Alpinia oxyphylla polysaccharide with high antioxidant activity and extraction and preparation method thereof

Abstract

The present invention relates to a polysaccharide of Alpinia oxyphylla with high antioxidant activity and an extraction and preparation method thereof, and relates to the technical field of extraction of polysaccharides of Alpinia oxyphylla. The polysaccharide of Alpinia oxyphylla is made from Alpinia oxyphylla powder, and is firstly extracted with a deep eutectic solvent to obtain an Alpinia oxyphylla solution, and then the Alpinia oxyphylla solution is subjected to low-temperature water extraction to obtain a crude extract of polysaccharides of Alpinia oxyphylla, and finally the crude extract of polysaccharides of Alpinia oxyphylla is subjected to a refining treatment to obtain the polysaccharide. The present invention extracts polysaccharides of Alpinia oxyphylla by combining deep eutectic solvent with low-temperature water extraction. Compared with the existing extraction and preparation methods, the extraction rate and total sugar content of polysaccharides of Alpinia oxyphylla are significantly improved. In addition, the antioxidant capacity and iron ion reducing capacity of polysaccharides of Alpinia oxyphylla extracted by the extraction and preparation method are further improved, and the polysaccharide has excellent antioxidant activity.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction from alpinia oxyphylla, and particularly relates to alpinia oxyphylla polysaccharide with high antioxidant activity and an extraction and preparation method thereof. Background Art

[0002] Alpinia oxyphylla is one of the four major southern Chinese medicines with a long history of medicinal use. It is recorded in the classical monographs on Chinese medicinal materials, such as "Supplement to Materia Medica", "Illustrated Materia Medica" and "Compendium of Materia Medica". Alpinia oxyphylla is pungent in taste and warm in nature. It enters the spleen and kidney meridians. It has the effects of warming the spleen, appetizing, relieving saliva, warming the kidney, consolidating essence and reducing urine. Polysaccharides, as an important active ingredient in medicinal plants, have attracted much attention in recent years. Studies have shown that medicinal plant polysaccharides have multiple biological activities such as antiviral, antioxidant, anti-tumor and immunomodulatory. However, due to the very complex composition of polysaccharides in plants, the separation and extraction process of polysaccharides is complicated and inefficient, which greatly limits the further research and development of medicinal plant polysaccharides.

[0003] Traditional methods for extracting plant polysaccharides mainly include mechanical crushing, microwave extraction, ultrasonic extraction, enzymatic extraction, etc. Mechanical crushing has high energy consumption, long processing time, and high heat generation, resulting in a large loss of effective components in cells. Auxiliary methods such as microwaves, ultrasound, and enzymes can increase the yield of polysaccharides. However, microwaves may degrade polysaccharide molecules and damage the polysaccharide structure, thereby affecting the extraction of polysaccharides; the ultrasonic method usually requires ice bath cooling and intermittent ultrasound to reduce the temperature of the processing liquid due to its high heat generation, making this method energy-intensive and time-consuming; the enzymatic method has the disadvantages of high cost and strong specificity. Therefore, it is very important to find a green and efficient method for extracting polysaccharides from Alpinia oxyphylla.

[0004] Deep eutectic solvent (DES) is a new type of extractant, a liquid system composed of two or more components (biodegradable, renewable, low-cost), which can act as a hydrogen bond acceptor or donor. Due to its excellent stability, non-toxicity, reusability and other characteristics, it is considered to be an ideal green alternative to common ionic liquids and organic volatile solvents. In recent years, it has been widely used in the extraction of plant active ingredients. The study of the extraction of plant polysaccharides with deep eutectic solvents in aqueous solution has many advantages, such as the high hydrogen bonding ability between deep eutectic solvents and polysaccharides, which can improve the solubility and extraction efficiency of macromolecular polysaccharides in solvents. At present, there is no report on the extraction of polysaccharides from Alpinia oxyphylla using deep eutectic solvents. Summary of the invention

[0005] The purpose of the present invention is to provide a polysaccharide from Alpinia oxyphylla with high antioxidant activity and a method for extracting and preparing the polysaccharide.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] As the first aspect of the present invention, a polysaccharide with high antioxidant activity is provided. The polysaccharide is made from polysaccharide powder, firstly extracted with a deep eutectic solvent to obtain a polysaccharide solution, then the polysaccharide solution is subjected to low-temperature water extraction to obtain a polysaccharide crude extract, and finally the polysaccharide crude extract is refined to obtain the polysaccharide.

[0008] As a further optimization scheme of the present invention, the deep eutectic solvent comprises 1,4-butanediol and choline chloride, and the molar ratio of choline chloride to 1,4-butanediol is 1:2 to 1:5, and the water content of the deep eutectic solvent is 20% to 40%.

[0009] As a further optimization scheme of the present invention, the temperature of the low-temperature water extraction is 40°C to 60°C.

[0010] As a second aspect of the present invention, there is also provided a method for extracting and preparing polysaccharides from Alpinia oxyphylla with high antioxidant activity as described above, comprising the following steps:

[0011] (1) Pretreatment: crushing the Alpinia oxyphylla seeds, soaking them in ethanol and drying them to obtain Alpinia oxyphylla seeds powder;

[0012] (2) Extraction: using a deep eutectic solvent to extract the Alpinia oxyphylla powder obtained in step (1) to obtain an Alpinia oxyphylla solution, subjecting the Alpinia oxyphylla solution to low-temperature water extraction, collecting the extract, filtering and concentrating it to obtain a crude extract of Alpinia oxyphylla polysaccharide;

[0013] (3) Purification: The crude extract of Alpinia oxyphylla polysaccharide obtained in step (2) is placed in a dialysis bag for dialysis to obtain a dialysis solution; Sevage reagent is added to deproteinize the dialysis solution, centrifuged, the supernatant is concentrated, ethanol precipitated, filtered, and freeze-dried to obtain the Alpinia oxyphylla polysaccharide with high antioxidant activity.

[0014] As a further optimization scheme of the present invention, in the step (1), the soaking time of 95% by volume ethanol is 36 to 72 hours, and it is replaced every 24 hours.

[0015] As a further optimization scheme of the present invention, in the step (2), the material-liquid ratio of the Alpinia oxyphylla powder to the deep eutectic solvent is 1:15 to 1:25.

[0016] As a further optimization scheme of the present invention, in the step (2), the Alpinia oxyphylla solution is placed in a magnetic stirring constant temperature water bath for extraction, the extraction temperature is 40°C to 60°C, and the extraction time is 60 to 120 minutes.

[0017] As a further optimization scheme of the present invention, in the step (3), the molecular weight cutoff of the dialysis bag is 6000-8000Da; and the dialysis time is 48-72h.

[0018] As a further optimization scheme of the present invention, in step (3), the Sevage reagent is a mixed solvent of chloroform and n-butanol in a volume ratio of 4:1.

[0019] The beneficial effects of the present invention are:

[0020] (1) The present invention uses a deep eutectic solvent coupled with low-temperature water extraction to extract Alpinia oxyphylla polysaccharides. Compared with the existing extraction and preparation methods, the extraction rate and total sugar content of Alpinia oxyphylla polysaccharides are significantly improved. In addition, the antioxidant capacity and iron ion reducing capacity of the Alpinia oxyphylla polysaccharides extracted by the extraction and preparation method are further improved, and the polysaccharides have excellent antioxidant activity.

[0021] (2) The present invention further improves the extraction rate and total sugar content of Alpinia oxyphylla polysaccharide by adjusting the composition ratio and water content of the deep eutectic solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the total antioxidant capacity determination result of the polysaccharide of Alpinia oxyphylla provided by the present invention;

[0023] Figure 2 This is the result of measuring the iron ion reducing ability of the Alpinia oxyphylla polysaccharide provided by the present invention. DETAILED DESCRIPTION

[0024] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0025] Example 1

[0026] This embodiment provides a method for extracting and preparing polysaccharides from Alpinia oxyphylla with high antioxidant activity, comprising the following steps:

[0027] (1) Pretreatment: 100 g of Alpinia oxyphylla kernels were crushed with a grinder, and soaked in 95% ethanol by volume for 72 h, with the 95% ethanol replaced every 24 h. After soaking, the kernels were dried in a blast dryer at 45° C. for 48 h to obtain Alpinia oxyphylla kernel powder;

[0028] (2) Extraction: Weigh 20 g of Alpinia oxyphylla powder and 400 mL of deep eutectic solvent, place them in a glass beaker and mix them to obtain an Alpinia oxyphylla solution, the deep eutectic solvent contains 1,4-butanediol and choline chloride, the molar ratio of choline chloride to 1,4-butanediol is 1:3, the water content of the deep eutectic solvent is 30%, the solid-liquid ratio of Alpinia oxyphylla powder to the deep eutectic solvent is 1:20 (g / mL), place the Alpinia oxyphylla solution in a magnetic stirring constant temperature water bath for low-temperature water extraction, the extraction temperature is 50°C, the extraction time is 60 min, the supernatant is collected by centrifugation, and filtered to obtain an extract, the extract is concentrated in a rotary evaporator, and the concentration temperature is 50°C to obtain a crude extract of Alpinia oxyphylla polysaccharide;

[0029] (3) Purification: The crude extract of Alpinia oxyphylla polysaccharide is placed in a dialysis bag (molecular weight cutoff is 8000Da), dialyzed in pure water for 72 hours to obtain a dialysate, added with Sevage reagent (chloroform: n-butanol = 4:1 v / v) 4 times the volume of the dialysate, shaken, deproteinized, centrifuged, and the supernatant is collected. The supernatant is concentrated in a rotary evaporator at a concentration temperature of 50°C to obtain an Alpinia oxyphylla polysaccharide refined extract, 95% by volume ethanol is added to the Alpinia oxyphylla polysaccharide refined extract until the alcohol concentration is 80%, allowed to stand at 4°C for 12 hours, and filtered to obtain an Alpinia oxyphylla polysaccharide extract. The Alpinia oxyphylla polysaccharide extract is dried in a freeze dryer for 72 hours at a drying temperature of -80°C and a pressure of 10 MPa to obtain Alpinia oxyphylla polysaccharide.

[0030] Example 2

[0031] This embodiment provides a method for extracting and preparing polysaccharides from Alpinia oxyphylla with high antioxidant activity, comprising the following steps:

[0032] (1) Pretreatment: 100 g of Alpinia oxyphylla kernels were crushed with a grinder, and soaked in 95% ethanol by volume for 72 h, with the 95% ethanol replaced every 24 h. After soaking, the kernels were dried in a blast dryer at 45° C. for 48 h to obtain Alpinia oxyphylla kernel powder;

[0033] (2) Extraction: Weigh 20 g of Alpinia oxyphylla powder and 400 mL of deep eutectic solvent, place them in a glass beaker and mix them to obtain an Alpinia oxyphylla solution, the deep eutectic solvent contains 1,4-butanediol and choline chloride, the molar ratio of choline chloride to 1,4-butanediol is 1:4, the water content of the deep eutectic solvent is 40%, the solid-liquid ratio of Alpinia oxyphylla powder to the deep eutectic solvent is 1:20 (g / mL), place the Alpinia oxyphylla solution in a magnetic stirring constant temperature water bath for low-temperature water extraction, the extraction temperature is 50°C, the extraction time is 60 min, the supernatant is collected by centrifugation, and filtered to obtain an extract, the extract is concentrated in a rotary evaporator, and the concentration temperature is 50°C to obtain a crude extract of Alpinia oxyphylla polysaccharide;

[0034] (3) Purification: The crude extract of Alpinia oxyphylla polysaccharide is placed in a dialysis bag (molecular weight cutoff is 8000Da), dialyzed in pure water for 72 hours to obtain a dialysate, added with Sevage reagent (chloroform: n-butanol = 4:1 v / v) 4 times the volume of the dialysate, shaken, deproteinized, centrifuged, and the supernatant is collected. The supernatant is concentrated in a rotary evaporator at a concentration temperature of 50°C to obtain an Alpinia oxyphylla polysaccharide refined extract, 95% by volume ethanol is added to the Alpinia oxyphylla polysaccharide refined extract until the alcohol concentration is 80%, allowed to stand at 4°C for 12 hours, and filtered to obtain an Alpinia oxyphylla polysaccharide extract. The Alpinia oxyphylla polysaccharide extract is dried in a freeze dryer for 72 hours at a drying temperature of -80°C and a pressure of 10 MPa to obtain Alpinia oxyphylla polysaccharide.

[0035] Example 3

[0036] This embodiment provides a method for extracting and preparing polysaccharides from Alpinia oxyphylla with high antioxidant activity, comprising the following steps:

[0037] (1) Pretreatment: 100 g of Alpinia oxyphylla kernels were crushed with a grinder, and soaked in 95% ethanol by volume for 72 h, with the 95% ethanol replaced every 24 h. After soaking, the kernels were dried in a blast dryer at 45° C. for 48 h to obtain Alpinia oxyphylla kernel powder;

[0038] (2) Extraction: Weigh 20 g of Alpinia oxyphylla powder and 400 mL of deep eutectic solvent, place them in a glass beaker and mix them to obtain an Alpinia oxyphylla solution, the deep eutectic solvent contains 1,4-butanediol and choline chloride, the molar ratio of choline chloride to 1,4-butanediol is 1:5, the water content of the deep eutectic solvent is 50%, the solid-liquid ratio of Alpinia oxyphylla powder to the deep eutectic solvent is 1:20 (g / mL), place the Alpinia oxyphylla solution in a magnetic stirring constant temperature water bath for low-temperature water extraction, the extraction temperature is 50°C, the extraction time is 60 min, the supernatant is collected by centrifugation, and filtered to obtain an extract, the extract is concentrated in a rotary evaporator, and the concentration temperature is 50°C to obtain a crude extract of Alpinia oxyphylla polysaccharide;

[0039] (3) Purification: The crude extract of Alpinia oxyphylla polysaccharide is placed in a dialysis bag (molecular weight cutoff is 8000Da), dialyzed in pure water for 72 hours to obtain a dialysate, added with Sevage reagent (chloroform: n-butanol = 4:1 v / v) 4 times the volume of the dialysate, shaken, deproteinized, centrifuged, and the supernatant is collected. The supernatant is concentrated in a rotary evaporator at a concentration temperature of 50°C to obtain an Alpinia oxyphylla polysaccharide refined extract, 95% by volume ethanol is added to the Alpinia oxyphylla polysaccharide refined extract until the alcohol concentration is 80%, allowed to stand at 4°C for 12 hours, and filtered to obtain an Alpinia oxyphylla polysaccharide extract. The Alpinia oxyphylla polysaccharide extract is dried in a freeze dryer for 72 hours at a drying temperature of -80°C and a pressure of 10 MPa to obtain Alpinia oxyphylla polysaccharide.

[0040] Comparative Example 1

[0041] This comparative example discloses a method for extracting polysaccharides from Alpinia oxyphylla using a physical ultrasonic method, and the specific steps are as follows:

[0042] (1) Pretreatment: 100 g of Alpinia oxyphylla kernels were crushed with a grinder, and soaked in 95% ethanol by volume for 72 h, with the 95% ethanol replaced every 24 h. After soaking, the kernels were dried in a blast dryer at 45° C. for 48 h to obtain Alpinia oxyphylla kernel powder;

[0043] (2) Extraction: Weigh 20 g of Alpinia oxyphylla powder and 400 mL of ultrapure water, place in a glass beaker and mix, obtain an Alpinia oxyphylla solution, the solid-liquid ratio of Alpinia oxyphylla powder to ultrapure water is 1:20 (g / mL), place the Alpinia oxyphylla solution in an ultrasonic cleaner for ultrasonic extraction, the extraction temperature is 50°C, the extraction power is 300 W, the extraction time is 60 min, the supernatant is collected by centrifugation, and filtered to obtain an extract; the extract is concentrated in a rotary evaporator at a concentration temperature of 50°C to obtain a crude extract of Alpinia oxyphylla polysaccharide;

[0044] (3) Purification: Place the crude extract of Alpinia oxyphylla polysaccharide in a dialysis bag (molecular weight cutoff of 8000Da), dialyze in pure water for 72 hours to obtain a dialysate; add 4 times the volume of the dialysate Sevage reagent (chloroform: n-butanol = 4:1 v / v), shake, deproteinize, centrifuge, take the supernatant, concentrate the supernatant in a rotary evaporator at a concentration temperature of 50°C to obtain an Alpinia oxyphylla polysaccharide extract, add 95% ethanol to the Alpinia oxyphylla polysaccharide extract to an alcohol concentration of 80%, let stand at 4°C for 12 hours, filter, and obtain an Alpinia oxyphylla polysaccharide extract; dry the Alpinia oxyphylla polysaccharide extract in a freeze dryer for 72 hours at a drying temperature of -80°C and a pressure of 10 MPa to obtain Alpinia oxyphylla polysaccharide.

[0045] Comparative Example 2

[0046] This comparative example discloses a method for extracting and preparing polysaccharides from Alpinia oxyphylla using only a low-temperature water extraction method, and the specific steps are as follows:

[0047] (1) Pretreatment: 100 g of Alpinia oxyphylla kernels were crushed with a grinder, and soaked in 95% ethanol by volume for 72 h, with the 95% ethanol replaced every 24 hours. After soaking, the kernels were dried in a blast dryer at 45° C. for 48 h to obtain Alpinia oxyphylla kernel powder;

[0048] (2) Weighing 20 g of Alpinia oxyphylla powder and 400 mL of ultrapure water, placing them in a glass beaker and mixing them, obtaining an Alpinia oxyphylla solution, wherein the solid-liquid ratio of the Alpinia oxyphylla powder to the ultrapure water is 1:20 (g / mL); placing the Alpinia oxyphylla solution in a magnetic stirring constant temperature water bath for low-temperature water extraction, the extraction temperature is 50° C., the extraction time is 60 min, the supernatant is collected by centrifugation, and filtered to obtain an extract; the extract is concentrated in a rotary evaporator, the concentration temperature is 50° C., and a crude extract of Alpinia oxyphylla polysaccharide is obtained;

[0049] (3) Purification: The crude extract of Alpinia oxyphylla polysaccharide is placed in a dialysis bag (molecular weight cutoff is 8000Da), dialyzed in pure water for 72 hours to obtain a dialysate, and Sevage reagent (chloroform: n-butanol = 4:1 v / v) with a volume of 4 times the dialysate is added, shaken, deproteinized, centrifuged, and the supernatant is collected. The supernatant is concentrated in a rotary evaporator at a concentration temperature of 50°C to obtain an Alpinia oxyphylla polysaccharide refined extract, 95% ethanol is added to the Alpinia oxyphylla polysaccharide refined extract to a concentration of 80%, and the extract is allowed to stand at 4°C for 12 hours, and filtered to obtain an Alpinia oxyphylla polysaccharide extract. The Alpinia oxyphylla polysaccharide extract is dried in a freeze dryer for 72 hours at a drying temperature of -80°C and a pressure of 10 MPa to obtain Alpinia oxyphylla polysaccharide.

[0050] Comparative Example 3

[0051] This comparative example discloses a method for extracting and preparing polysaccharides from Alpinia oxyphylla using only deep eutectic solvent extraction, and the specific steps are as follows:

[0052] (1) Pretreatment: 100 g of Alpinia oxyphylla kernels were crushed with a grinder, and soaked in 95% ethanol by volume for 72 h, with the 95% ethanol replaced every 24 h. After soaking, the kernels were dried in a blast dryer at 45° C. for 48 h to obtain Alpinia oxyphylla kernel powder;

[0053] (2) Extraction: Weigh 20 g of Alpinia oxyphylla powder and 400 mL of deep eutectic solvent, place them in a glass beaker and mix them to obtain an Alpinia oxyphylla solution, the deep eutectic solvent comprises 1,4-butanediol and choline chloride, the molar ratio of choline chloride to 1,4-butanediol is 1:4, the water content of the deep eutectic solvent is 0%, the solid-liquid ratio of Alpinia oxyphylla powder to the deep eutectic solvent is 1:20 (g / mL), centrifuge the Alpinia oxyphylla solution to collect the supernatant, filter it to obtain an extract, concentrate the extract in a rotary evaporator at a concentration temperature of 50°C to obtain a crude extract of Alpinia oxyphylla polysaccharide;

[0054] (3) Purification: The crude extract of Alpinia oxyphylla polysaccharide is placed in a dialysis bag (molecular weight cutoff is 8000Da), dialyzed in pure water for 72 hours to obtain a dialysate, added with Sevage reagent (chloroform: n-butanol = 4:1 v / v) 4 times the volume of the dialysate, shaken, deproteinized, centrifuged, and the supernatant is collected. The supernatant is concentrated in a rotary evaporator at a concentration temperature of 50°C to obtain an Alpinia oxyphylla polysaccharide refined extract, 95% by volume ethanol is added to the Alpinia oxyphylla polysaccharide refined extract until the alcohol concentration is 80%, allowed to stand at 4°C for 12 hours, and filtered to obtain an Alpinia oxyphylla polysaccharide extract. The Alpinia oxyphylla polysaccharide extract is dried in a freeze dryer for 72 hours at a drying temperature of -80°C and a pressure of 10 MPa to obtain Alpinia oxyphylla polysaccharide.

[0055] Comparative Example 4

[0056] This comparative example discloses a method for extracting and preparing polysaccharides from Alpinia oxyphylla using a deep eutectic solvent extraction method coupled with a hot water extraction method, and the specific steps are:

[0057] (1) Pretreatment: 100 g of Alpinia oxyphylla kernels were crushed with a grinder, and soaked in 95% ethanol by volume for 72 h, with the 95% ethanol replaced every 24 h. After soaking, the kernels were dried in a blast dryer at 45° C. for 48 h to obtain Alpinia oxyphylla kernel powder;

[0058] (2) Extraction: Weigh 20 g of Alpinia oxyphylla powder and 400 mL of deep eutectic solvent, place them in a glass beaker and mix them to obtain an Alpinia oxyphylla solution, the deep eutectic solvent contains 1,4-butanediol and choline chloride, the molar ratio of choline chloride to 1,4-butanediol is 1:4, the water content of the deep eutectic solvent is 40%, the solid-liquid ratio of Alpinia oxyphylla powder to the deep eutectic solvent is 1:20 (g / mL), place the Alpinia oxyphylla solution in a magnetic stirring constant temperature water bath for hot water extraction, the extraction temperature is 90°C, the extraction time is 60 min, the supernatant is collected by centrifugation, and filtered to obtain an extract, the extract is concentrated in a rotary evaporator, and the concentration temperature is 50°C to obtain a crude extract of Alpinia oxyphylla polysaccharide;

[0059] (3) Purification: The crude extract of Alpinia oxyphylla polysaccharide is placed in a dialysis bag (molecular weight cutoff is 8000Da), dialyzed in pure water for 72 hours to obtain a dialysate, added with Sevage reagent (chloroform: n-butanol = 4:1 v / v) 4 times the volume of the dialysate, shaken, deproteinized, centrifuged, and the supernatant is collected. The supernatant is concentrated in a rotary evaporator at a concentration temperature of 50°C to obtain an Alpinia oxyphylla polysaccharide refined extract, 95% by volume ethanol is added to the Alpinia oxyphylla polysaccharide refined extract until the alcohol concentration is 80%, allowed to stand at 4°C for 12 hours, and filtered to obtain an Alpinia oxyphylla polysaccharide extract. The Alpinia oxyphylla polysaccharide extract is dried in a freeze dryer for 72 hours at a drying temperature of -80°C and a pressure of 10 MPa to obtain Alpinia oxyphylla polysaccharide.

[0060] Verification test

[0061] 1. Determination of polysaccharide yield and total sugar content

[0062] (1) Polysaccharide yield = mass of polysaccharide from Alpinia oxyphylla kernel / mass of powder from Alpinia oxyphylla kernel.

[0063] (2) Determination of total sugar content

[0064] Neutral sugar determination: phenol-sulfuric acid method, with glucose dried to constant weight as the standard, prepare 0, 20, 40, 60, 80, 100 μg / mL standard solutions for drawing the standard curve, aspirate 400 μL of the standard solution and the sample solution diluted to the appropriate concentration, add 400 μL of the freshly prepared 6% phenol solution and 2 mL of concentrated sulfuric acid, vortex to mix, place for 10 min, bathe at 30°C for 20 min, vortex to mix, and measure the OD value at 490 nm; the results are expressed as the glucose equivalent in each gram of Alpinia oxyphylla extract.

[0065] Uronic acid determination: m-hydroxybiphenyl method, with galacturonic acid dried to constant weight as the standard, prepared 0, 20, 40, 60, 80, 100 μg / mL galacturonic acid solutions for drawing the calibration curve, aspirated the standard solution and 0.5 mL of the sample solution diluted to the appropriate concentration, added 3 mL of sodium tetraborate-concentrated sulfuric acid solution in an ice bath, vortexed to mix, heated in a boiling water bath for 5 min, cooled to room temperature, added 50 μL of m-hydroxybiphenyl reagent, and measured OD at 524 nm; the results were expressed as the galacturonic acid equivalent per gram of Alpinia oxyphylla extract.

[0066] Total sugar content = neutral sugar content + uronic acid content. The total sugar content is used to indicate the purity of the extracted polysaccharide.

[0067] The yield and total sugar content of Alpinia oxyphylla polysaccharide extracted by the methods disclosed in Examples 1-3 and Comparative Examples 1-4 were measured, and the results are shown in Table 1.

[0068] Table 1 Extraction rate and total sugar content of polysaccharides from Alpinia oxyphylla

[0069]

[0070]

[0071] As can be seen from Table 1, by comparing Examples 1-3 with Comparative Examples 1-4, the extraction rate of polysaccharides from Alpinia oxyphylla is between 5.5% and 7.6%, and the total sugar content is between 70.7% and 88.2% by using the deep eutectic solvent coupled with low-temperature water extraction method. Compared with the physical ultrasonic method, low-temperature water extraction method, anhydrous deep eutectic solvent extraction method and deep eutectic solvent coupled with hot water extraction method, the extraction rate and total sugar content of polysaccharides from Alpinia oxyphylla are significantly improved. The anhydrous eutectic solvent extraction method does not extract polysaccharides due to the high viscosity of the solution.

[0072] In addition, from the data comparison of Examples 1-3, it can be seen that the extraction rate and total sugar content of Alpinia oxyphylla polysaccharide in Example 2 are better than those in Examples 1 and 3. The reason is that the molar ratio of choline chloride and 1,4-butanediol in the deep eutectic solvent used in Example 2 is 1:4, and the water content is 40%. When the composition ratio of choline chloride and 1,4-butanediol is relatively low, the hydrogen bonding and polarity of the solution system are weak, which affects the dissolution of polysaccharides. When the composition ratio of choline chloride and 1,4-butanediol is too high, the composition The interactions such as the van der Waals force between the components increase, the viscosity of the system increases, and the polysaccharide dissolution is not utilized. The water content of the deep eutectic solvent system will also affect the polysaccharide extraction rate. When the water content is low, the viscosity in the system is high, which is not conducive to polysaccharide dissolution; when the water content is too high, the hydrogen bonds in the system are destroyed and the extraction efficiency is reduced. It can be seen that when the deep eutectic solvent is selected with a molar ratio of choline chloride and 1,4-butanediol of 1:4 and a water content of 40%, the optimal extraction yield and total sugar content of Alpinia oxyphylla polysaccharides can be obtained.

[0073] 2. Determination of total antioxidant capacity

[0074] Preparation of 1 / 1000 hydrogen peroxide solution: Take 10 μL of hydrogen peroxide solution, add it to 9.99 mL of double distilled water, and mix well to obtain 1 / 1000 hydrogen peroxide solution;

[0075] Preparation of ABTS working solution: Add 15.2 mL of buffer and 800 μL of 1 / 1000 hydrogen peroxide solution to 1 mL of ABTS solution to obtain ABTS working solution.

[0076] Sample determination: Add 20 μL peroxidase working solution to each detection well of the 96-well plate; add 10 μL PBS solution to the blank control well; add 10 μL 0, 0.005, 0.1, 0.2, 0.3, 0.4, 0.5 mM Trolox standard solution to the standard curve detection well; add 10 μL 40, 80, 100, 200, 300 μg / mL Alpinia oxyphylla polysaccharide sample solution to the sample detection well and mix gently; add 170 μL ABTS working solution to each well and mix gently; incubate at room temperature for 6 min and then measure A 414 ; Calculate ΔA for each point of the standard 414 :ΔA 414 =A 414 Blank Control-A 414 Standard, use ΔA 414 A standard curve was prepared using the concentration of Trolox standard solution.

[0077] The standard curve is represented by the formula y = ax + b (y is ΔA 414 , x is the concentration of Trolox standard solution, a is the slope, and y is the intercept). 414 =A414 Blank Control-A 414 The sample is calculated according to the standard curve formula to determine how many mM of Trolox standard the sample is equivalent to, which is the total antioxidant capacity of the sample. Each group is set up with 3 parallels to obtain the antioxidant capacity.

[0078] Expression of total antioxidant capacity: When using Trolox as a standard for total antioxidant capacity detection, the antioxidant capacity of the sample can be expressed by Trolox-Equivalent Antioxidant Capacity (TEAC). For example, if the concentration of a sample is 0.1 mg / mL, the ΔA 414 The same as 0.5 mM Trolox, the total antioxidant capacity of the sample is 0.5 mM / 0.1 mg / mL, which is 5 mmol / g.

[0079] The antioxidant capacity of the polysaccharides extracted from Alpinia oxyphylla by the methods disclosed in Example 2 and Comparative Examples 1, 2, and 4 was determined (no polysaccharide was extracted from Comparative Example 3, and no antioxidant capacity determination test was performed). The results are as follows: Figure 1 shown.

[0080] The results show that, by comparing Example 2 with Comparative Examples 1, 2, and 4, the use of deep eutectic solvent coupled with low-temperature water extraction significantly improves the total antioxidant capacity of Alpinia oxyphylla polysaccharides compared with physical ultrasonic method, low-temperature water extraction method, and deep eutectic solvent coupled with hot water extraction, and has a certain dose-effect relationship.

[0081] 3. Determination of iron ion reducing ability

[0082] Preparation of FRAP working solution: Add 15 mL of TPTZ diluent to 1.5 mL of TPTZ solution, mix thoroughly and add 1.5 mL of buffer to obtain FRAP working solution. After the FRAP working solution is prepared, incubate at 37°C and use it within 1-2 hours.

[0083] Sample determination: 180 μL FRAP working solution was added to each well of the 96-well plate; 5 μL PBS solution was added to the blank control well; 5 μL 0, 20, 50, 80, 110, 140, 170 μg / mL FeSO4 standard solution was added to the standard curve detection well; 5 μL 40, 80, 100, 200, 300 μg / mL Alpinia oxyphylla polysaccharide sample solution was added to the sample detection well and gently mixed; incubate at 37°C for 3-5 min and then determine A 593 ; Calculate the iron ion reducing ability of the sample based on the standard curve.

[0084] Expression of total antioxidant capacity: For the FRAP method, the total antioxidant capacity is expressed by the concentration of the FeSO4 standard solution. For example, if the absorbance obtained is the same as that of the 0.3mM FeSO4 standard solution, and the concentration of the polysaccharide sample is 0.15mg / mL, then the iron ion reducing capacity of the polysaccharide sample is 0.3mM / 0.15mg / mL, i.e. 2mmoL / g.

[0085] The iron ion reducing ability test was performed on the polysaccharides extracted from Alpinia oxyphyllae by the methods disclosed in Example 2 and Comparative Examples 1, 2, and 4 (Comparative Example 3 did not extract polysaccharides, so the iron ion reducing ability test was not performed). Figure 2 shown.

[0086] The results show that, by comparing Example 2 with Comparative Examples 1, 2, and 4, the deep eutectic solvent coupled with low-temperature water extraction significantly improves the iron ion reducing ability of Alpinia oxyphylla polysaccharide compared with the physical ultrasonic method, low-temperature water extraction method, and deep eutectic solvent coupled with hot water extraction, and has a certain dose-effect relationship.

[0087] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A polysaccharide from Alpinia oxyphylla with high antioxidant activity, characterized in that: The Alpinia oxyphylla polysaccharide is prepared by using Alpinia oxyphylla powder as a raw material, firstly extracting the Alpinia oxyphylla solution by using a deep eutectic solvent, then extracting the Alpinia oxyphylla solution by low-temperature water to obtain a crude extract of the Alpinia oxyphylla polysaccharide, and finally refining the crude extract of the Alpinia oxyphylla polysaccharide. The deep eutectic solvent comprises 1,4-butanediol and choline chloride, and the molar ratio of choline chloride to 1,4-butanediol is 1:2 to 1:5, and the water content of the deep eutectic solvent is 20% to 40%; The temperature of the low-temperature water extraction is 40°C to 60°C.

2. A method for extracting and preparing polysaccharides from Alpinia oxyphylla with high antioxidant activity as claimed in claim 1, characterized in that: The following steps are involved: (1) Pretreatment: crushing the Alpinia oxyphylla seeds, soaking them in ethanol and drying them to obtain Alpinia oxyphylla seeds powder; (2) Extraction: using a deep eutectic solvent to extract the Alpinia oxyphylla powder obtained in step (1) to obtain an Alpinia oxyphylla solution, subjecting the Alpinia oxyphylla solution to low-temperature water extraction, collecting the extract, filtering and concentrating it to obtain a crude extract of Alpinia oxyphylla polysaccharide; (3) Purification: The crude extract of Alpinia oxyphylla polysaccharide obtained in step (2) is placed in a dialysis bag for dialysis to obtain a dialysis solution; Sevage reagent is added to deproteinize the dialysis solution, centrifuged, the supernatant is concentrated, ethanol precipitated, filtered, and freeze-dried to obtain the Alpinia oxyphylla polysaccharide with high antioxidant activity.

3. The method for extracting and preparing Alpinia oxyphylla polysaccharide with high antioxidant activity according to claim 2, characterized in that: In the step (1), the immersion time in 95% by volume ethanol is 36 to 72 hours, and the ethanol is replaced every 24 hours.

4. The method for extracting and preparing Alpinia oxyphylla polysaccharide with high antioxidant activity according to claim 2, characterized in that: In the step (2), the material-liquid ratio of the Alpinia oxyphylla powder to the deep eutectic solvent is 1:15 to 1:

25.

5. The method for extracting and preparing Alpinia oxyphylla polysaccharide with high antioxidant activity according to claim 2, characterized in that: In the step (2), the Alpinia oxyphylla solution is placed in a magnetic stirring constant temperature water bath for extraction, the extraction temperature is 40° C. to 60° C., and the extraction time is 60 to 120 min.

6. The method for extracting and preparing Alpinia oxyphylla polysaccharide with high antioxidant activity according to claim 2, characterized in that: In the step (3), the molecular weight cutoff of the dialysis bag is 6000-8000Da; and the dialysis time is 48-72h.

7. The method for extracting and preparing Alpinia oxyphylla polysaccharide with high antioxidant activity according to claim 2, characterized in that: In the step (3), the Sevage reagent is a mixed solvent of chloroform and n-butanol in a volume ratio of 4:1.

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