Method for extracting polysaccharide from herb residues of cichorium glandulosum by response surface method and application of polysaccharide in oxidation resistance and blood sugar reduction

Abstract

The invention relates to research of cichorium glandulosum extracts, in particular to an extraction method of polysaccharide in cichorium glandulosum herb residues and application of the polysaccharide in resisting oxidation and reducing blood glucose, the herb residues obtained after alcohol extraction of cichorium glandulosum serve as raw materials, and the polysaccharide in the cichorium glandulosum herb residues is extracted through an enzyme method. The optimal extraction condition of the polysaccharide is determined by setting a single factor experiment and response surface optimization, and the application of resisting oxidation and reducing blood sugar is provided.

Description

Technical Field

[0001] The invention relates to research on chicory extract, and in particular to a method for extracting polysaccharides from chicory residues and applications of the polysaccharide in anti-oxidation and hypoglycemic activities. Background Art

[0002] Hairy chicory is 1-1.2m tall and covered with rough hairs. The stem is erect and branched. The leaves are alternate, ovate or oblong, deeply lobed, and half-amplexicaul at the base. The capitula grows in the axils of leaves or branches. The small flowers are blue, bisexual, and all are ligulate. The achenes are obovate, truncate at the top, gray-brown, about 2mm long, and with short pappus at the top. The roots of hairy chicory are conical, with lateral roots and many fibrous roots, 10-20cm long, and 0.5-1.5cm in diameter. The surface is brownish yellow with fine irregular longitudinal wrinkles. It is hard and not easy to break. The outer side of the cross section is yellowish-white, the middle part is off-white, and sometimes hollow. It has a slight odor and tastes bitter.

[0003] Hairy chicory is widely distributed in Xinjiang. It is a kind of Chinese medicinal material that is both a medicine and food. It tastes slightly bitter and salty, and is cool in nature. It belongs to the liver, gallbladder, and stomach meridians. It has the effects of clearing away heat and detoxification, clearing the liver and promoting bile secretion, strengthening the stomach and digestion, and diuresis and swelling. In recent years, hairy chicory has been developed into a Chinese patent medicine with significant efficacy, such as Hugan Buzure Granules, Fufang Muniziqi Granules, Qingre Kasen Granules, Yanxiao Dinaer Syrup, etc., which are widely used in the clinic to treat various liver and gallbladder diseases. Modern research shows that hairy chicory is rich in active ingredients such as polysaccharides, flavonoids, and terpenes, and has significant pharmacological activities such as lowering lipids, lowering blood sugar, protecting the liver, and antibacterial.

[0004] The extraction of Chinese medicinal substances is mainly fat-soluble components. The residues after extraction are mostly burned as fuel or discarded as garbage, which not only wastes resources but also causes great pollution to the environment. In the early stage, our research group focused on the active ingredients of the alcohol extract of chicory root. After repeated soaking in 95% ethanol, the extract was collected for subsequent research. The residues after extraction are often treated as waste. In fact, there are still a large number of active ingredients in the discarded residues, which still have medicinal and nutritional value. At present, there are many studies on polysaccharide extraction technology, mainly solvent extraction, enzymatic extraction, ultrasonic extraction, microwave-assisted extraction, etc. These methods have some problems, such as high cost, complicated process, and waste of fat-soluble components. Summary of the invention

[0005] In order to solve the above problems in the prior art, the present invention has invented a new method for extracting polysaccharides from chicory residues and an application thereof in vitro in terms of antioxidant activity and hypoglycemic activity.

[0006] The scheme of the present invention is a method for extracting polysaccharides from chicory residues, comprising the following steps:

[0007] (1) Slice the chicory root, soak it repeatedly in 90-95% ethanol, and then air-dry the residue;

[0008] (2) taking the chicory residue obtained in step (1), crushing it, and then passing it through a 20-60 mesh sieve;

[0009] (3) Enzymatic constant temperature shaking extraction for 1.5 to 2.5 hours, extraction temperature of 45 to 58°C, solid-liquid ratio of 1: (30 to 50) g / mL, enzyme amount of 0.5 to 3%, followed by cooling and separation.

[0010] Another preferred embodiment of the present invention is that the raw material for extracting hairy chicory is the residue after alcohol extraction of hairy chicory roots.

[0011] Another preferred embodiment of the present invention is that the amount of the enzyme is 2%.

[0012] The present invention also provides an application of the chicory polysaccharide obtained by the extraction method, and the application is to use the chicory polysaccharide obtained in medicine as an antioxidant.

[0013] The present invention also provides another application of the extract obtained by the extraction method, which is to use the obtained chicory polysaccharide in a hypoglycemic agent. More preferably, the obtained chicory polysaccharide is prepared into granules with a pharmaceutically acceptable auxiliary material.

[0014] Antioxidants are mostly used in food preservation, medicine and other fields. Although synthetic antioxidants such as BHT and TBHQ have strong antioxidant capacity, they have been found to have certain toxicity through research. Therefore, finding safe and natural food antioxidants has become a research hotspot. The antioxidant of the present invention has the advantages of being safe and non-toxic, and can be widely used in food additives as an antioxidant.

[0015] There is no ideal treatment for diabetes. Traditional sulfonylurea and biguanide hypoglycemic drugs will cause dependence and toxic side effects if taken for a long time. α-Glucosidase inhibitors are often used to treat type 2 diabetes, which can effectively reduce postprandial blood glucose levels and reduce the occurrence of diabetic complications. Therefore, finding efficient α-glucosidase inhibitors from natural products has become a research hotspot in recent years, and it has been reported that natural products such as citrus peel, licorice, mulberry leaves, dandelion, pomelo, dalbergia odorifera, Pu'er tea, bitter melon, ginkgo, etc. are mostly rich in flavonoids, polyphenols, polysaccharides and other ingredients. Therefore, studying the optimization process of extracting polysaccharides from chicory roots has high practical significance and broad application prospects for improving the economic value of chicory.

[0016] To this end, this experiment used enzymatic extraction of polysaccharides, and optimized the response surface experimental design (Box-Behnken experimental design) based on the single-factor experiment to obtain the best extraction conditions of polysaccharides. The antioxidant activity of chicory polysaccharides was studied by observing the ability of chicory polysaccharides to scavenge DPPH free radicals and the ability to scavenge the absorbance change caused by ABTS·+ through the DPPH and ABTS methods; the in vitro hypoglycemic activity of chicory polysaccharides was studied by the inhibition rate of α-glucosidase and α-amylase, providing important data for the evaluation and development and utilization of the health care function of chicory. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a curve diagram showing the effect of extraction time on polysaccharide yield in an embodiment of the present invention;

[0018] Figure 2 is a curve diagram showing the effect of the material-liquid ratio on the polysaccharide yield in the embodiment of the present invention;

[0019] Figure 3 is a curve diagram showing the effect of the amount of cellulase on the yield of polysaccharides in an embodiment of the present invention;

[0020] Figure 4 is a curve diagram showing the effect of extraction temperature on polysaccharide yield in an embodiment of the present invention;

[0021] Figure 5a is a contour map of the interaction between extraction time and cellulase amount in an embodiment of the present invention;

[0022] Figure 5b is an interaction surface diagram of the extraction time and the amount of cellulase in the embodiment of the present invention;

[0023] Figure 5c is a contour map of the interaction between extraction time and material-liquid ratio in an embodiment of the present invention;

[0024] Figure 5d is an interaction surface diagram of extraction time and material-liquid ratio in an embodiment of the present invention;

[0025] Figure 5e is a contour map of the interaction between extraction time and extraction temperature of an embodiment of the present invention;

[0026] Figure 5f is an interaction surface diagram of extraction time and extraction temperature in an embodiment of the present invention;

[0027] Figure 5g is a contour diagram of the interaction between the material-liquid ratio and the amount of cellulase in the embodiment of the present invention;

[0028] Figure 5h is an interaction surface diagram of the material-liquid ratio and the amount of cellulase in the embodiment of the present invention;

[0029] Figure 5i is a contour map of the interaction between extraction temperature and cellulase amount in an embodiment of the present invention;

[0030] Figure 5j is an interaction surface diagram of the extraction temperature and the amount of cellulase in an embodiment of the present invention;

[0031] Figure 5k is a contour diagram of the interaction between extraction temperature and material-liquid ratio in an embodiment of the present invention;

[0032] Figure 5l is an interaction surface diagram of extraction temperature and material-liquid ratio in an embodiment of the present invention;

[0033] Figure 6 It is the scavenging rate curve of DPPH by different concentrations of L-ascorbic acid and chicory polysaccharide;

[0034] Figure 7 It is the clearance curve of ABTS by different concentrations of L-ascorbic acid and chicory polysaccharide;

[0035] Figure 8 It is the inhibition rate curve of different concentrations of acarbose and chicory polysaccharide on α-glucosidase;

[0036] Fig. 9 It is the inhibition rate curve of different concentrations of acarbose and chicory polysaccharide on α-amylase; DETAILED DESCRIPTION

[0037] The present invention is described in detail below in conjunction with the accompanying drawings. The description in this part is only exemplary and explanatory and should not have any limiting effect on the scope of protection of the present invention. In addition, those skilled in the art can make corresponding combinations of the features in the embodiments and in different embodiments in this document based on the description of this document.

[0038] Example

[0039] 1 Experimental part

[0040] 1.1 Experimental instruments and drugs

[0041] 1.1.1 Experimental instruments

[0042] BJ-150 multifunctional pulverizer (Deqing Baijie Electric Co., Ltd.); GB / T6003 1-2012 standard test sieve (Shaoxing Shangyu Huafeng Hardware Instrument Co., Ltd.); 101-1AB electric blast drying oven (Tianjin Test Instrument Co., Ltd.); electric constant temperature water bath (Beijing Yongguangming Medical Instrument Co., Ltd.); circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd.); BP211D analytical balance (Sartorius, Germany, sensitivity: 0.01 mg); CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); VORTEX0 vortex mixer (Shanghai Dam Industrial Co., Ltd.); FA210B electronic balance (Shanghai Precision Scientific Instrument Co., Ltd.); SKY-100B constant temperature shaker (Shanghai Tiancheng Experimental Instrument Manufacturing Co., Ltd.); Varioskan LUX multifunctional microplate reader (Thermo Fisher Scientific, USA)

[0043] 1.1.2 Experimental drugs

[0044] Hairy chicory was purchased from Hotan County, Xinjiang Uygur Autonomous Region, and was identified by Professor Qin Dongmei of the School of Pharmacy of Shihezi University as the underground root part of hairy chicory (Cichorium glandulosum Boiss.et Huet) of the genus Cichorium in the Asteraceae family. Concentrated sulfuric acid, phenol, glucose (Shanghai Aladdin Biochemical Technology Co., Ltd.); cellulase (Shanghai MacLean Biochemical Technology Co., Ltd.); 95% ethanol (Hongyan Reagent Factory, Hedong District, Tianjin); potassium persulfate (Shanghai MacLean Biochemical Technology Co., Ltd.); 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (Shanghai Yuanye Biotechnology Co., Ltd.); 1,1-diphenyl-2-picrylhydrazyl (Shanghai Yuanye Biotechnology Co., Ltd.); L-ascorbic acid (Shanghai MacLean Biochemical Technology Co., Ltd.); α-glucosidase (Shanghai Yuanye Biotechnology Co., Ltd.); 0.1 mol / L phosphate buffer (pH 6.8); anhydrous sodium carbonate (Shanghai Yuanye Biotechnology Co., Ltd.); α-amylase (Shanghai Yuanye Biotechnology Co., Ltd.); soluble starch; DNS reagent (Beijing Regen Biotechnology Co., Ltd.)

[0045] 1.2 Experimental methods

[0046] 1.2.1 Extraction of polysaccharides from chicory residues

[0047] The dried chicory residue after alcohol extraction was crushed using a multifunctional crusher and passed through a 24-mesh sieve. Experiments were conducted according to four factors: extraction time (h), cellulase amount (%), extraction temperature (℃), and solid-liquid ratio (g / mL). After extraction, the residue was filtered and the filtrate was dried to calculate the yield.

[0048] 1.2.2 Determination of polysaccharide content

[0049] Prepare glucose solutions with concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, and distilled water as the blank group. Use a pipette to draw 100 μL of each solution and add it to a 1.5 mL centrifuge tube. Then add 100 μL of 6% phenol and 500 μL of concentrated sulfuric acid to each test tube. Mix well with a vortex mixer, let stand for 30 minutes, and use an enzyme marker to measure the absorbance at 490 nm. Draw a standard curve and regression equation with the polysaccharide mass concentration as the horizontal axis and the absorbance value as the vertical axis.

[0050] The standard curve equation is: Y = 5.8738x + 0.1748, R 2 =0.9993.

[0051] The polysaccharide content in the sample was determined as above, and the polysaccharide content was calculated as follows:

[0052] Polysaccharide content = (C × D / W) × 100%

[0053] Where: C is the sample glucose concentration / (mg / mL); D: sample dilution multiple; W: sample concentration / (mg / mL).

[0054] 1.2.3 Single Factor Experiment

[0055] (1) Effect of extraction time on polysaccharide yield

[0056] Take 1g of sample, under the conditions of solid-liquid ratio of 1:30, cellulase amount of 1.5%, extraction temperature of 50℃, and constant temperature oscillation extraction time of 1.5h, 1.75h, 2h, 2.25h, and 2.5h in sequence, calculate the polysaccharide yield, and investigate the effect of extraction time on the polysaccharide yield.

[0057] (2) Effect of the solid-liquid ratio on polysaccharide yield

[0058] Take 1g sample, add 20mL, 30mL, 40mL, 50mL, and 60mL of distilled water for constant temperature shaking extraction under the conditions of 2h, 1.5% cellulase amount, and 50℃ extraction temperature, calculate the polysaccharide yield, and investigate the effect of the solid-liquid ratio on the polysaccharide yield.

[0059] (3) Effect of cellulase dosage on polysaccharide yield

[0060] Take 1g sample, under the conditions of solid-liquid ratio of 1:30, extraction temperature of 50℃, extraction time of 2h, extract with cellulase dosage of 1%, 1.5%, 2%, 2.5%, and 3% in sequence, calculate the polysaccharide yield, and investigate the effect of cellulase dosage on the polysaccharide yield.

[0061] (4) Effect of extraction temperature on polysaccharide yield

[0062] Take 1g of sample, under the conditions of solid-liquid ratio of 1:30, cellulase amount of 1.5%, extraction time of 2h, and extraction at extraction temperatures of 42℃, 46℃, 50℃, 54℃, and 58℃ in sequence, calculate the polysaccharide yield, and investigate the effect of extraction temperature on the polysaccharide yield.

[0063] 1.2.4 Response surface optimization experiment

[0064] According to the results of the single factor experiment, a Box-Behnken experimental design was conducted on the four factors that affect the yield of polysaccharides from chicory residues: extraction time (A), cellulase content (B), solid-liquid ratio (C) and extraction temperature (D). The factor coding and level are shown in Table 1.

[0065] Table 1 Response surface factors and levels

[0066]

[0067] 1.2.5DPPH free radical scavenging method

[0068] (1) Preparation of DPPH test solution: accurately weigh 1.00 mg DPPH, dissolve it in 95% ethanol to 10 mL, mix well with a vortex mixer and dissolve with a digitally controlled ultrasonicator, and store in a dark place.

[0069] (2) Preparation of sample solution

[0070] The chicory polysaccharide and L-ascorbic acid were prepared into solutions with concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL, respectively.

[0071] (3) Sample determination: The test solution and DPPH solution were mixed in a ratio of 1:4, reacted in the dark at room temperature for 20 min, and the absorbance at 517 nm was measured using an ELISA reader.

[0072] A0: The absorbance of DPPH at 517nm

[0073] A: The absorbance of the test solution at 517nm

[0074] DPPH free radical scavenging rate (%) = [(A0-A) / A0] × 100%

[0075] 1.2.6 ABTS free radical scavenging method

[0076] (1) Configuration of ABTS working fluid

[0077] ① ABTS stock solution: Mix 2.5 mL of 7 mmol / L ABTS solution with 44 μL of 140 mmol / L potassium persulfate solution, mix well with a vortex mixer, and react in the dark at room temperature for 15 h to obtain ABTS stock solution.

[0078] ② ABTS working solution dilutes ABTS stock solution, and it can be used when the absorbance is 0.7±0.2

[0079] (2) Preparation of sample solution

[0080] The chicory polysaccharide and L-ascorbic acid were prepared into solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively.

[0081] (3) Sample measurement

[0082] Mix with the diluted ABTS stock solution at a ratio of 1:9, react in the dark at room temperature for 8 minutes, and measure the absorbance at 734 nm using an ELISA reader

[0083] A a : Absorbance of ABTS at 734 nm

[0084] A b : The absorbance of the test solution at 734nm

[0085] ABTS free radical scavenging rate (%) = [(A a —A b ) / A a ]×100%

[0086] 1.2.7 Determination of α-glucosidase inhibitory activity

[0087] (1) Solution preparation

[0088] ①0.1mol / LpH6.9 phosphate buffer solution: weigh 40.2000g of NaH2PO4 and 40.2300g of Na2HPO4 into a 100mL volumetric flask, and make up to volume with deionized water.

[0089] ②4.8mmol / LPNPG (4-nitrobenzene-aD-pyranoglucoside): Weigh 0.0483g of glucoside and dissolve it in pH 6.9 phosphate buffer solution, add to a 10mL volumetric flask and make up to volume with buffer solution.

[0090] ③1U / mL glucosidase: Dissolve a total of 100U of glucosidase in a buffer solution, add to a 10mL volumetric flask, and dilute to a concentration of 10U / mL with buffer solution. Then transfer 1mL of the 10U / mL glucosidase solution to a small sample bottle, add 9mL of phosphate buffer solution, and the concentration is now 1U / mL.

[0091] ④0.2mol / LNa2CO3 solution: Weigh 0.21199g of anhydrous sodium carbonate and dissolve it in deionized water. Add to a 10mL volumetric flask and make up to volume with deionized water.

[0092] (2) Sample measurement

[0093] Take 50 μL of 1U / mL α-glucosidase, 50 μL of chicory polysaccharide and acarbose solution with concentrations of 1, 2, 3, 4, and 5 mg / mL, and 280 μL of 0.1 mol / L phosphate buffer (pH 6.8), set the temperature of the water bath to 37 ° C, and water bath for 10 minutes. After the water bath, add 50 μL of 16 mmol / LPNPG, react in the water bath for 10 minutes, and finally add 200 μL of 0.2 mol / L Na2CO3 stop solution. After mixing with a vortex mixer, the absorbance is measured at 405 nm using an enzyme marker. As the sample experimental group. To avoid interference, the α-glucosidase solution is replaced by deionized water as a sample control. At the same time, in order to eliminate other factors that may affect the experimental results, the sample is replaced by deionized water as a blank control group. The other steps are the same as the sample experimental group to determine the difference between the sample blank group and the sample control group. The experiment was repeated three times to ensure the reliability of the results.

[0094] α-glucosidase inhibition rate (%) = [1-(A 样 -A 对 ) / A 空 ]×100%

[0095] A 样 : Absorbance of the sample solution, α-glucosidase and pNPG after reaction

[0096] A 对 : Absorbance value of α-glucosidase replaced by phosphate buffer

[0097] A 空 : Phosphate buffer replaces the absorbance of the sample solution

[0098] 1.2.8 Determination of α-amylase inhibitory activity

[0099] (1) Solution preparation

[0100] 0.5U / mL α-amylase: Dissolve 1mg of 35U / mg α-amylase in buffer solution, add to a 10mL volumetric flask, and dilute to 10U / mL with buffer solution. Then transfer 200μL of 10U / mL α-amylase solution to a small sample bottle, add 1200μL of phosphate buffer solution, and the concentration is 0.5U / mL.

[0101] (2) Sample measurement

[0102] Take 100 μL of 0.5U / mL α-amylase and 200 μL of the solution of the test product with a concentration of 1, 2, 3, 4, and 5 mg / mL, mix well, set the temperature of the water bath to 37°C, water bath for 10 minutes, add 200 μL of 2.0% soluble starch, use a vortex mixer to mix well, react in a water bath for 10 minutes, add 300 μL DNS reagent, react in water that has been preheated to boiling for 5 minutes, add 5 mL of distilled water for dilution, and use an enzyme marker to measure the absorbance at 540 nm after cooling, as the sample experimental group. To avoid interference, the α-amylase solution is replaced by phosphate buffer as a sample control. At the same time, in order to eliminate other factors that may affect the experimental results, the sample is replaced by deionized water as a blank control group. In order to determine the difference between the sample blank group and the sample control group, the experiment was repeated three times to ensure the reliability of the results.

[0103] α-amylase inhibition rate (%) = [1-(A 样 -A 对 ) / A 空 ]×100%

[0104] A 样 : Absorbance of the solution after the reaction of sample solution, 2.0% soluble starch and α-amylase

[0105] A 对 : Absorbance value of α-amylase instead of phosphate buffer

[0106] A 空 : Phosphate buffer replaces the absorbance of the sample solution

[0107] 2 Results and discussion

[0108] 2.1 Single factor test results

[0109] 2.1.1 Effect of extraction time on polysaccharide yield

[0110] Depend on Figure 1 It can be seen that: with the increase of the extraction time of chicory polysaccharide, the polysaccharide yield gradually increased. When the extraction time reached 2h, the yield of chicory polysaccharide reached 19.95%, and the polysaccharide yield began to decrease after 2h. The reason may be that chicory polysaccharide takes a certain amount of time to diffuse from the powder into the water. Therefore, within 2h of polysaccharide extraction, the polysaccharide concentration in the water is low. When the extraction time exceeds 2h, the chicory polysaccharide in the water reaches saturation, and the yield of chicory polysaccharide no longer increases with the increase of extraction time. In addition, the structure of the polysaccharide may change with the increase of time, resulting in a decrease in the polysaccharide yield. Considering the energy consumption and efficiency, the extraction time of 2h was selected for subsequent research.

[0111] 2.1.2 Effect of solid-liquid ratio on polysaccharide yield

[0112] Depend on Figure 2 It can be seen that as the solid-liquid ratio increases, the yield of hairy chicory polysaccharides shows a trend of first increasing and then decreasing. When the solid-liquid ratio reaches 1:40 (g / mL), the polysaccharide yield reaches a maximum value of 20.00%. The reason for this result may be that a large amount of water will increase the concentration difference between the intracellular fluid and the extracellular fluid of hairy chicory cells, accelerate the diffusion of the solution into the hairy chicory cells, thereby increasing the amount of polysaccharide dissolution and the yield of hairy chicory polysaccharides. However, if the water content is too high during the extraction process, it will lead to a wide variety of dissolved substances and confusion. These impurities may affect the effective release and full utilization of polysaccharides, and affect the yield of hairy chicory polysaccharides. Considering both energy consumption and efficiency, a solid-liquid ratio of 1:40 (g / mL) was selected for subsequent research.

[0113] 2.1.3 Effect of cellulase dosage on polysaccharide yield

[0114] like Figure 3 It can be seen that when the amount of cellulase increases, the yield of chicory polysaccharide shows a trend of first increasing and then decreasing. The yield of chicory polysaccharide reaches its peak after adding 2% cellulase, with a yield of 20.34%, and then gradually decreases; the reasons why the amount of cellulase affects the polysaccharide extraction rate may be the following: when the amount of cellulase is low, it mainly acts on the cell wall, destroys the cell wall, and releases the substances in the cell wall. When the cellulase is excessive, it can not only directly destroy the structure on the cell wall, but also penetrate into the cell wall to decompose the polysaccharides, proteins, lipids and other components, which reduces the yield of chicory polysaccharide. Considering the two aspects of energy consumption and efficiency, 2% cellulase was selected for subsequent research.

[0115] 2.1.4 Effect of extraction temperature on polysaccharide yield

[0116] like Figure 4 As shown in the figure, with the increase of extraction temperature, the extraction of chicory polysaccharide first increased and then decreased. When the extraction temperature of chicory polysaccharide was 50℃, the extraction rate reached the maximum, with a yield of 19.50%; when the extraction temperature was 50℃, the extraction rate reached a peak and then gradually decreased. This indicates that the optimum temperature of cellulase may be around 50℃. When the temperature is higher than 50℃, the activity of cellulase will decrease significantly. Cellulase may be less active in an environment above 50℃, thus failing to effectively decompose cellulose, affecting the yield of chicory polysaccharide. Considering energy consumption and efficiency, the extraction time of 50℃ was selected for subsequent research.

[0117] 2.2 Response surface optimization experimental results

[0118] 2.2.1 Model and ANOVA

[0119] Taking the yield of chicory polysaccharide as the response value, the Box-Behnken experimental design principle was applied, and the extraction time (A), cellulase content (B), solid-liquid ratio (C) and extraction temperature (D) were selected as influencing factors to obtain the experimental results shown in Table 2. The variance analysis of the data model for the yield of chicory polysaccharide extracted by enzyme-assisted extraction is shown in Table 3.

[0120] 2.2.2 Model establishment and analysis Design-Expert 13 software was used to analyze the experimental data in the table. The correlation fitting regression equation between polysaccharide yield (Y) and extraction time (A), cellulase amount (B), solid-liquid ratio (C), and extraction temperature (D) was: Y = 20.46 + 0.5054A + 0.4503B-0.1103C + 0.5933D-0.0454AB + 0.0524AC + 0.0271AD + 0.3824BC-0.3712BD + 0.1294CD-1.03A 2 -1.30B 2 -0.8994C 2 -0.9557D 2 .

[0121] Table 2 Response surface experiment scheme and experimental results

[0122]

[0123] Table 3 Analysis of variance

[0124] Note: ** indicates extremely significant difference (p < 0.01)

[0125] According to the response surface experiment results, the P value of the model is <0.0001, indicating that the regression equation is very significant and the experimental design is reliable. The P value of the lack of fit term is 0.0925 (P>0.05), indicating that the regression equation of the binary multiple terms has a good fit. The coefficient of variation is 0.9680%, indicating that the degree of dispersion between the variables is small and the data is relatively stable. The signal-to-noise ratio is one of the important indicators to measure the accuracy of the model. Usually the signal-to-noise ratio is greater than 4. The signal-to-noise ratio obtained in this experiment is 27.7988, indicating that the model has a good fit. The experimental fitting coefficient (R 2 ) is 0.9845, and the correction fitting coefficient (R 2Adj) is 0.9678, indicating that the linear relationship between the variables is closer. The P values ​​of the interaction terms C, AB, AC, AD and CD in the regression equation are greater than 0.05, indicating that the effect of each interaction term on the polysaccharide yield is not significant. The interaction of the four factors may be weak, or other influencing factors may offset these interaction effects, making their interaction insignificant. The P values ​​of A, B, D, BC, BD, A2, B2, C2, and D2 in Table 3 are less than 0.05. These interaction terms may significantly affect the yield of chicory polysaccharides. In addition, the F values ​​of the four factors A, B, C, and D are 93.96, 74.59, 4.48, and 129.49, respectively, indicating that the order of influence of each factor on the polysaccharide yield is extraction temperature (D) > extraction time (A) > enzyme amount (B) > solid-liquid ratio (C).

[0126] 2.2.3 Response surface plot analysis

[0127] Figure 5a-5l Design-Expert 13 software was used to draw the contour lines and response surfaces of each factor according to the model. The shape of the contour line is a powerful indicator that can intuitively reveal the strength of the interaction effect between two or more factors. By analyzing the contour lines, it can be found that when there is a strong interaction between two factors, the contour lines will appear elliptical. The stronger the interaction between them, the more contact points of the elliptical contour lines will increase. This phenomenon is called the "amplification effect" in physics. On the contrary, if the contour lines are circular, it means that the interaction between the two factors is relatively weak, or the degree of independence of the two factors is relatively high. In addition, the strength of the interaction between the factors can also be shown by the shape of the response surface. The more obvious the interaction between the two factors, the steeper the corresponding response surface. A steep response surface may imply a bidirectional, nonlinear interaction mode, while a gentle surface may reveal a unidirectional, linear influence relationship. As can be seen from Figure 5, the response surfaces of AB, AC, AD, and CD are relatively gentle, and the contour lines do not show obvious ellipses, indicating that the interaction between the factors is not significant. The response surfaces of BC and BC were steeper, and the contour lines showed obvious ellipse, indicating that the interaction between the factors was significant, which was consistent with the results of regression equation analysis.

[0128] 2.2.4 Optimal Condition Verification

[0129] The optimal extraction process parameters of hairy chicory polysaccharide predicted by Design-Expert 13 software are: extraction time 2.062h, cellulase amount 2.064%, solid-liquid ratio 1:39.933 (g / mL), extraction temperature 51.176℃. In order to ensure the accuracy of the experimental results, we adjusted and optimized various factors in the experimental process: extraction time 2.0h, cellulase amount 2.0%, solid-liquid ratio 1:40 (g / mL), extraction temperature 51℃. According to this extraction process, hairy chicory polysaccharide was extracted, and the yield of hairy chicory polysaccharide was measured to be (20.598±0.24)%, which is consistent with the predicted value of 20.633%, indicating that this extraction process is reliable and reasonable.

[0130] 2.3DPPH free radical scavenging rate

[0131] The scavenging rate of DPPH free radicals by chicory polysaccharides of different mass concentrations is shown in Figure 6 When the scavenging rate of DPPH free radicals reached the maximum value of 64.50% of the test concentration, when the mass concentration of hairy chicory polysaccharide was 5 mg / mL, compared with the scavenging ability of ascorbic acid at the same concentration of 5 mg / mL (the scavenging rate of 5 mg / mL ascorbic acid on DPPH free radicals was 96.40%), the antioxidant capacity of hairy chicory polysaccharide was weaker than that of ascorbic acid, but it still showed that it had a certain antioxidant capacity.

[0132] 2.4 ABTS free radical scavenging rate

[0133] The scavenging rate of ABTS free radicals by chicory polysaccharide of different mass concentrations is shown in Figure 7 When the scavenging rate of ABTS free radicals reached the maximum value of 96.58% of the test concentration, when the mass concentration of hairy chicory polysaccharide was 5 mg / mL, the antioxidant capacity was lower than that of ascorbic acid at the same concentration of 5 mg / mL (the scavenging rate of ascorbic acid at 5 mg / mL was 99.88%), but it still showed a certain antioxidant capacity.

[0134] 2.5α-glucosidase inhibition rate

[0135] Figure 8 It can be seen that the inhibition rate of acarbose reached the maximum at a concentration of 1 mg / mL, which was 99.4%±0.23%. As the concentration of acarbose increased, the inhibition rate of α-glucosidase tended to be flat. The inhibition rate of chicory polysaccharide reached the maximum at 5 mg / mL, which was 51.77%±0.29%. As the concentration increased, the inhibition rate of chicory polysaccharide on α-glucosidase gradually increased.

[0136] 2.6α-amylase inhibition rate

[0137] Fig. 9 It can be seen that the inhibition rate of acarbose on α-amylase reaches the maximum at 1 mg / mL of acarbose, with an inhibition rate of 99.36% ± 0.15%. As the concentration increases, the inhibition rate of acarbose on α-amylase tends to be flat. The inhibition rate of chicory polysaccharide on α-amylase reaches the maximum at 5 mg / mL of chicory polysaccharide, with an inhibition rate of 52.32% ± 0.28%. As the concentration increases, the inhibition rate of chicory polysaccharide on α-amylase gradually increases.

[0138] 3 Conclusion

[0139] The experimental results show that the extraction temperature, liquid-to-solid ratio, extraction time and cellulase amount all have an effect on the yield of chicory polysaccharide, among which temperature and time have the greatest influence. The optimal process conditions obtained by response surface design are extraction time 2.062h, cellulase amount 2.064%, liquid-to-solid ratio 1:39.933 (g / mL), and extraction temperature 51.176℃. Under these conditions, the yield of chicory polysaccharide is (20.598±0.24)%, which is consistent with the predicted value of 20.633%. This shows that the model of this experiment has a high degree of fitting, is accurate and effective, and can be used to optimize the extraction process of polysaccharide from chicory residues.

[0140] In the in vitro antioxidant activity experiment, the scavenging rate of 5mg / mL for DPPH free radicals was 64.50%, which was lower than the scavenging rate of 96.40% for DPPH free radicals by VC solution; the scavenging rate for ABTS free radicals reached the highest at 96.58% in 10 minutes, which was close to the scavenging rate of 99.88% for L-ascorbic acid in 10 minutes. The polysaccharide in chicory root may be its main active substance, but chicory root is rich in impurities such as protein, pigment, and small molecule compounds, which have a certain impact on the experiment. It can be developed into a natural antioxidant after further separation and purification. The polysaccharides extracted from the residues of chicory were tested for their hypoglycemic activity in vitro. The results showed that the inhibition rate of chicory polysaccharides on α-glucosidase reached 51.77%±0.29%, which was slightly lower than the inhibition rate of acarbose on α-glucosidase of 99.4%±0.23%; the inhibition rate of chicory polysaccharides on α-amylase reached 52.32%±0.28%, which was lower than the inhibition rate of acarbose on α-amylase of 99.36%±0.15%, but it also had a good inhibitory effect. It can become a hypoglycemic active drug and be prepared into a hypoglycemic agent with pharmaceutically acceptable excipients.

[0141] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A chicory polysaccharide having antioxidant and hypoglycemic effects, characterized in that: The extraction process includes the following steps: (1) Slice the chicory root, soak it repeatedly in 90-95% ethanol, and then air-dry the residue; (2) taking the chicory residue obtained in step (1), crushing it, and then passing it through a 20-60 mesh sieve; (3) Enzymatic extraction for 1.5 to 2.5 hours, extraction temperature 45 to 55°C, solid-liquid ratio 1: (30 to 50) g / mL, cellulase amount 1.5 to 2.5%, followed by cooling and separation to obtain a filtrate, which is then dried in an oven at 45 to 50°C.

2. The optimization method adopted in the extraction process according to claim 1, characterized in that: According to the Box-Behnken experimental design principle, the Design-Expert software was used to optimize the extraction time, solid-liquid ratio, extraction temperature and cellulase amount of polysaccharides from chicory residues. Constant temperature shaking was used to extract chicory polysaccharides.

3. The chicory according to claim 1, characterized in that: The hairy chicory originates from Hotan County, Xinjiang, China.

4. The chicory polysaccharide according to claim 1, characterized in that: Soak the chicory roots in 90-95% ethanol for more than 5 times, dry them and then extract them.

5. According to claim 4, the dried medicinal residues need to be sieved through a 24-mesh sieve.

6. The chicory polysaccharide according to claim 1, characterized in that: The extraction time is 1.5 to 2.5 hours.

7. The chicory polysaccharide according to claim 1, characterized in that: The extraction temperature is 45-55°C.

8. The chicory polysaccharide according to claim 1, characterized in that: The material-liquid ratio is 1: (30-50) g / mL, wherein the solvent is distilled water.

9. The chicory polysaccharide according to claim 1, characterized in that: The amount of the cellulase is 1.5-2.5%, wherein the enzyme used is cellulase.

10. The use of the chicory polysaccharide having antioxidant and hypoglycemic effects according to claim 1 in the preparation of antioxidant and hypoglycemic drugs, characterized in that: The obtained chicory polysaccharide and pharmaceutically acceptable auxiliary materials are prepared into granules.

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