Polygonatum polysaccharide extraction method

Abstract

The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a rhizoma polygonati polysaccharide extraction method which comprises the following steps: (1) slicing fresh rhizoma polygonati, drying, crushing and sieving to obtain rhizoma polygonati powder; (2) mixing polygonatum sibiricum powder with deionized water, heating and extracting for multiple times, combining extracting solutions, and reserving polygonatum sibiricum filter residues for later use; (3) performing microwave treatment on the rhizoma polygonati filter residues, adding deionized water, adding compound enzyme, adjusting the pH value and the temperature, performing enzymolysis treatment, and collecting supernate; and (4) mixing and concentrating the extracting solution in the step (2) and the supernate in the step (3), mixing the concentrated solution with an ethanol solution, standing in a low-temperature environment for alcohol precipitation, then centrifugally separating, collecting precipitates, and freeze-drying to obtain the polygonatum polysaccharide. The method is simple and easy to operate, and the prepared polysaccharide is high in yield, resistant to oxidation, capable of reducing blood sugar and high in tyrosinase inhibitory activity.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for extracting polygonatum polysaccharide. Background Art

[0002] Polygonatum sibiricum, a Chinese medicine. It is Polygonatum kingianum Coll.et Hemsl., Polygonatum sibiricum Red. or Polygonatum cyrtonema H, a plant of the Liliaceae family.

[0003] Polygonatum sibiricum has pharmacological effects such as enhancing immune system function, delaying aging, lowering blood sugar and blood lipids, antibacterial and anti-inflammatory, and antiviral and anti-tumor. Polygonatum sibiricum contains a variety of active ingredients that are beneficial to the human body, such as polysaccharides, saponins, lignans, cardiac glycosides, alkaloids, vitamins, and anthraquinone compounds. Polygonatum sibiricum polysaccharide, one of the main medicinal ingredients in Polygonatum sibiricum, generally has a content of more than 10.00% in Polygonatum sibiricum. Because of its high content and wide range of effects, it is considered to be the most important chemical component in Polygonatum sibiricum that exerts its pharmacological effects. Polygonatum sibiricum polysaccharide has good medicinal effects in antibacterial and anti-inflammatory, anti-oxidation, hypoglycemic, anti-tumor and immunomodulation. Therefore, how to efficiently extract Polygonatum sibiricum polysaccharide from Polygonatum sibiricum is a key process control point. Dried rhizome of Rhizoma Polygonati ua. According to different shapes, it is commonly called "big rhubarb", "chicken head rhubarb" and "ginger-shaped rhubarb". It has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs and benefiting the kidneys. It is often used for spleen and stomach qi deficiency, fatigue, insufficient stomach yin, dry mouth and poor appetite, dry cough due to lung deficiency, cough due to fatigue and hemoptysis, insufficient essence and blood, soreness of waist and knees, premature graying of hair, internal heat and thirst.

[0003] The Chinese patent with application number CN202410956079.9 discloses a process for extracting polygonatum polysaccharide, comprising the following steps: S1, pretreatment: ultrafine grinding is adopted to grind polygonatum to micrometer or nanometer level; S2, composite extraction: a low eutectic solvent is prepared by choline chloride and urea in a molar ratio of 1:2; after adjusting and optimizing the water content and temperature, the preliminary extraction of polygonatum powder is carried out to efficiently release polygonatum polysaccharide; combined with ultrasound-assisted enzymolysis, ultrasound destroys the cell wall, and enzyme-specific degradation of the connecting bonds synergistically improves the dissolution efficiency and quality of polysaccharide; S3, efficient purification stage. The present invention proposes a process for extracting polygonatum polysaccharide, which realizes efficient and high-purity extraction of polygonatum polysaccharide through pretreatment, composite extraction, efficient purification and drying and preservation steps. The process not only improves the extraction efficiency and purity of polygonatum polysaccharide, but also significantly enhances its antioxidant activity.

[0004] The Chinese patent application number CN202410733363.X discloses a polysaccharide of Polygonatum cyrtonema and its extraction method and application, wherein the extraction method of the polysaccharide of Polygonatum cyrtonema comprises the following steps: step 1, slicing, drying and powdering the polysaccharide sample to obtain polysaccharide powder; step 2, subjecting the polysaccharide powder obtained in step 1 to petroleum ether degreasing, ultrasonic extraction, alcohol precipitation, Sevag method deproteinization and dialysis treatment to obtain polysaccharide crude polysaccharide, wherein the mass ratio of polysaccharide powder to petroleum ether is 1: (10-15); step 3, subjecting the polysaccharide crude polysaccharide in step 2 to separation and purification treatment by DEAE-52 cellulose column and Sephadex G-100 column successively, and obtaining refined polysaccharide of Polygonatum cyrtonema after freeze drying. The present invention aims to obtain pharmaceutically usable polysaccharide of Polygonatum cyrtonema.

[0005] However, in the prior art, there are few reports on the extraction of polygonatum polysaccharide by combining hot water extraction with complex enzyme-assisted extraction. Summary of the invention

[0006] The present invention aims to solve the above technical problems and provides a method for extracting polygonatum polysaccharide. The method is simple and easy to operate, and the prepared polysaccharide has a high yield and high antioxidant, blood sugar lowering and tyrosinase inhibitory activities.

[0007] The technical solution of the present invention is:

[0008] A method for extracting polygonatum polysaccharide comprises the following steps:

[0009] (1) Slice fresh polygonatum, dry, crush, and sieve to obtain polygonatum powder;

[0010] (2) Mixing polygonatum powder with deionized water, heating and extracting for multiple times, combining the extracts, and filtering the polygonatum residue for later use;

[0011] (3) After the Polygonatum sibiricum residue is subjected to microwave treatment, deionized water is added, and then a composite enzyme is added, the pH value and temperature are adjusted, enzymatic hydrolysis is performed, and the supernatant is collected;

[0012] (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated, the concentrate is mixed with an ethanol solution, and the mixture is placed in a low temperature environment for alcohol precipitation, and then centrifuged and separated, the precipitate is collected, and freeze-dried to obtain polygonatum polysaccharide.

[0013] In order to dissolve the polygonatum polysaccharide component better, preferably, in step (2) of the present invention, the liquid ratio of polygonatum powder to deionized water is 1:25-35 g / mL.

[0014] The cell walls of the polygonatum polysaccharide are destroyed by heating and extraction, so that the polysaccharide in the cell can be better extracted. During the heating and extraction, the extraction time needs to be controlled. Preferably, in the step (2) of the present invention, the extraction is heated to 95-100° C. for 1.5-2.5 hours, and the heating and extraction are performed twice. If the extraction time is too long, the polysaccharide structure is destroyed, the polysaccharide yield decreases, and its hypoglycemic, antioxidant and whitening activities also decrease.

[0015] Microwave radiation can generate heat through molecular vibration and collision, causing the sample temperature to rise rapidly. This temperature rise can accelerate the enzymatic hydrolysis reaction, thereby improving the efficiency of subsequent enzymatic hydrolysis reactions and shortening the enzymatic hydrolysis cycle. At the same time, before enzymatic hydrolysis, the Polygonatum sibiricum filter residue is subjected to microwave treatment, which can change the physical and chemical properties of the Polygonatum sibiricum filter residue and lay the foundation for improving the activity of the enzymatic hydrolysis product. Preferably, in step (3) of the present invention, the Polygonatum sibiricum filter residue is subjected to microwave treatment at 600-700W for 60-90s.

[0016] In order to optimize the performance of polygonatum sibiricum polysaccharide and improve the biological activity of polygonatum sibiricum polysaccharide, after hydrothermal extraction, enzymatic extraction is also combined to further decompose macromolecular compounds into small molecular substances, improve the extraction rate, and improve the comprehensive performance of polygonatum sibiricum polysaccharide. Preferably, in step (3) of the present invention, 2.5-4% of the weight of the polygonatum sibiricum filter residue is added, and the composite enzyme is a mixture of cellulase and pectinase in a weight ratio of 1-3:1. During enzymatic hydrolysis, the solid-liquid ratio of the polygonatum sibiricum filter residue to deionized water is 1:25-35 g / mL, and the enzymatic hydrolysis is carried out for 50-60 min at a pH value of 4.5-5.0 and a temperature of 45-50°C.

[0017] In order to better purify the extract and further improve the activity of Polygonatum sibiricum polysaccharide, preferably, in step (3) of the present invention, the extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, and the concentrated solution is mixed with an ethanol solution with a volume concentration of 90-95%, and placed in an environment of 4°C for alcohol precipitation for 12 hours.

[0018] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0019] 1. The present invention extracts polygonatum polysaccharide by hot water extraction combined with complex enzyme-assisted extraction. The method is simple and easy to operate, and the prepared polysaccharide has a high yield and high antioxidant, blood sugar-lowering and tyrosinase inhibitory activities.

[0020] 2. After extracting polygonatum polysaccharide by hot water extraction, the present invention further treats polygonatum filter residue by microwave, which significantly improves the subsequent enzymatic hydrolysis efficiency, shortens the enzymatic hydrolysis time, and greatly shortens the entire preparation cycle of polygonatum polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a picture of the Polygonatum sibiricum polysaccharide prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] A method for extracting polygonatum polysaccharide comprises the following steps:

[0025] (1) Slice fresh white polygonatum cyrtonema, place in a drying oven at 60° C., grind and pass through a 60-mesh sieve to obtain polygonatum cyrtonema powder;

[0026] (2) Mixing polygonatum powder and deionized water at a solid-liquid ratio of 1:30 g / mL, heating to 100° C. and leaching for 2 h, leaching twice, combining the two extracts, and using the polygonatum residue for later use;

[0027] (3) After the Polygonatum sibiricum residue is treated with a 650W microwave for 750s, deionized water is added at a solid-liquid ratio of 1:30g / mL, mixed evenly, and then a composite enzyme of 3.5% by weight of the Polygonatum sibiricum residue is added, and enzymolysis is performed at a pH value of 4.5 and a temperature of 48°C for 55min, and the supernatant is collected; the composite enzyme is a mixture of cellulase and pectinase in a weight ratio of 1:1.

[0028] (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, the concentrate is mixed with a 95% ethanol solution at a solid-liquid ratio of 1:30 g / mL, and the mixture is allowed to stand at 4°C for 12 hours for alcohol precipitation. The mixture is centrifuged at 4800 r / min for 15 minutes, the precipitate is collected, and freeze-dried to obtain Polygonatum sibiricum polysaccharide.

[0029] Example 2

[0030] A method for extracting polygonatum polysaccharide comprises the following steps:

[0031] (1) Slice fresh white polygonatum cyrtonema, place in a drying oven at 60° C., grind and pass through a 60-mesh sieve to obtain polygonatum cyrtonema powder;

[0032] (2) Mixing polygonatum powder and deionized water at a solid-liquid ratio of 1:25 g / mL, heating to 100° C. and leaching for 1.5 h, leaching twice, combining the two extracts, and using the polygonatum residue for later use;

[0033] (3) After the Polygonatum sibiricum residue is treated with a 700 W microwave for 60 seconds, deionized water is added at a solid-liquid ratio of 1:35 g / mL, the mixture is mixed evenly, and then a composite enzyme of 4% by weight of the Polygonatum sibiricum residue is added, and enzymolysis is performed at a pH value of 4.5 and a temperature of 50° C. for 50 minutes, and the supernatant is collected; the composite enzyme is a mixture of cellulase and pectinase in a weight ratio of 3:1.

[0034] (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, the concentrate is mixed with a 95% ethanol solution at a solid-liquid ratio of 1:35 g / mL, and the mixture is allowed to stand at 4°C for 12 hours for alcohol precipitation. The mixture is centrifuged at 4800 r / min for 15 minutes, the precipitate is collected, and freeze-dried to obtain Polygonatum sibiricum polysaccharide.

[0035] Example 3

[0036] A method for extracting polygonatum polysaccharide comprises the following steps:

[0037] (1) Slice fresh white polygonatum cyrtonema, place in a drying oven at 60° C., grind and pass through a 60-mesh sieve to obtain polygonatum cyrtonema powder;

[0038] (2) Mixing polygonatum powder and deionized water at a solid-liquid ratio of 1:35 g / mL, heating to 95° C. and leaching for 2.5 h, leaching twice, combining the two extracts, and using the polygonatum residue for later use;

[0039] (3) After the Polygonatum sibiricum residue is treated with a 600 W microwave for 90 seconds, deionized water is added at a solid-liquid ratio of 1:25 g / mL, the mixture is mixed evenly, and then a composite enzyme of 2.5% by weight of the Polygonatum sibiricum residue is added, and enzymolysis is performed at a pH value of 5.0 and a temperature of 45° C. for 60 minutes, and the supernatant is collected; the composite enzyme is a mixture of cellulase and pectinase in a weight ratio of 2:1.

[0040] (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, the concentrate is mixed with a 90% ethanol solution at a solid-liquid ratio of 1:25 g / mL, and the mixture is allowed to stand at 4°C for 12 hours for alcohol precipitation. The mixture is centrifuged at 4800 r / min for 15 minutes, the precipitate is collected, and freeze-dried to obtain Polygonatum sibiricum polysaccharide.

[0041] Comparative Example 1 (Potassium sibiricum filter residue is not first treated with filter residue)

[0042] A method for extracting polygonatum polysaccharide comprises the following steps:

[0043] (1) Slice fresh white polygonatum cyrtonema, place in a drying oven at 60° C., grind and pass through a 60-mesh sieve to obtain polygonatum cyrtonema powder;

[0044] (2) Mixing polygonatum powder and deionized water at a solid-liquid ratio of 1:30 g / mL, heating to 100° C. and leaching for 2 h, leaching twice, combining the two extracts, and using the polygonatum residue for later use;

[0045] (3) After adding deionized water to the Polygonatum sibiricum residue at a solid-liquid ratio of 1:30 g / mL, the mixture is evenly mixed, and then a composite enzyme of 3.5% by weight of the Polygonatum sibiricum residue is added, and enzymolysis is performed at a pH value of 4.5 and a temperature of 48° C. for 55 minutes, and the supernatant is collected; the composite enzyme is a mixture of cellulase and pectinase in a weight ratio of 1:1.

[0046] (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, the concentrate is mixed with a 95% ethanol solution at a solid-liquid ratio of 1:30 g / mL, and the mixture is allowed to stand at 4°C for 12 hours for alcohol precipitation. The mixture is centrifuged at 4800 r / min for 15 minutes, the precipitate is collected, and freeze-dried to obtain Polygonatum sibiricum polysaccharide.

[0047] Comparative Example 2 (Potassium sibiricum filtration residue is not subjected to microwave treatment first)

[0048] A method for extracting polygonatum polysaccharide comprises the following steps:

[0049] (1) Slice fresh white polygonatum cyrtonema, place in a drying oven at 60° C., grind and pass through a 60-mesh sieve to obtain polygonatum cyrtonema powder;

[0050] (2) Mixing polygonatum powder and deionized water at a solid-liquid ratio of 1:30 g / mL, heating to 100° C. and leaching for 2 h, leaching twice, combining the two extracts, and using the polygonatum residue for later use;

[0051] (3) After adding deionized water to the Polygonatum sibiricum residue at a solid-liquid ratio of 1:30 g / mL, the mixture is evenly mixed, and then a composite enzyme of 3.5% by weight of the Polygonatum sibiricum residue is added, and enzymolysis is performed at a pH value of 4.5 and a temperature of 48° C. for 120 minutes, and the supernatant is collected; the composite enzyme is a mixture of cellulase and pectinase in a weight ratio of 1:1.

[0052] (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, the concentrate is mixed with a 95% ethanol solution at a solid-liquid ratio of 1:30 g / mL, and the mixture is allowed to stand at 4°C for 12 hours for alcohol precipitation. The mixture is centrifuged at 4800 r / min for 15 minutes, the precipitate is collected, and freeze-dried to obtain Polygonatum sibiricum polysaccharide.

[0053] Comparative Example 3 (Hot Water Method and Ultrasound Combination)

[0054] (1) Slice fresh white polygonatum cyrtonema, place in a drying oven at 60° C., grind and pass through a 60-mesh sieve to obtain polygonatum cyrtonema powder;

[0055] (2) Mixing polygonatum powder and deionized water at a solid-liquid ratio of 1:30 g / mL, heating to 100° C. and leaching for 2 h, leaching twice, combining the two extracts, and using the polygonatum residue for later use;

[0056] (3) After adding deionized water to the Polygonatum sibiricum residue at a solid-liquid ratio of 1:30 g / mL, the mixture was evenly mixed, and ultrasonic extraction was performed at a power of 300 W and a temperature of 50° C. for 0.5 h, the extraction was repeated twice, the two extracts were combined, and the supernatant was collected; the complex enzyme was a mixture of cellulase and pectinase at a weight ratio of 1:1.

[0057] (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, the concentrate is mixed with a 95% ethanol solution at a solid-liquid ratio of 1:30 g / mL, and the mixture is allowed to stand at 4°C for 12 hours for alcohol precipitation. The mixture is centrifuged at 4800 r / min for 15 minutes, the precipitate is collected, and freeze-dried to obtain Polygonatum sibiricum polysaccharide.

[0058] Comparative Example 4 (combination of hot water method and alkaline composite hot water extraction method)

[0059] (1) Slice fresh white polygonatum cyrtonema, place in a drying oven at 60° C., grind and pass through a 60-mesh sieve to obtain polygonatum cyrtonema powder;

[0060] (2) Mixing polygonatum powder and deionized water at a solid-liquid ratio of 1:30 g / mL, heating to 100° C. and leaching for 2 h, leaching twice, combining the two extracts, and using the polygonatum residue for later use;

[0061] (3) After adding NaOH solution (pH=8) to the Polygonatum sibiricum residue at a solid-liquid ratio of 1:30 g / mL, the mixture is evenly mixed, heated to 100° C. and extracted for 2 h, extracted twice, combined the two extracts, and collected the supernatant; the composite enzyme is a mixture of cellulase and pectinase at a weight ratio of 1:1.

[0062] (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, the concentrate is mixed with a 95% ethanol solution at a solid-liquid ratio of 1:30 g / mL, and the mixture is allowed to stand at 4°C for 12 hours for alcohol precipitation. The mixture is centrifuged at 4800 r / min for 15 minutes, the precipitate is collected, and freeze-dried to obtain Polygonatum sibiricum polysaccharide.

[0063] Test Example 1: Effect of the method of the present invention on the yield of Polygonatum cyrtonema polysaccharide

[0064] The calculation formula of Polygonatum sibiricum polysaccharide yield is as follows.

[0065]

[0066] In the formula: m1 is the mass of Polygonatum cyrtonema polysaccharide, g; m2 is the mass of Polygonatum cyrtonema raw material, g.

[0067] The weighing calculation results are shown in Table 1.

[0068] Table 1 Yield of Polygonatum cyrtonema polysaccharide in each group

[0069] Group Yield of Polygonatum sibiricum polysaccharide / % Example 1 31.23±1.34 Example 2 30.91±0.93 Example 3 30.21±0.78 Comparative Example 1 25.77±0.47 Comparative Example 2 30.73±0.52 Comparative Example 3 28.41±1.01 Comparative Example 4 27.95±0.61

[0070] As can be seen from Table 1, the yield of polygonatum polysaccharide prepared by the method of the present invention is higher. However, from the data of comparative examples 1 and 2, it can be seen that the enzymatic hydrolysis of polygonatum polysaccharide to achieve the yield of Example 1 requires a longer time. This shows that the present invention can reduce the time of enzymatic hydrolysis by subjecting the polygonatum filter residue to microwave treatment after hydrothermal extraction and then subjecting it to enzymatic hydrolysis.

[0071] Experimental Example 2: Effect of the method of the present invention on the in vitro functional activity of Polygonatum cyrtonema polysaccharide

[0072] 1. DPPH free radical scavenging ability

[0073] (1) Test method: The antioxidant activity of Polysaccharide from Yunnan Polygonatum was evaluated by in vitro DPPH free radical scavenging ability, referring to the method of Tang Yayuan et al. (TANG Yayuan, WANG Xuefeng, WEI Zhen, et al. Antioxidant activity and stability of polysaccharide from banana flower [J]. Science and Technology of Food Industry, 2024, 45 (7): 100-107.). 0.2 mg / mL Polysaccharide from Yunnan Polygonatum solution was thoroughly mixed with 0.2 mmol / L DPPH free radical solution, reacted in the dark for 30 min, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rate was calculated by formula (2).

[0074]

[0075] Wherein: A0 is the absorbance value of anhydrous ethanol; A1 is the absorbance value after the reaction of anhydrous ethanol and the sample solution; A2 is the absorbance value after the reaction of DPPH free radical solution and the sample solution.

[0076] (2) See 2 for the results.

[0077] Table 2 DPPH free radical scavenging ability of polygonatum polysaccharide in each group

[0078] Group DPPH free radical scavenging ability / % Example 1 92.98±1.24 Example 2 91.24±2.01 Example 3 91.87±2.42 Comparative Example 1 73.15±1.78 Comparative Example 2 79.52±1.67 Comparative Example 3 68.25±2.23 Comparative Example 4 65.47±2.15

[0079] As can be seen from Table 2, the DPPH free radical scavenging ability of the polygonatum polysaccharide prepared by the method of the present invention is strong, indicating that its antioxidant activity is high, while the enzymatic hydrolysis time of comparative example 1 is insufficient, the prepared polysaccharide content is low, and the overall antioxidant activity is low, and although the polysaccharide content of comparative example 2 is not much different from that of the embodiment, the polygonatum filter residue is not subjected to microwave treatment before enzymatic hydrolysis, and the subsequent enzymatic hydrolysis effect is not as good as that of the embodiment, thereby affecting the overall antioxidant activity of the final polygonatum polysaccharide, and when the polygonatum polysaccharide of the present invention is prepared by hot water method and ultrasonic extraction in comparative example 3, the antioxidant activity of the obtained polygonatum polysaccharide is not as high as that of the embodiment, and the antioxidant activity of comparative example 4 is the lowest, which may be that the polygonatum filter residue is in a high temperature environment for a short time, and the structure of some of its antioxidant active substances is destroyed, resulting in its relatively low antioxidant activity. In summary, the method of the present invention can well improve the DPPH free radical scavenging ability of polygonatum polysaccharide.

[0080] 2. α-Glucosidase Inhibition Ability

[0081] (1) Experimental method: The hypoglycemic activity of Polygonatum cyrtonema polysaccharide was evaluated by in vitro α-glucosidase inhibition ability, referring to the method of Tang et al. (TANG Y, HE X, LIU G, et al. Effects of different extraction methods on the structural, antioxidant and hypoglycemic properties of red pitaya stem polysaccharide [J]. Food chemistry, 2023, 405: 134804.). 2 mg / mL Polygonatum cyrtonema polysaccharide solution was mixed with 8 U / mL α-glucosidase and 0.05 mmol / L sodium phosphate buffer (pH 6.8), and then incubated in a 37°C water bath for 10 min. Then, 8 mmol / L PNPG was added and mixed, and then incubated in a 37°C water bath for 10 min. Finally, 0.1 mol / L Na2CO3 solution was added to terminate the reaction. The absorbance value was measured at a wavelength of 405 nm. The α-glucosidase inhibition rate was calculated by formula (3).

[0082]

[0083] Wherein: A0 is the absorbance value after the reaction when distilled water replaces the sample; A1 is the absorbance value after the reaction when distilled water replaces α-glucosidase; A2 is the absorbance value after the reaction when the sample to be tested is added.

[0084] (2) See 3 for the results.

[0085] Table 3 α-glucosidase inhibition ability of Polygonatum sibiricum polysaccharide in each group

[0086] Group α-Glucosidase inhibition ability / % Example 1 93.78±2.65 Example 2 92.47±0.97 Example 3 92.14±1.27 Comparative Example 1 76.24±2.14 Comparative Example 2 81.52±0.73 Comparative Example 3 72.64±1.02 Comparative Example 4 70.21.±1.77

[0087] As can be seen from Table 3, the α-glucosidase inhibitory ability of the polygonatum polysaccharide prepared by the method of the present invention is strong, indicating that it has a strong hypoglycemic ability. The enzymolysis time of comparative example 1 is short, and the final polysaccharide is less than that of the embodiment. In addition, the enzymolysis time is not enough, and some effective substances with α-glucosidase inhibitory ability may not be extracted in the polygonatum filter residue, so the α-glucosidase inhibitory ability of comparative example 1 is weak; while comparative example 2 is not microwave treated, and the enzymolysis is stopped after the polysaccharide yield equivalent to the embodiment is reached, and some effective substances with α-glucosidase inhibitory ability may not be extracted, so its overall α-glucosidase inhibitory ability is also weaker than that of the embodiment; when the polygonatum polysaccharide of the present invention is prepared by hot water method and ultrasonic extraction in comparative example 3, although the polysaccharide yield is close to that of the embodiment, the polysaccharide structure and composition obtained are different due to the difference in the method, so the polysaccharide α-glucosidase inhibitory ability obtained is poorer than that of the embodiment; the polysaccharide in the polygonatum filter residue of comparative example 4 is in a high temperature state for too long, and its part of the polysaccharide structure may be destroyed, so the α-glucosidase inhibitory ability is poorer than that of the embodiment. In summary, the polygonatum polysaccharide prepared by the method of the present invention has a high blood sugar lowering activity.

[0088] 3. Tyrosinase inhibitory activity

[0089] (1) Experimental method: refer to the method of He Xuemei et al. (He Xuemei, Yue Jian, Qiu Furong, et al. Effect of dynamic high-pressure microfluidization assisted extraction on the structure and activity of polysaccharides from Polygonatum kingianum Coll. et Hemsl. [J]. Food Science, 2022, 43 (19): 95-101.). To a 2 mg / mL solution of Polygonatum kingianum polysaccharide, pH 6.8 phosphate buffer and 100 μg / mL tyrosinase solution were added in sequence, mixed thoroughly, preheated at 37°C for 5 min, and then 2.5 mmol / L L-tyrosine solution was added. The mixture was kept in a 37°C water bath for 10 min, and the absorbance of the reaction solution was measured at 490 nm. The tyrosinase inhibition of the samples was calculated according to formula (4).

[0090]

[0091] Wherein: A1 is the absorbance value after the reaction when distilled water replaces the sample; A2 is the absorbance value after the reaction when distilled water replaces the sample and buffer replaces tyrosinase; A3 is the absorbance value after the reaction when the sample to be tested is added; A4 is the absorbance value after the reaction when buffer replaces tyrosinase.

[0092] (2) See 4 for the results.

[0093] Table 4 Inhibitory effect of Polygonatum sibiricum polysaccharide on tyrosinase in each group

[0094] Group Tyrosinase inhibition ability / % Example 1 92.14±0.88 Example 2 91.24±1.26 Example 3 92.02±2.35 Comparative Example 1 78.21±1.26 Comparative Example 2 83.64±1.14 Comparative Example 3 81.24±0.84 Comparative Example 4 78.24.±2.11

[0095] Tyrosinase is the key rate-limiting enzyme in the melanin production process. By reducing the activity of tyrosinase in the body, melanin production is reduced, thereby achieving a whitening effect. As can be seen from Table 4, the polygonatum polysaccharide prepared by the method of the present invention has a strong tyrosinase inhibitory ability, indicating that it has a high whitening activity. Comparative Example 1 may be due to insufficient enzymatic hydrolysis time, and the obtained polysaccharide contains less polysaccharide components with tyrosinase inhibitory ability. Comparative Example 2 may not have microwave treatment, and the effect of enzymatic hydrolysis may not be able to completely extract the polysaccharide components with strong tyrosinase inhibitory ability. Comparative Examples 3 and Comparative Examples 4 have changes in the extraction combination, resulting in changes in the structure and content of the extracted polysaccharide components, that is, the overall polygonatum polysaccharide obtained has insufficient tyrosinase inhibitory activity. In summary, the polygonatum polysaccharide prepared by the method of the present invention has a very high whitening activity.

[0096] Based on the data of Experimental Examples 1 and 2, it can be seen that the yield of Polygonatum sibiricum polysaccharide prepared by the method of the present invention is high, and its DPPH free radical scavenging ability, α-glucosidase inhibitory ability and tyrosinase inhibitory activity are also high.

[0097] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for extracting polygonatum polysaccharide, characterized in that: The following steps are involved: (1) Slice fresh polygonatum, dry, crush, and sieve to obtain polygonatum powder; (2) Mixing polygonatum powder with deionized water, heating and extracting for multiple times, combining the extracts, and filtering the polygonatum residue for later use; (3) After the Polygonatum sibiricum residue is subjected to microwave treatment, deionized water is added, and then a composite enzyme is added, the pH value and temperature are adjusted, enzymatic hydrolysis is performed, and the supernatant is collected; (4) The extract of step (2) and the supernatant of step (3) are mixed and concentrated, the concentrate is mixed with an ethanol solution, and the mixture is placed in a low temperature environment for alcohol precipitation, and then centrifuged and separated, the precipitate is collected, and freeze-dried to obtain polygonatum polysaccharide.

2. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (2), the liquid ratio of polygonatum sibiricum powder to deionized water is 1:25-35 g / mL.

3. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (2), the mixture is heated to 95-100° C. and leached for 1.5-2.5 hours.

4. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (2), heating and leaching are performed twice.

5. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (3), the Polygonatum sibiricum filter residue is treated with a microwave at 600-700W for 60-90s.

6. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (3), the solid-liquid ratio of the polygonatum sibiricum filter residue to deionized water is 1:25-35 g / mL.

7. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (3), 2.5-4% of the weight of the Polygonatum sibiricum residue is added as the complex enzyme.

8. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (3), the complex enzyme is a mixture of cellulase and pectinase in a weight ratio of 1-3:

1.

9. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (3), the enzymatic hydrolysis is carried out for 50-60 minutes at a pH value of 4.5-5.0 and a temperature of 45-50°C.

10. The method for extracting polygonatum polysaccharide according to claim 1, characterized in that: In the step (3), the extract of step (2) and the supernatant of step (3) are mixed and concentrated to 1 / 10 of the original volume, the concentrate is mixed with an ethanol solution having a volume concentration of 90-95%, and placed in an environment of 4°C for alcohol precipitation for 12 hours.

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