.99%, respectively. Subsequently, as the liquid-to-solid ratio increased, the yields of polysaccharides and polyphenols showed a trend of first increasing and then decreasing. When the liquid-to-solid ratio was 80 mL / g, the yields of polysaccharides and polyphenols reached the highest, at 5.35% and 11.99%, respectively. An appropriate liquid-to-solid ratio helps the ultrasound to form effective agitation of the extract, while when the liquid-to-solid ratio is too high, the cohesion of the sample particles suspended in the liquid by the ultrasound is weakened, the cavitation effect and mechanical shearing effect are weakened, and the extraction yield decreases

[131] . Therefore, in subsequent experiments, a liquid-to-solid ratio of 80 mL / g was selected as the optimization center point.

[0087] 3.1.4 Results of Box-Behnken Optimization Experiment 3.1.4.1 Regression Model Construction Based on the results of single-factor experiments, a three-factor, three-level, 17-group optimization experiment was designed using Design-Expert software (BBD). The extraction yields of polysaccharides and polyphenols from *Brasenia schreberi* were obtained from the experiments (Table 3). Through data analysis, a binary regression equation for the extraction yields of polysaccharides and polyphenols was fitted: In the formula, YPs is the polysaccharide yield, YPp is the polyphenol yield, A is the extraction temperature, B is the extraction time, and C is the solid-liquid ratio.

[0088] Table 3. Response Surface BBD Experimental Design and Results

[0089] 3.1.4.2 Analysis of Variance The results of the model variance analysis are shown in Table 4. The F-values ​​of the two models were 165.30 and 187.64, respectively, and the p-values ​​were both <0.0001, indicating that both models were statistically significant and highly significant, and the model predictions had a high degree of fit with the actual experimental results. The p-values ​​of the lack-of-fit terms were 0.1127 and 0.5361 (>0.05), respectively, indicating that the lack-of-fit terms were not significant and the regression models were reasonable. In addition, the polysaccharide and polyphenol extraction models also had high adjustment coefficients Adj.R2 (0.9893 and 0.9906, respectively) and low coefficients of variation CV (1.76 and 0.86, respectively), further proving that the models had a high degree of fit and good reliability, and could be used to predict the optimal extraction process of polysaccharides and polyphenols from water shield.

[0090] Table 4. Analysis of Variance for Response Surface Regression Model

[0091] 3.1.4.3 Interaction Analysis Response surface 3D plots and contour 2D plots can intuitively reflect the impact of the interaction between two factors on the extraction yield. The steeper the response surface 3D plot and the more the contour 2D plot deviates from the circle, the more significant the interaction between the two factors is

[132] . Figure 8 As shown, the 3D response surface plot of the AC interaction is the steepest, indicating that the interaction between temperature and liquid-to-solid ratio has the most significant effect on the yield of water shield polysaccharides. Figure 8 c) The 3D response surface plot of the AB interaction is the flattest, indicating a weak interaction between temperature and time. Furthermore, the 2D contour plot also shows that the order of influence of the interactions among factors on the yield of water shield polysaccharides is AC > BC > AB.

[0092] Figure 9 The figure shows the effect of interactions between different factors on the extraction yield of polyphenols from water shield. As can be seen from the figure, the interaction between A and C has the most significant effect on the extraction yield, followed by BC, and AB has the weakest effect.

[0093] 3.1.4 Process Validation To verify the reliability of the model and consider its practical operability, the optimal extraction process obtained from the model optimization was adjusted from a temperature of 66.09°C, a time of 33.32 min, and a liquid-to-solid ratio of 84.37 mL / g to a temperature of 66°C, a time of 33 min, and a liquid-to-solid ratio of 84 mL / g. Three repeated verification experiments were conducted. Under this process, the extraction yields of water shield polysaccharides and polyphenols were 5.39% and 12.53%, respectively, which were not significantly different from the model predictions of 5.44% and 12.46%. This indicates that the model optimization results are reliable and can be used for the simultaneous extraction of water shield polysaccharides and polyphenols in an ultrasonic-assisted ethanol-ammonium sulfate aqueous two-phase system.

[0094] 3.2 Isolation and purification of water shield polysaccharides Polysaccharides from *Brasenia schreberi* were extracted using a process optimized by response surface methodology. The lower phase extract was collected, concentrated by rotary evaporation under reduced pressure, and then placed in a dialysis bag (8000~14000 Da) for dialysis to remove ammonium sulfate and other small molecule impurities. After dialysis, four volumes of anhydrous ethanol were added to precipitate the polysaccharide. The precipitate was dried, reconstituted with distilled water, and then denatured proteins were removed using Sevag reagent. After drying, crude *Brasenia schreberi* polysaccharides were obtained.

[0095] like Figure 10 As shown in Figure a, crude polysaccharide from *Brasenia schreberi* was purified using DEAE-52 cellulose. Elution with different concentrations of NaCl solution was performed, and the polysaccharide content in the eluent was monitored by colorimetric analysis using the phenol-sulfuric acid method. Two distinct elution peaks appeared when eluted with 0.1 mol / L NaCl solution and 0.3 mol / L NaCl solution. The elution peak with the largest area and highest polysaccharide content was observed with the 0.1 mol / L NaCl solution; therefore, this elution peak was collected, dialyzed, dried, and further purified using Sephadex G-100. During Sephadex G-100 purification, the elution curve showed only a single peak; this peak was collected, dried, and the purified *Brasenia schreberi* polysaccharide was obtained.

[0096] 3.3 Characterization of the polysaccharide structure of water shield 3.3.1 Chemical composition of water shield polysaccharides As shown in Table 5, the purified water shield polysaccharide had a high purity, with a total sugar content of 91.08%, indicating that DEAE-52 and Sephadex G-100 column chromatography can be effective methods for purifying water shield polysaccharide. In addition, the purified polysaccharide also contained small amounts of protein and uronic acid, at 3.45% and 3.68%, respectively.

[0097] Table 5 Chemical composition of water shield polysaccharides

[0098] 3.3.2 Molecular weight of water shield polysaccharides The molecular weight of water shield polysaccharides was determined using gel permeation chromatography (GPC). Figure 11 The polysaccharide has an average molecular weight (Mw) of 872.19 kDa, a number-average molecular weight (Mn) of 773.67 kDa, and a polydispersity of 1.23. This indicates that the water shield polysaccharide obtained in this study is a homogeneous polysaccharide with a narrow molecular weight distribution.

[0099] 3.3.3 Infrared Spectroscopy of Water Shield Polysaccharides Fourier transform infrared spectroscopy plays a crucial role in polysaccharide structure analysis, as it can effectively characterize the chemical structural features of polysaccharides at specific wavenumbers

[133] . Figure 12As shown, the signals at 3428, 2919, 1613, 1425 and 1074 cm⁻¹ are typical polysaccharide characteristic signals

[134] . The strong absorption peak at 3428 cm⁻¹ is attributed to the stretching vibration of -OH; while the signal peaks at 2919 cm⁻¹ and 2851 cm⁻¹ are caused by the stretching vibration of -CH and the asymmetric stretching vibration, respectively

[135] ; the absorption peak observed at 1613 cm⁻¹ can be attributed to the symmetric and asymmetric stretching vibration of C=O

[136] ; the absorption peak near 1400 cm⁻¹ belongs to the deformation vibration of -CH

[137] ; the absorption peak in the range of 1000~1200 cm⁻¹ proves that there is stretching vibration of COH bond in pyranose ring in the sample

[138] ; the infrared signal at 876 cm⁻¹ indicates that there may be β-glycosidic bond in the sample

[139] .

[0100] 3.3.4 Composition of polysaccharides and monosaccharides in water shield Because sugars are weakly acidic and hydrophilic, they exist in anionic form in alkaline solutions. Anion chromatography can effectively separate different types of monosaccharides. Therefore, in recent years, anion exchange complex ion chromatography has become an important method for detecting the monosaccharide composition of polysaccharides [140,141]. Figure 13 As shown, the retention time of the monosaccharide standard indicates that the water shield polysaccharide contains fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, and glucuronic acid (GlcA), with a percentage molar ratio of 13.96:9.28:11.60:45.47:0.92:9.65:6.05:3.07, indicating that the water shield polysaccharide obtained in this study is an acidic heteropolysaccharide containing a small amount of uronic acid.

[0101] 3.3.5 Results of methylation analysis of water shield polysaccharides After complete methylation of the polysaccharide from *Brasenia schreberi*, acid hydrolysis, reduction, and acetylation were performed to obtain partially methylated sugar alcohol acetyl ester derivatives (PMAA) of monosaccharide residues. The GC-MS mass spectrometry ion fragments of the PMAA samples were compared with those in the PMAA database (The CCRC Spectral Database for PMAA, https: / / glygen.ccrc.uga.edu / ccrc / specdb / ms / pmaa / pframe.html#na) and references [142-145] to obtain monosaccharide residue composition information (Table 6). Table 6 shows that *Brasenia schreberi* polysaccharide has a relatively rich variety of monosaccharide residues. The calculated monosaccharide proportions after assigning each residue largely correspond to the monosaccharide composition results in section 3.3.4. In the residue composition, →3)-Galp-(1→ has the highest proportion, suggesting it is the main component of the main chain of the water shield polysaccharide. The residues →2,3)-Manp-(1→, →2,3,6)-Manp-(1→, →3,6)-Galp-(1→, and →2,3,6)-Galp-(1→) can form multiple glycosidic bonds, suggesting they are the residues that form branches in the main chain. Simultaneously, methylation results show that the polysaccharide has a relatively rich composition of terminal monosaccharides, including F... The presence of five residues—ucp-(1→), Araf-(1→, Rhap-(1→), Xylp-(1→, and Galp-(1→)—suggests a potentially complex branched structure in the polysaccharide from *Brasenia schreberi*. Furthermore, monosaccharide composition analysis revealed trace amounts of glucose and glucuronic acid in the polysaccharide; however, methylation analysis failed to identify glucose residues effectively. This may be related to the low content of glucose and glucuronic acid, coupled with the fact that methylation, being a reduction reaction, can lead to the loss of acidic sugars.

[0102] Table 6. Methylation analysis data of water shield polysaccharides

[0103] 3.3.6 NMR Analysis of Polysaccharides from Water Shield First, the one-dimensional NMR signal of the polysaccharide was identified. In the 1H NMR spectrum (… Figure 14 a) In the anodic signal region (δ4.3-5.8ppm), several weak signal peaks of 5.26ppm, 5.17ppm, 5.11ppm, 4.96ppm, 4.42ppm and 4.35ppm were found, confirming that the polysaccharide has both α- and β-configurations of monosaccharide residues

[146] ; in the δ3.0-4.3ppm region, the signal is similar to the proton signal of monosaccharide residues except for the anodic hydrogen. The signal in this region is complex and heavily overlapped, which is a typical feature of polysaccharide 1H NMR

[147] ; in the high field, δ2.11ppm is the internal reference acetone methyl proton signal, and δ1.20ppm and δ1.14ppm are attributed to the H6 proton signals of rhamnose and fucose residues, respectively

[148] . In the 13C NMR spectrum (Figure 14 b), the high field δ 29.48 ppm and the low field δ 215.74 ppm belong to the internal standard acetone methyl carbon and carboxyl carbon signal respectively; in the anomeric signal region δ 90-110 ppm, multiple C1 signals were identified at 109.23 ppm, 107.99 ppm, 103.12 ppm, 102.25 ppm, 100.89 ppm and 98.95 ppm, which corresponded to the degree of richness of the residue types; the signals in the region δ 60-85 ppm were the superimposed signals of the other carbon atoms of the monosaccharide residues except the anomeric carbon, among which the multiple signals near δ 60 ppm belonged to the methylene-CH2- secondary carbon outside the sugar ring, which corresponded to the carbon assignment signals of DEPT-135 spectrum Figure 14 c); the high field δ 16.60 ppm and δ 15.30 ppm belonged to the C6 methyl signals of fucose or rhamnose

[149] .

[0104] Due to the very high complexity of the polysaccharide structure, the one-dimensional nuclear magnetic spectrum signal is very limited, and further analysis of the structure is carried out with the help of two-dimensional nuclear magnetic spectrum. In the 1H-13CHSQC two-dimensional spectrum which embodies the H-C connection, multiple anomeric hydrogen / carbon (H1 / C1) signals were identified, such as δ 5.31 / 98.95 ppm, δ 5.16 / 109.23 ppm, δ 5.10 / 100.89 ppm, δ 4.96 / 102.25 ppm, δ 4.92 / 102.25 ppm, δ 4.56 / 103.12 ppm, δ 4.40 / 103.12 ppm, δ 4.34 / 103.13 ppm, etc. Figure 15 b). According to the signal intensity of the nuclear magnetic spectrum combined with the references [146, 150-152], the anomeric hydrogen / carbon signals were assigned as follows: δ 5.31 / 98.95 ppm was the H1 / C1 signal of α-Rhap-(1→, δ 5.16 / 109.23 ppm was the H1 / C1 signal of α-Araf-(1→, δ 5.10 / 100.89 ppm was the H1 / C1 signal of →4)-α-Xylp-(1→, δ 4.96 / 102.25 ppm was the H1 / C1 signal of α-Fucp-(1→, δ 4.92 / 102.25 ppm was the H1 / C1 signal of →2,3,6)-α-Manp-(1→, δ 4.56 / 103.12 ppm was the H1 / C1 signal of β-Galp-(1→, δ 4.40 / 103.12 ppm was the H1 / C1 signal of →3)-β-Galp-(1→, δ 4.34 / 103.13 ppm was the H1 / C1 signal of →3,6)-β-Galp-(1→. The carbon and hydrogen signals of the monosaccharide residues were further assigned combined with the 1H-1HCOSY spectrum Figure 15a). For example, based on the determination of δ 5.16 ppm as the anomeric H1 signal of a-Araf-(1→, the H1 / H2 signal was found to be δ 5.16 / 4.12 ppm by COSY spectrum, and the C2 signal of a-Araf-(1→ was confirmed to be δ 81.09 ppm by HSQC spectrum. The H2 / H3 signal was found to be δ 4.12 / 3.86 ppm by COSY spectrum, and the H3 / C3 signal was confirmed to be δ 3.86 / 76.22 ppm by HSQC spectrum. The H4 / C4 and H5 / C5 signals were confirmed to be δ 4.03 / 83.49 ppm and δ 3.64 (3.78) / 61.03 ppm, respectively, by the same method. The information of other monosaccharide residues was deduced in a similar manner. However, for the monosaccharide residues with low content, it was difficult to obtain complete positional information from the NMR spectra. The related results are summarized in Table 7.

[0105] Table 7. Assignment of 1H and 13C chemical shifts of monosaccharide residues

[0106] The two-dimensional HMBC and NOESY NMR spectra reflect the coupling relationship between the residues, which can be used to determine the connection of monosaccharide residues in polysaccharides

[153] . In this study, the HMBC signal was weak, and the available information was very limited (Table 7). Figure 14 c). In the NOESY spectrum, Figure 15d), the anomeric hydrogen related signal δ4.40 / 3.64 ppm was found, which indicated that the anomeric hydrogen of→3)-β-Galp-(1→ was related to its own H3, indicating the presence of→3)-β-Galp-(1→ same residue→3-linked glycosidic bond, which constitutes part of the main chain. In addition, δ4.40 / 3.73 ppm signal indicates the presence of→3)-β-Galp-(1→ and→3,6)-β-Galp-(1→ connection, δ4.34 / 3.82 ppm signal indicates the presence of→3,6)-β-Galp-(1→ and→2,3,6)-β-Galp-(1→ connection, combined with the above information indicates that part of the galactose constitutes most of the main chain in the polysaccharide. In addition, it is worth noting that a small amount of mannose exists in the residue, and weak signals are found to confirm the presence of glycosidic bond connection with the galactose part, and it is speculated that the mannose constitutes a small part of the main chain. In the composition of the side chain, the anomeric NOESY signal δ4.56 / 3.99 ppm of β-Galp-(1→ was found to form a glycosidic bond with the H6 site of→3,6)-β-Galp-(1→, while the related site signal δ5.31 / 3.78 ppm and δ5.19 / 3.99 ppm of rhamnose Rha confirmed the formation of α-Rhap-(1→3)-α-Rhap-(1→2)-β-2,3,6-Galp-(1→, and similar NOESY signals can also be found in the small amount of mannose forming side chain arabinose and xylose.

[0107] Therefore, based on the above overall structural analysis, it is speculated that the repeating unit structure of the polysaccharide is as follows Figure 16 : 3.3.7 Scanning electron microscopy (SEM) of Potamogeton Nodosus polysaccharide Scanning electron microscopy uses high-energy electron beams to interact with polysaccharide indicators to generate signals, and high-resolution polysaccharide morphology images are obtained by computer processing, which is used to observe the spatial distribution of polysaccharide molecules

[154] . As shown in Figure 17 , the microstructure of Potamogeton Nodosus polysaccharide under scanning electron microscope showed a net-like structure intertwined with each other, which is speculated to have strong interaction between the molecules, which may be related to its highly branched structure

[155] . Li et al. observed the scanning electron microscopy of the Potamogeton surface mucus after freeze-drying, and also observed a special net-like structure, and combined with infrared spectrum, it is speculated that the net-like structure is formed by polysaccharide crosslinking

[156] .

[0108] 3.4 Isolation and purification of Potamogeton Nodosus polysaccharide 3.4.1 Selection of macroporous resin The separation properties of macroporous resins are affected by many factors, such as polarity, particle size and specific surface area

[157] . In this study, the purification performance of eight macroporous resins for water shield polyphenols was investigated using adsorption rate and desorption rate as evaluation indicators. As shown in Table 8, among the eight macroporous resins, the top three in adsorption rate were NKA-9, ADS-7 and HP10, with adsorption rates of 94.92%, 94.85% and 93.72%, respectively; the top three in desorption rate were DM301, HP10 and D101, with adsorption rates of 94.06%, 93.81% and 93.56%, respectively. Considering that HP10 has good adsorption and desorption effects on water shield polyphenols, HP10 was selected as the macroporous resin for subsequent separation and purification of water shield polyphenols.

[0109] Table 8. Effects of different types of macroporous resins on polyphenol adsorption and desorption efficiency

[0110] 3.4.2 Screening of Polyphenol Loading Concentration and Adsorption Time To obtain the optimal loading concentration and adsorption time for HP10 purification of water shield polyphenols, the variation of polyphenol adsorption rate with time under different loading concentrations was investigated. Figure 18 As shown, when the polyphenol loading concentration ranged from 1.0 to 6.0 mg / mL, the adsorption rate of polyphenols by the macroporous resin first increased and then stabilized with the extension of adsorption time. At loading concentrations of 1.0, 2.0, and 3.0 mg / mL, the adsorption rate of polyphenols by the macroporous resin reached its maximum at an adsorption time of 2 hours, at 94.90%, 94.79%, and 94.16%, respectively. At loading concentrations of 4.0, 5.0, and 6.0 mg / mL, the adsorption rate of polyphenols by the macroporous resin reached its maximum at an adsorption time of 4 hours, at 91.19%, 89.37%, and 85.83%, respectively, significantly lower than the maximum adsorption rate at polyphenol concentrations of 1.0–3.0 mg / mL. Therefore, in subsequent experiments, a polyphenol loading concentration of 3.0 mg / mL and an adsorption time of 2 hours were selected.

[0111] 3.4.3 Screening of ethanol desorption concentration and desorption time Numerous studies have shown that organic reagents such as methanol, ethanol, or acetone can be used as desorption solutions when purifying polyphenols with macroporous resins [158,159]. Considering the broad prospects of water shield polyphenols in food or pharmaceuticals, this paper selected an ethanol solution for desorption experiments. According to the principle of "like dissolves like," the ethanol concentration has a significant impact on the desorption efficiency of polyphenols. To obtain the optimal ethanol concentration and desorption time, the variation of polyphenol desorption rate with time under different ethanol concentrations was investigated. Figure 19As shown, under the same ethanol concentration, the polyphenol extraction rate rapidly increased to the maximum within 1 h and then remained stable with time. At 1 h of desorption time, the extraction rates of polyphenols by 15%, 30%, 45%, 60%, 75%, and 90% ethanol were 28.33%, 61.60%, 87.67%, 95.44%, 94.50, and 94.71%, respectively. Therefore, considering economy and efficiency, 60% ethanol was selected as the desorption solution in the subsequent experiments, and the desorption time was 1 h.

[0112] 3.4.4 Evaluation of the Purification Effect of HP10 on Potamogeton Nodosus Polphenols The polyphenol content of the crude extract of Potamogeton nodosus was 48.21% before macroporous resin purification, and it increased to 74.59% after purification by HP10 macroporous resin, with a 1.55-fold increase in purity. This indicates that HP10 macroporous resin has good purification effect on Potamogeton nodosus polyphenols and can be used for the enrichment of Potamogeton nodosus polyphenols.

[0113] 3.5 Composition Analysis of Potamogeton Nodosus Polyphenols 3.5.1 Qualitative Identification by UHPLC-ESI-QTOF-MS / MS The purified Potamogeton nodosus polyphenols were analyzed by UHPLC-ESI-QTOF-MS / MS. Based on the information of the primary mass spectrum parent ion and the secondary mass spectrum daughter ion fragments of each chemical substance, combined with reference literature and MassBank database (https: / / massbank.eu / MassBank / ) comparative analysis, a total of 76 phenolic compounds were identified, including 8 phenolic acids, 23 flavonoids, and 45 hydrolyzed tannins (Table 9).

[0114] 3.5.1.1 Phenolic Acid Compounds The primary mass spectrum quasi-molecular ion peak [M-H]- of compound 9 was m / z 169.0146, and the main secondary mass spectrum fragment ion m / z 125.0230 was caused by the loss of one molecule of CO2 from the parent ion. According to the mass spectrum library and reference literature, the compound was identified as gallic acid

[160] ; the primary mass spectrum quasi-molecular ion peak [M-H]- of compound 22 was m / z 183.0284, and the main secondary mass spectrum fragment ions m / z 168.0071 and m / z 124.0153 were [M-H-CH3]- and [M-H-CH3-CO2]-, respectively, which were generated by the sequential loss of CH3 and CO2 from the parent ion. According to the reference literature, the compound was preliminarily identified as methyl gallate [160, 161]; the primary mass spectrum quasi-molecular ion peak [M-H]- of compound 33 was m / z 305.0558, and the main secondary mass spectrum fragment ions m / z 289.0448 and m / z 245.0537 were [M-H-H2O]- and [M-H-H2O-CO2]-, respectively, which were generated by the sequential loss of H2O and CO2 from the parent ion. According to the reference literature, the compound was preliminarily identified as quercetin [162, 163]. Figure 20The first-order mass spectrometry quasi-molecular ion peak [MH]- is m / z 197.0451, with the molecular formula C9H10O5. It loses one molecule of C2H4 to form a fragment ion [MH-C2H4]- (m / z 169.0146), and continues to lose one molecule of CO2 to produce a fragment [MH-C2H4-CO2]- (m / z 124.0153). According to the reference, it is identified as ethyl gallate

[162] .

[0115] Table 9. Chemical constituents of water shield polyphenols: UHPLC-ESI-QTOF-MS / MS mass spectrometry information

[0116] Compound 21 has a primary mass spectrometry quasi-molecular ion peak [MH]- at m / z 321.0334, with the molecular formula C14H10O9. Its secondary mass spectrometry fragment ions are mainly m / z 169.0119 and m / z 125.0230. Based on the references, it is preliminarily identified as digalic acids [163,164]. Compound 63 has a primary mass spectrometry quasi-molecular ion peak [MH]- at m / z 335.0415 and is identified as methyl digallate. Its main fragment ion m / z 183.084 is the methyl digallate ion produced by collision-induced dissociation, resulting in the loss of a C7H4O4 molecule ([MH-C7H4O4]-) [165,166]. The primary mass spectrometry (MS) precursor ion [MH]- of compounds 68 and 69 is m / z 349.0579, and they share the same secondary fragment ions m / z 197.0451, m / z 169.0119, and m / z 124.0153. Based on literature, they have been identified as ethyl-m-digallate and its isomers [167,168]. The primary mass spectrometry (MS) quasi-molecular ion peak [MH]- of compound 44 is 300.9995, and the main secondary mass spectrometry fragment ions are [MH-OH]- (m / z 283.9975), [MH-CO2]- (m / z 257.0108), [MH-CO2-CO]- (m / z 229.0161), and [MH-CO2-CO-2CO]- (m / z 173.0235). Figure 21 ), which is consistent with the cleavage pathway of ellagic acid reported in the literature, so it was identified as ellagic acid [161,169].

[0117] 3.5.1.2 Flavonoids The primary mass spectrometry mother ions [M-H]- of compounds 34, 36 and 42 were all m / z 563.1408, and the molecular formula was speculated to be C26H28O14. The main secondary mass spectrometry fragment ions were [M-H-C3H6O3]- (m / z 473.1082), [M-H-C4H8O4]- (m / z 443.0929), [M-H-C4H8O4-C2H4O2]- (m / z 383.0734), [M-H-C4H8O4-C3H6O3]- (m / z 353.0637), [M-H-C4H8O4-C3H6O3-CO]- (m / z 325.0638) and [M-H-C4H8O4-C3H6O3-2CO]- (m / z 297.0758), which were consistent with the mass spectrometry library and the reported mass spectrometry fragmentation pathway of shahmatoside, so the three compounds were preliminarily identified as shahmatoside and its isomers

[170] ; the primary mass spectrometry mother ion [M-H]- of compound 38 was m / z 447.0892, and the secondary ions were mainly m / z 357.0574, m / z 327.0458 and m / z 299.0557, which were consistent with the mass spectrometry library and the reported mass spectrometry fragmentation pathway of oxymatrine

[171] ; compound 46 was preliminarily identified as gossypin, and its primary mass spectrometry mother ion [M-H]- was m / z 479.0823, and the main secondary ion m / z 317.0283 was the residual part after the loss of a hexose group[172, 173]. The primary mass spectrometry mother ion [M-H]- of compound 73 was m / z 301.0351, and the molecular formula was C15H10O7, which was identified as quercetin. As a typical flavonol compound, quercetin has strong stability of the A and B ring phenyl rings, and is not easy to crack in the mass spectrometry fragmentation process, and mainly undergoes reverse Diels-Alder / RDA cracking of the C ring, and the cracking products mainly have the C ring with a part of the A and B rings[174, 175]. For example, the secondary ion m / z 151.0231 of compound 73 was the A ring with a part of the C ring, and the secondary ion m / z 133.0231 was the B ring with a part of the C ring. Figure 22RDA cleavage at 0,4 position generated 0,4 A fragment ion m / z 107.0091, 1,2 A fragment ion m / z 178.9977 and 1,2 B fragment ion m / z 121.0278, and 1,3 A fragment ion m / z 151.0029 and 1,3 B fragment ion m / z 149.0251 [176, 177]. Compound 76 was 3-O-methylquercetin, which primary mass spectrum mother ion [M-H]- (m / z 315.0478) lost [M-H-CH2]- to generate quercetin ion m / z 301.0351

[178] . Compound 54 was quercetin-3-O-glucoside, which primary mass spectrum mother ion [M-H]- (m / z 463.0908) lost [M-H-C6H10O5]- to generate quercetin ion m / z 301.0351

[179] . Compound 71 primary mass spectrum mother ion [M-H]- was m / z 639.1394, and its molecular formula was C27H28O18. Its main daughter ion m / z 477.1032 was mother ion lost [M-HC6H10O5]-, m / z 463.0864 was mother ion lost [M-H-C6H8O6]-, and m / z 301.0351 implied the existence of quercetin structure, so it was determined as quercetin glucoside glucuronides [180, 181]. Compounds 49 and 70 were determined as rutin and its isomer according to the literature

[179] . Compounds 45, 53 and 65, primary mass spectrum mother ion [M-H]- was m / z 615.0993, and its molecular formula was C28H24O16. Its main daughter ions m / z 463.0908 and m / z 300.0268 were mother ion lost galloyl and glucose, respectively, and combined with the literature, the three compounds were determined as quercetin-O-galloyl-glucopyranoside and its isomer

[182] . Compound 74 primary mass spectrum mother ion [M-H]- was m / z 285.0407, and its molecular formula was C15H10O6. It was identified as kaempferol according to the mass spectrum database and the literature [183, 184]. Compound 64 primary mass spectrum mother ion [M-H]- (m / z 447.0908) lost [M-H-C6H10O5]- to generate kaempferol ion m / z 284.0320, so it was identified as kaempferol-3-O-glucose

[179] .The primary mass spectrum of compound 61 was m / z 599.1046 [M-H]-, and the molecular formula was C28H24O15. The main daughter ions were m / z 447.0948 and m / z 285.0373, which were generated by the successive loss of galloyl and glucose from the parent ion, respectively. According to the literature, these three compounds were identified as Kaempferol-O-galloyl-glucopyranoside

[185] .

[0118] 3.5.1.3 Hydrolyzable tannins Hydrolyzable tannins are a class of polyphenols widely present in plants. In the process of mass spectrometry fragmentation, hydrolyzable tannins often undergo neutral loss of galloyl (152 Da), galloyl glucose (332 Da), HHDP (Hexahydroxydiphenoyl, 302 Da), and HHDP-glucose (482 Da) and galloyl-HHDP-glucose (634 Da) [186, 187]. The presence of ion fragments of m / z 169 in the secondary mass spectrum of hydrolyzable tannins indicates the presence of galloyl groups in the compound, and the presence of ion fragments of m / z 301 indicates the presence of HHDP groups in the compound

[188] . For example, Figure 23As shown, the ion of penta-galloyl-glucose lost one galloyl group to form the ion of tetra-galloyl-glucose m / z 787.1058, the ion of tetra-galloyl-glucose lost one galloyl group to form the ion of tri-galloyl-glucose m / z 635.0870, the ion of tri-galloyl-glucose lost one galloyl group to form the ion of di-galloyl-glucose m / z 483.0798, the ion of di-galloyl-glucose lost one galloyl group to form the ion of galloyl-glucose m / z 331.0651, and the ion of galloyl-glucose lost one glucose to form the ion of gallic acid m / z 169.0119; m / z 769.0895, m / z 769.0895, 617.0816 and 465.0671 were the fragments of the ion of tetra-galloyl-glucose, tri-galloyl-glucose and di-galloyl-glucose lost H2O, respectively. Therefore, the mass spectrum chart can identify compound 1, 6 and 8 as galloyl-glucose and its isomers, compound 3, 10, 11, 13, 14, 15, 16, 17, 18 and 19 as di-galloyl-glucose and its isomers, compound 24, 25, 26, 27, 29, 30, 32 and 35 as tri-galloyl-glucose and its isomers, compound 31, 39, 41, 47, 51, 56, 57 and 59 as tetra-galloyl-glucose and its isomers, and compound 48, 52, 55, 58, 60 and 62 as penta-galloyl-glucose and its isomers [189, 190]. The secondary mass spectrum ion of compound 2, 4, 7, 12, 20, 28, 37, 40 and 43 all have m / z 300.9995 fragments, indicating that they all contain HHDP groups.The primary mass spectrometry mother ion [M-H]- of compound 2 was m / z 481.0565, which was identified as HHDP-glucose; the primary mass spectrometry mother ion [M-H]- of compounds 4, 7 and 12 was m / z 633.0759, which was identified as Galloyl-HHDP-glucose; the primary mass spectrometry mother ion [M-H]- of compounds 5, 20 and 28 was m / z 785.0847, which was identified as Di-galloyl-HHDP-glucose; and the primary mass spectrometry mother ion [M-H]- of compounds 37, 40 and 43 was m / z 937.0959, which was identified as Tri-galloyl-HHDP-glucose[191,192].

[0119] 3.5.2 HPLC quantitative analysis According to the qualitative identification results of UHPLC-ESI-QTOF-MS / MS, 13 kinds of polyphenols such as gallic acid, methyl gallate and rutin were selected for quantitative analysis. As shown in Table 10, the baseline of the 13 standard samples was smooth and the separation degree was good. The linear equation R2 obtained by taking the polyphenol concentration as x and the peak area as y was greater than 0.999, and the linear relationship was good (Table 10). When the sample of the purified polyphenols of Potamogeton was analyzed, 11 kinds of compounds were detected except that hyperoside and kaempferol were below the detection limit and had no response. According to the calculation of the linear equation of the standard sample, it was found that the contents of kaempferol 3-O-(6''-galloyl)-glucoside and ethyl gallate in the polyphenols of Potamogeton were the highest, which were 205.74 mg / g and 117.88 mg / g, respectively. Figure 24

[0120] Table 10 Content analysis of main components of Potamogeton polyphenols

[0121] Different extraction processes were used to extract polysaccharides and polyphenols from Potamogeton. The yields of polysaccharides and polyphenols were measured after extraction, and the results are shown in the table below. It can be seen that the yields of polysaccharides and polyphenols obtained by the application examples were lower than those obtained under the optimized conditions of the application (extraction temperature 66℃, extraction time 33min, liquid-solid ratio 84mL / g, under which the yields of polysaccharides and polyphenols were 5.39% and 12.53%, respectively).

[0122]

[0123] ​The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement within the technical range disclosed by the present application and within the spirit and principle of the present application should be covered within the protection scope of the present application.

Claims

1. A method for simultaneous extraction of polysaccharides and polyphenols from Potamogeton crispus based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy, characterized in that, The method comprises the following steps: preparing a two-water-phase extraction solution with a total volume of 8 ml, the extraction solution being prepared by mixing anhydrous ethanol with a mass fraction of 25-75% and an ammonium sulfate solution with a mass fraction of 20-40%, and adding 0.10 g of a powder of the plant into the extraction solution; extracting at 60 DEG C under the action of ultrasonic power of 320 W for 20 min; centrifuging at 13,000 rpm for 5 min; collecting the supernatant and the lower clear liquid as a polyphenol extraction solution and a polysaccharide extraction solution respectively, and determining the yield after diluting to 100 ml.

2. The method of claim 1, wherein, Further comprising: The polysaccharide concentration is determined by the phenol-sulfuric acid method with glucose as a standard, and the polyphenol concentration is determined by the Folin-phenol method with gallic acid as a standard.

3. A system for simultaneous extraction of polysaccharides and polyphenols from Potamogeton crispus based on ethanol-ammonium sulfate aqueous two-phase ultrasonic synergy, characterized in that, The system comprises: a preparation unit for mixing anhydrous ethanol and an ammonium sulfate solution to prepare a two-water-phase extraction solution with a mass fraction of 25-75% and 20-40%; an ultrasonic extraction unit for ultrasonic treatment of the mixture of the extraction solution and the powder of the plant at 60 DEG C and ultrasonic power of 320 W; a phase separation unit for centrifugal separation of a polyphenol phase and a polysaccharide phase at 13,000 rpm after ultrasonic treatment at 320 W; and a detection unit for determining the yield after diluting the obtained two phases.

4. The system of claim 3, wherein, Further comprising: A calculation module for calculating the extraction yield according to the determined polysaccharide concentration, polyphenol concentration and raw material mass.

5. A computer device for performing the method of claim 1, wherein, The computer device comprises a memory and a processor, the memory stores a computer program, and the processor can execute the computer program to sequentially execute: extraction solution preparation parameter setting, ultrasonic extraction condition control, centrifugal separation control and yield calculation.

6. A two-aqueous phase simultaneous extraction kit, characterized in that, The kit comprises: anhydrous ethanol; ammonium sulfate solid; a pre-calibrated volume bottle; and an instruction manual, which clearly indicates that a two-water-phase extraction solution is prepared with a mass fraction of 25-75% and 20-40%, and the ultrasonic extraction is performed at 60 DEG C, 320 W, 20 min and 13,000 rpm for 5 min.

7. A polysaccharide-polyphenol complex based on the method according to claim 1, characterized by The complex is obtained by two-water-phase ultrasonic synergistic and simultaneous extraction of the powder of the plant, the polysaccharide purity is not less than 90%, and the total polyphenol content is not less than 5%.

8. A functional health food based on the complex as claimed in claim 7, characterized by, The complex is added to the health food in a proportion of 1-5 g per serving.

9. A data processing flow based on the method according to any one of claims 1 to 2, characterized in that, The flow comprises: standard curve drawing; sample absorbance determination; concentration calculation and extraction yield statistics; single factor analysis and response surface optimization evaluation.

10. A laboratory automation extraction device based on the method according to claim 1, characterized in that, Comprising: A solution preparation module; an ultrasonic extraction module; A centrifugal separation module; An automatic dilution and sampling module; An online detection and data output module.

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