Preparation method of Chinese mesona herb polysaccharide capable of regulating activity of glucose metabolism

Abstract

The invention belongs to the technical field of polysaccharide extraction, and particularly relates to mesona chinensis polysaccharide with glycometabolism regulating activity, which is prepared by the steps of micro-fermentation drying, raw material pretreatment, pulping, targeted extraction of polysaccharide, primary separation of polysaccharide, secondary separation of polysaccharide, polysaccharide purification, polysaccharide drying and the like. The mesona chinensis polysaccharide prepared by the method disclosed by the invention has good effects of inhibiting the activity of alpha-glucosidase, improving pancreas beta cell injury and the like, i.e., the mesona chinensis polysaccharide has relatively strong glucosyl metabolism regulating activity.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and in particular relates to a method for preparing agar-agar polysaccharide with the activity of regulating sugar metabolism. Background Art

[0002] Agar-agar grass, known as "fairy grass", is an annual herbaceous plant of the genus Agar-agar of the Lamiaceae family. According to the "Chinese Materia Medica", "National Compilation of Chinese Herbal Medicines", "Chinese Medicine Dictionary", etc., Agar-agar grass is cold, sweet, light and cool, has the effects of clearing away heat and relieving summer heat, relieving fever and diuresis, and is mainly used to treat diseases such as heatstroke, erysipelas, and diabetes. As a special food and medicine resource, Agar-agar grass is cultivated and promoted on a large scale in the southeastern regions of my country, such as Guangdong, Guangxi, and southern Fujian. It has become an important pillar industry for increasing farmers' income in the production areas and has played a positive role. Therefore, its development and utilization are of great significance. However, in processing and utilization, Agar-agar grass is mainly developed into traditional or low-end foods such as herbal tea and jelly, lacking innovative or high-end products, which seriously restricts the healthy development of the Agar-agar grass industry.

[0003] Polysaccharides are the most important and abundant active ingredients in agar-agar. Previous studies have found that agar-agar polysaccharides have a strong ability to improve sugar metabolism disorders, but are easily affected by steps such as drying fresh grass, polysaccharide extraction, and polysaccharide separation and purification. On the other hand, due to the complexity of the structure of agar-agar polysaccharides themselves, it is difficult to clarify the relationship between the structure of the sugar chain fragments in the "active center" and the regulation of sugar metabolism, which hinders the development of polysaccharide functional foods.

[0004] In view of this, it has become very necessary to provide a method for the targeted preparation of agar-agar polysaccharides with the goal of improving abnormal glucose metabolism, and use it as a raw material for functional food formulas that has the effect of regulating glucose metabolism, so as to solve the problems of low comprehensive utilization of agar-agar polysaccharides and short industrial chain, and provide consumers with more choices. Summary of the invention

[0005] The present invention aims to solve the above technical problems and provide a polysaccharide from Agarwood having the activity of regulating sugar metabolism. The polysaccharide from Agarwood has good effects of inhibiting α-glucosidase activity and improving pancreatic β cell damage, that is, it has a stronger sugar metabolism regulating activity.

[0006] The technical solution of the present invention is:

[0007] A polysaccharide from Agarwood having the activity of regulating sugar metabolism, the structural formula of which is:

[0008]

[0009] The present invention also provides a method for preparing polysaccharides from Agar-agar grass having the activity of regulating sugar metabolism, comprising the following steps:

[0010] (1) Micro-fermentation drying: fresh agar-agar grass is dried outdoors during the day, collected and piled in the evening, and covered with a film to keep it stuffy; the process is repeated until the agar-agar grass is dried to a moisture content of less than 5 wt %, thereby obtaining dried agar-agar grass;

[0011] (2) Raw material pretreatment: crushing the dried agar-agar grass, sieving, adding anhydrous ethanol to the agar-agar grass powder, stirring, extracting, centrifuging, filtering, collecting the precipitate, and obtaining decolorized agar-agar grass powder;

[0012] (3) pulping: adding deionized water to the decolorized agar-agar powder, crushing and grinding, and collecting agar-agar pulp;

[0013] (4) Targeted extraction of polysaccharides: Mix the agar-agar grass slurry with distilled water, continue to add the complex enzyme, mix well, and obtain a mixture; ultrasonicate the mixture, centrifuge it, collect and concentrate the supernatant, and obtain a concentrated solution;

[0014] (5) First separation of polysaccharides: adding anhydrous ethanol to the concentrate for alcohol precipitation, and filtering to obtain crude polysaccharides of Agar-agar grass;

[0015] (6) Second separation of polysaccharides: The crude polysaccharides of Agar-agar were initially separated by using an anion exchange chromatography column, and gradient elution was performed in sequence with ultrapure water and 0.1, 0.2 and 0.3 M NaCl solutions; the best elution peak was selected according to the elution curve, and the eluate was combined and collected according to the peak, concentrated by rotary evaporation, dialyzed with a 3000-3500 Da dialysis bag for 2-3 days, and freeze-dried for later use;

[0016] (7) Polysaccharide purification: The component with the highest yield obtained by separation and purification in step (6) is dissolved in ultrapure water, further eluted and purified by gel chromatography column combined with pure water, and all eluates of the symmetrical peak are collected; concentrated by rotary evaporator to obtain a gel column purified MCP-1b polysaccharide concentrate;

[0017] (8) Polysaccharide drying: The concentrated solution of the polysaccharide MCP-1b from the herb Auricularia auricula was freeze-dried in vacuum to obtain the refined herb Auricularia auricula polysaccharide.

[0018] In the method of the present invention, the micro-fermentation method (also known as the sweating method) in the primary processing link is a unique drying processing method, that is, the raw materials are heated after being stacked in a closed manner, so that the internal moisture overflows, which is not only conducive to the drying of the raw materials, but also can change certain chemical components of the raw materials during the "sweating" process, change the color of the raw materials, increase the aroma of the raw materials, reduce the irritation of the raw materials, and have an important impact on the formation of the quality of the raw materials. During the "sweating" process, the temperature inside the jelly grass gradually increases, a series of enzymatic reactions occur, and the chemical components of the jelly grass are transformed; as the "sweating" processing continues, the temperature inside the jelly grass continues to rise, the plant's heat stress resistance reaction is activated, and some secondary metabolites are generated, thereby increasing the content of the active ingredients of the jelly grass. After the jelly grass "sweats", it can increase the source of its active precursor compounds, and at the same time play a role in reducing toxicity and increasing efficacy, converting ineffective chemical components into effective chemical components, promoting the absorption of active ingredients, reducing the production cost of the jelly grass, and providing auxiliary means for the study of the metabolic mechanism of the jelly grass components.

[0019] Preferably, in step (2) of the present invention, the solid-liquid ratio of agar-agar grass powder to anhydrous ethanol is 1:10-12 g / mL, and the extraction is performed for 3-6 hours.

[0020] Preferably, in step (3) of the present invention, the material-liquid ratio of the decolorized agar-agar powder to deionized water is 1:30-40 g / mL, and the grinding pressure is 30-40 MPa.

[0021] Preferably, in step (4) of the present invention, the amount of the complex enzyme added is 0.1%-0.3% of the total volume of the mixture of the jelly grass slurry and distilled water.

[0022] In order to obtain the polysaccharide of the structure of the present invention, preferably, in the step (4) of the present invention, the complex enzyme is a mixture of cellulase, pectinase and papain in a mass ratio of 2:1:1.

[0023] Preferably, in step (4) of the present invention, the liquid ratio of the agar-agar slurry to distilled water is 10-20 g / mL.

[0024] Preferably, in step (4) of the present invention, the ultrasonic extraction conditions are: temperature of 50-60°C, time of 1-1.5h, and power of 150-225W.

[0025] Preferably, in step (4) of the present invention, the supernatant is concentrated to 1 / 10 of the original volume.

[0026] Preferably, in step (5) of the present invention, anhydrous ethanol is added to the concentrated solution at a volume ratio of 1:3-5 and the concentrated solution is allowed to stand at 4-6°C for 20-24 hours for alcohol precipitation.

[0027] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0028] (1) The method of the present invention uses a micro-fermentation method to dry fresh grass, which can improve the inhibitory effect of polysaccharides of agar-agar grass on α-glucosidase activity and improve the efficacy of improving pancreatic β-cell damage.

[0029] (2) The method of the present invention adopts wet pulverization to process the agar-agar powder. Through strong shearing and high-speed impact, the agar-agar powder is dispersed more evenly in water, and at the same time, the breakage of the agar-agar cell wall is accelerated, the permeability of the cell is increased, and the solvent can more easily enter the cell interior, which is beneficial to increase the yield of agar-agar polysaccharides.

[0030] (3) The method of the present invention uses a micro-fermentation method, wet crushing and a characteristic complex enzyme system to carry out targeted extraction of agar-agar grass pulp. The prepared polysaccharide has the activity of regulating sugar metabolism, and the polysaccharide yield and purity are both high.

[0031] (4) The preparation process of the present invention is simple, easy to operate, and reduces energy consumption.

[0032] (5) The present invention makes full use of resources, effectively improves the processing efficiency of agar-agar grass, and increases the economic value of the agar-agar grass planting industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a chromatographic column diagram for separation and purification of crude polysaccharides from Agar-agar Grass in Example 1 of the present invention;

[0034] Figure 2 This is an absolute molecular weight analysis chart of the polysaccharide MCP-1b of Auricularia aurantifolia in Example 1 of the present invention;

[0035] Figure 3 This is the ion chromatogram of the polysaccharide MCP-1b of Agar-agar grass in Example 1 of the present invention;

[0036] Figure 4 This is the total ion current diagram of the polysaccharide MCP-1b of Agar-agar grass in Example 1 of the present invention;

[0037] Figure 5 The polysaccharide MCP-1b of Example 1 of the present invention 1 H NMR one-dimensional hydrogen spectrum;

[0038] Figure 6 The polysaccharide MCP-1b of Example 1 of the present invention 13 C NMR one-dimensional carbon spectrum;

[0039] Figure 7 This is the COSY hydrogen-hydrogen correlation spectrum of the polysaccharide MCP-1b of Auricularia aurantifolia in Example 1 of the present invention;

[0040] Figure 8This is the NOESY hydrogen-hydrogen correlation spectrum of the polysaccharide MCP-1b of Auricularia aurantifolia in Example 1 of the present invention;

[0041] Fig. 9 This is the HSQC carbon-hydrogen correlation spectrum of the polysaccharide MCP-1b of Agar-agar grass in Example 1 of the present invention;

[0042] Fig.10 This is the HMBC carbon-hydrogen correlation spectrum of the Agarwood polysaccharide MCP-1b of Example 1 of the present invention;

[0043] Fig.11 This is an analysis diagram of the regulating sugar metabolism activity of the Agar-agar polysaccharide MCP-1b and its fermentation products in Example 1 of the present invention. DETAILED DESCRIPTION

[0044] 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.

[0045] In the following embodiments:

[0046] Cellulase comes from Nanning Pangbo Bioengineering Co., Ltd., with a specification of 20,000 u / g, food grade;

[0047] The specification of papain is 30,000 u / g, food grade;

[0048] Pectinase comes from Nanning Pangbo Bioengineering Co., Ltd., with a specification of 20,000 u / g and food grade.

[0049] Example 1

[0050] (1) Micro-fermentation drying: fresh agar-agar grass is spread out, dried outdoors in the sun for 1 day, and then collected and piled in the evening, covered with film to keep it stuffy; spread out during the day, and repeat the process. After two days, the agar-agar grass is completely dry, the color turns to brown or tan, and the moisture content is less than 5wt%, thereby obtaining dried agar-agar grass;

[0051] (2) Raw material pretreatment: the dried agar-agar grass was crushed and passed through a 60-mesh sieve. Anhydrous ethanol was added to the agar-agar grass powder at a solid-liquid ratio of 1:10 g / mL and stirred for extraction for 3 h. The fat-soluble pigment and some impurities were extracted by stirring. The mixture was centrifuged and filtered. The precipitate was collected to obtain decolorized agar-agar grass powder.

[0052] (3) Pulping: deionized water was added to the decolorized agar-agar powder at a solid-liquid ratio of 1:30 g / mL, and the powder was ground by wet grinding at a grinding pressure of 30 MPa to collect the agar-agar slurry;

[0053] (4) Targeted extraction of polysaccharides: Mix agar-agar slurry and distilled water at a solid-liquid ratio of 1:10 g / mL, continue to add 0.1% of the total volume of the mixture of agar-agar slurry and distilled water (the complex enzyme is a mixture of cellulase, pectinase, and papain in a mass ratio of 2:1:1), mix well, and obtain a mixture; extract the mixture by ultrasonication at 50°C and 150W for 1 hour, and then centrifuge at 6000 rpm / min for 10 minutes, collect and concentrate the supernatant to 1 / 10 of the original volume, and obtain a concentrated solution;

[0054] (5) First separation of polysaccharides: anhydrous ethanol was added to the concentrate at a volume ratio of 1:3 (v / v) and the concentrate was allowed to stand at 4°C for 20 h for alcohol precipitation, and the crude polysaccharides of Agar-agar were obtained by filtration;

[0055] (6) Second separation of polysaccharides: A DEAE seplife FF weak anion exchange chromatography column was used to perform preliminary separation of crude polysaccharides from the herb. Ultrapure water and 0.1, 0.2 and 0.3 M NaCl solutions were used for gradient elution in sequence at a flow rate of 4 mL / min. The best elution peak was selected according to the elution curve, and the eluates were combined and collected according to the peaks. After rotary evaporation and concentration, the eluate was dialyzed for 2 days using a 3000 Da dialysis bag and freeze-dried for later use. Figure 1 As shown, four polysaccharide components (MCP-1, MCP-2, MCP-3 and MCP-4) were obtained, among which MCP-1 had the highest yield of 47.3% and a purity of 70.2%.

[0056] (7) Polysaccharide purification: The MCP-1 obtained by separation and purification in (6) was dissolved in ultrapure water, and further eluted and purified using a Sephacryl S-400HR gel chromatography column combined with pure water. All eluates of the symmetrical peak were collected and concentrated using a rotary evaporator to obtain a gel column purified agar-agar polysaccharide MCP-1b concentrate;

[0057] (8) Polysaccharide drying: The concentrated solution of polysaccharide MCP-1b of Auricularia aurantifolia was placed in a vacuum freeze dryer for freeze drying to obtain the refined polysaccharide MCP-1b component with a polysaccharide purity of 84.1%.

[0058] Example 2

[0059] (1) Micro-fermentation drying: fresh agar-agar grass is spread out, dried outdoors in the sun for 1 day, and then collected and piled in the evening, covered with film to keep it stuffy; spread out during the day, and repeat the process. After two days, the agar-agar grass is completely dry, the color turns to brown or tan, and the moisture content is less than 5wt%, thereby obtaining dried agar-agar grass;

[0060] (2) Raw material pretreatment: the dried agar-agar grass was crushed and passed through a 60-mesh sieve, and anhydrous ethanol was added to the agar-agar grass powder at a solid-liquid ratio of 1:12 g / mL, and stirred and extracted for 6 h, and the fat-soluble pigment and some impurities were extracted by stirring, and the mixture was centrifuged and filtered, and the precipitate was collected to obtain decolorized agar-agar grass powder;

[0061] (3) Pulping: deionized water was added to the decolorized agar-agar powder at a solid-liquid ratio of 1:40 g / mL, and the powder was ground by wet grinding at a grinding pressure of 40 MPa to collect the agar-agar slurry;

[0062] (4) Targeted extraction of polysaccharides: Mix the agar-agar slurry with distilled water at a solid-liquid ratio of 1:20 g / mL, continue to add 0.3% of the total volume of the agar-agar slurry and distilled water (the complex enzyme is a mixture of cellulase, pectinase, and papain in a mass ratio of 2:1:1), mix well, and obtain a mixture; extract the mixture by ultrasonication at 50°C and 225W for 1.5h, and then centrifuge at 6000rpm / min for 15min, collect and concentrate the supernatant to 1 / 10 of the original volume to obtain a concentrate;

[0063] (5) First separation of polysaccharides: anhydrous ethanol was added to the concentrate at a volume ratio of 1:5 (v / v) and the concentrate was allowed to stand at 6°C for 24 h for alcohol precipitation, and the crude polysaccharides of Agar-agar were obtained by filtration;

[0064] (6) Second separation of polysaccharides: A DEAE seplife FF weak anion exchange chromatography column was used to perform preliminary separation of crude polysaccharides from the herb Agar-agar. Ultrapure water and 0.1, 0.2, and 0.3 M NaCl solutions were used for gradient elution in sequence at a flow rate of 4 mL / min. The best elution peak was selected according to the elution curve, and the eluates were combined and collected according to the peaks. After rotary evaporation and concentration, the eluate was dialyzed for 3 days using a 3500 Da dialysis bag and freeze-dried for later use.

[0065] (7) Polysaccharide purification: The component with the highest yield obtained by separation and purification in (6) was dissolved in ultrapure water, and further eluted and purified using a Sephacryl S-400HR gel chromatography column combined with pure water, and all the eluates of the symmetrical peak were collected and concentrated using a rotary evaporator to obtain a gel column purified polysaccharide concentrate of Herba Aurantii;

[0066] (8) Polysaccharide drying: The concentrated polysaccharide solution of Agar-agar was placed in a vacuum freeze dryer and freeze-dried to obtain a refined Agar-agar polysaccharide component with a polysaccharide purity of 81.6%.

[0067] The ion purification elution curve of the sample is plotted with the number of eluent tubes as the horizontal axis and the total sugar content (blue) and the NaCl concentration in the eluent (green) as the vertical axis, as shown in Figure 2. Figure 1(Left) As shown. In Example 1, the ion purification obtained two main elution peak components, ion elution peak 1 (tubes 7-16), recorded as MCP-1 (yield 47.3%, purity 70.4%); ion elution peak 2 (tubes 28-37), recorded as MCP-2 (yield 36.9%, purity 37.1%).

[0068] The gel purification elution curve of the sample is plotted with the number of elution tubes as the horizontal axis and the total sugar content (blue) as the vertical axis, as shown in Figure 1 (right) As shown in the figure, in Example 1, a main elution peak component was obtained by gel purification, namely, gel elution peak 1 (tubes 37-51), which was recorded as MCP-1b, and the polysaccharide purity was 84.1%.

[0069] The identification of the polysaccharide MCP-1b of the herb Auricularia aurantifolia in Example 1 is as follows:

[0070] (1) Molecular weight analysis of oxalic acid polysaccharide in jelly

[0071] The molecular weight (Mw) of the polysaccharide MCP-1b of Example 1 was studied by gel chromatography-differential refractive index-multi-angle laser light scattering system. The results showed that the component was a relatively pure and uniform component (see Figure 2 ), and the Mw was measured to be 77.69 kDa.

[0072] (2) Monosaccharide composition of oxalic acid polysaccharide in jelly

[0073] The monosaccharide composition of the polysaccharide MCP-1b of the herb Auricularia aurantifolia in Example 1 was analyzed by ion chromatography. Figure 3 As shown. According to the absolute quantitative method, the masses of different monosaccharides were determined, and the molar ratio was calculated based on the molar mass of the monosaccharides. The MCP-1b of the agar grass is a heteropolysaccharide, mainly composed of galacturonic acid, galactose, glucose, arabinose, mannose (molar mass ratios of 35.48%, 20.72%, 12.71%, 11.50%, 10.40%), etc., and also contains a small amount of glucuronic acid, rhamnose, and xylose (molar mass ratios of 3.27%, 3.10%, 2.82%).

[0074] (3) Analysis of methylation of oxalic acid polysaccharides in jelly

[0075] The polysaccharide MCP-1b of Example 1 was derivatized by methylation and the like, and then analyzed by gas chromatography-mass spectrometry. The results are as follows: Figure 4 And as shown in Table 1.

[0076] Table 1 Analysis of bonding structure of polysaccharide MCP-1b

[0077]

[0078] As can be seen from Table 1, the connection modes of MCP-1b components include t-Araf, t-Xylp, 2-Rhap, t-Glcp, 3-Araf, t-Galp, 5-Araf, 3-Glcp, 3-Manp, 4-Manp, 4-Galp, 4-Galp-UA, 4-Glcp, 6-Galp, 3,4-Glcp and 3,6-Manp.

[0079] (4) Fine chemical structure of oxalic acid polysaccharide in jelly

[0080] The MCP-1b sample of the polysaccharide of the herb Auricularia auricula in Example 1 was analyzed by multidimensional nuclear magnetic resonance scanning. Specifically: (1) an appropriate amount of the polysaccharide of the herb Auricularia auricula MCP-1b sample was fully dissolved in D2O to prepare a polysaccharide solution with a concentration of greater than or equal to 40 mg / mL; the dissolved solution was transferred to a nuclear magnetic resonance tube, and 0.5 mL was added; the nuclear magnetic resonance tube was placed in a nuclear magnetic resonance spectrometer and scanned in one dimension. 1 H spectrum, 13 C spectrum, two-dimensional COSY, HSQC, HMBC, NOESY spectra. The target was quantitatively analyzed using a Bruker (Germany) 500MHz NMR spectrometer with a scanning temperature of 25°C. Liquid probe QXI 1H / 31P / 13C / 15N 5mm quadruple resonance reverse detection probe (Z-gradient, ATMAcc), technical parameters: signal-to-noise ratio (1H): 888; resolution (Hz): 0.32 (rotating) BBFO1H-19F, 31P-15N, 1H decoupling / observe multi-nuclear forward detection probe (Z-gradient, ATM) technical parameters: signal-to-noise ratio (1H): 798; resolution (Hz): 0.26 (rotating); signal-to-noise ratio (13C): 328; resolution (Hz): 0.1.

[0081] Specifically, according to Figure 5-Figure 10 , the assignment of sugar residue displacement can be completed, and the results are as follows:

[0082] Table 2 Sugar residues 1 H and 13 Chemical shift of C

[0083]

[0084] Note: nd is the abbreviation of “not detected”, which means not recognized.

[0085] Therefore, based on the one-dimensional and two-dimensional NMR information and the methylation analysis, and further combined with the molecular weight, monosaccharide composition and methylation analysis data, the fine chemical structure of the polysaccharide MCP-1b of the present embodiment 1 was finally determined as follows:

[0086]

[0087] Experimental Example 1: Effects of different drying methods on the activity of crude polysaccharides from Agar-agar grass

[0088] Fresh agar-agar grass was used as the object to compare the effects of five drying methods, including natural drying, hot air drying, frozen vacuum drying, microwave vacuum drying and micro-fermentation, on the in vitro antioxidant and pancreatic β-cell damage-improving effects of crude polysaccharides from agar-agar grass. A comprehensive quantitative evaluation method was used to determine that the crude polysaccharide MCP prepared from micro-fermented dried agar-agar grass had the highest activity score, and its effect on improving pancreatic β-cell damage ranked first. The results are shown in Table 3. Specifically:

[0089] (1) Natural drying is different from Example 1 in that step (1) is to spread out the fresh agar grass, dry it outdoors in the sun for two days, and then dry it indoors for two days until the agar grass is completely dry and its color changes from green to brown or tan; the rest is the same as Example 1;

[0090] (2) Hot air drying is different from Example 1 in that step (1) is to spread the fresh agar-agar grass on a plate and place it in an oven at 60° C. for drying. After six hours, the agar-agar grass is completely dried and taken out. The rest is the same as Example 1.

[0091] (3) Freeze vacuum drying is as follows: The difference from Example 1 is that step (1) is to spread fresh agar grass flat on a freeze drying plate, pre-freeze for one day and then put it into a freeze dryer. After two days, the agar grass is completely dried and taken out; the rest is the same as Example 1;

[0092] (4) Microwave vacuum drying is as follows: The difference from Example 1 is that step (1) is to spread the fresh agar-agar grass on a plate and place it in a microwave vacuum dryer. After 30 minutes, the agar-agar grass is completely dried and then taken out; the rest is the same as Example 1;

[0093] (5) Micro-fermentation drying is the same as in Example 1.

[0094] Table 3 Comprehensive quantitative evaluation of five drying methods

[0095]

[0096]

[0097] Note: Y1 is T-AOC (Y1), total antioxidant capacity; Y2 is DPPH free radical scavenging capacity; Y3 is hydroxyl free radical scavenging capacity·OH; Y4 is superoxide anion free radical scavenging capacity O2 ·- ; Y5 is inhibiting apoptosis and improving the ability of pancreatic β-cell damage; ΔE is the comprehensive score of the sample.

[0098] As can be seen from Table 3: the crude polysaccharide MCP prepared with micro-fermented dried agar-agar grass as raw material has the highest activity score, and its effect on improving pancreatic β-cell damage ranks first, indicating that the micro-fermentation method combined with wet method and targeted enzymatic extraction can effectively improve the activity of agar-agar grass polysaccharides in regulating sugar metabolism.

[0099] Experimental Example 2: Inhibition of α-glucosidase by different sugar components in agar-agar grass

[0100] The α-glucosidase inhibition test was used to compare the glucose metabolism activities of crude polysaccharides of Agarwood, MCP-1, MCP-1b and MCP-2, as shown in Table 4. The α-glucosidase inhibition ability IC 50 The results are shown in Table 5. The specific experimental steps are as follows:

[0101] Take a 2 mL centrifuge tube and add the reagents in the order listed in the table below. Measure the absorbance at a wavelength of 405 nm and calculate the inhibition rate according to formula (1).

[0102]

[0103] Where, W is the α-glucosidase inhibition rate, A c is the blank absorbance value, A S is the sample or positive absorbance value, A b is the blank absorbance of the sample.

[0104] Table 4. Table of reagent addition for determining the ability to inhibit α-glucosidase activity

[0105]

[0106]

[0107] Table 5 Comparison of α-glucosidase inhibition ability of different sugar components in agar-agar grass

[0108] Group <![CDATA[α-glucosidase inhibitory capacity IC 50 (μg / mL)]]> Polysaccharide from Herba Agarwood <![CDATA[30.13±1.76 c ]]> MCP-1 <![CDATA[34.60±2.38 b ]]> MCP-1b <![CDATA[36.91±2.91 b ]]> MCP-2 <![CDATA[66.57±3.64 a ]]> Acarbose (positive control) <![CDATA[10.47±0.88 d ]]>

[0109] Note: Different letters indicate significant differences (P<0.05).

[0110] As can be seen from Table 5, the glucose metabolism regulating activities of MCP-1 and MCP-1b are significantly better than those of MCP-2, and the glucose metabolism regulating activities of MCP-1 and MCP-1b are close to those of acarbose, indicating that the MCP-1 and MCP-1b prepared by the present invention have higher glucose metabolism regulating activities.

[0111] Experimental Example 3: Analysis of the activity of MCP-1b and its fermentation product D-MCP-1b in regulating glucose metabolism

[0112] Pancreatic β cells (RIN-5mF cells) were cultured at 1.5 x 10 5 / ml density was inoculated into a 96-well plate, 100ul per well (blank group without cells), and after adherence, proceed according to Table 6.

[0113] Table 6 Dosing treatment

[0114]

[0115]

[0116] CCK8 detection was performed after 24 h of incubation.

[0117] The results are as follows Fig.11 As shown, through the high sugar combined with STZ-induced pancreatic β-cell injury model, it was compared and determined that MCP-1b had a good activity in improving glucose metabolism before and after fermentation by intestinal flora, and the glucose control advantage of D-MCP-1b obtained by in vitro simulation of human colon fermentation was more significant.

[0118] 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 polysaccharide from Herba Aurantii having the activity of regulating sugar metabolism, characterized in that: Its structural formula is:

2. The method for preparing the polysaccharide from Artemisia argyi having the activity of regulating sugar metabolism as claimed in claim 1, characterized in that: The following steps are involved: (1) Micro-fermentation drying: fresh agar-agar grass is dried outdoors during the day, collected and piled in the evening, and covered with a film to keep it stuffy; the process is repeated until the agar-agar grass is dried to a moisture content of less than 5 wt %, thereby obtaining dried agar-agar grass; (2) Raw material pretreatment: crushing the dried agar-agar grass, sieving, adding anhydrous ethanol to the agar-agar grass powder, stirring, extracting, centrifuging, filtering, collecting the precipitate, and obtaining decolorized agar-agar grass powder; (3) pulping: adding deionized water to the decolorized agar-agar powder, crushing and grinding, and collecting agar-agar pulp; (4) Targeted extraction of polysaccharides: Mix the agar-agar grass slurry with distilled water, continue to add the complex enzyme, mix well, and obtain a mixture; ultrasonicate the mixture, centrifuge it, collect and concentrate the supernatant, and obtain a concentrated solution; (5) First separation of polysaccharides: adding anhydrous ethanol to the concentrate for alcohol precipitation, and filtering to obtain crude polysaccharides of Agar-agar grass; (6) Second separation of polysaccharides: The crude polysaccharides of Agar-agar were initially separated by using an anion exchange chromatography column, and gradient elution was performed in sequence with ultrapure water and 0.1, 0.2 and 0.3 M NaCl solutions; the best elution peak was selected according to the elution curve, and the eluate was combined and collected according to the peak, concentrated by rotary evaporation, dialyzed with a 3000-3500 Da dialysis bag for 2-3 days, and freeze-dried for later use; (7) Polysaccharide purification: The component with the highest yield obtained by separation and purification in step (6) is dissolved in ultrapure water, further eluted and purified by gel chromatography column combined with pure water, and all eluates of the symmetrical peak are collected; concentrated by rotary evaporator to obtain a gel column purified MCP-1b polysaccharide concentrate; (8) Polysaccharide drying: The concentrated solution of the polysaccharide MCP-1b from the herb Auricularia auricula was freeze-dried in vacuum to obtain the refined herb Auricularia auricula polysaccharide.

3. The polysaccharide from Herba Aurantii having the activity of regulating sugar metabolism as claimed in claim 2, characterized in that: In the step (2), the solid-liquid ratio of agar-agar powder to anhydrous ethanol is 1:10-12 g / mL, and the extraction is performed for 3-6 hours.

4. The method for preparing the polysaccharide from Agarwood having the activity of regulating sugar metabolism as claimed in claim 2, characterized in that: In the step (3), the material-liquid ratio of the decolorized agar-agar powder to deionized water is 1:30-40 g / mL, and the grinding pressure is 30-40 MPa.

5. The method for preparing the polysaccharide from Agarwood having the activity of regulating sugar metabolism as claimed in claim 2, characterized in that: In the step (4), the amount of the complex enzyme added is 0.1%-0.3% of the total volume of the mixture of the jelly grass slurry and distilled water.

6. The method for preparing the polysaccharide from Artemisia argyi having the activity of regulating sugar metabolism as claimed in claim 2, characterized in that: In the step (4), the complex enzyme is a mixture of cellulase, pectinase and papain in a mass ratio of 2:1:

1.

7. The method for preparing the polysaccharide from Artemisia argyi having the activity of regulating sugar metabolism as claimed in claim 2, characterized in that: In the step (4), the liquid ratio of the jelly grass slurry to distilled water is 1:10-20 g / mL.

8. The method for preparing the polysaccharide from Agarwood having the activity of regulating sugar metabolism as claimed in claim 2, characterized in that: In the step (4), the ultrasonic extraction conditions are: temperature of 50-60°C, time of 1-1.5h, and power of 150-225W.

9. The method for preparing the polysaccharide from Agarwood having the activity of regulating sugar metabolism as claimed in claim 2, characterized in that: In the step (4), the supernatant is concentrated to 1 / 10 of the original volume.

10. The method for preparing the polysaccharide from Artemisia argyi having the activity of regulating sugar metabolism as claimed in claim 2, characterized in that: In the step (5), anhydrous ethanol is added to the concentrated solution at a volume ratio of 1:3-5 and the concentrated solution is allowed to stand at 4-6°C for 20-24 hours for alcohol precipitation.

Images