Extraction method of rhododendron simsii polysaccharide and application thereof in reducing blood sugar

Abstract

The application discloses a kind of extraction method of Rhododendron simsii polysaccharide and its application in reducing blood sugar, belongs to the technical field of plant active ingredient extraction, including the dry crushing of Rhododendron simsii branch leaf, pass 60 mesh screen, obtain Rhododendron simsii powder;The obtained powder is extracted with ethanol to extract fat-soluble ingredients, after separating ethanol extract, the remaining residue is dried for later use;The obtained residue is mixed with eutectic solvent, ultrasonic-assisted extraction is carried out, filtration, centrifugation is carried out, and the supernatant obtained is the crude extract of Rhododendron simsii polysaccharide;Rhododendron simsii polysaccharide crude extract and anhydrous ethanol are mixed uniformly, alcohol precipitation, centrifugation, anhydrous ethanol washing are carried out, and the crude extract of seaweed polysaccharide is obtained;The crude extract of seaweed polysaccharide is freeze-dried, and the fluffy material is the Rhododendron simsii polysaccharide.The above-mentioned extraction method of Rhododendron simsii polysaccharide is used, and after ethanol-DES gradient combined technology treatment, the polysaccharide yield is improved to 8.09%.

Description

Technical Field

[0001] This invention relates to the field of plant active ingredient extraction technology, and in particular to a method for extracting polysaccharides from Loropetalum chinense and its application in lowering blood sugar. Background Technology

[0002] Loropetalum chinense var. rubrum is a common garden plant, and its pruned branches and leaves are often considered waste. Studies have shown that Loropetalum chinense is rich in polysaccharides and polyphenols, among which polysaccharides possess antioxidant, immunomodulatory, and hypoglycemic effects. Traditional water extraction methods for polysaccharides suffer from low yields and long processing times. Demodulated eutectic solvents (DES) are used for natural product extraction due to their green and efficient characteristics, but have not yet been applied to the industrial extraction of Loropetalum chinense polysaccharides. Furthermore, existing research focuses primarily on the flavonoids in Loropetalum chinense, lacking systematic verification of the hypoglycemic activity of its polysaccharides. Summary of the Invention

[0003] The purpose of this invention is to provide a method for extracting polysaccharides from Loropetalum chinense and their application in lowering blood sugar. The polysaccharides prepared by this method have high yield and purity and good hypoglycemic activity.

[0004] This invention provides a method for extracting polysaccharides from Loropetalum chinense, characterized by comprising the following steps:

[0005] S1. Dry and pulverize the branches and leaves of Loropetalum chinense, and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0006] S2. Extract the fat-soluble components from the powder obtained in step S1 by soaking it in ethanol. After separating the ethanol extract, dry the remaining residue for later use.

[0007] S3. The residue obtained in step S2 is mixed with a eutectic solvent and subjected to ultrasonic-assisted extraction. After filtration and centrifugation, the supernatant is taken to obtain the crude extract of Loropetalum chinense polysaccharide. The eutectic solvent is composed of choline chloride and acetic acid in a molar ratio of 1:2.

[0008] S4. Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, perform alcohol precipitation, centrifugation, and wash with anhydrous ethanol to obtain the crude extract of seaweed polysaccharide.

[0009] S5. Freeze-dry the crude extract of seaweed polysaccharide to obtain a fluffy substance, which is Loropetalum chinense polysaccharide.

[0010] Preferably, in the above-mentioned method for extracting polysaccharides from Loropetalum chinense, the hydrogen bond acceptor and hydrogen bond donor of the eutectic solvent are choline chloride and acetic acid, respectively, mixed in a molar ratio of 1:2.

[0011] According to the above-described method for extracting polysaccharides from Loropetalum chinense, the water content of the eutectic solvent in step S3 is 0-80%. Preferably, the water content of the eutectic solvents choline chloride and acetic acid is 35%.

[0012] According to the above-described method for extracting Loropetalum chinense polysaccharides, the liquid-to-solid ratio of the eutectic solvents choline chloride and acetic acid to Loropetalum chinense powder in step S3 is 10-60 mL / g. Preferably, the liquid-to-solid ratio of the eutectic solvents choline chloride and acetic acid to Loropetalum chinense powder is 60 mL / g.

[0013] According to the above-described method for extracting polysaccharides from Loropetalum chinense, the extraction temperature in step S3 is 30-70℃. Preferably, the extraction temperature is 57℃.

[0014] According to the above-described method for extracting polysaccharides from Loropetalum chinense, the extraction time in step S3 is 10-35 minutes. Preferably, the extraction time is 25°C.

[0015] According to the above-mentioned method for extracting polysaccharides from Loropetalum chinense, the centrifugation speed in step S3 is higher than 5000 rpm, and the centrifugation speed in step S4 is lower than 3000 rpm.

[0016] This invention provides the application of Loropetalum chinense polysaccharide prepared by the extraction method described above in the preparation of hypoglycemic products. The hypoglycemic products prepared using Loropetalum chinense polysaccharide include health products or pharmaceutical compositions.

[0017] Therefore, the present invention employs the above-mentioned method for extracting polysaccharides from Loropetalum chinense and its application in lowering blood sugar, and the beneficial effects include at least the following:

[0018] The prepared Loropetalum chinense polysaccharides exhibit high yield and strong activity, and the process is green and efficient. Loropetalum chinense branches and leaves contain abundant fat-soluble components such as polyphenols and pigments. During preparation, ethanol pretreatment was used to preferentially extract these fat-soluble components, reducing impurity interference. Simultaneously, a eutectic solvent (DES) system (choline chloride / acetic acid, 35% water content) was used for ultrasonic-assisted extraction of the residue. To avoid high-temperature damage to the polysaccharide structure, extraction parameters were optimized using response surface methodology: temperature 60℃, time 25 minutes, and liquid-to-solid ratio 60:1, ensuring efficient extraction while preserving polysaccharide activity. After treatment with the ethanol-DES gradient technique, the polysaccharide yield increased to 8.09%, nearly 13 times higher than the traditional water extraction method.

[0019] During the extraction process, the DES system itself possesses selective solubility properties, reducing the co-extraction of impurities such as flavonoids and tannins. Simultaneously, ethanol pretreatment removes most lipid-soluble interfering substances, further reducing the difficulty of subsequent purification and ensuring polysaccharide purity. Gradient centrifugation removes large molecules such as proteins while ensuring that the polysaccharide does not undergo structural changes under high-speed centrifugation, thus preserving its activity. In vitro activity verification showed that the obtained polysaccharide exhibited an IC50 inhibitory effect on α-glucosidase. 50 The value was 8.433 mg / mL.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 The graph shows the extraction rates of Loropetalum chinense polysaccharides obtained in Examples 1-12 under the same conditions using different low eutectic solvent systems and water.

[0022] Figure 2 The 3D response surface plot shows the effect of the interaction of various factors on the extraction rate of polysaccharides from Loropetalum chinense leaves in low co-soluble solvent extraction. In the plot, (A) is the extraction temperature, (B) is the extraction time, (C) is the liquid-to-solid ratio, and (D) is the water content. Detailed Implementation

[0023] To better understand the above technical solutions, a detailed description of the solutions will be provided below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0024] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms, and “multiple” generally includes at least two unless the context clearly indicates otherwise.

[0025] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the article or device that includes said element.

[0026] Example 1

[0027] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0028] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0029] (c) Select choline chloride and glycerol in a molar ratio of 1:2 and place them in a beaker. Heat and stir until a transparent and homogeneous liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0030] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0031] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0032] Example 2

[0033] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0034] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0035] (c) Select choline chloride and acetic acid in a molar ratio of 1:2 and place them in a beaker. Heat and stir until a transparent and homogeneous liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0036] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0037] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0038] Example 3

[0039] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0040] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0041] (c) Select choline chloride and urea in a molar ratio of 1:2 and place them in a beaker. Heat and stir until a transparent and uniform liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the low eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0042] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0043] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0044] Example 4

[0045] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0046] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0047] (c) Select choline chloride and lactic acid in a molar ratio of 1:2 and place them in a beaker. Heat and stir until a transparent and uniform liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0048] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0049] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0050] Example 5

[0051] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0052] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0053] (c) Select choline chloride and 1,3-butanediol in a molar ratio of 1:2 and place them in a beaker. Heat and stir until a transparent and homogeneous liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0054] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0055] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0056] Example 6

[0057] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0058] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0059] (c) Select choline chloride and malic acid in a molar ratio of 1:2 and place them in a beaker. Heat and stir until a transparent and uniform liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0060] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0061] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0062] Example 7

[0063] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0064] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0065] (c) Select choline chloride and acrylic acid in a molar ratio of 1:2 and place them in a beaker. Heat and stir until a transparent and homogeneous liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0066] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0067] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0068] Example 8

[0069] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0070] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0071] (c) Select betaine and acrylic acid in a molar ratio of 1:3 and place them in a beaker. Heat and stir until a transparent and uniform liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0072] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0073] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0074] Example 9

[0075] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0076] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0077] (c) Select betaine and acetic acid in a molar ratio of 1:3 and place them in a beaker. Heat and stir until a transparent and uniform liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the low eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0078] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0079] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0080] Example 10

[0081] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0082] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0083] (c) Select betaine and ethylene glycol in a molar ratio of 1:3 and place them in a beaker. Heat and stir until a transparent and uniform liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 20%.

[0084] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 20mL / g. Extract the solution in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0085] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0086] Example 11

[0087] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0088] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0089] (c) Weigh 0.5g of pretreated Loropetalum chinense powder, add deionized water at a liquid-to-solid ratio of 20mL / g, and extract in an ultrasonic cleaner at 28℃ for 15 minutes. After extraction, filter and let the filtrate cool to room temperature. Centrifuge the filtrate at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides by the phenol-sulfuric acid method.

[0090] (d) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate for 12 hours, then centrifuge at 3000 rpm for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, and centrifuge again at 3000 rpm for 20 minutes. Collect the precipitate and freeze-dry it to obtain Loropetalum chinense polysaccharide.

[0091] Example 12

[0092] (a) Pulverize the dried branches and leaves of Loropetalum chinense and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder;

[0093] (b) The Loropetalum chinense powder was impregnated with anhydrous ethanol at a material-to-liquid ratio of 1 g / mL, ultrasonically treated for 30 minutes, filtered, and the precipitate was dried to obtain the pretreated Loropetalum chinense powder.

[0094] (c) Select choline chloride and acetic acid in a molar ratio of 1:2 and place them in a beaker. Heat and stir until a transparent and homogeneous liquid is formed. Remove and let it cool naturally to room temperature. If no precipitate is formed, it indicates that the eutectic solvent has been successfully synthesized. Then add deionized water according to the mass ratio to make the water content 35%.

[0095] (d) Weigh 0.5g of pretreated Loropetalum chinense powder and add it to the low eutectic solvent prepared in step (c) at a liquid-to-solid ratio of 60mL / g. Extract the solution in an ultrasonic cleaner at 57℃ for 25 minutes. After extraction, filter the solution and let it cool to room temperature. Centrifuge the solution at 5000r / min for 20 minutes. After centrifugation, take the supernatant to obtain the crude extract of Loropetalum chinense polysaccharides. Then, determine the content of Loropetalum chinense branch and leaf polysaccharides using the phenol-sulfuric acid method.

[0096] (e) Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, refrigerate in a low-temperature refrigerator for 12 hours, then centrifuge at 3000 r / min for 20 minutes, discard the supernatant, add 10 mL of 95% ethanol for washing, continue centrifuging at 3000 r / min for 20 minutes, take the precipitate and freeze-dry it, and the dried product is Loropetalum chinense polysaccharide.

[0097] Examples 1-11 compare different low eutectic solvent systems and water extraction. Using the polysaccharide extraction rate as the response value, the choline chloride / acetic acid system was found to be the best for extracting polysaccharides from Loropetalum chinense (6.43%), which is about 10 times higher than that of pure water extraction (0.64%).

[0098] The study investigated four influencing factors—extraction temperature, extraction time, liquid-to-solid ratio, and water content of the low co-soluble solvent—through single-factor experiments and Box-Behnken response surface methodology. Figure 2As shown, the optimal extraction process was obtained: 57.590 degrees Celsius, extraction time 24.632 min, liquid-to-solid ratio 60:1, and water content 35.393%. The actual operating conditions were: 60 degrees Celsius, extraction time 25 min, liquid-to-solid ratio 60:1, and water content 35%. The final polysaccharide yield was calculated to be 8.09%, close to the theoretical value of 8.034%. This represents a 120% improvement compared to the original process's 6.43%.

[0099] In vitro hypoglycemic activity assay

[0100] The polysaccharide obtained in Example 12 was used as the test sample, and the degree of inhibition of α-glucosidase activity was used to measure the in vitro hypoglycemic activity of the polysaccharide.

[0101] Prepare phosphate buffer solution by mixing equal volumes of 0.1 mol / L sodium dihydrogen phosphate and 0.1 mol / L disodium hydrogen phosphate solution. Prepare PNPG solution by dissolving PNPG in 0.1 mol / L PBS to a concentration of 2.5 mmol / L. Prepare 0.2 mol / L sodium carbonate solution and 10% DMSO. Dissolve acarbose and the test sample in 10% DMSO, then dilute with PBS to a concentration of 2.5 mg / mL. The 96-well plate contains a blank control, negative control, positive control, experimental group, and background group. Blank control: 80 μL PBS solution + 20 μL 10% DMSO; Negative control: 70 μL PBS solution + 10 μL 10% DMSO + 20 μL α-glucosidase; Positive control: 70 μL PBS solution + 10 μL acarbose + 20 μL α-glucosidase; Experimental group: 70 μL PBS solution + 10 μL compound + 20 μL α-glucosidase; Background group: 90 μL PBS solution + 10 μL compound. Each group had 3 parallel wells. After adding the above groups, incubate the 96-well plate at 37℃ for 15 min. Remove the plate and add 20 μL of PNPG solution to each well, then incubate at 37℃ for 30 min. Remove the plate again and add 80 μL of sodium carbonate solution to each well. Read the data using a microplate reader at 405 nm. Calculate the average value for each group and calculate the inhibition rate using the formula: [Inhibition rate = (Negative control - Blank control) - (Experimental group - Background group) / (Negative control - Blank control)] * 100%. Continue to dilute the active compound to test the inhibition rate, and calculate the IC50 using SPSS software. 50 .

[0102] The experimental results are shown in Table 1:

[0103] Table 1 Results of polysaccharide α-glucosidase inhibitory activity

[0104] polysaccharides 8.433 Acarbose 0.00226

[0105] Therefore, this invention employs the aforementioned method for extracting Loropetalum chinense polysaccharides, resulting in polysaccharides with high yield and strong activity, and the process is green and efficient. Loropetalum chinense branches and leaves contain abundant fat-soluble components such as polyphenols and pigments. During the preparation process, ethanol pretreatment is used to preferentially extract fat-soluble components, reducing impurity interference. Simultaneously, a low-melting-point solvent (DES) system (choline chloride / acetic acid, 35% water content) is used for ultrasonic-assisted extraction of the residue. To avoid high-temperature damage to the polysaccharide structure, extraction parameters are optimized using response surface methodology: temperature 60℃, time 25 minutes, and liquid-to-solid ratio 60:1, ensuring efficient extraction while preserving polysaccharide activity. After treatment with an ethanol-DES gradient, the polysaccharide yield increases to 8.09%, nearly 13 times higher than the traditional water extraction method. During extraction, the DES system itself has selective dissolution characteristics, reducing the co-extraction of impurities such as flavonoids and tannins. Furthermore, ethanol pretreatment removes most fat-soluble interfering substances, further reducing the difficulty of subsequent purification and ensuring polysaccharide purity. Gradient centrifugation removes large molecules such as proteins while ensuring that the polysaccharide does not undergo structural changes during high-speed centrifugation, thus preserving its activity. In vitro activity verification showed that the obtained polysaccharide exhibited an IC50 inhibitory effect on α-glucosidase. 50 The value was 8.433 mg / mL.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for extracting polysaccharides from Loropetalum chinense, characterized in that, Includes the following steps: S1. Dry and pulverize the branches and leaves of Loropetalum chinense, and pass them through a 60-mesh sieve to obtain Loropetalum chinense powder; S2. Extract the fat-soluble components from the powder obtained in step S1 by soaking it in ethanol. After separating the ethanol extract, dry the remaining residue for later use. S3. The residue obtained in step S2 is mixed with a eutectic solvent and subjected to ultrasonic-assisted extraction. After filtration and centrifugation, the supernatant is taken to obtain the crude extract of Loropetalum chinense polysaccharide. The hydrogen bond acceptor and hydrogen bond donor of the eutectic solvent are choline chloride and acetic acid mixed in a molar ratio of 1:

2. S4. Mix the crude extract of Loropetalum chinense polysaccharide with anhydrous ethanol at a ratio of 1:4, perform alcohol precipitation, centrifugation, wash with anhydrous ethanol, and then freeze-dry to obtain a fluffy substance, which is Loropetalum chinense polysaccharide.

2. The method for extracting polysaccharides from Loropetalum chinense according to claim 1, characterized in that, In step S3, the water content of the eutectic solvent is 0-80%.

3. The method for extracting polysaccharides from Loropetalum chinense according to claim 1, characterized in that, The liquid-to-solid ratio of the eutectic solvent choline chloride and acetic acid to the Loropetalum chinense powder in step S3 is 10-60 mL / g.

4. The method for extracting polysaccharides from Loropetalum chinense according to claim 1, characterized in that, The extraction temperature in step S3 is 30-70℃.

5. The method for extracting polysaccharides from Loropetalum chinense according to claim 1, characterized in that, The extraction time in step S3 is 10-35 minutes.

6. The method for extracting polysaccharides from Loropetalum chinense according to claim 1, characterized in that, The centrifugation speed in step S3 is higher than 5000 r / min, and the centrifugation speed in step S4 is lower than 3000 r / min.

7. The application of the Loropetalum chinense polysaccharide prepared by the extraction method according to any one of claims 1-6 in the preparation of hypoglycemic products, characterized in that, Hypoglycemic products prepared using Loropetalum chinense polysaccharides include health supplements or pharmaceutical compositions.

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