CN115124629B - Preparation and application of seaweed polysaccharide calcium - Google Patents

Abstract

The invention belongs to the field of seaweed polysaccharide research, and discloses a wakame polysaccharide UPPS-1 capable of inhibiting α-glucosidase activity, a preparation method and application thereof. UPPS‑1 is a homogeneous polysaccharide obtained by leaching wakame with calcium chloride solution, separated and purified by ion exchange column and gel column, with a weight average molecular weight of 174.88kDa. According to the research of α-glucosidase activity, UPPS-1 inhibits α-glucosidase on the intestinal mucosa, slows down the decomposition of starch into glucose, thereby reducing and delaying the absorption of glucose in the small intestine, and improving abnormal blood sugar in diabetic patients. The polysaccharide of the invention can be used as a potential diabetes inhibitor for the development of functional food or medicine.

Description

Preparation and application of a kind of seaweed polysaccharide calcium

technical field

The invention relates to a preparation method and application of seaweed extract.

Background technique

Brown algae contain polysaccharides, proteins, vitamins, minerals and other nutrients. Polysaccharides are a class of polymer compounds with various biological activities such as anti-oxidation, anti-tumor, immune regulation, and anti-virus, so they have application value in drug development.

Wakame is the stalk part of Undaria pinnatifida, a large-scale economical brown algae, mainly distributed in Liaoning, Shandong, Jiangsu, Zhejiang and other places in my country, belonging to Phaeophyta and Phaeophyta. It has high water content, hard tissue and thick brown color. It is widely used in food cooking because of its good taste. Wakame has good effects in anti-virus, anti-tumor, lowering blood pressure, immune regulation, and treatment of cardiovascular and cerebrovascular diseases. At present, there is no in-depth research on the extraction, separation and biological activity of wakame polysaccharides.

Contents of the invention

The primary purpose of the present invention is to provide a wakame polysaccharide that can regulate diabetes.

Another object of the present invention is to provide the above-mentioned preparation method of wakame polysaccharides capable of regulating diabetes; the present invention takes the wakame entity as the research object, separates and purifies it through ion exchange columns and molecular sieves through calcium chloride leaching and alcohol precipitation, and studies Its biological activity, molecular weight and monosaccharide composition of wakame polysaccharide were analyzed.

Another object of the present invention is to provide the application of the above-mentioned crony crema polysaccharide that can regulate diabetes.

The object of the present invention is achieved through the following technical solutions:

A wakame polysaccharide UPPS-1 capable of regulating diabetes, with a weight average molecular weight of 174.880kDa

The sugar content of the wakame point polysaccharide is 37.95%.

The point wakame polysaccharide UPPS-1 is mainly composed of mannuronic acid, glucosamine, rhamnose, glucuronic acid, galacturonic acid with a molar ratio of 4.35:1.86:2.32:4.50:0.94:2.07:48.42:16.01 , glucose, galactose and fucose.

The preparation method of the above-mentioned point wakame polysaccharide comprises the following steps:

(1) Desalination of wakame, dried in the sun and beaten into powder.

(2) adding CaCl2 solution for extraction, collecting the supernatant, and concentrating under reduced pressure.

(3) The concentrated solution is subjected to ethanol precipitation, decolorization, protein removal, dialysis, and freeze-drying to obtain crude polysaccharides.

(4) The crude polysaccharide was prepared into a solution and eluted by an ion-exchange column, and the target peak product was collected for tracking and monitoring by the phenol-sulfuric acid method, and the crude fraction was obtained after concentration, dialysis, and freeze-drying.

(5) The crude components were further separated by a gel column, and the target peak product was collected for tracking and monitoring by the phenol-sulfuric acid method, and the subdivided components were obtained after concentration, dialysis, and freeze-drying.

The step (1) specifically follows the following steps: wash the salt on the surface of the wakame with tap water, change the water every 12 hours, soak it for three days, drain it, dry it naturally, and then pulverize it to obtain the wakame powder.

The step (2) specifically follows the following steps: the wakame powder is extracted at 0.15mol/LCaCl2, the ratio of solid to liquid is 1:15, and extracted at 70° C. for 3 hours. The filtrate was collected and concentrated under reduced pressure to obtain a wakame concentrate.

The step (3) specifically follows the following steps: use 80% ethanol to precipitate the wakame concentrate overnight, collect the precipitate by centrifugation, redissolve it with a small amount of deionized water, decolorize 717 anion resin at 55°C for 12 hours, and remove protein by Sevage method (Chloroform:n-butanol=4:1) more than three times until there is no white floc at the boundary between the two phases. Dialyzed at 3500Da for 48h and freeze-dried to obtain crude polysaccharide.

The step (4) specifically follows the following steps: dissolving the crude polysaccharide of wakame peduncle with deionized water, configuring a polysaccharide solution with a concentration of 30 mg/mL and separating it through a DEAE-52 ion exchange column, using 0, 0.5, 1, 2 mol /LNacl solution for elution, the flow rate was controlled at 1mL/min, the elution time was 10min/tube, and the phenol-sulfuric acid method was used for tracking and monitoring, and the main peak was collected according to the absorbance. The 0.5mol/L Nacl solution eluted fractions were collected, concentrated, dialyzed for 48 hours with a 3500Da dialysis bag, and freeze-dried to obtain the crudely divided wakame polysaccharide UPPS-1.

The step (5) specifically follows the following steps: dissolving the crude polysaccharides of wakame stalks with pure water, and configuring a polysaccharide solution with a concentration of 30 mg/mL to be separated by a Sephadex-G100 gel column, and eluted with pure water, and the flow rate is controlled at 0.5mL/min, elution time 10min/tube, track and monitor by phenol sulfuric acid method, collect the main peak according to the absorbance. The fractions eluted with pure water were collected, concentrated under reduced pressure, dialyzed with a 3500 Da dialysis bag for 48 hours, and freeze-dried to obtain subdivided wakame polysaccharide UPPS-1.

Application of the above-mentioned wakame polysaccharide UPPS-1 in the preparation and regulation of diabetes.

The α-glucosidase inhibitor slows down the decomposition of starch into glucose by inhibiting α-glucosidase on the intestinal mucosa, thereby reducing and delaying the absorption of glucose by the small intestine and improving abnormal blood sugar in diabetic patients.

In the above method, compared with the traditional water solvent extraction method, the extraction of the wakame polysaccharide by calcium chloride in the present invention reduces the generation of alginate and improves the solubility.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1. The present invention takes wakame as the research object, extracts with calcium chloride solution, separates and purifies through DEAE-52 ion exchange column and molecular sieve to prepare wakame polysaccharide UPPS-1, and determines its specific active use.

2. Compared with the traditional water solvent extraction method, the extraction method of wakame polysaccharide of the present invention has the advantage of using calcium chloride solution, which removes part of the alginate and improves the solubility of the polysaccharide.

3. It has been determined that the weight-average molecular weight of wakame polysaccharide UPPS-1 is 174.880kDa, mainly composed of mannuronic acid, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and fucose composition.

4. Undaria UPPS and UPPS-1 can inhibit α-glucosidase and slow down the decomposition of starch into glucose in the concentration range of 100-1000 μg/mL, thereby inhibiting diabetes.

Description of drawings

Fig. 1 is the elution figure of wakame polysaccharide through DEAE-52 ion exchange column chromatography;

Fig. 2 is the elution figure of wakame polysaccharide through Sephadex-G100 gel column chromatography;

Fig. 3 is the ultraviolet spectrogram of wakame polysaccharide UPPS-1;

Fig. 4 is the infrared spectrogram of wakame polysaccharide UPPS-1;

Fig. 5 is a standard sugar peak time table;

Figure 6 is a standard sugar HPLC figure;

Fig. 7 is a monosaccharide composition diagram of wakame polysaccharide UPPS-1;

Fig. 8 is a graph showing the influence of wakame polysaccharide UPPS and UPPS-1 on inhibiting α-glucosidase;

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, equipment and methods used in the present invention are conventional commercially available reagents, equipment and conventional methods of use in the technical field.

Example 1

Wash the salt on the surface of the crucian stalk with tap water, change the water every 12 hours, soak it for three days, drain it, dry it naturally and then powder it to obtain the crony stalk powder;

Extract wakame powder at 0.15mol/LCaCl2, solid-liquid ratio 1:15, and extract at 70°C for 3h. Collect the filtrate and concentrate under reduced pressure to obtain the wakame concentrate;

Precipitate the wakame concentrate with 80% ethanol overnight, collect the precipitate by centrifugation, redissolve it with a small amount of deionized water, decolorize the chlorinated 717 anion resin at 55°C for 12 hours, and remove the protein by Sevage method (chloroform:n-butanol=4:1) More than three times, until there is no white floc at the boundary between the two phases. Dialysis at 3500Da for 48 hours and freeze-drying to obtain crude polysaccharide;

Dissolve wakame crude polysaccharides in deionized water, configure a polysaccharide solution with a concentration of 30 mg/mL, separate it through a DEAE-52 ion exchange column, and use 0, 0.5, 1, 2 mol/L Nacl solution to elute in sequence, and the flow rate is controlled at 1 mL/L min, the elution time is 10 min/tube, and the phenol-sulfuric acid method is used for tracking and monitoring, and the main peak is collected according to the absorbance. Collect 0.5mol/L Nacl solution eluted fractions, concentrate, dialyze with 3500Da dialysis bag for 48h, and freeze-dry to obtain the crudely divided wakame polysaccharide UPPS-1;

Dissolve the crude polysaccharides of wakame peduncle in pure water, prepare a polysaccharide solution with a concentration of 30mg/mL, separate it on a Sephadex-G100 gel column, and use pure water for elution, the flow rate is controlled at 0.5mL/min, and the elution time is 10min/tube , using the phenol-sulfuric acid method to track and monitor, and collect the main peak according to the absorbance. Collect the eluted fractions in pure water, concentrate under reduced pressure, dialyze with a 3500Da dialysis bag for 48 hours, and freeze-dry to obtain subdivided wakame polysaccharide UPPS-1;

Determination of Sugar Content of Undaria pinnatifera Polysaccharide UPPS-1

Weigh 10mg of glucose, dilute to 100mL, pipette 0.1, 0.2, 0.4, 0.6, 0.8, 1mL of glucose solution, add distilled water to dilute to 1mL. Dissolve phenol in a 60°C water bath, take 5mL and dilute to 100mL. Add 1mL of 5% phenol solution and shake well. Add 5mL of concentrated sulfuric acid, shake well, react at room temperature, and let stand for 30min. Absorbance was measured at 490 nm. Draw the standard curve with the glucose concentration as the abscissa and the absorbance as the ordinate. The wakame polysaccharide UPPS-1 was configured into a solution with a concentration of 1mg/mL, and 1mL was taken for detection by the phenol-sulfuric acid method. According to the standard curve y=6.6798x-0.034 (R2=0.9997), the sugar content of UPPS-1 was calculated to be 37.95%.

Ultraviolet spectrum analysis of crony UPPS-1

Take a certain amount of the above-mentioned wakame polysaccharide and dissolve it in distilled water at a concentration of 1mg/mL, and scan it on a spectrophotometer with a wavelength range of 200-800nm. The results are shown in Figure 3, and there are no characteristic absorption peaks at 260nm and 280nm , indicating that wakame polysaccharide does not contain nucleic acid and protein.

Infrared spectrum analysis of crony UPPS-1

Mix 2 mg of wakame polysaccharide obtained above with 50 mg of dried potassium bromide powder, grind it thoroughly and press it into tablets, and scan the infrared spectrum in the range of 4000-400 cm-1, the results are shown in Figure 4, at 3444.20 cm-1 The characteristic absorption peak of sugar appears, which is produced by O-H stretching vibration. There is an absorption peak at 2930.90cm-1, which is produced by C-H stretching vibration. The absorption peak at 1420.07cm-1 is produced by the stretching vibration of C-O. 1643.99cm-1 is the absorption peak produced by the asymmetric stretching vibration of C=O. It is deduced that UPPS has a carboxyl group and contains uronic acid. There is asymmetric stretching vibration of S=O at 1243.76cm-1, and asymmetric stretching vibration of C-O-S at 821.28cm-1. UPPS may contain sulfuric acid groups. The stretching vibration of C-C is at 1055.07cm-1.

Monosaccharide composition analysis of crony UPPS-1

Take 10 mg of wakame UPPS-1 obtained from the appeal, hydrolyze with 1mol/L trifluoroacetic acid at 110°C for 6 hours, and then derivatize with 0.5mol/LPMP methanol solution, and analyze the derivatives on Agilent 1260 high performance liquid chromatography.

Chromatographic conditions: BDS HYPERSIL C18 column (5μm×4.6mm×250mm), detection wavelength 250nm, column temperature 30°C, flow rate 0.8mL/min, UV detector, mobile phase is phosphate buffer: acetonitrile (87:17) .

The standard products are: glucose, galactose, fucose, glucuronic acid, glucosamine, galactosamine, mannose, rhamnose, xylose, galacturonic acid, and arabinose. The results are shown in Figures 5 and 6.

From the results of chromatographic detection, it can be seen that the crony polysaccharide is mainly composed of mannuronic acid, glucosamine, rhamnose, glucuronic acid, and galacturonic acid with a molar ratio of 4.35:1.86:2.32:4.50:0.94:2.07:48.42:16.01. , glucose, galactose and fucose.

Determination of α-glucosidase activity of crony UPPS and UPPS-1

0.2U/mL α-glucosidase 0.3mL and 0.4mL polysaccharide samples with different concentrations were incubated in a constant temperature water bath at 37°C for 10 minutes, and 0.3mL of 5mmol/LPNPG solution was added to continue to incubate in a constant temperature water bath at 37°C for 20min. At a wavelength of 405nm, the absorbance value was measured as Ai, 0.2mol/L (ph=6.8) phosphate buffer saline instead of the sample to measure the absorbance is A0, and the phosphate buffer saline instead of the enzyme is Aj. The formula for α-glucosidase inhibition rate is:

T=[1-(Ai-Aj)/A0]×100%, the results are shown in Figure 6, crony UPPS and UPPS-1 showed good inhibition rate at 100-1000ug/mL.

Claims (6)

1. A wakame polysaccharide UPPS-1 with α-glucosidase activity is characterized in that: the weight average molecular weight is 174.88kDa; the molar ratio of described wakame polysaccharide UPPS-1 is 4.35:1.86:2.32:4.50 :0.94:2.07:48.42:16.01 of mannuronic acid, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, fucose. 2. A wakame polysaccharide UPPS-1 having α-glucosidase activity according to claim 1, characterized in that: the sugar content of the wakame polysaccharide UPPS-1 is 37.95%. 3. according to the preparation method of wakame polysaccharide UPPS-1 described in any one of claim 1-2, it is characterized in that, comprising the following steps: (1) Wakame is desalted, dried and beaten into powder; (2) adding _CaCl solution for extraction, collecting the supernatant, and concentrating under reduced pressure; (3) Precipitating the concentrated solution with ethanol, decolorizing, removing protein, dialysis, and freeze-drying to obtain crude polysaccharide; (4) The crude polysaccharide is prepared into a solution and eluted by an ion exchange column, and the target peak product is collected for tracking and monitoring by the phenol-sulfuric acid method, and the crude fraction is obtained after concentration, dialysis, and freeze-drying; (5) The crude components are further separated through a gel column, and the target peak product is collected for tracking and monitoring by the phenol-sulfuric acid method, and the subdivided components are obtained after concentration, dialysis, and freeze-drying; The step (4) specifically follows the following steps: dissolving the crude polysaccharide of wakame peduncle with deionized water, configuring a polysaccharide solution with a concentration of 30 mg/mL and separating it through a DEAE-52 ion exchange column, using 0, 0.5, 1, 2 mol /LNacl solution elution, the flow rate is controlled at 1mL/min, the elution time is 10min/tube, and the phenol-sulfuric acid method is used for tracking and monitoring. The main peak is collected according to the absorbance, and the eluted components of 0.5mol/LNacl solution are collected. After concentration, use a 3500Da dialysis bag Dialyzed for 48 hours and freeze-dried to obtain crudely divided undaria polysaccharide UPPS-1; The step (5) specifically follows the following steps: dissolving the crude polysaccharides of wakame stalks with pure water, and configuring a polysaccharide solution with a concentration of 30 mg/mL to be separated by a Sephadex-G100 gel column, and eluted with pure water, and the flow rate is controlled at 0.5mL/min, elution time 10min/tube, track and monitor by phenol-sulfuric acid method, collect the main peak according to the absorbance, collect the eluted components in pure water, concentrate under reduced pressure, dialyze with 3500Da dialysis bag for 48h, freeze-dry to obtain subdivision The crony polysaccharide UPPS-1. 4. the preparation method of wakame polysaccharide UPPS-1 according to claim 3, is characterized in that: The step (1) specifically follows the following steps: wash the salt on the surface of the wakame with tap water, replace the water every 12 hours, soak it for three days, drain it, dry it naturally, and then powder it to obtain the wakame powder; The step (2) specifically follows the following steps: extract wakame powder 0.15mol/LCaCl ₂ , solid-liquid ratio 1:15, and extract at 70°C for 3 hours, collect the filtrate, concentrate under reduced pressure, and obtain a concentrated solution of wakame; The step (3) specifically follows the following steps: use 80% ethanol to precipitate the wakame concentrate overnight, collect the precipitate by centrifugation, redissolve it with a small amount of deionized water, decolorize 717 anion resin at 55°C for 12 hours, and remove protein by Sevage method More than three times, until there is no white floc at the boundary between the two phases, dialyze at 3500Da for 48 hours, freeze-dry to obtain crude polysaccharide, chloroform:n-butanol=4:1. 5. The use of a wakame polysaccharide UPPS-1 having α-glucosidase activity according to any one of claims 1-2 in the preparation of diabetes inhibitor drugs. 6. The application according to claim 5, characterized in that: the diabetes inhibitor drug has the function of inhibiting the α-glucosidase on the intestinal mucosa, so that the decomposition of starch into glucose slows down, thereby reducing and delaying the reaction of the small intestine to glucose. Absorption, improve the activity of abnormal blood sugar in diabetic patients.

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