CN117210519A - Polygonatum cyrtonema polysaccharide oligosaccharide tablet, and preparation method and application thereof - Google Patents
Polygonatum cyrtonema polysaccharide oligosaccharide tablet, and preparation method and application thereof Download PDFInfo
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- CN117210519A CN117210519A CN202310927816.8A CN202310927816A CN117210519A CN 117210519 A CN117210519 A CN 117210519A CN 202310927816 A CN202310927816 A CN 202310927816A CN 117210519 A CN117210519 A CN 117210519A
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- Prior art keywords
- polysaccharide
- reaction
- polygonatum cyrtonema
- fructosidase
- enzymolysis
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Abstract
The invention discloses a polygonatum cyrtonema polysaccharide oligosaccharide tablet segment with multiple flowers, a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) Dissolving polygonatum cyrtonema polysaccharide in buffer solution of enzymolysis reaction; (2) adding fructosidase to carry out enzymolysis reaction; (3) After terminating the reaction, the reaction solution is centrifuged, dialyzed, purified and freeze-dried in sequence, thus obtaining the product. According to the invention, the degradation fragments of the polygonatum cyrtonema polysaccharide are prepared by an enzymatic degradation method, the activity of promoting GLP-1 secretion is used as a guide, various parameters of enzymolysis reaction are optimized, and the oligosaccharide fragments with high activity in the polygonatum cyrtonema polysaccharide are obtained by screening, so that the method has the advantages of simplicity in operation, strong specificity, environment friendliness and the like, and provides scientific basis and theoretical basis for development of natural GLP-1 agonist hypoglycemic drugs.
Description
Technical Field
The invention relates to a plant polysaccharide oligosaccharide fragment, in particular to a polygonatum cyrtonema polysaccharide oligosaccharide fragment and a preparation method thereof, and further relates to application of the prepared polygonatum cyrtonema polysaccharide oligosaccharide fragment in preparation of a medicament for promoting GLP-1 secretion, belonging to the field of plant polysaccharide oligosaccharide fragments and application thereof.
Background
Polygonatum cyrtonema Fall is sweet and neutral in taste and has the effects of tonifying qi and yin, strengthening spleen, moistening lung and tonifying kidney. Polygonatum cyrtonema Fall mainly contains polysaccharide, steroid saponin, flavone, phenylpropanoids, anthraquinone compounds, vitamins, various amino acids and other compounds. The polysaccharide is the main functional component in the polygonatum cyrtonema, and is one of important indexes for judging the quality of the polygonatum cyrtonema in pharmacopoeia. The rhizoma polygonati polysaccharide has a plurality of good biological activities: enhancing immunity, resisting tumor, resisting bacteria, resisting aging, resisting virus, reducing blood sugar and blood lipid, improving atherosclerosis, inhibiting osteoporosis, resisting depression, protecting myocardial cells, etc. Studies show that polygonatum cyrtonema polysaccharide can play a role in reducing blood sugar by promoting intestinal epithelial L cells to secrete GLP-1. However, the polysaccharide structure is complex, the structure is difficult to accurately determine, and the deep research on the action mechanism of the polygonatum cyrtonema polysaccharide is limited.
The molecular modification of polysaccharide means structural modification of polysaccharide molecules by chemical, physical, biological and other methods, and mainly comprises the steps of changing the molecular mass of the polysaccharide, the types, the numbers, the positions, the spatial conformation and the like of substituents, so as to influence the biological activity of the polysaccharide. At present, polysaccharide structure modification methods proposed by scholars at home and abroad are mainly divided into chemical modification, physical modification and biological modification. However, chemical modification and physical modification often require chemical reagents and a severe reaction environment, and are easy to cause environmental pollution, resource waste and other conditions. The biological modification is carried out by adopting specific enzyme, and the method has the advantages of strong specificity, environment protection, mild condition, no toxic or side reaction and the like. The research shows that the Polygonatum cyrtonema polysaccharide mainly consists of fructose and glucose in the proportion of 28:1, and the structure is characterized in that the main chain consists of beta-D-fructose. The existing Polygonatum cyrtonema Fall polysaccharide has poor secretion promotion activity on GLP-1, and needs to be improved.
Disclosure of Invention
The invention aims at providing a polygonatum cyrtonema polysaccharide oligosaccharide fragment with GLP-1 secretion promoting activity and a preparation method thereof;
the second purpose of the invention is to apply the polysaccharide oligosaccharide fragment of Polygonatum cyrtonema Fabricius provided by the invention to the preparation of a medicament for promoting GLP-1 secretion or the preparation of a hypoglycemic medicament;
in order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:
in one aspect, the invention provides a polysaccharide oligosaccharide fragment of Polygonatum cyrtonema Fabricius, which comprises the following steps: (1) Dissolving Polygonatum Cyrtonema Polysaccharide (PCP) in buffer solution of enzymolysis reaction; (2) adding fructosidase to carry out enzymolysis reaction; (3) After the reaction is terminated, the reaction solution is centrifugated, dialyzed, purified and freeze-dried to obtain the product.
In a preferred embodiment of the present invention, the buffer solution in step (1) is preferably a citric acid-disodium hydrogen phosphate buffer solution; further preferably, polygonatum cyrtonema polysaccharide is dissolved in a citric acid-disodium hydrogen phosphate buffer solution to a final concentration of 0.67mg/mL.
In a preferred embodiment of the invention, the fructosidase in the step (2) is added in an amount of 30-150U/mL; the content of reducing sugar in each reaction system is rapidly increased within 0.5-2 h from the beginning of the reaction, and the content of reducing sugar in the system is gradually increased along with the extension of time; and the content of reducing sugar in each reaction system is maintained relatively stable within 2-12 hours after the reaction starts, and no obvious rising trend exists. As is clear from the graph, the amount of reducing sugar in the reaction system was the largest when the enzyme addition amount was 120U/mL, and the amount of reducing sugar in the system was decreased when the enzyme addition amount was increased to 150U/mL. It is presumed that the presence of an excessive amount of fructosidase inhibits the enzyme activity, resulting in a decrease in the content of reducing sugar in the reaction system. Therefore, the optimum enzyme concentration for degrading PCP by fructosidase in the enzymolysis reaction is 120U/mL.
In a preferred embodiment of the present invention, the pH value of the reaction system of the enzymolysis reaction in the step (2) is 2-6; the pH is one of the important factors affecting the enzyme activity, and the content of reducing sugar in each system gradually increases within 2 hours of the enzymolysis reaction. When the reaction time was fixed at 12 hours, the content of reducing sugar in the reaction system at pH3 was the highest, and it was 0.493mg/mL. The minimum reducing sugar content at pH 2 was 0.293mg/mL. The reducing sugar content of the pH 4, the pH 5 and the pH 6 is lower than the pH3 and higher than the pH 2. It is explained that pH3 is most suitable for the degradation of PCP by fructosidase, and therefore, the pH of the reaction system for the enzymatic hydrolysis reaction is most preferably 3.
In a preferred embodiment of the invention, the temperature of the enzymolysis reaction in the step (2) is 30-70 ℃; the enzymolysis reaction is greatly affected by temperature. The temperature rise can promote the enzymolysis reaction and increase the enzymolysis rate; whereas an excessively high temperature may inactivate and denature the enzyme, thereby decreasing the enzymatic hydrolysis rate. As can be seen from FIG. 3, the reaction system has lower reducing sugar content at 30 ℃ and 70 ℃, which indicates that fructosidase cannot perform better degradation at lower or higher temperature; when the reaction temperature is 60 ℃, the content of reducing sugar in the reaction system is the highest, which indicates that 60 ℃ is the proper temperature for the fructosidase to perform degradation; therefore, the temperature of the enzymatic hydrolysis reaction of the present invention is most preferably 60 ℃.
In a preferred embodiment of the present invention, the time of the enzymolysis reaction in step (2) is 0.5h-12h, preferably the time of the enzymolysis reaction is 4h.
In a preferred embodiment of the present invention, the centrifugation in step (3) is preferably performed at a rotational speed of 5000g, and the centrifugation time is preferably 10min; the dialysis preferably adopts a dialysis bag with the molecular weight cut-off of more than 500Da, and comprises the steps of dialyzing with flowing water for 24-48 hours and then with deionized water for 12-48 hours; the purification is preferably performed by Sephadex chromatography G-25.
In order to screen and obtain the optimum parameters of enzymolysis reaction, the invention designs a response surface experiment by adopting the parameters of the content of reducing sugar, the enzyme adding amount, the reaction pH value and the enzymolysis reaction temperature, and discovers that when the reaction temperature is fixed at 60 ℃, the content of reducing sugar in a reaction system is increased along with the increase of the enzyme adding amount and the reaction pH value within the range of 90U/mL-128U/mL and the reaction pH value of 2-3.14 and is slowly reduced along with the increase of the enzyme adding amount and the reaction pH value when the highest point is exceeded. When the reaction pH is 3, the enzyme adding amount is 90U/mL-128U/mL, the enzymolysis temperature is 50-58.5 ℃, the content of reducing sugar in the reaction system is gradually increased, and when the enzyme adding amount exceeds 128U/mL, the enzymolysis temperature is higher than 58.5 ℃, the content of reducing sugar in the system begins to be reduced. When the fixed enzyme adding amount is 120U/mL, the content of reducing sugar in the reaction system is also increased and then reduced along with the increase of the reaction pH and the enzymolysis temperature; and finally determining optimal reaction parameters of the enzymolysis reaction through response surface analysis as follows: the enzyme adding amount of glycosidase is 128U/mL, the pH value of the enzymolysis reaction is 3.14, and the temperature of the enzymolysis reaction is 58.5 ℃.
The invention provides a polygonatum cyrtonema polysaccharide oligosaccharide fragment with optimal promoting activity for intestinal epithelial cell secretion GLP-1, which is named EPCP-4h and consists of 13.94% of glucose and 86.06% of fructose, and the molecular weight of the polygonatum cyrtonema polysaccharide oligosaccharide fragment is 2470.51Da.
Further preferably, the carbohydrate of EPCP-4h is 99.97+ -8.21%, reducing sugar 52.56 + -0.16%; the average height distribution is-2.0 nm.
In another aspect, the invention provides a method for preparing a polysaccharide oligosaccharide fragment (EPCP-4 h) of Polygonatum cyrtonema, comprising the following steps: (1) Dissolving Polygonatum cyrtonema polysaccharide in citric acid-disodium hydrogen phosphate buffer solution; (2) adding fructosidase to carry out enzymolysis reaction; wherein, parameters of enzymolysis reaction are as follows: the enzyme concentration is 128U/mL, the pH value of the enzymolysis reaction is 3.14, the temperature of the enzymolysis reaction is 58.5 ℃, and the enzymolysis reaction time is 4 hours; (3) After the enzymolysis reaction is terminated, the reaction solution is centrifuged, dialyzed, purified and freeze-dried to obtain the product.
In the step (3), the reaction solution is centrifuged (the rotation speed is 5000xg,10 min), and a dialysis bag with the molecular weight cut-off of more than 500Da is adopted to dialyze for 24-48 hours by flowing water and then dialyzed for 12-48 hours by deionized water; the purification is preferably performed by using Sephadex chromatography G-25
Experiments show that the Polygonatum Cyrtonema Polysaccharide (PCP) and the polygonatum cyrtonema polysaccharide fragment degraded by fructosidase can promote NCI-H716 cells to secrete GLP-1, wherein the EPCP-4H has the best activity of promoting NCI-H716 cells to secrete GLP-1: EPCP-4H acts on NCI-H716 cells with a GLP-1 secretion of 1.4 times that of PCP, 1.1 times that of EPCP-8H; thus, EPCP-4h is the best active fragment for promoting secretion of GLP-1 by intestinal epithelial cells.
In still another aspect, the invention provides the use of various polygonatum cyrtonema polysaccharide fragments degraded by fructosidase in preparing a medicament for promoting GLP-1 secretion or preparing a medicament for reducing blood sugar.
Therefore, the invention provides a pharmaceutical composition for promoting GLP-1 secretion or reducing blood sugar, which consists of an effective amount of various polygonatum cyrtonema polysaccharide fragments degraded by fructosidase and pharmaceutically acceptable auxiliary materials or carriers.
The person skilled in the art can prepare the pharmaceutical composition into a conventional pharmaceutical preparation according to a conventional method in the field of pharmaceutical preparations, and the dosage form of the pharmaceutical preparation can be solid, semisolid or liquid; preferably lyophilized powder, tablet, capsule, granule, pill, oral liquid, dry suspension, dripping pill, dry extract or infusion.
The administration mode of the pharmaceutical preparation is oral administration or injection administration.
The auxiliary materials or carriers in the invention refer to auxiliary materials or carriers conventional in the pharmaceutical field, such as: diluents, disintegrants, lubricants, excipients, binders, glidants, fillers, surfactants, and the like; in addition, other adjuvants such as flavoring agents and sweeteners may be added to the composition. The diluent can be one or more components for increasing the weight and volume of the tablet, and common diluents include lactose, starch, pregelatinized starch, microcrystalline cellulose, sorbitol, mannitol, inorganic calcium salt and the like; of these, lactose, starch, microcrystalline cellulose are most commonly used. The disintegrating agent can be one or a mixture of several of crosslinked polyvinylpyrrolidone (2-6% of the total weight), crosslinked sodium carboxymethyl cellulose (2-6% of the total weight), alginic acid (2-5% of the total weight) and microcrystalline cellulose (5-15% of the total weight). The lubricant comprises one or a mixture of more of stearic acid, sodium stearate, magnesium stearate, calcium stearate, polyethylene glycol, talcum powder and hydrogenated vegetable oil. The amount of lubricant (to total weight) is in the range of 0.10 to 1%, typically 0.25 to 0.75%. The binder may be one or more ingredients that facilitate granulation; can be starch slurry (10-30% by weight of binder), hydroxypropyl methylcellulose (2-5% by weight of binder), polyvinylpyrrolidone (2-20% by weight of binder), and preferably ethanol aqueous solution of polyvinylpyrrolidone. The glidant can be one or a mixture of a plurality of micropowder silica gel, talcum powder and magnesium trisilicate. The surfactant can be one or more components capable of improving wettability and increasing drug dissolution, and is commonly sodium dodecyl sulfate (commonly used range is 0.2-6% and total weight ratio).
According to the invention, the degradation fragments of the polygonatum cyrtonema polysaccharide are prepared by an enzymatic degradation method, the activity of promoting GLP-1 secretion is used as a guide, various parameters of enzymolysis reaction are optimized, and the oligosaccharide fragments with high activity in the polygonatum cyrtonema polysaccharide are obtained by screening, so that the method has the advantages of simplicity in operation, strong specificity, environment friendliness and the like, and provides scientific basis and theoretical basis for development of natural GLP-1 agonist hypoglycemic drugs.
Drawings
FIG. 1 shows the effect of enzyme concentration on fructosidase degradation modified PCP in an embodiment of the present invention;
FIG. 2 shows the effect of pH on fructosidase degradation modified PCP in an embodiment of the present invention;
FIG. 3 shows the effect of temperature on fructosidase degradation modified PCP in an embodiment of the present invention;
FIG. 4 is a response surface analysis chart showing the design of the preparation process of the polygonatum cyrtonema oligosaccharide fragments in the embodiment of the invention;
FIG. 5 shows the effect of a polygonatum cyrtonema oligosaccharide fragment on GLP-1 secretion by intestinal epithelial L cells in an embodiment of the invention;
FIG. 6 shows an ultraviolet spectrum of an oligosaccharide fragment of Polygonatum cyrtonema Fabricius in an embodiment of the present invention;
FIG. 7 shows an infrared spectrum of an oligosaccharide fragment of Polygonatum cyrtonema Fabricius in an embodiment of the present invention;
FIG. 8 shows a particle size distribution of oligosaccharide fragments of Polygonatum cyrtonema in an embodiment of the present invention;
FIG. 9 shows Congo red experiment results of oligosaccharide fragments of Polygonatum cyrtonema in an embodiment of the present invention;
FIG. 10 shows an SEM image of oligosaccharide fragments of Polygonatum cyrtonema Fabricius in an embodiment of the invention;
FIG. 11 shows AFM spectra of oligosaccharide fragments of Polygonatum cyrtonema in an embodiment of the present invention;
FIG. 12 shows the effect of a polygonatum cyrtonema oligosaccharide fragment in the examples of the present invention on GLP-1 content in portal plasma when administered directly to the jejunum or ileum;
FIG. 13 shows the effect of polygonatum cyrtonema oligosaccharide fragments on portal blood glucose levels when administered directly to the jejunum or ileum in an embodiment of the present invention.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Example 1 preparation of Polygonatum cyrtonema polysaccharide oligosaccharide fragment (EPCP-4 h)
The purified Polygonatum cyrtonema polysaccharide is dissolved in 0.05M citric acid-disodium hydrogen phosphate buffer solution with pH of 3.14 to prepare sugar solution with final concentration of 0.67mg/mL. Adding fructosidase with the enzyme concentration of 128U/mL, and carrying out enzymolysis reaction for 4h at 58.5 ℃; boiling and inactivating after the reaction is finished; centrifuging the reaction solution (the centrifugal rotation speed is 5000xg, and the centrifugation is 10 min), dialyzing for 24-48 h by using flowing water and then dialyzing for 12-48 h by using a dialysis bag with the molecular weight cut-off of more than 500 Da; and finally purifying by using a sephadex chromatography G-25, and freeze-drying the purified product to obtain the final product.
Example 2 preparation of Polygonatum cyrtonema polysaccharide oligosaccharide fragment (EPCP-8 h)
The purified Polygonatum cyrtonema polysaccharide is dissolved in 0.05M citric acid-disodium hydrogen phosphate buffer solution with pH of 3.14 to prepare sugar solution with final concentration of 0.67mg/mL. Fructosidase with an enzyme concentration of 128U/mL was added and reacted at 58.5℃for 8 hours. After the reaction is finished, boiling and inactivating. Centrifuging the reaction solution (the centrifugal rotation speed is 5000xg, and the centrifugation is 10 min), and dialyzing the reaction solution for 12-48 h by using flowing water and deionized water by using a dialysis bag with the molecular weight cut-off of more than 500 Da; and finally purifying by using a sephadex chromatography G-25, and freeze-drying the purified product to obtain the final product.
EXAMPLE 3 preparation of polysaccharide oligosaccharide fragments of Polygonatum cyrtonema
The purified Polygonatum cyrtonema polysaccharide is dissolved in 0.05M citric acid-disodium hydrogen phosphate buffer solution with pH of 2 to prepare sugar solution with final concentration of 0.67mg/mL. Fructosidase with an enzyme concentration of 30U/mL was added and reacted at 30℃for 12 hours. After the reaction is finished, boiling and inactivating. Centrifuging the reaction solution (the centrifugal rotation speed is 5000xg, and the centrifugation is 10 min), and dialyzing the reaction solution for 12-48 h by using flowing water and deionized water by using a dialysis bag with the molecular weight cut-off of more than 500 Da; and finally purifying by using a sephadex chromatography G-25, and freeze-drying the purified product to obtain the final product.
EXAMPLE 4 preparation of polysaccharide oligosaccharide fragments of Polygonatum cyrtonema
The purified Polygonatum cyrtonema polysaccharide is dissolved in 0.05M citric acid-disodium hydrogen phosphate buffer solution with pH of 6 to prepare sugar solution with final concentration of 0.67mg/mL. Fructosidase with an enzyme concentration of 150U/mL was added and reacted at 70℃for 0.5h. After the reaction is finished, boiling and inactivating. Centrifuging the reaction solution (the centrifugal rotation speed is 5000xg, and the centrifugation is 10 min), and dialyzing the reaction solution for 12-48 h by using flowing water and deionized water by using a dialysis bag with the molecular weight cut-off of more than 500 Da; and finally purifying by using a sephadex chromatography G-25, and freeze-drying the purified product to obtain the final product.
EXAMPLE 5 preparation of polysaccharide oligosaccharide fragments of Polygonatum cyrtonema
The purified Polygonatum cyrtonema polysaccharide is dissolved in 0.05M citric acid-disodium hydrogen phosphate buffer solution with pH of 3 to prepare sugar solution with final concentration of 0.67mg/mL. Fructosidase with an enzyme concentration of 120U/mL was added and reacted at 60℃for 4 hours. After the reaction is finished, boiling and inactivating. Centrifuging the reaction solution (the centrifugal rotation speed is 5000xg, and the centrifugation is 10 min), and dialyzing the reaction solution for 12-48 h by using flowing water and deionized water by using a dialysis bag with the molecular weight cut-off of more than 500 Da; and finally purifying by using a sephadex chromatography G-25, and freeze-drying the purified product to obtain the final product.
Experimental example 1 optimization, identification and Performance analysis of polysaccharide enzymolysis Process of Polygonatum cyrtonema
1. Experimental method
1.1 response surface optimization fructosidase degradation Polygonatum cyrtonema polysaccharide enzymolysis technology
Step one: preparation of polysaccharide oligosaccharide fragment of Polygonatum cyrtonema Fabricius
Polygonatum Cyrtonema Polysaccharide (PCP) is dissolved in a citric acid-disodium hydrogen phosphate buffer solution with a corresponding pH value to prepare a sugar solution with a final concentration of 0.67mg/mL. The appropriate dose of fructosidase is added and the reaction is carried out at the corresponding temperature. After the reaction of the reaction system is finished, the mixture is boiled in boiling water for 15min to denature and inactivate the added enzyme preparation. Dialyzing the reaction solution, and freeze-drying to obtain the polysaccharide oligosaccharide fragment of Polygonatum cyrtonema Falcatum.
Step two: determination of reducing sugar content of Polygonatum cyrtonema polysaccharide oligosaccharide fragments
Accurately preparing 1.0mg/mL of polygonatum cyrtonema oligosaccharide fragment solution, taking 1.0mL of the solution into a 15mL test tube, and measuring the content of reducing sugar in the sample according to a DNS method.
Step three: design of single factor experiment
(1) Influence of enzyme addition on fructosidase degradation modification of PCP
The enzyme addition amounts of each experimental group were set to be 30, 60, 90, 120 and 150U/mL, respectively. The enzymolysis temperature and the pH value of the reaction system are respectively set to 50 ℃ and 3, 8 time points (0.5 h, 1h, 2h, 4h, 6h, 8h, 10h and 12 h) are selected, and 1mL of solution is respectively sucked from the reaction system to detect the change condition of the reducing sugar content in the reaction system.
(2) Influence of pH on fructosidase degradation modified PCP
Setting the enzyme adding amount in the reaction system as the optimal experimental value, setting the reaction temperature to be 50 ℃, respectively selecting five levels of reaction pH 2, 3, 4, 5 and 6, and examining the influence of the pH value on the fructosidase degradation modified polygonatum cyrtonema polysaccharide, wherein the operation steps are the same.
(3) Influence of the enzymolysis temperature on the modification of PCP by fructosidase degradation
Based on the above experimental condition investigation results, the enzymolysis temperature is set to be 30 ℃, 40 ℃, 50 ℃,60 ℃ and 70 ℃, and the influence of the enzymolysis temperature on the degradation and modification of the polygonatum cyrtonema polysaccharide by fructosidase is investigated according to the above experimental steps.
Step four: response surface optimization design
The effect of three factors of enzyme addition, reaction pH value and enzymolysis temperature and interaction thereof on the degradation modification of PCP (fructosidase) is studied by using a Box-Benhnken center combined test and a response surface analysis method (Response Surface Methodology, RSM).
TABLE 1 response surface design analysis factors and levels
1.2 screening for secretion of GLP-1 Activity by Enteromorpha L cells
According to the optimal enzymolysis preparation process, the polygonatum cyrtonema polysaccharide enzymolysis fragments (EPCPs) with different time periods (0.5H, 1H, 2H, 4H and 8H) are prepared and are administrated to NCI-H716 cells. NCI-H716 cell density was adjusted to 1X 10 6 And each mL. 0.5mL of the cell suspension was added to each well of the 48-well plate, and incubated in an incubator for 48 hours. After 48h EPCP solution with a final concentration of 200. Mu.g/mL is added respectively, the culture is incubated for 2h, the negative control is complete culture solution, after the incubation is finished, centrifugation is carried out for 15min at 1000rpm and 4 ℃, PMSF (with a final concentration of 50. Mu.g/mL) is added into the supernatant, and the mixture is preserved at 80 ℃. GLP-1 content in the supernatant was measured by ELISA kit method.
1.3 physical and chemical Properties determination and Structure analysis of Polygonatum cyrtonema polysaccharide oligosaccharide fragments
1.3.1 Total carbohydrate, protein, uronic acid, reducing sugar content determination
The contents of total carbohydrate, protein, uronic acid and reducing sugar are determined by phenol sulfuric acid method, coomassie brilliant blue method, m-hydroxybiphenyl method and DNS method.
1.3.2 UV Spectrometry and IR Spectrometry
And (3) taking a proper amount of polygonatum cyrtonema oligosaccharide solution (0.1 mg/mL), setting the wavelength range to be 200-800 nm, and scanning on an ultraviolet-visible spectrophotometer.
Weighing 2mg of five kinds of fully dried Polygonatum cyrtonema Fabricius oligosaccharides, adding 200mg of dried KBr powder into an agate mortar respectively, grinding and fully mixing. After tabletting by a tablet press, setting the scanning range of a Fourier infrared spectrum scanner to 4000cm -1 -400cm -1 Scanning analysis was performed.
1.3.3 molecular weight measurement and particle size measurement
Molecular weight distribution of Polygonatum cyrtonema oligosaccharides was determined by gel permeation chromatography (HPGPC), standard curves were established with different concentrations of dextran and molecular weights were calculated. Detection conditions: the Shimadzu LC-10A high performance liquid chromatography system, shimadzu RID-10A detector, TSK G4000PWXL column (7.8 mm. Times.300 mm), mobile phase of ultrapure water, flow rate of 0.6mL/min, column temperature of 25 ℃.
The particle size of the five enzymatic polysaccharide were measured using a Markov particle size meter. The polysaccharide (0.5 mg/mL) was diluted with ultrapure water to avoid multiple scattering effects. The measurement was performed at 25℃and repeated three times.
1.3.4 Congo Red experiments
Preparing 2.5mg/mL oligosaccharide solution, mixing 2mL polysaccharide solution with 2mL Congo red reagent 80 μmol/L, gradually adding NaOH solution 1mol/L to gradually increase NaOH final concentration from 0.0mol/L to 0.5mol/L, scanning with ultraviolet full wavelength scanner, and measuring maximum absorption wavelength (lambda) with NaOH final concentration as abscissa max ) Plotted on the ordinate.
1.3.5 scanning electron microscope experiments
The dried polysaccharide powder (3 mg) was spread on a conductive gel, and then a conductive film was sprayed on the surface of the polysaccharide sample by a vacuum spraying apparatus, and the microscopic morphology of the five polysaccharides was observed by a scanning electron microscope.
1.3.6 monosaccharide composition
A clean chromatographic vial was weighed 5mg of the dried oligosaccharide sample, 1mL of 2M TFA acid solution was added, and the mixture was heated at 60℃for 1 hour. And (5) introducing nitrogen and drying. Adding 99.99% methanol for cleaning, drying, and repeating cleaning for 2-3 times. Dissolving with appropriate amount of sterile water, and transferring into chromatographic bottle for testing.
The chromatographic system employs a Thermo ICS5000 ion chromatographic system (ICS 5000, thermo Fisher Scientific, USA), and Dionex is employed TM CarboPac TM PA20 (150.0 mm,10 μm) liquid chromatographic column, sample injection amount of 5. Mu.L, mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH,0.2M NaAc), flow rate of 0.5mL/min, column temperature of 30 ℃, and analysis and detection of monosaccharide composition by electrochemical detector.
1.3.7 atomic force microscope
An oligosaccharide solution of 10. Mu.g/mL was prepared, the solution was dropped onto mica flakes, dried and further fixed overnight with absolute ethanol. The microstructure of the oligosaccharide fragment was observed by AFM system (Germany-Broker-BRUKER Dimension Icon).
1.4 in situ detection experiments
After the end of adaptive feeding, 18 SD rats were fasted overnight (6 per group). The rats were anesthetized with uratam (0.5 mL/100 g), the abdominal cavity was opened, and the jejunum or ileum of the rats were ligated after cannulation of the hepatic portal vein, respectively. After measuring the fasting blood glucose level and collecting fasting blood (0 min), physiological saline, a low-concentration EPCP-4h solution (0.5 g/4 mL/kg), and a high-concentration EPCP-4h solution (2 g/4 mL/kg) were directly administered to the ligated jejunum or ileum, respectively. After 15, 30, 60, 90, 120min of administration, blood was collected from portal vein using a syringe containing EDTA (final concentration of 1 mg/mL), aprotinin (final concentration of 500 klU/mL), and DPP-IV inhibitor (final concentration of 100 mM), and centrifuged at 1600g at 4deg.C for 15min to collect plasma, which was stored at-80deg.C. And the blood glucose value of the rat at each time point was measured by a blood glucose meter. During the experiment, the rat status was observed, the rats were anesthetized by increasing the injection of anesthetic in time, and the body temperature of the rats was maintained by a heating pad.
1.5 statistical methods
Drawing and one-way analysis of variance were performed using graphpad8.0 software. Results are expressed as mean±sd; p values less than 0.05 are considered statistically significant.
2. Experimental results
2.1 Single factor Experimental design results
2.1.1 Effect of enzyme addition on modification of PCP by fructosidase degradation
As shown in FIG. 1, the content of reducing sugar in each reaction system is rapidly increased within 0.5-2 h from the beginning of the reaction, and the content of reducing sugar in the system is gradually increased along with the extension of time; and the content of reducing sugar in each reaction system is maintained relatively stable within 2-12 hours after the reaction starts, and no obvious rising trend exists. As is clear from FIG. 1, the amount of reducing sugar in the reaction system was the largest when the enzyme addition amount was 120U/mL, and the amount of reducing sugar in the system was decreased when the enzyme addition amount was increased to 150U/mL. It is presumed that the presence of an excessive amount of fructosidase inhibits the enzyme activity, resulting in a decrease in the content of reducing sugar in the reaction system. Therefore, the optimum enzyme concentration for fructosidase to degrade PCP is 120U/mL.
2.1.2 Effect of pH on fructosidase degradation modification of PCP
pH is one of the important factors affecting enzyme activity. As is clear from FIG. 2, the content of reducing sugar in each system gradually increased within 2 hours of the reaction. When the reaction time was fixed at 12 hours, the content of reducing sugar in the reaction system at pH3 was the highest, and it was 0.493mg/mL. The minimum reducing sugar content at pH 2 was 0.293mg/mL. The reducing sugar content of the pH 4, the pH 5 and the pH 6 is lower than the pH3 and higher than the pH 2. It is indicated that pH3 is most suitable for fructosidase degradation of PCP.
2.1.3 Effect of the reaction temperature on the modification of PCP by fructosidase degradation
The enzymolysis reaction is greatly affected by temperature. The temperature rise can promote the enzymolysis reaction and increase the enzymolysis rate; whereas an excessively high temperature may inactivate and denature the enzyme, thereby decreasing the enzymatic hydrolysis rate. As can be seen from FIG. 3, the reaction system has lower reducing sugar content at 30 ℃ and 70 ℃, which indicates that fructosidase cannot perform better degradation at lower or higher temperature; when the reaction temperature is 60 ℃, the content of reducing sugar in the reaction system is the highest, which indicates that 60 ℃ is a proper temperature for the fructosidase to perform degradation.
2.2 response surface Experimental design and data analysis
TABLE 2 response surface Experimental design and results
Regression equation fitting and analysis of variance
Regression equation fitting and variance analysis of polyglucosidase degradation polygonatum cyrtonema polysaccharide: the regression analysis results are shown in Table 3, after regression fitting of each factor, data fitting is carried out on Y by taking A, B, C as an independent variable, and the following multiple quadratic regression equation is established:
Y＝0.5140+0.0269A+0.0763B-0.0129C+0.022AB+0.0003AC-0.01BC-0.0539A 2 -0.2946B 2 -0.0489C 2
wherein Y is the content of reducing sugar in the reaction system, A is the enzyme adding amount, B is the reaction pH, and C is the enzymolysis temperature.
TABLE 3 regression equation analysis of variance
Note that: * P < 0.05 is a significant difference, P < 0.01 is a very significant difference
And performing variance analysis on the regression equation, wherein the F value of the obtained model is 66.97, and the P value of the model is less than 0.0001, which indicates that the model is obvious and has certain rationality. Determining the coefficient R 2 0.9885, a coefficient of variation C.V. of 8.46%, shows that the experiment has very good weightRenaturation, the test results can be predicted and interpreted during the test. A, B, A in model regression equation 2 、B 2 、C 2 P values of less than 0.05, wherein B, B 2 The P value of (2) is less than 0.0001, which indicates that the enzyme addition amount, the reaction pH and the enzymolysis temperature have a significant effect on the process of degrading PCP by fructosidase, and the effect of the reaction pH is very significant. While the P value of C, AB, AC, BC is greater than 0.05, it is shown that interaction between the three factors has no significant effect on the fructosidase degradation modified PCP.
As can be seen from fig. 4: when the reaction temperature is fixed at 60 ℃, the content of reducing sugar in the reaction system increases with the increase of the enzyme adding amount and the reaction pH in the range of 90U/mL-128U/mL and the reaction pH of 2-3.14, and when the maximum temperature is exceeded, the content of reducing sugar slowly decreases with the increase of the enzyme adding amount and the reaction pH. When the reaction pH is 3, the enzyme adding amount is 90U/mL-128U/mL, the enzymolysis temperature is 50-58.5 ℃, the content of reducing sugar in the reaction system is gradually increased, and when the enzyme adding amount exceeds 128U/mL, the enzymolysis temperature is higher than 58.5 ℃, the content of reducing sugar in the system begins to be reduced. When the fixed enzyme adding amount is 120U/mL, the content of reducing sugar in the reaction system is increased and then reduced along with the increase of the reaction pH and the enzymolysis temperature. Through response surface analysis, the optimal system for degrading and modifying polygonatum cyrtonema polysaccharide by fructosidase is as follows: the enzyme amount is 128U/mL, the reaction pH is 3.14, and the enzymolysis temperature is 58.5 ℃.
2.3 screening for secretion of GLP-1 Activity by Enteromorpha L cells
As can be seen from FIG. 5, both PCP and fructosidase-degraded polysaccharide fragments can promote the secretion of GLP-1 by NCI-H716 cells, wherein EPCP-4H has optimal activity in promoting the secretion of GLP-1 by NCI-H716 cells. EPCP-4H secreted GLP-1 into NCI-H716 cells in PCP
1.4 times, 1.1 times that of EPCP-8 h. Thus, EPCP-4h is the best active fragment for promoting secretion of GLP-1 by intestinal epithelial cells.
2.4 determination of physicochemical Properties and structural analysis of Polygonatum cyrtonema polysaccharide oligosaccharide fragments
2.4.1 physical and chemical Properties determination of Polygonatum cyrtonema polysaccharide oligosaccharide fragments
The results of measuring the physicochemical properties of PCP and EPCP-4h are shown in Table 4: both the carbohydrate content is above 95%, the protein and uronic acid are not contained, and the reducing sugar content in EPCP-4h is far greater than that of PCP.
TABLE 4 physicochemical Properties of Polygonatum cyrtonema oligosaccharide fragments
2.4.2 ultraviolet and Infrared spectra
Fig. 6 shows: at 260nm and 280nm, there was no absorption peak in both PCP and EPCP-4 h. Indicating that the purified enzymolysis polysaccharide does not contain nucleic acid and protein. Fig. 7 shows: PCP and EPCP-4h have similar spectra, 3389cm -1 And 2930cm -1 The nearby signal peaks are the telescopic vibration absorption peaks of O-H and C-H respectively, and the first two peaks are characteristic absorption peaks of polysaccharide. Asymmetric stretching vibrations of the carboxylic acid anion (COO-) of C=O cause 1598cm -1 Peaks appear nearby. 1418cm -1 The nearby peaks are due to stretching vibrations of COO-. 1020cm -1 The two peaks in the vicinity are C-O stretching vibrations of the sugar ring. 927-845 cm -1 The nearby absorption peaks are due to fructose with beta configuration, indicating that both contain beta-D-fructofuranose.
2.4.3 molecular weight measurement and particle size measurement
The molecular weight distribution of PCP and EPCP-4h is shown in Table 5, and the presence of fructosidase reduced the molecular weight of Polygonatum cyrtonema polysaccharide from 8995.12Da to 2470.51Da. As can be seen from fig. 8, the particle size of the polysaccharide gradually decreases with the increase of the degradation time, which indicates that the polysaccharide from polygonatum cyrtonema having a larger original molecular weight is degraded into fragments having a smaller molecular weight under the action of fructosidase.
TABLE 5 molecular weight distribution of oligosaccharide fragments of Polygonatum cyrtonema
2.4.4 Congo Red experiments
As shown in FIG. 9, it is evident from FIG. 9 that as the concentration of NaOH increases from 0mol/L to 0.5mol/L, the concentration of PCP and EPCP-4h are increased max Red shift occurs, indicating that both PCP and EPCP-4h have triple helix structures.
2.4.5 scanning electron microscope
As can be seen from fig. 10, the natural PCP has a smooth and compact surface and shows characteristic aggregation. The PCP (EPCP-4 h) treated by fructosidase damages the original smooth and compact surface of polysaccharide, and uneven and rough structural fragments gradually increase along with the prolongation of enzymolysis time. Indicating that the presence of the enzyme degraded the PCP into smaller fragments.
2.4.6 monosaccharide composition
As shown in Table 6, analysis of monosaccharide composition showed EPCP-4h to be a heteropolysaccharide. Consists of 13.94% glucose and 86.06% fructose. Studies have shown that the molar ratio of fructose to glucose in PCP is 28:1, while the molar ratio of fructose to glucose in EPCP-4h is 6:1. It is presumed that the glucose ratio is increased by decreasing the fructose ratio in Polygonatum cyrtonema polysaccharide due to the degradation of fructosidase.
TABLE 6 monosaccharide composition analysis of oligosaccharide fragments of Polygonatum cyrtonema
2.4.7 atomic force microscope
As shown in FIG. 11, the average height of EPCP-4h was distributed between-2.0 nm and 2.0nm. Studies show that the height of the single-chain polysaccharide is about 0.1 nm-1.0 nm, which indicates that aggregation occurs between EPCP-4h molecules. And the height distribution of PCP is 0.0 nm-30.0 nm. Indicating that EPCP-4h has a lower degree of polymerization and a lower height.
2.5 in situ detection experiments
EPCP-4h solutions were directly administered to jejunum or ileum, respectively, to monitor the effect of the enzymatic fragment on GLP-1 content in the portal plasma of SD rats in real time. As shown in fig. 12, the direct administration of the physiological saline administration was able to significantly increase the GLP-1 content in portal plasma in jejunum or ileum, and the increase in GLP-1 content gradually became gentle after 30min of administration, compared to the normal group to which physiological saline was administered. As can be seen from FIG. 13, the direct administration of EPCP-4h solution to the jejunum or ileum of rats had no significant effect on the blood glucose level in the portal plasma.
Claims (10)
1. The preparation method of the polygonatum cyrtonema polysaccharide oligosaccharide tablet is characterized by comprising the following steps of: (1) Dissolving polygonatum cyrtonema polysaccharide in buffer solution of enzymolysis reaction; (2) adding fructosidase to carry out enzymolysis reaction; (3) After the reaction is terminated, the reaction solution is centrifugated, dialyzed, purified and freeze-dried to obtain the product.
2. The polysaccharide oligosaccharide fragment of Polygonatum cyrtonema Falcatum according to claim 1, wherein the buffer solution in step (1) is a citric acid-disodium hydrogen phosphate buffer solution; preferably, polygonatum cyrtonema polysaccharide is dissolved in a citric acid-disodium hydrogen phosphate buffer solution to a final concentration of 0.67mg/mL.
3. The polysaccharide oligosaccharide fragment of Polygonatum cyrtonema-nature of claim 1, wherein the fructosidase added in step (2) is 30-150U/mL; preferably, the fructosidase is added in step (2) in an amount of 120U/mL.
4. The polysaccharide oligosaccharide fragment of Polygonatum cyrtonema Falcatum according to claim 1, wherein the temperature of the enzymolysis reaction in step (2) is 30-70 ℃; preferably, the temperature of the enzymolysis reaction in the step (2) is 60 ℃.
5. The polysaccharide oligosaccharide fragment of Polygonatum cyrtonema of claim 1, wherein the time of the enzymatic hydrolysis in step (2) is 0.5h to 12h, preferably the time of the enzymatic hydrolysis in step (2) is 4h.
6. The Polygonatum cyrtonema polysaccharide oligosaccharide fragment according to claim 1, wherein the dialysis in step (3) is performed by using a dialysis bag with a molecular weight cut-off of more than 500Da, which is dialyzed by running water and then by deionized water; the purification is carried out by adopting sephadex chromatography G-25.
7. A polysaccharide oligosaccharide fragment of Polygonatum cyrtonema Fabricius is characterized by comprising 13.94% of glucose and 86.06% of fructose, and has a molecular weight of 2470.51Da.
8. A method for preparing the polysaccharide oligosaccharide fragment of polygonatum cyrtonema of claim 7, which is characterized by comprising the following steps: (1) Dissolving polygonatum cyrtonema polysaccharide in buffer solution of enzymolysis reaction; (2) adding fructosidase to carry out enzymolysis reaction; the reaction parameters of the enzymolysis reaction are as follows: the enzyme adding amount of the fructosidase is 128U/mL, the pH value of the enzymolysis reaction is 3.14, and the temperature of the enzymolysis reaction is 58.5 ℃; (3) After the reaction is terminated, the reaction solution is centrifugated, dialyzed, purified and freeze-dried to obtain the product.
9. The method of claim 8, wherein the buffer solution in step (1) is a citric acid-disodium hydrogen phosphate buffer solution; preferably, the polygonatum cyrtonema polysaccharide is dissolved in a citric acid-disodium hydrogen phosphate buffer solution until the final concentration is 0.67mg/mL; step (3), dialyzing the reaction solution by using deionized water after dialyzing the reaction solution by using flowing water by using a dialysis bag with the molecular weight cut-off of more than 500 Da; the purification is performed by using sephadex chromatography G-25.
10. Use of a polysaccharide oligosaccharide fragment of any one of claims 1-7 of polygonatum cyrtonema for the preparation of a medicament for promoting GLP-1 secretion or for the preparation of a hypoglycemic medicament.
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