Eutectic solvent extraction method of yellowed green tea polysaccharide

Abstract

The invention provides a deep-eutectic solvent extraction method of yellow green tea polysaccharide, which comprises the following steps: crushing yellow green tea, decoloring, and drying for later use; preparing the eutectic solvent by adopting a direct heating method, and preparing the eutectic solvent with specific water content; in ultrasonic equipment, mixing and extracting the yellowed green tea powder and a deep-eutectic solvent, taking supernate, adding a Sevage reagent to remove protein, centrifuging, taking supernate, repeatedly removing protein for multiple times, and combining the supernate to obtain a second mixed solution; and adding absolute ethyl alcohol for alcohol precipitation, centrifuging to obtain crude polysaccharide precipitate, redissolving the crude polysaccharide precipitate with distilled water, performing rotary evaporation, desalting through a membrane dialysis method, and finally freeze-drying to obtain the yellowing green tea crude polysaccharide. The invention explores an efficient, green and safe extraction method of the yellow green tea polysaccharide, and solves the problems of low extraction rate and low purity of tea polysaccharide. In addition, the extract has typical tea polysaccharide structural characteristics, and has good antioxidation and hypoglycemic potential.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, and in particular to a method for extracting polysaccharides from xanthocyanin green tea using a eutectic solvent. Background Technology

[0002] Tea polysaccharides are a general term for a class of acidic polysaccharides or acidic glycoproteins tightly bound to proteins, extracted from tea leaves. They possess various biological activities and have complex structures, with molecular weights ranging from 40,000 to 100,000. The origin, variety, maturity, and age of the tea tree all affect the content of tea polysaccharides. The monosaccharide components of tea polysaccharides mainly include rhamnose, arabinose, galactose, and glucose. Various factors such as tea type, fermentation degree, and tea processing techniques can all influence the monosaccharide composition of tea polysaccharides.

[0003] Tea polysaccharides have a variety of bioactive functions, such as anti-oxidation, hypoglycemia, anti-tumor and regulation of intestinal flora, and have been widely used in the food industry.

[0004] Tea polysaccharides can enhance the antioxidant capacity of cells by increasing the scavenging rate of hydroxyl free radicals, reducing malondialdehyde content, and increasing the activity of superoxide dismutase (SOD) in erythrocytes. In the adjunctive treatment of diabetes, tea polysaccharides have a non-competitive inhibitory effect on α-glucosidase and a certain inhibitory capacity on α-amylase activity, effectively improving symptoms of hypertension and hyperlipidemia during diabetes treatment.

[0005] Animal experiments have shown that tea polysaccharides can enhance the phagocytic index of peritoneal macrophages, increase serum hemolysin levels in mice, and promote antibody formation in spleen cells, thereby enhancing the immune function of tumor-bearing mice. Furthermore, it can inhibit tumor growth by increasing erythrocyte catalase activity by inhibiting serum lactate dehydrogenase activity.

[0006] Regarding the improvement of gut microbiota by tea polysaccharides, tea polysaccharides can effectively improve gut microbiota imbalance, increase the abundance of Lactobacillus and Heterobacterium, and inhibit the growth of potentially pathogenic microorganisms. Tea polysaccharides can also exert positive effects on host physiology and immune regulation by altering gut microbiota metabolites.

[0007] Traditional extraction methods for tea polysaccharides, such as water extraction and alcohol precipitation, enzymatic extraction, ultrasound-assisted extraction, and microwave extraction, generally suffer from high consumption, safety hazards, and environmental pollution. Furthermore, these methods are time-consuming and inefficient. Therefore, exploring a novel, efficient, green, and safe extraction method has become a pressing issue in tea polysaccharide extraction, aiming to address the problems of low extraction rates and purity.

[0008] Eutectic solvents possess physicochemical properties similar to traditionally used ionic liquids, and are characterized by non-toxicity, biodegradability, recyclability, and relatively low cost. Compared to traditional methods of extracting tea polysaccharides using organic solvents, ultrasound-assisted eutectic solvent extraction effectively avoids potential damage to the structure and activity of tea polysaccharides caused by excessively high temperatures and unsuitable pH environments. Considering extraction efficiency, product purity, and environmental safety, eutectic solvents are ideal solvent systems.

[0009] Huang Jian et al.'s patent (CN117069873A) "An extraction process for high-purity tea polysaccharides based on camellia seed meal" uses a combination of papain and cellulase enzymes combined with microwave-assisted extraction of tea polysaccharides, but the extraction process is cumbersome and involves a large amount of solvent with environmental pollution risks.

[0010] The patent (CN202211287717.X) by Shao Jinhua et al. of Hunan University of Science and Technology, entitled "A method for extracting polysaccharides from black tea using an ultrasonic-assisted eutectic solvent", constructs a eutectic solvent system of choline chloride and lactic acid to extract tea polysaccharides from Qiyang black tea, with a higher extraction rate than the traditional water extraction method.

[0011] The patent (CN202311263142.2) by Lu Weihong et al. of Harbin Institute of Technology, entitled "A method for synergistic extraction of bitter tea polysaccharides and its radiation protection application", uses ultra-high pressure synergistic microwave countercurrent assisted extraction of bitter tea polysaccharides, but the purity of the obtained polysaccharides is limited.

[0012] The patents mentioned above for tea polysaccharide extraction focus on using single or combined polysaccharide extraction methods to extract polysaccharides from different types of tea, and all of them take environmental factors into consideration to some extent. However, the polysaccharide extraction rate is limited, the subsequent purification process is complex, and there are problems such as relatively complex operating equipment and technology, and high actual production costs. Therefore, there is an urgent need for a low-eutectic solvent extraction method for xanthated green tea polysaccharides. Summary of the Invention

[0013] The purpose of this invention is to provide a method for extracting polysaccharides from xanthocyanin green tea using a eutectic solvent.

[0014] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0015] This invention provides a method for extracting polysaccharides from xanthated green tea using a eutectic solvent, comprising the following steps:

[0016] S1. Pulverize the yellowed green tea, decolorize it, and dry it for later use;

[0017] S2. A eutectic solvent is prepared by direct heating, and a eutectic solvent with a specific water content is formulated.

[0018] S3. In an ultrasonic device, the yellowed green tea powder is mixed with a eutectic solvent for extraction, and the supernatant is taken to obtain the first mixture.

[0019] S4. Add Sevage reagent to the first mixture to remove protein and obtain a deproteinized mixture. Then centrifuge the deproteinized mixture, take the supernatant, repeat the process several times to remove protein, and combine the supernatants to obtain the second mixture.

[0020] S5. The obtained second mixture was added to anhydrous ethanol for precipitation, centrifuged to obtain crude polysaccharide precipitate, the crude polysaccharide precipitate was reconstituted with distilled water, rotary evaporated, desalted by membrane dialysis, and finally freeze-dried to obtain yellowed green tea crude polysaccharide.

[0021] Preferably, in S2, the method for preparing the eutectic solvent is as follows: choline chloride and urea are mixed in a molar ratio of 1:1 to 1:6, and heated and stirred in a water bath at 90°C for 30 to 50 minutes to obtain the eutectic solvent.

[0022] Preferably, in step S2, distilled water is added to the eutectic solvent to obtain a eutectic solvent with a water content of 20-70%.

[0023] Preferably, in step S3, the yellowed green tea powder and a eutectic solvent are extracted for 10-60 minutes at a solid-liquid ratio of 1:20-60 and an extraction temperature of 60-80°C to obtain the first mixture.

[0024] Preferably, in S3, the power of the ultrasound is 39~451W.

[0025] Preferably, in S4, the volume ratio of n-butanol to chloroform in the Sevage reagent is 1:(4~5).

[0026] Preferably, in S5, the volume ratio of the second mixture to anhydrous ethanol is 1:(4~5).

[0027] Preferably, in S5, the centrifugation speed is 4000~5000 r / min, and the centrifugation time is 10~15 min.

[0028] Preferably, in step S1, the yellowed green tea is pulverized and passed through a 40-mesh sieve to obtain yellowed green tea powder. The yellowed green tea powder is then decolorized overnight with four times its volume of ethanol and then air-dried naturally.

[0029] This invention also provides a xanthocyanin polysaccharide, which is prepared by a eutectic solvent extraction method.

[0030] The beneficial effects of this invention are:

[0031] (1) Traditional extraction methods for tea polysaccharides, such as water extraction and alcohol precipitation, enzymatic methods, and ultrasound-assisted methods, generally suffer from problems such as high solvent consumption, safety hazards, and environmental pollution. In addition, the extraction process is time-consuming and has low extraction efficiency. Therefore, this invention explores a novel, efficient, green, and safe method for extracting polysaccharides from xanthated green tea, providing a solution to the problem of low extraction rate and purity of tea polysaccharides.

[0032] (2) This invention uses polysaccharides from etiolated green tea as raw material and employs an ultrasound-assisted choline chloride-urea eutectic solvent system to extract the polysaccharides, aiming to explore an extraction method with a high polysaccharide extraction rate. The effects of six factors—molar ratio of eutectic solvent components, extraction temperature, ultrasonic power, extraction time, water content, and solid-liquid ratio—on the extraction rate of tea polysaccharides were investigated in single-factor experiments. Based on the single-factor experiments, response surface methodology combined with Box-Behnken design was used to optimize the optimal extraction process for etiolated green tea polysaccharides. The optimized process flow was as follows: extraction temperature 78℃, water content in the DES system 52%, choline chloride:urea molar ratio 1:4.63, solid-liquid ratio 1:46 (g / mL), ultrasonic power 369 W, and extraction time 40 min. Under these extraction conditions, the polysaccharide yield was 11.02±0.47%.

[0033] (3) The polysaccharide extracted from the yellowed green tea obtained by this invention is dark brown and has a reflective luster under light. The total sugar content of the isolated and purified yellowed green tea polysaccharide is 39.09%±0.72%, the uronic acid content is 16.00%±0.59%, the reducing sugar content is 9.49%±0.36%, and the protein content is 2.91%±0.21%.

[0034] (4) The isolated and purified xanthant green tea polysaccharides were analyzed by UV-Vis absorption spectroscopy and Fourier transform infrared spectroscopy. UV-Vis results showed that the extracted polysaccharides had no obvious peaks at 260 nm and 280 nm, indicating that the protein was effectively removed. FT-IR results showed that the extracted polysaccharides had stretching vibration peaks of hydroxyl (OH) and amino (NH), stretching vibration peaks of carbonyl (C=O), bending vibration peaks of CH, bending vibration peaks of hydroxyl (OH), stretching vibration peaks of CO, and stretching vibration peaks of ether bond (COC), which are consistent with the characteristics of tea polysaccharides. Ultrasonic treatment did not significantly change the functional group structure of the tea polysaccharides.

[0035] (5) In vitro antioxidant activity and in vitro hypoglycemic activity were analyzed in the isolated and purified xanthocyanin green tea polysaccharide. The antioxidant activity results showed that the scavenging ability of xanthocyanin green tea polysaccharide against both free radicals increased with increasing concentration, with a more significant scavenging ability against DPPH free radicals, but still lagging behind vitamin C. The hypoglycemic activity results showed that the inhibitory ability of xanthocyanin green tea polysaccharide against α-amylase and α-glucosidase increased with increasing concentration, with a better inhibitory ability against α-amylase. From a pharmaceutical perspective, xanthocyanin green tea polysaccharide has good application prospects as a natural hypoglycemic component. Attached Figure Description

[0036] Figure 1 A schematic diagram showing the effect of different molar ratios of eutectic solvent components on the extraction rate of tea polysaccharides;

[0037] Figure 2 This is a schematic diagram illustrating the effect of the solid-liquid ratio on the polysaccharide extraction rate of etiolated green tea.

[0038] Figure 3 A schematic diagram showing the effect of DES moisture content on the extraction rate of polysaccharides from yellowed green tea.

[0039] Figure 4 This is a schematic diagram illustrating the effect of ultrasonic power on the extraction rate of polysaccharides from xanthated green tea.

[0040] Figure 5 A schematic diagram illustrating the effect of extraction temperature on the extraction rate of polysaccharides from yellowed green tea.

[0041] Figure 6 A schematic diagram illustrating the effect of extraction time on the extraction rate of polysaccharides from etiolated green tea.

[0042] Figure 7 Response surface plot and contour plot showing the effect of the interaction between temperature and moisture content on the yield of polysaccharides in yellowed green tea;

[0043] Figure 8 The response surface plot and contour plot show the effect of the interaction between temperature and solid-liquid ratio on the yield of polysaccharides in etiolated green tea.

[0044] Figure 9 The response surface plot and contour plot show the effect of the interaction between solid-liquid ratio and water content on the yield of polysaccharides in yellowed green tea.

[0045] Figure 10 This is the standard curve for total sugar.

[0046] Figure 11 For the uronic acid standard curve;

[0047] Figure 12 This is the standard curve for reducing sugars;

[0048] Figure 13This is a standard curve for proteins.

[0049] Figure 14 The UV-Vis absorption spectrum of polysaccharides from yellowed green tea;

[0050] Figure 15 Fourier transform infrared spectrum of polysaccharides from yellowed green tea;

[0051] Figure 16 The scavenging effect of xanthocyanin green tea polysaccharides on ABTS+·;

[0052] Figure 17 The scavenging effect of polysaccharides from yellowed green tea on DPPH·;

[0053] Figure 18 The inhibitory effect of polysaccharides from yellowed green tea on α-glucosidase;

[0054] Figure 19 This study investigated the inhibitory effect of polysaccharides from yellowed green tea on α-amylase. Detailed Implementation

[0055] This invention provides a method for extracting polysaccharides from xanthated green tea using a eutectic solvent, comprising the following steps:

[0056] S1. Pulverize the yellowed green tea, decolorize it, and dry it for later use;

[0057] S2. A eutectic solvent is prepared by direct heating, and a eutectic solvent with a specific water content is formulated.

[0058] S3. In an ultrasonic device, the yellowed green tea powder is mixed with a eutectic solvent for extraction, and the supernatant is taken to obtain the first mixture.

[0059] S4. Add Sevage reagent to the first mixture to remove protein and obtain a deproteinized mixture. Then centrifuge the deproteinized mixture, take the supernatant, repeat the process several times to remove protein, and combine the supernatants to obtain the second mixture.

[0060] S5. The obtained second mixture was added to anhydrous ethanol for precipitation, centrifuged to obtain crude polysaccharide precipitate, the crude polysaccharide precipitate was reconstituted with distilled water, rotary evaporated, desalted by membrane dialysis, and finally freeze-dried to obtain yellowed green tea crude polysaccharide.

[0061] In this invention, in step S2, the method for preparing the eutectic solvent is as follows: choline chloride and urea are mixed in a molar ratio of 1:1 to 1:6, and heated and stirred in a water bath at 90°C for 30 to 50 minutes to prepare the eutectic solvent.

[0062] In this invention, the molar ratio of choline chloride to urea is 1:1 to 1:6, preferably 1:1.5 to 1:5.5, more preferably 1:2 to 1:5, and even more preferably 1:3 to 1:4.

[0063] In this invention, in step S2, distilled water is added to the eutectic solvent to obtain a eutectic solvent with a water content of 20-70%, preferably 30-60%, more preferably 35-55%, and even more preferably 40-50%.

[0064] In this invention, in step S3, the yellowed green tea powder and a eutectic solvent are extracted for 10 to 60 minutes at an extraction temperature of 60 to 80°C with a solid-liquid ratio of 1:20 to 60 to obtain the first mixture.

[0065] In this invention, the solid-liquid ratio of the yellowed green tea powder to the eutectic solvent is 1:20~60, preferably 1:25~55, more preferably 1:30~50, and even more preferably 1:35~45.

[0066] In this invention, the extraction temperature is 60~80℃, preferably 63~78℃, more preferably 65~75℃, and even more preferably 68~73℃.

[0067] In this invention, the extraction time is 10-60 min, preferably 15-55 min, more preferably 20-50 min, and even more preferably 30-40 min.

[0068] In this invention, in S3, the power of the ultrasound is 39~451W, preferably 50~400W, more preferably 60~350W, and even more preferably 70~300W.

[0069] In this invention, in S4, the volume ratio of n-butanol to chloroform of the Sevage reagent is 1:(4~5), preferably 1:(4.2~4.8), more preferably 1:(4.3~4.7), and even more preferably 1:(4.4~4.6).

[0070] In this invention, in step S5, the volume ratio of the second mixture to anhydrous ethanol is 1:(4~5), preferably 1:(4.2~4.8), more preferably 1:(4.3~4.7), and even more preferably 1:(4.4~4.6).

[0071] In this invention, in S5, the centrifugal speed is 4000~5000 r / min, preferably 4100~4900 r / min, more preferably 4200~4800 r / min, and even more preferably 4300~4700 r / min.

[0072] In this invention, the centrifugation time is 10-15 min, preferably 11-14 min, more preferably 12-13 min, and even more preferably 12.2-12.8 min.

[0073] In this invention, in step S1, the yellowed green tea is pulverized and passed through a 40-mesh sieve to obtain yellowed green tea powder. The yellowed green tea powder is then decolorized overnight with four times its volume of ethanol and then air-dried naturally.

[0074] This invention also provides a xanthocyanin polysaccharide, which is prepared by a eutectic solvent extraction method.

[0075] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0076] Example 1

[0077] A method for extracting polysaccharides from xanthocyanin green tea using a eutectic solvent includes the following steps:

[0078] S1. The finished green tea (provided by Sichuan Sanshan Tea Industry Co., Ltd.) processed from the yellowing variety "Zhonghuang No. 1" is pulverized and passed through a 40-mesh sieve. It is then decolorized with four times its volume of ethanol overnight and naturally air-dried to obtain tea powder.

[0079] S2. Choline chloride and urea are mixed at a molar ratio of 1:4.63 and heated and stirred in a 90°C water bath for 50 min to prepare a eutectic solvent. Distilled water is added to the eutectic solvent to obtain a eutectic solvent with a water content of 52%.

[0080] S3. In an ultrasonic device, the yellowed green tea powder and a eutectic solvent are extracted at a solid-liquid ratio of 1:46 and an extraction temperature of 78°C for 40 min. The supernatant is collected to obtain a crude polysaccharide extract. The ultrasonic power is 369W.

[0081] S4. Add Sevage reagent to the crude polysaccharide extract to remove proteins. Mix n-butanol and chloroform at a volume ratio of 1:4 to prepare the Sevage working solution. Add 1 / 5 volume of Sevage working solution to the polysaccharide extract, shake continuously for 15 min, centrifuge at 5000 r / min for 15 min to separate the supernatant, discard the protein layer and organic phase, add another 1 / 5 volume of Sevage working solution to the resulting upper polysaccharide solution, and repeat this protein removal process six times.

[0082] S5. After protein removal, the combined supernatant was precipitated overnight with four volumes of anhydrous ethanol, followed by centrifugation at 5000 r / min for 15 min to obtain crude polysaccharide precipitate. The crude polysaccharide was then reconstituted with distilled water, rotary evaporated, and desalted by membrane dialysis (MWCO 7000 Da, washing with distilled water for 48 h). Finally, the lyophilized crude polysaccharide from xanthocyanin green tea was obtained. The lyophilized xanthocyanin green tea polysaccharide was dark brown with a reflective luster under light; the solution was transparent dark brown with some insoluble precipitate. The polysaccharide yield after lyophilization was 11.02 ± 0.47%.

[0083] Example 2

[0084] A method for extracting polysaccharides from xanthocyanin green tea using a eutectic solvent includes the following steps:

[0085] S1. The finished green tea (provided by Sichuan Sanshan Tea Industry Co., Ltd.) processed from the yellowing variety "Zhonghuang No. 1" is pulverized and passed through a 40-mesh sieve. It is then decolorized with four times its volume of ethanol overnight and naturally air-dried to obtain tea powder.

[0086] S2. Choline chloride and urea are mixed at a molar ratio of 1:3 and heated and stirred in a 90°C water bath for 30 min to prepare a eutectic solvent. Distilled water is added to the eutectic solvent to obtain a eutectic solvent with a water content of 40%.

[0087] S3. In an ultrasonic device, the yellowed green tea powder and a eutectic solvent are extracted at a solid-liquid ratio of 1:50 and an extraction temperature of 70°C for 30 minutes. The supernatant is collected to obtain a crude polysaccharide extract. The ultrasonic power is 400W.

[0088] S4. Add Sevage reagent to the crude polysaccharide extract to remove proteins. Mix n-butanol and chloroform at a volume ratio of 1:4 to prepare the Sevage working solution. Add 1 / 5 volume of Sevage working solution to the polysaccharide extract, shake continuously for 15 min, centrifuge at 5000 r / min for 15 min to separate the supernatant, discard the protein layer and organic phase, add another 1 / 5 volume of Sevage working solution to the resulting upper polysaccharide solution, and repeat this protein removal process six times.

[0089] S5. After protein removal, the combined supernatant was precipitated overnight with four volumes of anhydrous ethanol, followed by centrifugation at 5000 r / min for 15 min to obtain crude polysaccharide precipitate. The crude polysaccharide was then reconstituted with distilled water, rotary evaporated, and desalted by membrane dialysis (MWCO 7000 Da, washing with distilled water for 48 h). Finally, it was freeze-dried to obtain the crude polysaccharide from xanthocyanin green tea.

[0090] Example 3

[0091] A method for extracting polysaccharides from xanthocyanin green tea using a eutectic solvent includes the following steps:

[0092] S1. The finished green tea (provided by Sichuan Sanshan Tea Industry Co., Ltd.) processed from the yellowing variety "Zhonghuang No. 1" is pulverized and passed through a 40-mesh sieve. It is then decolorized with four times its volume of ethanol overnight and naturally air-dried to obtain tea powder.

[0093] S2. Choline chloride and urea are mixed at a molar ratio of 1:2 and heated and stirred in a 90°C water bath for 40 min to prepare a eutectic solvent. Distilled water is added to the eutectic solvent to obtain a eutectic solvent with a water content of 60%.

[0094] S3. In an ultrasonic device, the yellowed green tea powder and a eutectic solvent are extracted at a solid-liquid ratio of 1:30 and an extraction temperature of 80°C for 50 min. The supernatant is collected to obtain a crude polysaccharide extract. The ultrasonic power is 300W.

[0095] S4. Add Sevage reagent to the crude polysaccharide extract to remove proteins. Mix n-butanol and chloroform at a volume ratio of 1:4 to prepare the Sevage working solution. Add 1 / 5 volume of Sevage working solution to the polysaccharide extract, shake continuously for 15 min, centrifuge at 5000 r / min for 15 min to separate the supernatant, discard the protein layer and organic phase, add another 1 / 5 volume of Sevage working solution to the resulting upper polysaccharide solution, and repeat this protein removal process six times.

[0096] S5. After protein removal, the combined supernatant was precipitated overnight with four volumes of anhydrous ethanol, followed by centrifugation at 5000 r / min for 15 min to obtain crude polysaccharide precipitate. The crude polysaccharide was then reconstituted with distilled water, rotary evaporated, and desalted by membrane dialysis (MWCO 7000 Da, washing with distilled water for 48 h). Finally, it was freeze-dried to obtain the crude polysaccharide from xanthocyanin green tea.

[0097] The effect of different molar ratios of eutectic solvent components on the extraction rate of tea polysaccharides is as follows: Figure 1 As shown. The decrease in the melting point of DES is related to the strong hydrogen bonding between the donor and acceptor; hydrogen bonds can form between the chloride ions in choline chloride and the -NH2 groups in urea. The system has the lowest melting point when the molar ratio of choline chloride to urea is 1:2, and it remains a liquid at room temperature. The melting point of DES increases with the increase of the molar ratio of choline chloride to urea. When the molar ratio reaches 1:6, the system has high viscosity and a high melting point. To prepare a clear and transparent state using the direct heating method, heating at 100 °C for 75 min is required. Figure 1It was found that the highest polysaccharide extraction rate was achieved when the molar ratio of choline chloride to urea was 1:4. This is because the low proportion of urea resulted in strong hydrogen bonds in the system, limiting the mass transfer rate of polysaccharides. As the molar ratio increased, hydrogen bond interactions decreased, promoting polysaccharide dissolution. When the molar ratio reached 1:5, the system exhibited higher viscosity and surface tension, increasing the resistance to polysaccharide dissolution and diffusion, which was detrimental to the mass transfer process. Therefore, a molar ratio of 1:3 to 1:5 was selected as the subsequent optimization experimental conditions.

[0098] The effect of different solid-liquid ratios on the extraction rate of tea polysaccharides is as follows: Figure 2 As shown, the solid-liquid ratio affects the extraction rate by altering the partition coefficient of the extract in the extraction system. Within a certain range, the dissolution efficiency of the target substance increases with the increase of the solid-liquid ratio. However, an excessively high solid-liquid ratio not only leads to difficulties in subsequent extract separation but also weakens the intermolecular interactions of the target substance, thereby reducing the extraction rate. As shown in the figure, the extraction rate is highest at a solid-liquid ratio of 1:40 (g / mL). Both excessively low and excessively high solid-liquid ratios are detrimental to polysaccharide dissolution.

[0099] The effect of different water contents on the extraction rate of tea polysaccharides in eutectic solvent systems is as follows: Figure 3 As shown. Both components of DES, choline chloride and urea, are hygroscopic; differences in water content within the system may alter the hydrogen bond interactions between the two components. For example... Figure 3 As shown, the extraction rate increases with increasing moisture content in the DES system from 20% to 40%. However, when the moisture content is between 40% and 60%, the extraction rate of polysaccharides from yellowed green tea remains relatively stable. This may be because the decrease in extraction rate due to DES structural damage caused by increased moisture content balances with the increased mass transfer efficiency of polysaccharides in the system. However, with further increases in moisture content, the original structure of DES is destroyed, leading to a decrease in polysaccharide extraction rate. Therefore, a moisture content of 40%–60% was selected as the subsequent optimization experimental condition.

[0100] The effect of different ultrasonic powers on the extraction rate of tea polysaccharides is as follows: Figure 4 As shown, the cavitation effect generated by ultrasound causes the cells of the tea raw material to rupture through instantaneous high temperature and pressure, thereby promoting the dissolution of polysaccharides. Figure 4 As shown, the polysaccharide extraction rate of yellowed green tea generally showed a significant upward trend with increasing ultrasonic power in the range of 39–451 W. The change was not significant in the range of 121–287 W, but it increased significantly in the range of 287–451 W. Considering the significant increase in polysaccharide extraction rate in the range of 369–451 W...

[0101] The effect of different extraction temperatures on the extraction rate of tea polysaccharides is as follows: Figure 5As shown in the figure. Studies have shown that extraction temperature has a significant impact on polysaccharide extraction rate, mainly by promoting the thermal motion of target molecules, increasing solubility, and increasing exudation efficiency, thereby promoting mass transfer. Furthermore, the DES system used in this experiment is temperature-sensitive; the viscosity of DES decreases with increasing temperature, thus increasing the mass transfer efficiency of polysaccharides in the extract and leading to a higher extraction rate. Figure 5 It was found that the extraction rate steadily increased with increasing temperature within the range of 30~70℃, but decreased slightly at 80℃, suggesting that higher extraction temperatures may damage the polysaccharide structure. Therefore, this experiment selected 60~80℃ as the optimized experimental conditions.

[0102] The effect of different extraction times on the extraction rate of tea polysaccharides is as follows: Figure 6 As shown, the extraction rate of polysaccharides from yellowed green tea reached its maximum between 30 and 40 min with increasing extraction time, and then showed a downward trend. While the dissolution rate of polysaccharides increased with longer extraction time, the osmotic pressure of the solution system reached equilibrium after a period of time, and the properties of the DES system, such as viscosity, water content, and conductivity, changed, leading to a decrease in the extraction rate. Furthermore, prolonged exposure to high temperature and ultrasonic environments also resulted in partial degradation of the polysaccharides. Therefore, a fixed extraction time of 40 min was used as the subsequent optimization experimental condition.

[0103] Based on the previous single-factor experimental results, and according to the Box-Behnken design, four factors were optimized: the molar ratio of choline chloride to urea, the moisture content of DES, the extraction temperature, and the solid-liquid ratio. The polysaccharide extraction rate was used as the response value. The Box-Behnken experimental design and results are shown in Table 1.

[0104] Table 1. Box-Behnken Experimental Design and Results

[0105] Serial Number Temperature (°C) B. Moisture content (%) C molar ratio D (solid-to-material ratio, g / mL) Y polysaccharide extraction rate (%) 1 60 40 1:4 40 5.38±0.06 2 80 40 1:4 40 6.44±0.15 3 60 60 1:4 40 4.66±0.26 4 80 60 1:4 40 7.02±0.32 5 70 50 1:3 30 7.25±0.40 6 70 50 1:5 30 6.99±0.26 7 70 50 1:3 50 7.72±0.30 8 70 50 1:5 50 7.65±0.63 9 60 50 1:4 30 5.69±0.67 10 80 50 1:4 30 5.88±0.47 11 60 50 1:4 50 4.79±0.72 12 80 50 1:4 50 7.25±0.70 13 70 40 1:3 40 7.21±0.40 14 70 60 1:3 40 7.33±0.60 15 70 40 1:5 40 7.43±0.48 16 70 60 1:5 40 7.63±0.32 17 60 50 1:3 40 5.63±0.78 18 80 50 1:3 40 6.64±0.36 19 60 50 1:5 40 5.66±0.33 20 80 50 1:5 40 7.06±0.80 21 70 40 1:4 30 6.32±0.55 22 70 60 1:4 30 6.86±0.43 23 70 40 1:4 50 7.14±0.27 24 70 60 1:4 50 6.91±0.44 25 70 50 1:4 40 8.42±0.53 26 70 50 1:4 40 8.11±0.24 27 70 50 1:4 40 7.94±0.52 28 70 50 1:4 40 8.29±0.54 29 70 50 1:4 40 7.77±0.20

[0106] Analysis of variance was performed on the experimental data using Design-Expert 13 software, yielding the quadratic multiple regression equation: Y = 8.105 + 0.706 A + 0.04 B + 0.053C + 0.206 D + 0.3256 AB + 0.098 AC + 0.566AD + 0.019 BC - 0.192 BD + 0.046 CD - 1.646 A 2 - 0.614 B 2 - 0.133 C 2 - 0.605D 2 .

[0107] Table 2 shows that the p-value of the model is <0.0001, indicating that the model has a highly significant correlation. Furthermore, the p-value lacking fit is 0.7587 > 0.05, indicating that the model is highly feasible and can accurately predict the impact of different combinations of the four factors on the polysaccharide extraction rate. The correlation coefficient R of the binary regression equation is... 2 =0.9752, indicating that the regression equation is significant. Analysis of the F-values ​​shows that the order of influence of the four factors on the extraction rate of polysaccharides from yellowed green tea is: extraction temperature (A) > material-to-liquid ratio (D) > molar ratio (C) > moisture content (B). Among them, extraction temperature and material-to-liquid ratio have extremely significant effects on the extraction rate of polysaccharides from yellowed green tea (p < 0.01). Analysis of the correlation coefficients yields B, C, AC, BC, BD, CD, and C. 2 The p-values ​​of all terms are greater than 0.05, indicating no significant impact. Therefore, the above seven terms are removed from the regression equation, resulting in the optimized regression model:

[0108] Y= 8.105 + 0.706 A + 0.206 D + 0.3256 AB + 0.566 AD -1.646 A 2 -0.614B 2 -0.605 D 2 .

[0109] Table 2. Correlation coefficients and significance analysis of the quadratic model

[0110]

[0111] Note: ** indicates extremely significant difference (P<0.01); * indicates significant difference (P>0.05)

[0112] To visually represent how the four factors interact and affect the extraction rate, Design-Expert software was used to create contour plots and response surface plots between each factor and the response value. The density and slope of the contour lines in the response surface model represent the degree of mutual influence between the factors.

[0113] Depend on Figure 7 The steep slope indicates a significant interaction between temperature and moisture content, which in turn affects the extraction rate. Furthermore, the near-elliptical center of the contour surface further confirms this interaction, with temperature having a more pronounced effect.

[0114] Depend on Figure 8 It can be seen that the response surface plot has a clear slope, indicating that there is a significant interaction between temperature and solid-liquid ratio. Both factors have an important impact on the mass transfer efficiency of polysaccharides in the extract, but temperature is still the main factor affecting the extraction rate.

[0115] Depend on Figure 9It can be seen that there is an interaction between the solid-liquid ratio and the water content. The polysaccharide extraction rate first increases and then decreases as the solid-liquid ratio and water content increase.

[0116] The model obtained through response surface methodology shows that the optimal process conditions for extracting polysaccharides from xanthan green tea using the ultrasound-assisted eutectic solvent method are: extraction temperature 78℃, DES system water content 52%, choline chloride:urea molar ratio 1:4.63, solid-liquid ratio 1:46 (g / mL), ultrasonic power 369 W, and extraction time 40 min.

[0117] The total sugar content in the extracted xanthan green tea polysaccharides was determined using the phenol-sulfuric acid method, and the results are as follows: Figure 10 As shown in Table 3, the glucose standard curve was obtained as Y = 0.00324X + 0.016, with a correlation coefficient R² = 0.9913. Substituting the absorbance values ​​of the tea polysaccharide determination into this equation yielded the tea polysaccharide content per unit extract, as shown in Table 3.

[0118] The content of uronic acid in the extracted tea polysaccharides was determined by the m-hydroxyphenol method, and the results are as follows: Figure 11 As shown in Table 3, the obtained uronic acid standard curve is Y = 0.007294X + 0.0138, with a correlation coefficient R² = 0.999. Substituting the absorbance values ​​of tea polysaccharides into this equation yields the uronic acid content per unit extract, as shown in Table 3 below.

[0119] The reducing sugar content in the extracted tea polysaccharides was determined using the dinitrosalicylic acid method, and the results are as follows: Figure 12 As shown in Table 3. The obtained reducing sugar standard curve is Y = 0.0006X - 0.00258, with a correlation coefficient R² = 0.995. Substituting the absorbance values ​​of tea polysaccharides into this equation yields the reducing sugar content per unit extract, as shown in Table 3 below.

[0120] The protein content in the obtained tea polysaccharides was determined using the Coomassie brilliant blue method, and the results are as follows: Figure 13 As shown in Table 3 below, the obtained protein standard curve is Y = 0.00365X + 0.04011, with a correlation coefficient R² = 0.995. Substituting the absorbance values ​​of tea polysaccharides into this equation yields the protein content per unit extract, as shown in Table 3 below.

[0121] Experimental results showed that the total sugar content in the xanthocyanin polysaccharide obtained after extraction and preliminary purification was limited, possibly due to the disruption of glycosidic bonds by ultrasound. The uronic acid content was 16.00±0.59%, indicating that the obtained xanthocyanin polysaccharide was an acidic polysaccharide. After protein removal, the protein content in the tea polysaccharide was only 2.91±0.21%, proving the effectiveness of the Sevage method for protein removal.

[0122] Table 3. Content of Physicochemical Components of Tea Polysaccharides

[0123] sample Total sugar Glucuronic acid reducing sugars protein Yellowed green tea polysaccharides 39.09±0.72% 16.00±0.59% 9.49±0.36% 2.91±0.21%

[0124] The polysaccharide extract of yellowed green tea was diluted and its absorbance in the 200-400 nm range was scanned using a UV spectrophotometer. The complete UV-Vis absorption spectrum results are as follows: Figure 14 As shown in the figure, the sample showed no obvious peak shape at 280 nm, indicating that the Sevage protein removal method is effective and can remove most of the protein impurities in the extract.

[0125] A mixed powder of polysaccharide sample and KBr (2 mg / 150 mg) was prepared, and Fourier transform infrared spectroscopy was used to analyze the polysaccharide of xanthated green tea at 4000-500 cm⁻¹. -1 Infrared characterization was performed within the wavenumber range, and the results are as follows: Figure 15 As shown. At 3000-3500 cm -1 Within this range, the polysaccharide exhibits a strong and broad peak, indicating the presence of stretching vibration peaks of the hydroxyl (OH) and amino (NH) groups, at 1609 cm⁻¹. -1 The sharp, strong peak at 1442 cm⁻¹ is the carbonyl C=O stretching vibration peak. -1 The peak at 1360 cm⁻¹ represents the CH bending vibration peak. -1 The peak at 1222 cm⁻¹ represents the bending vibration of the hydroxyl group (OH). -1 and 1090 cm -1 The peaks at these locations represent CO stretching vibrations and COC stretching vibrations of the ether bond. Therefore, the obtained FT-IR spectrum conforms to the characteristics of tea polysaccharides, indicating that the extraction method essentially preserves the functional group characteristics of tea polysaccharides.

[0126] The antioxidant capacity of the extracted tea polysaccharides was preliminarily evaluated using ABTS free radical scavenging rate. This experiment used vitamin C as a reference to test the ABTS free radical scavenging capacity of xanthated green tea polysaccharides. Figure 16 It was found that within the concentration range of 3.0–18.0 mg / mL, the scavenging ability of polysaccharides from yellowed green tea against ABTS free radicals was enhanced. Higher polysaccharide concentrations resulted in better free radical scavenging effects, indicating a positive correlation between its antioxidant capacity and concentration. When the polysaccharide concentration reached 18.0 mg / mL, the free radical scavenging rate reached 53.98 ± 2.34%, which is still lower than that of ascorbic acid. Fitting the obtained data yielded the formula Y = 1.22X. 1.25 The IC50 value of ABTS free radical scavenging ability of xanthan green tea polysaccharides was calculated. 50 The concentration was 19.25 mg / mL.

[0127] The antioxidant capacity of the extracted tea polysaccharides was preliminarily evaluated using DPPH free radical scavenging rate. This experiment used vitamin C as a reference to test the ABTS free radical scavenging capacity of xanthocyanin green tea polysaccharides. Figure 17It was found that within the concentration range of 2.0–20.0 mg / mL, the scavenging ability of polysaccharides from etiolated green tea against DPPH free radicals was enhanced. With increasing polysaccharide concentration, the free radical scavenging rate generally showed an upward trend, with a small increase between 2.0 and 10.0 mg / mL, but a significant increase within the 10.0–20.0 mg / mL range. When the polysaccharide concentration reached 20.0 mg / mL, its free radical scavenging ability reached 63.45 ± 1.70%. Fitting the obtained data yielded the formula Y = 0.518X. 1.59 The IC50 value of DPPH free radical scavenging ability of xanthan green tea polysaccharides was calculated. 50 The concentration was 17.71 mg / mL. Experiments showed that the free radical scavenging capacity of tea polysaccharides was generally positively correlated with concentration, but there was a certain gap compared to ascorbic acid.

[0128] The in vitro hypoglycemic ability of the extracted tea polysaccharides was preliminarily evaluated using α-glucosidase inhibition rate. This experiment used acarbose as a reference to test the α-glucosidase inhibition ability of xanthan green tea polysaccharides. Figure 18 It was found that within the range of 4.0–20.0 mg / mL, the α-glucosidase inhibition rate steadily increased with increasing acarbose concentration. At the same concentration, xanthan green tea polysaccharides also showed a positive correlation between concentration and inhibition rate, but their inhibition rate against α-glucosidase was lower than that of the positive control, acarbose. When the tea polysaccharide concentration was 20.0 mg / mL, the enzyme inhibition rate reached 50.13 ± 4.27%. Fitting the obtained data yielded the formula Y = 7.17 X 0.65 The IC50 inhibitory activity of α-glucosidase in xanthan green tea polysaccharides was calculated. 50 The concentration was 19.84 mg / mL. Tea polysaccharides have potential value in the development and production of α-glucosidase inhibitors.

[0129] The in vitro hypoglycemic ability of the extracted tea polysaccharides was preliminarily evaluated using α-amylase inhibition rate. Acarbose was used as a reference in this experiment to test the α-amylase inhibition ability of xanthocyanin green tea polysaccharides. Figure 19 As shown, the inhibition rate of α-amylase increased with increasing polysaccharide concentration in yellowed green tea. When the polysaccharide concentration was 1.8 mg / mL, the inhibition rate reached 60.33 ± 3.26%, which is significantly lower than that of the clinically used drug acarbose (inhibition rate 85.16 ± 3.48%). Fitting the obtained data yielded the formula Y = 16.73 X 2.09 The IC50 inhibitory activity of α-amylase in xanthocyanin green tea polysaccharides was calculated. 50 With a concentration of 1.69 mg / mL, it can inhibit α-amylase to a certain extent even at a low concentration. Therefore, xanthan green tea polysaccharide has potential value in the development and production of α-amylase inhibitors.

[0130] This method yields a high extraction rate of polysaccharides from etiolated green tea, effectively removing proteins. The resulting etiolated green tea polysaccharides are dark brown and reflective under light. The tea polysaccharides retain their in vitro antioxidant and hypoglycemic activities, ensuring both high extraction rates and preservation of bioactivity, thus showing promising potential for deep processing and utilization of tea.

[0131] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting polysaccharides from xanthocyanin green tea using a eutectic solvent, characterized in that, Includes the following steps: S1. Pulverize the yellowed green tea, decolorize it, and dry it for later use; S2. A eutectic solvent is prepared by direct heating, and a eutectic solvent with a specific water content is formulated. S3. In an ultrasonic device, the yellowed green tea powder is mixed with a eutectic solvent for extraction, and the supernatant is taken to obtain the first mixture. S4. Add Sevage reagent to the first mixture to remove protein and obtain a deproteinized mixture. Centrifuge the deproteinized mixture, take the supernatant, repeat the process several times to remove protein, and combine the supernatants to obtain the second mixture. S5. The obtained second mixture was added to anhydrous ethanol for precipitation, centrifuged to obtain crude polysaccharide precipitate, the crude polysaccharide precipitate was reconstituted with distilled water, rotary evaporated, desalted by membrane dialysis, and finally freeze-dried to obtain yellowed green tea crude polysaccharide.

2. The method according to claim 1, characterized in that, In S2, the method for preparing the eutectic solvent is as follows: choline chloride and urea are mixed in a molar ratio of 1:1 to 1:6, and heated and stirred in a water bath at 90°C for 30 to 50 minutes to obtain the eutectic solvent.

3. The method according to claim 1, characterized in that, In S2, distilled water is added to the eutectic solvent to obtain a eutectic solvent with a water content of 20-70%.

4. The method according to claim 1, characterized in that, In S3, the yellowed green tea powder and a eutectic solvent are extracted for 10-60 minutes at a solid-liquid ratio of 1:20-60 and an extraction temperature of 60-80°C to obtain the first mixture.

5. The method according to claim 1, characterized in that, In S3, the power of the ultrasound is 39~451W.

6. The method according to claim 1, characterized in that, In S4, the volume ratio of n-butanol to chloroform in the Sevage reagent is 1:(4~5).

7. The method according to claim 1, characterized in that, In S5, the volume ratio of the second mixture to anhydrous ethanol is 1:(4~5).

8. The method according to claim 1, characterized in that, In S5, the centrifugation speed is 4000~5000 r / min, and the centrifugation time is 10~15 min.

9. The method according to claim 1, characterized in that, In S1, the yellowed green tea is pulverized and passed through a 40-mesh sieve to obtain yellowed green tea powder. The yellowed green tea powder is then decolorized overnight with four times its volume of ethanol and then air-dried naturally.

10. A polysaccharide from xanthocyanin green tea, characterized in that, It was prepared by the eutectic solvent extraction method of xanthocyanin green tea polysaccharide according to any one of claims 1 to 9.

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