Preparation method and application of potamogeton crispus alpha-glucosidase inhibitor for reducing blood sugar
By preparing crude protein PCTp from *Potamogeton crispus*, the problem of the lack of natural α-glucosidase inhibitors in existing technologies has been solved, achieving a highly efficient and safe α-glucosidase inhibition effect, which is suitable for the preparation of hypoglycemic drugs.
Patent Information
- Application Number
- CN202511404667.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-12-16
AI Technical Summary
Currently, there is no research on the preparation of hypoglycemic α-glucosidase inhibitors from *Potamogeton crispus*. Existing synthetic drugs have side effects, so it is necessary to screen for highly effective and safe α-glucosidase inhibitors from natural sources.
An ultrasonic-assisted extraction and pH adjustment method was used to prepare *Potamogeton crispus* extract from raw materials. The process included multi-step ultrasonic decolorization, heating extraction, and centrifugation to obtain crude protein PCTp from *Potamogeton crispus* as the main active ingredient.
The crude protein PCTp from *Potamogeton crispus* showed significant α-glucosidase inhibitory activity, with an IC50 value of 3.290 μg/mL, which was superior to acarbose. It also showed good stability under different conditions, making it suitable for the preparation of hypoglycemic drugs.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a preparation method and application of a Potamogeton crispus hypoglycemic alpha-glucosidase inhibitor. BACKGROUND
[0002] Diabetes is a metabolic disease, and defects in insulin action, insulin secretion, or both can cause diabetes, which is characterized by hyperglycemia. Diabetes has become a global health challenge, and its incidence is increasing. Studies have shown that type II diabetes can cause metabolic disorders of carbohydrates, proteins, and fats, oxidative stress, protein non-enzyme glycosylation, and other reactions in the body, thereby causing patients to develop various chronic complications such as cardiovascular disease, kidney failure, eye disease, nerve or vascular damage, foot complications, and the like. Globally, 11.3% of deaths are due to diabetes, almost half of which are under the age of 60. In 2019, there were approximately 463 million people worldwide with diabetes, and this number could reach 700 million by 2045. Therefore, it is necessary to find effective drugs and methods for treating diabetes.
[0003] One of the effective treatments for type II diabetes is to inhibit the absorption of glucose by inhibiting carbohydrate hydrolytic enzymes such as alpha-amylase and alpha-glucosidase in the digestive organs, thereby reducing postprandial blood glucose levels. Alpha-glucosidase mainly exists in the brush border of the human small intestinal mucosa and is a key enzyme that catalyzes the last step in the digestion of carbohydrates. It can hydrolyze alpha-(1,4) glycosidic bonds to release non-reducing end glucose. Alpha-glucosidase inhibitors can slow down the hydrolysis and absorption of carbohydrates in the diet and inhibit the increase in postprandial blood glucose. Synthetic alpha-glucosidase inhibitors used in clinical practice include miglitol, voglibose, and acarbose, but they have certain side effects such as gastrointestinal distension, diarrhea, abdominal cramps, and the like. Alpha-glucosidase inhibitors screened from natural sources have the advantages of high efficiency, safety, and no toxic side effects, and have become a research hotspot for the prevention and treatment of diabetes.
[0004] Potamogeton crispus, a perennial submerged plant of the family Potamogetonaceae, is a typical autumn germination and winter growth aquatic plant. It is widely distributed in freshwater areas such as lakes and rivers in winter and spring and is a dominant species of submerged plants in winter and spring. It has important values in maintaining the stability of the structure and function of aquatic ecosystems, controlling water eutrophication, and water ecological restoration. Potamogeton crispus has a unique life history. In late spring and early summer, it flowers and produces a large number of hair-like asexual propagules, turions, also known as scale branches, and abundant seeds. The turions accumulate rich storage substances after maturation. After the turions fall off from the mother plant, they can continue to carry out photosynthesis, and the storage substances continue to accumulate, the density gradually increases, and the turions sink to the bottom of the water. Turions have greater population expansion capacity than seeds.
[0005] Many compounds with α-glucosidase inhibitory effect have been isolated from natural plants, such as flavonoids, anthocyanins, alkaloids, terpenoids, glycosides, and phenolic acids. Ezzat et al. isolated quercetin-3, 6, 7-trimethyl ether, isohamnetin-4'-methyl ether, spinasterol, isohamnetin, and a new flavone compound from the plant of Asteraceae, which all have better inhibitory effect than acarbose. Lu et al. isolated polyphenols loddigesiinol G-J and crepidatuol B from the stems of Dendrobium officinale, which both have significant enzyme inhibitory activity with IC 50 values of 16.7, 10.9, 2.7, 3.2, and 18.9 μmol / L, respectively. Wang et al. isolated ursane triterpenoids from the stems of Uncaria gambier, which have good α-glucosidase inhibitory activity with an IC 50 value of 49.0 μmol / L. Kim
[30] extracted an indole alkaloid from coffee, which reversibly and non-competitively inhibits α-glucosidase with significant and concentration-dependent inhibitory activity.
[0006] Currently, there is no report on the preparation of a hypoglycemic α-glucosidase inhibitor from the raw material of Potamogeton crispus. SUMMARY
[0007] The present application aims to provide a preparation method and application of a hypoglycemic α-glucosidase inhibitor from Potamogeton crispus.
[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions. A preparation method of a Potamogeton crispus extract, comprising the following steps: S1: after the fresh Potamogeton crispus raw material is cleaned, the raw material powder is obtained by drying, crushing, and sieving; the raw material powder is mixed with 80% ethanol aqueous solution as a decolorizing agent, and the supernatant 1 and the precipitate 1 are obtained by ultrasonic-assisted decolorization, heat preservation decolorization, and centrifugation; the precipitate 1 is mixed with 80% ethanol aqueous solution as a decolorizing agent, and the supernatant 2 and the precipitate 2 are obtained by ultrasonic-assisted decolorization, heat preservation decolorization, and centrifugation; S2: the precipitate 2 is swelled with water, the pH is adjusted, and the supernatant 3 and the precipitate 3 are obtained by ultrasonic-assisted extraction, heating extraction, and centrifugation; the precipitate 3 is swelled with water, the pH is adjusted, and the supernatant 4 and the precipitate 4 are obtained by ultrasonic-assisted extraction, heating extraction, and centrifugation; the supernatant 4 is swelled with water, the pH is adjusted, and the supernatant 5 and the precipitate 5 are obtained by ultrasonic-assisted extraction, heating extraction, and centrifugation; the supernatant 3, the supernatant 4, and the supernatant 5 are combined, the pH is adjusted, and the concentrated solution is obtained by concentration under reduced pressure; ethanol is added to the concentrated solution for alcohol precipitation, and the supernatant 6 and the precipitate 6 are obtained by centrifugation; the precipitate 6 is dissolved in water, the pH is adjusted, and the supernatant 7 and the precipitate 7 are obtained by standing and centrifugation, and the precipitate 7 is the Potamogeton crispus extract.
[0009] The above step S1 is as follows: Take fresh *Potamogeton crispus* raw material, clean it, dry it with hot air at 60℃ to constant weight, pulverize it, and pass it through a 60-mesh sieve to obtain raw material powder; use 80% ethanol aqueous solution as decolorizing agent, mix it with the raw material powder at a material-to-liquid ratio of 1:5, and decolorize it with 480W ultrasonic assistance at room temperature for 20 minutes, then transfer it to a 50℃ environment for heat preservation and decolorization for 4 hours, centrifuge it, and separate it to obtain supernatant 1 and precipitate 1; use 80% ethanol aqueous solution as decolorizing agent, mix it with precipitate 1 at a material-to-liquid ratio of 1:5, and decolorize it with 480W ultrasonic assistance at room temperature for 20 minutes, then transfer it to a 50℃ environment for heat preservation and decolorization for 4 hours, centrifuge it, and separate it to obtain supernatant 2 and precipitate 2.
[0010] The fresh raw materials for the above step S1 include freshly harvested Potamogeton crispus and / or fresh Potamogeton crispus.
[0011] Step S2 above is specifically as follows: Precipitate 2 is soaked in water at a material-to-liquid ratio of 1:8 at room temperature until fully swollen. The pH is then adjusted to 9, and extraction is performed at room temperature using 480W ultrasound-assisted extraction for 20 minutes. Subsequently, it is transferred to a 60℃ environment for constant temperature extraction for 2 hours, centrifuged, and the supernatant 3 and precipitate 3 are separated. Precipitate 3 is soaked in water at a material-to-liquid ratio of 1:8 at room temperature until fully swollen. The pH is then adjusted to 9, and extraction is performed at room temperature using 480W ultrasound-assisted extraction for 20 minutes. Subsequently, it is transferred to a 60℃ environment for constant temperature extraction for 2 hours, centrifuged, and the supernatant 4 and precipitate 4 are separated. Precipitate 4 is soaked in water at a material-to-liquid ratio of 1:8 at room temperature until fully swollen, and the pH is adjusted to 9. 9. Extraction was performed at room temperature using 480W ultrasound-assisted extraction for 20 min, followed by constant temperature extraction at 60℃ for 2 h. After centrifugation, supernatant 5 and precipitate 5 were obtained. Supernatant 3, supernatant 4 and supernatant 5 were combined to obtain a combined solution. The pH of the combined solution was adjusted to 7, and then concentrated under reduced pressure at 60℃ to obtain a concentrated solution. Three volumes of anhydrous ethanol were added to the concentrated solution, and the solution was allowed to stand at 4℃ for 4 h for alcohol precipitation. After centrifugation, supernatant 6 and precipitate 6 were obtained. Precipitate 6 was dissolved in five times its mass of water, the pH was adjusted to 3, and the solution was allowed to stand at 4℃ for 4 h. After centrifugation, supernatant 7 and precipitate 7 were obtained. Precipitate 7 is the extract of *Potamogeton crispus*.
[0012] A type of herb extract, which is prepared by the above-described method.
[0013] The above-mentioned application of a *Potamogeton crispus* extract in the preparation of α-glucosidase inhibitors.
[0014] The above-mentioned application of a *Potamogeton crispus* extract in the preparation of drugs that improve insulin resistance.
[0015] The above-mentioned application of a *Potamogeton crispus* extract in the preparation of a drug for treating diabetes.
[0016] The above-mentioned application of a *Potamogeton crispus* extract in the preparation of hypoglycemic drugs. Attached Figure Description
[0017] Figure 1 IC50 of different extracts on the inhibitory activity of α-glucosidase 50 value.
[0018] Figure 2 Chromatogram of PCTp passing through a Sephacryl S-1000 column.
[0019] Figure 3 Establishment of IR-HepG2 cell model.
[0020] Figure 4 Effects of PCTp on glucose uptake in IR-HepG2 cells.
[0021] Figure 5 The mechanism of PCTp's inhibitory effect on α-glucosidase.
[0022] Figure 6 The type and constant of PCTp inhibition of α-glucosidase.
[0023] Figure 7 The effect of heating time on the stability of PCTp inhibition of α-glucosidase activity.
[0024] Figure 8 The effect of pH on the stability of PCTp's inhibition of α-glucosidase activity.
[0025] Figure 9 The effect of metal ions on the stability of PCTp's inhibition of α-glucosidase activity.
[0026] Figure 10 The effects of preservatives and antioxidants on the stability of PCTp's inhibition of α-glucosidase activity.
[0027] Figure 11 Effect of sucrose on the stability of PCTp-inhibited α-glucosidase activity. Detailed Implementation
[0028] To provide a clearer understanding of the present invention, the technical solution of the present invention will be further described below in conjunction with specific embodiments; it should be noted that the scope of protection of the present invention is not limited thereto.
[0029] Example 1: S1: Freshly harvested *Potamogeton crispus* buds were cleaned, dried with hot air at 60℃ to constant weight, pulverized, and passed through a 60-mesh sieve to obtain raw material powder. Using 80% ethanol aqueous solution as a decolorizing agent, the powder was mixed with the above raw material powder at a material-to-liquid ratio of 1:5 (mass-to-volume ratio, m / V). The mixture was then treated with ultrasonic assisted treatment at 480W for 20 min at room temperature, followed by incubation at 50℃ for 4 h. Finally, it was centrifuged at 7000 r / min for 20 min to separate the supernatant 1 and precipitate 1. Using 80% ethanol aqueous solution as a decolorizing agent, the mixture was mixed with the above precipitate 1 at a material-to-liquid ratio of 1:5 (mass-to-volume ratio, m / V). The mixture was then treated with ultrasonic assisted treatment at 480W for 20 min at room temperature, followed by incubation at 50℃ for 4 h. Finally, it was centrifuged at 7000 r / min for 20 min to separate the supernatant 2 and precipitate 2.
[0030] S2: Add water to the above precipitate 2 at a material-to-liquid ratio of 1:8 (mass-to-volume ratio, m / V), soak at room temperature until fully swollen, adjust the pH to 9 with NaOH solution, and extract with ultrasonic assistance at 480W for 20 min at room temperature. Then transfer to a 60℃ environment for constant temperature extraction for 2 h, and centrifuge at 7000 r / min for 20 min to separate the supernatant 3 and precipitate 3. Add water to the above precipitate 3 at a material-to-liquid ratio of 1:8 (mass-to-volume ratio, m / V), soak at room temperature until fully swollen, adjust the pH to 9 with NaOH solution, and extract with ultrasonic assistance at 480W for 20 min at room temperature. Then transfer to a 60℃ environment for constant temperature extraction for 2 h, and centrifuge at 7000 r / min for 20 min to separate the supernatant 4 and precipitate 4. The above precipitate 4 was soaked in water at a material-to-liquid ratio of 1:8 (m / V) at room temperature until fully swollen. The pH was adjusted to 9 with NaOH solution, and the mixture was extracted with ultrasound at 480W for 20 min at room temperature. Then, it was transferred to a 60℃ environment for constant temperature extraction for 2 h, followed by centrifugation at 7000 rpm for 20 min to separate the supernatant 5 and precipitate 5. The supernatants 3, 4, and 5 were combined to obtain a combined solution. The pH of the combined solution was adjusted to 7 with HCl solution, and then concentrated under reduced pressure at 60℃ to 1 / 8 of the original volume to obtain a concentrated solution. Three times the volume of anhydrous ethanol (based on the volume of the concentrated solution) was added to the concentrated solution, and the mixture was allowed to stand at 4℃ for 4 h for alcohol precipitation. Then, it was centrifuged at 7000 rpm for 20 min to separate the supernatant 6 and precipitate 6. Precipitate 6 is the extracted *Polygonum multiflorum* polysaccharide-protein mixture. The precipitate 6 was dissolved in 5 times its mass of water, the pH was adjusted to 3 with HCl solution, and the mixture was allowed to stand at 4°C for 4 hours. Then, it was centrifuged at 7000 rpm for 20 minutes to separate the supernatant 7 and the precipitate 7. Precipitate 7 is the crude protein from *Potamogeton crispus* Turion (PCTp), which is the *Potamogeton crispus* extract subsequently used to determine α-glucosidase inhibitory activity.
[0031] (1) Determination of the inhibitory activity of PCTp and raw material powder on α-glucosidase Add the same volume of samples of different concentrations to 30 μL of 0.5 U / mL α-glucosidase, incubate at 37 °C for 10 min, then add 70 μL of 5 mmol / L p-nitrophenyl-α-D-glucopyranoside (pNPG) to initiate the enzymatic reaction, and continue incubation for 20 min. After incubation, add 70 μL of 0.2 mol / L sodium carbonate to terminate the reaction, and measure the absorbance at 405 nm. Calculate the α-glucosidase inhibition rate based on the following formula: α-glucosidase inhibition rate / % = [1 - (A1 - A0) / (T1 - T0)] × 100, where: A1 is the absorbance of the sample group; A0 is the absorbance without α-glucosidase; T1 is the absorbance without sample; and T0 is the absorbance without both sample and α-glucosidase.
[0032] The results showed that the IC50 of PCTp on α-glucosidase inhibitory activity was [missing information]. 50 The value was 3.290±0.199μg / mL, and the IC50 of the raw material powder was... 50 The value was 9.50 ± 0.56 μg / mL, and the IC50 of acarbose was... 50 The value was 102.65±6.86μg / mL, indicating that PCTp had the strongest inhibitory activity against α-glucosidase, followed by the raw material powder, while acarbose had the weakest inhibitory activity.
[0033] (2) Determination of active ingredients in extracts Determination of IC50 of the inhibitory activity of *Potamogeton crispus* polysaccharide-protein mixture and PCTp against α-glucosidase 50 Value. (By) Figure 1 It can be seen that the IC50 of the polysaccharide-protein mixture from *Potamogeton crispus* inhibits α-glucosidase activity. 50 Value >20 μg / mL, IC50 of PCTp inhibitory activity against α-glucosidase 50 The value was 3.290 μg / mL, indicating that PCTp is the main active ingredient that inhibits α-glucosidase.
[0034] (3) Determination of PCTp purity PCTp was separated using a 1cm × 30cm propylene dextran gel column (Sephacryl S-1000) with water as the mobile phase, a flow rate of 1.0 mL / min, and a detection wavelength of 280 nm. Figure 2 As can be seen, PCTp has a relatively concentrated molecular weight distribution and high purity. Simultaneously, the results show that the IC50 of PCTp's inhibitory activity against α-glucosidase after column chromatography is [data missing]. 50The value was 2.089 ± 0.109 μg / mL, which is similar to the IC50 value before column chromatography. 50 The values are not significantly different.
[0035] Example 2: (1) Construction of IR-HepG2 cell model HepG2 cells were seeded in 96-well plates and cultured for 24 hours. The culture medium was then discarded, and 100 μL of DMEM high-glucose medium containing different concentrations of insulin and dexamethasone (including fetal bovine serum) was added to each well. The cells were cultured for another 48 hours to construct the IR-HepG2 cell model. The concentration combinations were: 0.01 μmol / L insulin + 5 μmol / L dexamethasone, 0.1 μmol / L insulin + 5 μmol / L dexamethasone, 1.0 μmol / L insulin + 5 μmol / L dexamethasone, and 10 μmol / L insulin + 5 μmol / L dexamethasone. A control group of normal HepG2 cells was also included. After culturing, the culture medium in each well was discarded, the cells were washed once with PBS, and then 100 μL of serum-free medium was added for another 48 hours. Glucose content was determined using the glucose oxidase (GOD) method of a glucose (GLU) assay kit, and cell viability was detected by the CCK-8 assay. The amount of glucose absorbed per unit cell was calculated. A significant decrease in glucose absorption per unit cell (P<0.05) was used as the criterion for successful establishment of the IR-HepG2 cell model.
[0036] Depend on Figure 3 It was observed that the lowest glucose uptake per unit cell was observed at an induction concentration of 0.01 μmol / L insulin + 5 μmol / L dexamethasone, which was 0.7477 ± 0.097 μmol / L, a decrease of 30.25% compared to the control group (1.072 ± 0.109 μmol / L). Therefore, 0.01 μmol / L insulin + 5 μmol / L dexamethasone was determined to be the optimal induction concentration for establishing the IR-HepG2 cell model.
[0037] (2) Effect of PCTp on glucose uptake in IR-HepG2 cells HepG2 cells were seeded in 96-well plates and cultured for 24 h. The culture medium was then discarded, and 100 μL of DMEM high-glucose medium containing 0.01 μmol / L insulin, 5 μmol / L dexamethasone, and fetal bovine serum was added. The cells were cultured for another 48 h to construct the IR-HepG2 cell model. A normal HepG2 cell control group was also included. After culturing, the culture medium in each well was discarded, and the cells were washed once with PBS. Then, 100 μL of serum-free medium containing different concentrations of the samples was added, and the cells were cultured for another 48 h. Glucose content was measured using the GLU assay kit (GOD method), and cell viability was detected using the CCK-8 assay to calculate the glucose uptake per unit cell.
[0038] Depend on Figure 4 It is evident that, compared to the control group, the glucose uptake per unit cell in the IR-HepG2 cell model group was reduced. After treatment with different concentrations of PCTp, the glucose uptake per unit cell in the IR-HepG2 cell model group increased with increasing PCTp concentration. Among them, the effect of 6 μg / mL PCTp on increasing the glucose uptake per unit cell in the IR-HepG2 cell model group was better than that of the acarbose (20 μg / mL) treatment group, and the effect of 18 μg / mL PCTp on increasing the glucose uptake per unit cell in the IR-HepG2 cell model group was more significant.
[0039] Example 3: (1) Mechanism of PCTp's inhibitory effect on α-glucosidase With the substrate PNPG concentration fixed at 5 mmol / L, the enzymatic reaction rates were measured at α-glucosidase concentrations of 0.125 U / mL, 0.25 U / mL, 0.5 U / mL, and 1.0 U / mL, with and without PCTp (0 μg / mL), and with the addition of PCTp (2 μg / mL, 4 μg / mL, and 8 μg / mL). An inhibition kinetic curve of α-glucosidase was plotted with enzyme concentration on the x-axis and enzyme reaction rate on the y-axis to determine the reversibility of the inhibition.
[0040] Depend on Figure 5 It is evident that, under a fixed substrate PNPG concentration, the enzymatic reaction rate of α-glucosidase increases with increasing α-glucosidase concentration, both with and without PCTp. After the addition of PCTs, the enzymatic reaction rate decreases, the slope of the curves becomes smaller, and all four curves pass through the origin, indicating that the inhibitory mechanism of PCTp on α-glucosidase activity is reversible.
[0041] (2) Determination of the type of inhibition and inhibition constant of PCTp on α-glucosidase With a fixed α-glucosidase concentration (0.5 U / mL), the enzymatic reaction rates were measured at substrate PNPG concentrations of 0.2 mmol / L, 0.4 mmol / L, 0.8 mmol / L, and 1.6 mmol / L, with and without PCTp (0 μg / mL), and with the addition of PCTp (2 μg / mL, 4 μg / mL, and 8 μg / mL). Lineweaver-Burk double reciprocal curves were plotted with the reciprocal of substrate concentration on the x and y axes and the reciprocal of the enzymatic reaction rate on the y-axis. The kinetic characteristics and the inhibitory constant K for the free enzyme were determined by plotting the intercept and slope. i The inhibition constant K of enzyme-substrate complex (ES) is .
[0042] Depend on Figure 6It is evident that, under a fixed α-glucosidase concentration, the reciprocal of the substrate concentration and the reciprocal of the enzyme-catalyzed reaction rate show a linear positive correlation between the absence of PCTp and the presence of PCTp; the four Lineweaver-Burk curves intersect at the same point in the second quadrant, and the maximum enzyme-catalyzed reaction rate decreases after the addition of PCTp, and the Michaelis constant K... m The value increases. Based on this, it can be determined that PCTp inhibits α-glucosidase activity in a mixed manner. Further calculations yield the inhibition constant K of PCTp on the free enzyme. i The inhibition constant K for the enzyme-substrate complex is 3.47 μg / mL. is It is 2.15 μg / mL, K is <K i This indicates that the inhibition is a mixed type of competitive and non-competitive inhibition. Therefore, the inhibitory effect of PCTp on α-glucosidase activity is a reversible mixed type of competitive and non-competitive inhibition.
[0043] Example 4: (1) Effect of heating time on the stability of PCTp inhibitory α-glucosidase activity A 4.5 μg / mL PCTp solution was incubated at 100℃ for 0, 5, 10, 20, and 40 min, respectively. After incubation, the changes in its inhibitory activity against α-glucosidase were measured. Figure 7 It is evident that within the incubation range of 0–40 min, the inhibition rate of PCTp against α-glucosidase showed a decreasing trend, but the overall decrease was relatively small. Specifically, compared to the control group, the inhibition rate in the 40 min incubation group decreased from 68.59±1.25% to 60.29±1.31%, a decrease of only 12.1%. These results indicate that at 100℃, heating time has little effect on the stability of PCTp's inhibition of α-glucosidase activity.
[0044] (2) Effect of pH on the stability of PCTp inhibitory α-glucosidase activity A 4.0 μg / mL PCTp solution was taken, and its pH was adjusted to 3.0, 5.0, 7.0, and 9.0, respectively. After shaking and reacting at room temperature for 2 hours, the pH was adjusted to 7.0, and the changes in inhibitory activity against α-glucosidase were then detected. Figure 8 It is evident that PCTp exhibits the highest inhibitory activity against α-glucosidase at pH 5.0, while the inhibitory activity is lowest at pH 9.0, although the decrease is not significant. Compared to the pH 5.0 group, the inhibition rate in the pH 9.0 group decreased from 59.40±0.87% to 45.75±3.81%, a reduction of 22.98%. These results indicate that pH value has a certain influence on the stability of PCTp's inhibition of α-glucosidase activity.
[0045] (3) Effects of metal ions on the stability of PCTp's inhibitory activity against α-glucosidase Take a 0.1 mg / mL PCTp solution and react it with a 0.05 mol / L K+ solution. + Solution, Na + Solution, Ca 2+ Solution, Mg 2+ Solution, Al 3+ Solution, Fe 3+ Solution, Zn 2+ After mixing the solutions, they were incubated at 37°C for 1 hour, then diluted 30-fold, and the changes in inhibitory activity against α-glucosidase were measured. Figure 9 It can be seen that Zn 2+ It has a certain inhibitory effect on the inhibitory activity of PCTp, K + Na + The effect on its inhibitory activity is minimal, with almost no significant inhibitory effect; while Fe 3+ The effect on PCTp inhibitory activity was most significant at 0.05 mol / L Fe. 3+ After treatment, the inhibitory activity of PCTp decreased by 96.2% compared to the control group (without metal ions); Ca 2+ Mg 2 + Al 3+ Its effect on PCTp inhibitory activity is secondary.
[0046] (4) Effects of preservatives and antioxidants on the stability of PCTp's inhibitory activity against α-glucosidase. A 0.1 mg / mL PCTp solution was mixed with 30 mg / mL benzoic acid solution, 30 mg / mL sorbic acid solution, 4.0 mg / mL tert-butylhydroxyanisole (BHA) solution, 4.0 mg / mL butylated hydroxytoluene (BHT) solution, and 4.0 mg / mL vitamin C solution, respectively. After incubation at 37℃ for 1 h, the mixture was diluted 30-fold, and the changes in its inhibitory activity against α-glucosidase were measured. Figure 10 It is evident that, at the experimental concentrations, benzoic acid, sorbic acid, BHA, BHT, and vitamin C have almost no effect on the inhibitory stability of PCTp on α-glucosidase.
[0047] (5) Effect of sucrose on the stability of PCTp's inhibitory activity against α-glucosidase A 0.1 mg / mL PCTp solution was mixed with sucrose solutions of concentrations of 5 wt%, 10 wt%, 20 wt%, 40 wt%, and 60 wt%, respectively. After incubation at 37°C for 1 hour, the mixture was diluted 30-fold, and the changes in inhibitory activity against α-glucosidase were measured. Figure 11It is evident that low-concentration sucrose solution has a certain activating effect on α-glucosidase. Within the concentration range of 5wt% to 60wt%, the inhibitory effect on PCTp activity increases with increasing sucrose concentration, but the overall inhibitory effect is weak. Treatment with 60wt% sucrose solution reduced PCTp activity by 23.6%.
[0048] (6) Effect of in vitro simulated digestion on the stability of PCTp's inhibitory activity against α-glucosidase: A PCTp solution with a concentration of 20 μg / mL was digested in simulated gastrointestinal fluid, and the changes in its inhibitory activity against α-glucosidase were measured. The results showed that after simulated gastrointestinal digestion, the inhibitory activity of PCTp against α-glucosidase was significantly lower than that of the control group (undigested PCTp solution). The inhibition rate of PCTp against α-glucosidase decreased from 86.35±0.30% before digestion to 77.48±3.03%, a decrease of 10.27%. This indicates that gastrointestinal digestion has a relatively small overall effect on the inhibitory activity of PCTp against α-glucosidase.
[0049] Example 5: (1) Preparation of Poria cocos PCTp tablets Take 10g of PCTp, 300g of sodium carboxymethyl cellulose, 70g of sodium bicarbonate, 30g of magnesium stearate, and 800g of starch. Mix the above materials thoroughly and evenly, and then compress them into 10,000 tablets using a tablet press at 60°C.
[0050] (2) Preparation of Poria cocos powder tablets Take 300g of freshly harvested Podocarpus sclerotium and / or fresh Podocarpus sclerotium raw material powder, 300g of sodium carboxymethyl cellulose, 70g of sodium bicarbonate, 30g of magnesium stearate, and 800g of starch. Mix the above materials thoroughly and evenly, and then compress them into 10,000 tablets using a tablet press at 60℃.
Claims
1. A method for preparing a *Potamogeton crispus* extract, characterized in that: Includes the following steps: S1: Fresh Potamogeton crispus raw material is cleaned, dried, crushed, and sieved to obtain raw material powder; 80% ethanol aqueous solution is used as a decolorizing agent and mixed with the raw material powder, and then subjected to ultrasonic-assisted decolorization, heat preservation decolorization, and centrifugation to obtain supernatant 1 and precipitate 1; 80% ethanol aqueous solution is used as a decolorizing agent and mixed with precipitate 1, and then subjected to ultrasonic-assisted decolorization, heat preservation decolorization, and centrifugation to obtain supernatant 2 and precipitate 2; S2: Add water to precipitate 2 to swell, adjust pH, and extract by ultrasonic-assisted extraction, heating extraction, and centrifugation to obtain supernatant 3 and precipitate 3; add water to precipitate 3 to swell, adjust pH, and extract by ultrasonic-assisted extraction, heating extraction, and centrifugation to obtain supernatant 4 and precipitate 4; add water to supernatant 4 to swell, adjust pH, and extract by ultrasonic-assisted extraction, heating extraction, and centrifugation to obtain supernatant 5 and precipitate 5; combine supernatant 3, supernatant 4, and supernatant 5, adjust pH, and concentrate under reduced pressure to obtain concentrated solution; add ethanol to concentrated solution for alcohol precipitation, centrifuge to obtain supernatant 6 and precipitate 6; dissolve precipitate 6 in water, adjust pH, let stand, and centrifuge to obtain supernatant 7 and precipitate 7, which is the extract of *Potamogeton crispus*.
2. The preparation method according to claim 1, characterized in that: Step S1 is as follows: Take fresh *Potamogeton crispus* raw material, wash it clean, dry it with hot air at 60℃ to constant weight, pulverize it, and pass it through a 60-mesh sieve to obtain raw material powder; use 80% ethanol aqueous solution as decolorizing agent, mix it with the raw material powder at a material-to-liquid ratio of 1:5, and decolorize it with 480W ultrasonic assistance at room temperature for 20 minutes, then transfer it to a 50℃ environment for 4 hours of heat preservation and decolorization, centrifuge it, and separate it to obtain supernatant 1 and precipitate 1; use 80% ethanol aqueous solution as decolorizing agent, mix it with precipitate 1 at a material-to-liquid ratio of 1:5, and decolorize it with 480W ultrasonic assistance at room temperature for 20 minutes, then transfer it to a 50℃ environment for 4 hours of heat preservation and decolorization, centrifuge it, and separate it to obtain supernatant 2 and precipitate 2.
3. The preparation method according to claim 1, characterized in that: The fresh raw materials for step S1 include freshly harvested Potamogeton crispus and / or fresh Potamogeton crispus.
4. The preparation method according to claim 1, characterized in that: Step S2 is as follows: Precipitate 2 is soaked in water at a material-to-liquid ratio of 1:8 at room temperature until fully swollen. The pH is then adjusted to 9, and extraction is performed at room temperature using 480W ultrasound-assisted extraction for 20 minutes. Subsequently, the mixture is transferred to a 60℃ environment for constant temperature extraction for 2 hours. After centrifugation, supernatant 3 and precipitate 3 are obtained. Precipitate 3 is soaked in water at a material-to-liquid ratio of 1:8 at room temperature until fully swollen. The pH is then adjusted to 9, and extraction is performed at room temperature using 480W ultrasound-assisted extraction for 20 minutes. Subsequently, the mixture is transferred to a 60℃ environment for constant temperature extraction for 2 hours. After centrifugation, supernatant 4 and precipitate 4 are obtained. Precipitate 4 is soaked in water at a material-to-liquid ratio of 1:8 at room temperature until fully swollen. The pH is then adjusted to 9, and extraction is performed at room temperature using 480W ultrasound-assisted extraction for 20 minutes. Subsequently, the mixture is transferred to a 60℃ environment for constant temperature extraction for 2 hours. After centrifugation, supernatant 5 and precipitate 5 are obtained. Supernatant 3, supernatant 4, and supernatant 5 are combined to obtain a combined solution. The combined solution was adjusted to pH 7 and then concentrated under reduced pressure at 60°C to obtain a concentrate. Three volumes of anhydrous ethanol were added to the concentrate, and the mixture was allowed to stand at 4°C for 4 hours for alcohol precipitation. After centrifugation, the supernatant 6 and precipitate 6 were separated. Precipitate 6 was dissolved in five times its mass of water, the pH was adjusted to 3, and the mixture was allowed to stand at 4°C for 4 hours. After centrifugation, the supernatant 7 and precipitate 7 were separated. Precipitate 7 is the extract of *Potamogeton crispus*.
5. A *Potamogeton crispus* extract, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 4.
6. The use of the Podocarpus extract according to claim 5 in the preparation of α-glucosidase inhibitors.
7. The use of the *Potamogeton crispus* extract according to claim 5 in the preparation of a medicament for improving insulin resistance.
8. The use of the *Potamogeton crispus* extract according to claim 5 in the preparation of a medicament for treating diabetes.
9. The use of the *Potamogeton crispus* extract according to claim 5 in the preparation of a hypoglycemic drug.