CN112409504B - Preparation method and application of Sipunculus nudus polysaccharide - Google Patents
Preparation method and application of Sipunculus nudus polysaccharide Download PDFInfo
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Abstract
The invention relates to the technical field of medicines, in particular to a preparation method and application of Sipunculus nudus polysaccharide, which is characterized in that Sipunculus nudus is processed to extract polysaccharide, and then the polysaccharide is mixed with Sipunculus nudus extract, turmeric powder, sodium bicarbonate, citric acid and maltodextrin to prepare effervescent tablets; the invention explores the new application of Sipunculus nudus polysaccharide, and the Sipunculus nudus polysaccharide is prepared into effervescent tablets which are convenient to carry and eat, so that the additional value of Sipunculus nudus is improved, and the process is simple and is suitable for large-scale production.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of Sipunculus nudus polysaccharide.
[ background of the invention ]
Sipunculus nudus L, also called Sipunculus nudus L, commonly called sandworm, is called marine cordyceps sinensis, not only has delicious taste and rich nutritional value, but also is an important marine product with homology of medicine and food. The medicinal value is recorded by various Chinese herbs: the Chinese medicinal animal record that it can invigorate the spleen and treat nocturia; the records of ocean medicine and effective prescription record that it clears lung heat, eliminates phlegm and cough; the record of Nanhai Marine medicinal biology records that the Chinese medicinal herb can clear away lung-heat and treat tooth swelling and pain and can replace Chinese caterpillar fungus; recorded in Chinese medicinal marine life, it is used for treating hectic fever due to yin deficiency, night sweat, cough due to pulmonary tuberculosis, chest distress, excessive phlegm, etc.; the manual of the commonly used Chinese herbal medicine in northern sea is recorded that it can not be cured for a long time. Recorded in Zhonghua ocean materia Medica, its biological activities are called its salty taste and cold nature. It enters lung and kidney meridians, and has effects of clearing lung-heat, relieving cough, invigorating spleen, nourishing yin, lowering fire, and relieving pain. It can be used for treating night sweat due to yin deficiency, hectic fever, cough due to pulmonary tuberculosis, chest distress, excessive phlegm, chronic dysentery, and nocturia.
At present, Sipunculus nudus products in the market are mainly dry products, and can be eaten only by further processing and cooking, so that certain cooking skill is required, a large amount of time is required, and the method is extremely inconvenient. Sipunculus nudus protein fiber is abundant, so that people with chewing disorder cannot effectively absorb the nutrition. Therefore, the development of an instant food which is convenient, has good taste, retains the nutritive value and has medicinal efficacy is extremely demanded in the market.
[ summary of the invention ]
In view of the above, the invention provides a preparation method and application of Sipunculus nudus polysaccharide, which develops new application of Sipunculus nudus polysaccharide, prepares the Sipunculus nudus polysaccharide into effervescent tablets convenient to carry and eat, improves the additional value of Sipunculus nudus, has simple process, and is suitable for large-scale production.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of Sipunculus nudus polysaccharide comprises the following steps:
(1) taking fresh nude Sipunculus nudus, removing silt and viscera, cleaning, and homogenizing to obtain homogenate;
(2) adding deionized water with the temperature of 95-105 ℃ into the homogenate obtained in the step (1), extracting for 4.5-5.5h, filtering to remove insoluble components, then carrying out vacuum concentration on the filtrate to 1/4 with the original volume, adding absolute ethanol with the volume 4 times that of the filtrate into the filtrate, standing overnight, then centrifuging for 4-6min under the conditions that the temperature is 4-6 ℃, the rotation speed is 6500 and 7500rpm, pouring out the supernatant and collecting the precipitate;
(3) washing the precipitate obtained in the step (3) with absolute ethyl alcohol for 2-3 times, centrifuging again, collecting the washed precipitate, dissolving the precipitate with distilled water, and adding an extracting agent chloroform-n-butyl alcohol to extract free protein;
(4) passing the crude sugar extract obtained in step (3) through DEAE-Sepharose ion exchange column (2.0 × 40cm), and purifying with ddH at flow rate of 0.5mL/min2Eluting with column O, and further passing through Sepharose CL-6B chromatography column (2.2 × 50cm), wherein the eluate is 0.3ml/min double distilled water; analyzing the absorption peak of the eluate by HPLC, measuring the absorbance of polysaccharide solution at 490nm by phenol-sulfuric acid method and 280nm by ultraviolet spectrophotometry, drawing elution curve, mixing eluates of the same chromatographic peak according to the elution curve, and analyzing with ddH2Desalting with oxygen, and dialyzing at 4 deg.C for 48 hr; dialyzing, purifying with Sephadex column chromatography, and measuring absorbance of polysaccharide solution at 490nm by phenol-sulfuric acid methodAnd drawing an elution curve, combining eluents with the same chromatographic peak, freeze-drying, crushing and sieving with a 100-mesh and 120-mesh sieve to obtain the sipunculus nudus polysaccharide.
The purity of the polysaccharide is 40% -60% at present, but the purity of the Sipunculus nudus polysaccharide exceeds 92%, and the Sipunculus nudus polysaccharide has the function of reducing blood sugar; can be used as hypoglycemic agent, and the optimal dosage is 100mg/kg body weight.
In the invention, furthermore, the weight of the Sipunculus nudus monomer selected in the step (1) is 10-15 g.
In the invention, further, the volume ratio of the homogenate in the step (2) to the deionized water is 1: 2-3.
The invention also provides an application of the Sipunculus nudus polysaccharide, wherein the Sipunculus nudus polysaccharide is used as a raw material to prepare an instant effervescent tablet, and the instant effervescent tablet is prepared from the following raw materials in parts by weight: 8-10 parts of Sipunculus nudus polysaccharide, 20-25 parts of Sipunculus nudus extract, 0.5-2 parts of turmeric powder, 20-25 parts of sodium bicarbonate, 18-23 parts of citric acid, 8-10 parts of maltodextrin, 1-2 parts of lubricant and 3-4 parts of adhesive.
In the invention, further, the preparation method of the instant effervescent tablet comprises the following steps:
1) preparing Sipunculus nudus extract: placing the cleaned naked Sipunculus nudus body wall in a vacuum drying oven at 55-60 ℃ for 3-5h, taking out, crushing the sample, sieving with a 50-70 mesh sieve, adding 10 times of distilled water by volume into the sieved Sipunculus nudus powder, performing hot reflux extraction in a boiling water bath for 2-4 times, each time for 85-95min, performing suction filtration, combining filtrates, performing rotary evaporation and concentration, freezing and drying the concentrated solution, crushing, and sieving with a 110-mesh and 130-mesh sieve to obtain a Sipunculus nudus extract for later use;
2) preparing turmeric powder: taking fresh turmeric, cleaning, juicing, performing rotary evaporation and concentration to 1/5 of the original volume, performing freeze drying on the concentrated solution, crushing, and sieving with a 115-mesh and 125-mesh sieve to obtain turmeric powder for later use;
3) preparing an effervescent tablet: respectively drying citric acid and sodium bicarbonate at 55-65 deg.C for 2-2.5h, dry mixing Sipunculus nudus polysaccharide, Sipunculus nudus extract, Curcuma rhizome powder and sodium bicarbonate, adding adhesive, and wet mixing to obtain alkaline granule; then, taking citric acid and maltodextrin as a filling agent for dry mixing, adding an adhesive for wet mixing, and preparing acidic particles; respectively drying the alkaline granules and the acidic granules at 40-50 ℃ until the water content is less than 3%, then mixing the alkaline granules and the acidic granules, adding a lubricant into the mixed granules, uniformly mixing, and tabletting to obtain the effervescent tablet.
In the invention, the adhesive is prepared by mixing PVP with the volume fraction of 3% and ethanol solution with the volume fraction of 75% according to the mass ratio of 1: 2-3.
The prepared effervescent tablet is an instant effervescent tablet, the total weight of the effervescent tablet is 2.5g, and the eating mode is as follows: mixing the raw materials in a mixing ratio of 1: 100 (tablet: drinking water).
Sensory indexes of the effervescent tablets are as follows: the finished product is milky yellow in color, round in tissue form, smooth in surface and regular in appearance; no fishy smell and bitter taste, slightly sandworm fragrance, agreeable sour and sweet taste, soft mouthfeel and moderate hardness;
the quality difference between effervescent tablets is as follows: randomly sampling 20 pieces of samples, weighing the total mass, and calculating the average mass, wherein the mass difference limit of the single piece is less than or equal to 5%;
the physical and chemical indexes of the effervescent tablets are as follows: the water content is less than or equal to 2.0 percent; the pH value is 5.2-6.2; disintegration time limit: 6 tablets are randomly sampled, 200mL of normal-temperature distilled water is respectively added to quickly generate a large amount of bubbles, and after the bubbles disappear completely, the effervescent tablets are dissolved completely without residues. The disintegration time is less than or equal to 200 s; the hygroscopicity is less than or equal to 1.5 percent; hardness: the hardness of the effervescent tablet is detected by a tablet hardness tester, and the hardness is 52-60N.
The invention has at least the following beneficial effects:
1. according to the Sipunculus nudus polysaccharide prepared by the invention, the in-vitro enzyme inhibition activity and animal experiment analysis results show that the Sipunculus nudus polysaccharide has an obvious blood sugar reducing effect, and animal experiment results show that the optimal blood sugar reducing effect dose of the Sipunculus nudus polysaccharide is 100mg/kg body weight, and the blood sugar reducing effect of the Sipunculus nudus polysaccharide is obviously superior to that of a positive control medicament, namely metformin. In addition, the purity of the polysaccharide prepared by the method is over 92 percent, and the purity of the polysaccharide is far higher than that of the polysaccharide prepared by the conventional method.
2. The novel health-care effervescent tablet is prepared by taking sipunculus nudus and turmeric as raw materials and adding adhesives and other raw materials, is convenient to carry and eat, and has the characteristics of good taste, physiological effect and high nutritional value. The Sipunculus nudus polysaccharide and the Sipunculus nudus powder are added into the effervescent tablet, so that the nutritional value of the Sipunculus nudus is reserved, the bioavailability of nutritional ingredients is improved, the effervescent tablet has a good market development prospect, and the variety of the effervescent tablet is enriched.
[ description of the drawings ]
FIG. 1 is a bar graph of the effect of Sipunculus nudus polysaccharide on α -glucosidase and α -amylase inhibitory activity;
FIG. 2 is a line graph showing the effect of Sipunculus nudus polysaccharide on fasting plasma glucose in diabetic mice;
FIG. 3 is a line graph showing the effect of Sipunculus nudus polysaccharide on serum triglycerides of diabetic mice;
FIG. 4 is a line graph showing the effect of Sipunculus nudus polysaccharide on total serum cholesterol in diabetic mice;
FIG. 5 is a line graph showing the effect of Sipunculus nudus polysaccharide on serum insulin of diabetic mice;
FIG. 6 is a bar graph of the effect of Sipunculus nudus polysaccharide on serum glucagon from diabetic mice;
FIG. 7 is a bar graph of the effect of Sipunculus nudus polysaccharide on serum epinephrine in diabetic mice;
FIG. 8 is a bar graph of the effect of Sipunculus nudus polysaccharide on hepatic glycogen content in diabetic mice;
FIG. 9 is a bar graph of the effect of Sipunculus nudus polysaccharide on the content of glycogen in the muscle of diabetic mice;
FIG. 10 is a bar graph of the effect of Sipunculus nudus polysaccharide on body weight in diabetic mice;
FIG. 11 is a bar graph of the effect of Sipunculus nudus polysaccharide on organ-to-body ratio index in diabetic mice.
[ detailed description ] embodiments
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1:
the embodiment provides a preparation method of Sipunculus nudus polysaccharide, which comprises the following steps:
(1) taking fresh Sipunculus nudus, wherein the weight of the selected Sipunculus nudus monomer is 15g, removing silt and internal organs, cleaning, and homogenizing to obtain homogenate;
(2) adding 105 ℃ deionized water into the homogenate obtained in the step (1), wherein the volume ratio of the homogenate to the deionized water is 1:3, extracting for 5.5h, filtering to remove insoluble components, then concentrating the filtrate in vacuum to 1/4 of the original volume, adding anhydrous ethanol which accounts for 4 times of the volume of the filtrate into the filtrate, standing overnight, then centrifuging for 6min at the temperature of 6 ℃ and the rotation speed of 7500rpm, pouring out the supernatant and collecting the precipitate;
(3) washing the precipitate obtained in the step (2) with absolute ethyl alcohol for 3 times, centrifuging again, collecting the washed precipitate, dissolving the precipitate with distilled water, adding an extracting agent chloroform-n-butyl alcohol for extraction to remove free protein, and obtaining a crude sugar extracting solution for later use;
(4) passing the crude sugar extract obtained in step (3) through DEAE-Sepharose ion exchange column (2.0 × 40cm), and purifying with ddH at flow rate of 0.5mL/min2Eluting with column O, and further passing through Sepharose CL-6B chromatography column (2.2 × 50cm), wherein the eluate is 0.3ml/min double distilled water; analyzing the absorption peak of the eluate by HPLC, measuring the absorbance of polysaccharide solution at 490nm by phenol-sulfuric acid method and 280nm by ultraviolet spectrophotometry, drawing elution curve, mixing eluates of the same chromatographic peak according to the elution curve, and analyzing with ddH2Desalting with oxygen, and dialyzing at 4 deg.C for 48 hr; dialyzing, purifying with Sephadex column chromatography, and measuring polysaccharide solution at 490nm by phenol-sulfuric acid methodAnd (3) drawing an elution curve, combining eluates with the same chromatographic peak, freeze-drying, crushing, and sieving with a 120-mesh sieve to obtain the sipunculus nudus polysaccharide.
Example 2
The embodiment provides a preparation method of Sipunculus nudus polysaccharide, which comprises the following steps:
(1) taking fresh Sipunculus nudus, wherein the weight of the selected Sipunculus nudus monomer is 12g, removing silt and internal organs, cleaning, and homogenizing to obtain homogenate;
(2) adding deionized water of 100 ℃ into the homogenate obtained in the step (1), wherein the volume ratio of the homogenate to the deionized water is 1:2, extracting for 5.0h, filtering to remove insoluble components, then concentrating the filtrate in vacuum to 1/4 of the original volume, adding absolute ethyl alcohol of 4 times of the volume of the filtrate into the filtrate, standing overnight, then centrifuging for 5min at the temperature of 5 ℃ and the rotation speed of 7000rpm, pouring out the supernatant and collecting the precipitate;
(3) washing the precipitate obtained in the step (2) with absolute ethyl alcohol for 2-3 times, centrifuging again, collecting the washed precipitate, dissolving the precipitate with distilled water, adding an extracting agent chloroform-n-butanol, and extracting to remove free protein to obtain a crude sugar extract for later use;
(4) passing the crude sugar extract obtained in step (3) through DEAE-Sepharose ion exchange column (2.0 × 40cm), and purifying with ddH at flow rate of 0.5mL/min2Eluting with column O, and further passing through Sepharose CL-6B chromatography column (2.2 × 50cm), wherein the eluate is 0.3ml/min double distilled water; analyzing the absorption peak of the eluate by HPLC, measuring the absorbance of polysaccharide solution at 490nm by phenol-sulfuric acid method and 280nm by ultraviolet spectrophotometry, drawing elution curve, mixing eluates of the same chromatographic peak according to the elution curve, and analyzing with ddH2Desalting with oxygen, and dialyzing at 4 deg.C for 48 hr; further purifying the polysaccharide with Sephadex chromatographic column after dialysis, measuring absorbance value of polysaccharide solution at 490nm by phenol-sulfuric acid method, drawing elution curve, combining eluates of the same chromatographic peak, freeze drying, pulverizing, and sieving with 110 mesh sieve to obtain Sipunculus nudus polysaccharide.
Example 3
The embodiment provides a preparation method of Sipunculus nudus polysaccharide, which comprises the following steps:
(1) taking fresh Sipunculus nudus, wherein the weight of the selected Sipunculus nudus monomer is 10-15g, removing silt and internal organs, cleaning, and homogenizing to obtain homogenate;
(2) adding deionized water with the temperature of 95 ℃ into the homogenate obtained in the step (1), wherein the volume ratio of the homogenate to the deionized water is 1:2, extracting for 4.5h, filtering to remove insoluble components, then concentrating the filtrate in vacuum to 1/4 of the original volume, adding anhydrous ethanol which accounts for 4 times of the volume of the filtrate into the filtrate, standing overnight, then centrifuging for 4min under the conditions that the temperature is 4-6 ℃ and the rotating speed is 6500rpm, pouring out supernatant and collecting precipitate;
(3) washing the precipitate obtained in the step (2) with absolute ethyl alcohol for 2 times, centrifuging again, collecting the washed precipitate, dissolving the precipitate with distilled water, adding an extracting agent chloroform-n-butyl alcohol for extraction to remove free protein, and obtaining a crude sugar extracting solution for later use;
(4) passing the crude sugar extract obtained in step (3) through DEAE-Sepharose ion exchange column (2.0 × 40cm), and purifying with ddH at flow rate of 0.5mL/min2Eluting with column O, and further passing through Sepharose CL-6B chromatography column (2.2 × 50cm), wherein the eluate is 0.3ml/min double distilled water; analyzing the absorption peak of the eluate by HPLC, measuring the absorbance of polysaccharide solution at 490nm by phenol-sulfuric acid method and 280nm by ultraviolet spectrophotometry, drawing elution curve, mixing eluates of the same chromatographic peak according to the elution curve, and analyzing with ddH2Desalting with oxygen, and dialyzing at 4 deg.C for 48 hr; further purifying the polysaccharide with Sephadex chromatographic column after dialysis, measuring absorbance value of polysaccharide solution at 490nm by phenol-sulfuric acid method, drawing elution curve, combining eluates of the same chromatographic peak, freeze drying, pulverizing, and sieving with 120 mesh sieve to obtain Sipunculus nudus polysaccharide.
Example 4
The embodiment provides an instant effervescent tablet, which is prepared from the following raw materials in parts by weight: 8 parts of Sipunculus nudus polysaccharide, 20 parts of Sipunculus nudus extract, 0.5 part of turmeric powder, 20 parts of sodium bicarbonate, 18 parts of citric acid, 8 parts of maltodextrin, 1 part of lubricant and 3 parts of adhesive; wherein the Sipunculus nudus polysaccharide is the Sipunculus nudus polysaccharide prepared in the embodiment 2;
the preparation method of the instant effervescent tablet comprises the following steps:
1) preparing Sipunculus nudus extract: placing the cleaned nude Sipunculus nudus body wall in a vacuum drying oven at 55 ℃ for 3h, taking out, crushing the sample, sieving with a 50-mesh sieve, adding 10 times of distilled water by volume into the sieved Sipunculus nudus powder, performing hot reflux extraction in a boiling water bath for 2 times, each time for 85min, performing suction filtration, combining filtrates, performing rotary evaporation concentration, freeze-drying the concentrated solution, crushing, and sieving with a 110-mesh sieve to obtain a Sipunculus nudus extract for later use;
2) preparing turmeric powder: cleaning fresh turmeric, juicing, rotary evaporating and concentrating to 1/5, freeze drying the concentrated solution, pulverizing, and sieving with 115 mesh sieve to obtain turmeric powder;
3) preparing an effervescent tablet: respectively drying citric acid and sodium bicarbonate at 55 deg.C for 2 hr, dry mixing Sipunculus nudus polysaccharide, Sipunculus nudus extract, Curcuma rhizome powder and sodium bicarbonate, adding adhesive, and wet mixing to obtain alkaline granule; then, taking citric acid and maltodextrin as a filling agent for dry mixing, adding an adhesive for wet mixing, and preparing acidic particles; respectively drying the alkaline granules and the acidic granules at 40 ℃ until the water content is less than 3%, then mixing the alkaline granules and the acidic granules, adding a lubricant into the mixed granules, uniformly mixing, and tabletting to obtain the effervescent tablet; the adhesive is prepared by mixing PVP with the volume fraction of 3% and ethanol solution with the volume fraction of 75% according to the mass ratio of 1: 2.
Example 5
The embodiment provides an instant effervescent tablet, which is prepared from the following raw materials in parts by weight: 10 parts of Sipunculus nudus polysaccharide, 25 parts of Sipunculus nudus extract, 2 parts of turmeric powder, 25 parts of sodium bicarbonate, 23 parts of citric acid, 10 parts of maltodextrin, 2 parts of lubricant and 4 parts of adhesive; wherein the Sipunculus nudus polysaccharide is the Sipunculus nudus polysaccharide prepared in the embodiment 2;
the preparation method of the instant effervescent tablet comprises the following steps:
1) preparing Sipunculus nudus extract: placing the cleaned nude Sipunculus nudus body wall in a vacuum drying oven at 60 ℃ for 5h, taking out, crushing the sample, sieving with a 70-mesh sieve, adding 10 times of distilled water by volume into the sieved Sipunculus nudus powder, performing hot reflux extraction in a boiling water bath for 4 times, performing suction filtration for 95min each time, combining filtrates, performing rotary evaporation concentration, freeze-drying the concentrated solution, crushing, and sieving with a 130-mesh sieve to obtain a Sipunculus nudus extract for later use;
2) preparing turmeric powder: cleaning fresh turmeric, juicing, rotary evaporating and concentrating to 1/5, freeze drying the concentrated solution, pulverizing, and sieving with 125 mesh sieve to obtain turmeric powder;
3) preparing an effervescent tablet: respectively drying citric acid and sodium bicarbonate at 65 deg.C for 2.5h, dry mixing Sipunculus nudus polysaccharide, Sipunculus nudus extract, Curcuma rhizome powder and sodium bicarbonate, adding adhesive, and wet mixing to obtain alkaline granule; then, taking citric acid and maltodextrin as a filling agent for dry mixing, adding an adhesive for wet mixing, and preparing acidic particles; respectively drying the alkaline granules and the acidic granules at 50 ℃ until the water content is less than 3%, then mixing the alkaline granules and the acidic granules, adding a lubricant into the mixed granules, uniformly mixing, and tabletting to obtain the effervescent tablet; the adhesive is prepared by mixing PVP with the volume fraction of 3% and ethanol solution with the volume fraction of 75% according to the mass ratio of 1: 3.
Test examples
Study on hypoglycemic action of Sipunculus nudus polysaccharide prepared in examples 1-3 of the present application
1. Study of inhibitory Activity on alpha-Amylase and alpha-glucosidase
The method comprises the following steps: acarbose was used as a positive control. Diluting a polysaccharide sample by using a PBS buffer solution, and preparing a series of different polysaccharide solutions with the concentration of 0.2-1.0 mg/mL. Adding 100 mu L of polysaccharide samples with different mass concentrations into a test tube, respectively adding 100 mu L of alpha-amylase solution (the specific enzyme activity is 1U/mL), adding 200 mu L of 1% starch solution, and shaking and mixing uniformly. And (3) carrying out constant-temperature water bath at 37 ℃ for 10min, adding 1mL of DNS reagent to stop the reaction, carrying out water bath at 100 ℃ for 10min, adding 7.5mL of ice distilled water to dilute, and measuring the absorbance at 520 nm. The sample control tube uses an equal amount of phosphate buffer solution to replace the enzyme solution, the blank control tube uses an equal amount of phosphate buffer solution to replace the sample solution, and the blank tube uses PBS buffer solution.
α -amylase inhibition rate ═ 100% (1- ((sample a + enzyme-a + PBS)/(APBS + enzyme-APBS)));
determination of α -glucosidase inhibitory activity: adding 200 mu L of polysaccharide samples with different mass concentrations into a test tube, respectively adding 200 mu L of alpha-glucosidase solution (the specific enzyme activity is 1U/mL), shaking and mixing uniformly, then adding 100 mu L of pNPG solution (5mmol/L, prepared by PBS buffer solution with pH 6.8), shaking and mixing uniformly. The reaction was quenched by addition of 200. mu.L Na2CO3 after 30min of water bath at 37 ℃ and absorbance was measured at 400 nm. The sample control tube uses PBS buffer solution to replace enzyme solution, the blank control tube uses PBS buffer solution to replace polysaccharide solution, and the blank tube uses PBS buffer solution. The IC50 value is the concentration of inhibitor at which 50% inhibition is achieved.
α -glucosidase inhibition rate ═ 1- ((sample a + enzyme-a sample + PBS)/(APBS + enzyme-APBS)) × 100%
As shown in FIG. 1, the inhibition rates of Sipunculus nudus polysaccharide on alpha-glucosidase and alpha-amylase increased in a concentration-dependent manner (p <0.05, n is 3) within a concentration range of 0.2-0.6 mg/mL. And when the polysaccharide concentration is increased from 0.6 to 1.0mg/mL, the inhibition rate changes less and tends to be stable. When the concentration of the polysaccharide is 1.0mg/mL, the inhibition rates of the sipunculus nudus polysaccharide and the acarbose on the alpha-glucosidase are respectively (80.25 +/-0.95)% and (89.06 +/-0.72)%. The inhibition rates of alpha-amylase are respectively (75.27 +/-1.09)% and (82.15 +/-1.09)%, thus the sipunculus nudus polysaccharide has obvious inhibition effect on alpha-amylase and alpha-glucosidase. Acarbose is a clinical drug for the treatment of postprandial hyperglycemia, but acarbose treatment is often accompanied by side effects such as abdominal distension and diarrhea.
2. The blood sugar reducing effect of Sipunculus nudus polysaccharide is analyzed by a diabetes mouse model constructed by streptozotocin.
Experimental mice: SPF grade C57BL/6 mice, female, weighing 18-22 g. Experimental mice were induced by intraperitoneal injection of freshly prepared streptozotocin solution at a dose of 80mg/kg body weight. After mice were treated with streptozotocin solution for 72h, blood was taken from the tail vein of the mice, and the fasting blood glucose level of each mouse was measured. Selecting mice with blood sugar value higher than 11.1 and lower than 21mmol/L as experimental groups, and regarding the mice as diabetic mouse models.
The positive control group was treated with the chemical metformin at a dose of 200mg/kg body weight and the sipunculus nudus polysaccharide was given treatment at low, medium and high doses, respectively low dose groups: 50 mg/kg; the medium dose group: 100 mg/kg; high dose group: 200mg/kg body weight. During the experiment, the stomach was gazed once a day at a fixed time. Tail vein blood was collected and fasting blood glucose levels (FBG) of each group of mice were measured. The assay was started one week after dosing and was assayed for 4 weeks.
The experimental groups were as follows:
1-Con (normal group), fed with normal feed;
2-Dia (model group) diabetic mice;
3-Pos (positive control group): 200mg/kg metformin;
4-Sipunculus nudus polysaccharide low dose group: 50 mg/kg;
5-Sipunculus nudus polysaccharide middle dose group: 100 mg/kg;
6-Sipunculus nudus polysaccharide high dose group: 200 mg/kg.
The effect of Sipunculus nudus polysaccharide on fasting plasma glucose in diabetic mice is shown in FIG. 2. The fasting blood glucose level in diabetic mice was effectively suppressed after 4 weeks of administration of the sipunculus nudus polysaccharide and metformin intervention therapy in uropathic mice. The blood sugar value of the Sipunculus nudus polysaccharide middle dose group (100mg/kg) tends to be stable after the third week of treatment and is reduced by 8.12 +/-1.78 mmol/L compared with that of the diabetes model group.
After the measurement of the fasting blood sugar value is completed, 0.5mL of blood is taken from the tail part, the blood is centrifuged for 10min at 4000r/min at 4 ℃, and the upper layer serum is taken after centrifugation, wherein the values of Triglyceride (TG) and Total Cholesterol (TC) in the serum are shown in a figure 3-4. The Sipunculus nudus polysaccharide group can effectively reduce the level of triglyceride and cholesterol, and the triglyceride and cholesterol values of the Sipunculus nudus polysaccharide dose group are close to the level of the normal group and are better than those of the positive metformin control group in the fourth week.
The serum insulin, glucagon, and epinephrine content of the mice were measured at week 4 after dosing. The results of the experiments are shown in FIGS. 5-7. The Sipunculus nudus polysaccharide can obviously improve the insulin level, and the effect is better than that of a positive metformin control group. The glucagon concentration of the diabetes model group is obviously increased, the positive metformin group and the Sipunculus nudus polysaccharide group can inhibit the glucagon concentration, and the glucagon concentration of the high-dose group of the Sipunculus nudus polysaccharide is equivalent to that of the normal group. The positive metformin group and the Sipunculus nudus polysaccharide group can inhibit epinephrine concentration and reduce blood sugar concentration, but the Sipunculus nudus polysaccharide group has obviously better effect than the positive control group.
3. Determination of influence on glycogen and myoglycogen content
Glycogen and muscle glycogen, which are stored mainly in the liver and muscle as reserve energy, are high-molecular polysaccharides composed of glucose units, and contain about 4% and 2% glycogen in the liver and muscle tissues, respectively. One of the main reasons why the body produces hyperglycemia is that the liver and muscle tissues cannot store glycogen, and the tissues cannot utilize glucose to provide energy for the body. The results of the experiments are shown in FIGS. 8-9. The content of the muscle glycogen and the liver glycogen of the mice in the diabetes model group is obviously lower than that of the liver glycogen and the muscle glycogen of the mice in the normal control group, (p < 0.05). After the medicine metformin and the sipunculus nudus polysaccharide are administrated for four weeks, the content of hepatic glycogen and myoglycogen is obviously increased (p is less than 0.05) compared with that of a diabetes model group, the effect of the sipunculus nudus polysaccharide group is obviously better than that of a positive control group, wherein the effect of increasing the hepatic glycogen content is the best in a medium-dosage group, and the effect of increasing the myoglycogen content is equivalent in a medium-high dosage group.
4. Influence on body weight and organ-to-body weight ratio of diabetic mice
Weight loss and increased relative organ weight ratios are considered to be typical features of diabetes, which are associated with defects in glucose metabolism and excessive breakdown of tissue proteins. The results of the experiments are shown in FIGS. 10-11. The body weight of mice in the diabetic group was significantly reduced compared to those in the normal group. After 4 weeks of administration, Sipunculus nudus polysaccharide can significantly increase the body weight of diabetic mice, and the ability thereof is significantly better than that of metformin. Of the three doses of polysaccharide, the medium dose group had the most significant effect on increasing body weight in diabetic mice.
The body ratio indexes of the liver, the spleen and the kidney of the diabetic mice are obviously improved compared with those of normal mice, and after 4 weeks of administration, the body ratio of the positive metformin control group and the body ratio of the mice of the diabetic model group are equivalent and not relieved. The Sipunculus nudus polysaccharide can obviously reduce the organ-to-body ratio of diabetic mice, and the organ-to-body ratio index of a medium-dose group is close to that of a normal mouse group.
The in vitro enzyme inhibition activity and animal experiment analysis result show that the Sipunculus nudus polysaccharide has obvious blood sugar reducing effect. Animal experiment results show that the optimal dosage of the Sipunculus nudus polysaccharide for reducing blood sugar is 100mg/kg body weight. The blood sugar reducing effect of the medicine is obviously better than that of a positive control medicament, namely metformin.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention.
Claims (5)
1. The application of Sipunculus nudus polysaccharide is characterized in that the Sipunculus nudus polysaccharide is used as a raw material to prepare an instant effervescent tablet, and the instant effervescent tablet is prepared from the following raw materials in parts by weight: 8-10 parts of Sipunculus nudus polysaccharide, 20-25 parts of Sipunculus nudus extract, 0.5-2 parts of turmeric powder, 20-25 parts of sodium bicarbonate, 18-23 parts of citric acid, 8-10 parts of maltodextrin, 1-2 parts of lubricant and 3-4 parts of adhesive;
the preparation method of the Sipunculus nudus extract comprises the following steps: placing the cleaned naked Sipunculus nudus body wall in a vacuum drying oven at 55-60 ℃ for 3-5h, taking out, crushing the sample, sieving with a 50-70 mesh sieve, adding 10 times of distilled water by volume into the sieved Sipunculus nudus powder, performing hot reflux extraction in a boiling water bath for 2-4 times, each time for 85-95min, performing suction filtration, combining filtrates, performing rotary evaporation and concentration, freezing and drying the concentrated solution, crushing, and sieving with a 110-mesh and 130-mesh sieve to obtain a Sipunculus nudus extract for later use;
the preparation method of the Sipunculus nudus polysaccharide comprises the following steps:
(1) taking fresh nude Sipunculus nudus, removing silt and viscera, cleaning, and homogenizing to obtain homogenate;
(2) adding deionized water with the temperature of 95-105 ℃ into the homogenate obtained in the step (1), extracting for 4.5-5.5h, filtering to remove insoluble components, then carrying out vacuum concentration on the filtrate to 1/4 with the original volume, adding absolute ethanol with the volume 4 times that of the filtrate into the filtrate, standing overnight, then centrifuging for 4-6min under the conditions that the temperature is 4-6 ℃, the rotation speed is 6500 and 7500rpm, pouring out the supernatant and collecting the precipitate;
(3) washing the precipitate obtained in the step (2) with absolute ethyl alcohol for 2-3 times, centrifuging again, collecting the washed precipitate, dissolving the precipitate with distilled water, adding an extracting agent chloroform-n-butanol, and extracting to remove free protein to obtain a crude sugar extract for later use;
passing the crude sugar extract obtained in step (3) through DEAE-Sepharose ion exchange column at flow rate of 0.5mL/min ddH2Eluting the column with 0.3ml/min double distilled water, and further passing through Sepharose CL-6B chromatographic column; analyzing the absorption peak of the eluate by HPLC, measuring the absorbance of polysaccharide solution at 490nm by phenol-sulfuric acid method and 280nm by ultraviolet spectrophotometry, drawing elution curve, mixing eluates of the same chromatographic peak according to the elution curve, and analyzing with ddH2Desalting with oxygen, and dialyzing at 4 deg.C for 48 hr; and (3) further purifying the polysaccharide by using a sephadex chromatographic column after dialysis, determining the absorbance value of the polysaccharide solution at 490nm by using a phenol-sulfuric acid method, drawing an elution curve, combining eluents with the same chromatographic peak, freeze-drying, and then crushing and sieving by using a 100-mesh and 120-mesh sieve to obtain the sipunculus nudus polysaccharide.
2. The use of Sipunculus nudus polysaccharide according to claim 1, wherein the weight of the Sipunculus nudus monomer selected in step (1) is 10-15 g.
3. The use of Sipunculus nudus polysaccharide according to claim 1, wherein the volume ratio of homogenate to deionized water in the step (2) is 1: 2-3.
4. The use of Sipunculus nudus polysaccharide according to any one of claims 1-3, wherein the instant effervescent tablet is prepared by the following steps:
1) preparing Sipunculus nudus extract: placing the cleaned naked Sipunculus nudus body wall in a vacuum drying oven at 55-60 ℃ for 3-5h, taking out, crushing the sample, sieving with a 50-70 mesh sieve, adding 10 times of distilled water by volume into the sieved Sipunculus nudus powder, performing hot reflux extraction in a boiling water bath for 2-4 times, each time for 85-95min, performing suction filtration, combining filtrates, performing rotary evaporation and concentration, freezing and drying the concentrated solution, crushing, and sieving with a 110-mesh and 130-mesh sieve to obtain a Sipunculus nudus extract for later use;
2) preparing turmeric powder: taking fresh turmeric, cleaning, juicing, performing rotary evaporation and concentration to 1/5 of the original volume, performing freeze drying on the concentrated solution, crushing, and sieving with a 115-mesh and 125-mesh sieve to obtain turmeric powder for later use;
3) preparing an effervescent tablet: respectively oven drying citric acid and sodium bicarbonate at 55-65 deg.C for 2-2.5h, dry mixing Sipunculus nudus polysaccharide, Sipunculus nudus extract, Curcuma rhizome powder and sodium bicarbonate, adding adhesive, and wet mixing to obtain alkaline granule; then, taking citric acid and maltodextrin as a filling agent for dry mixing, adding an adhesive for wet mixing, and preparing acidic particles; respectively drying the alkaline granules and the acidic granules at 40-50 ℃ until the water content is less than 3%, then mixing the alkaline granules and the acidic granules, adding a lubricant into the mixed granules, uniformly mixing, and tabletting to obtain the effervescent tablet.
5. The use of Sipunculus nudus polysaccharide according to claim 4, wherein the binder is prepared by mixing 3 vol.% PVP and 75 vol.% ethanol solution at a mass ratio of 1: 2-3.
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