A method for determining astragalus polysaccharide

Abstract

This invention relates to the field of polysaccharide determination technology, providing a method for determining Astragalus polysaccharides. It employs a code-free glucometer to measure glucose content, offering convenient operation, high specificity, and minimal interference. It eliminates the need for polysaccharide purification such as protein removal, reducing polysaccharide loss. Using a sample as a blank effectively reduces interference from glucose naturally present in Astragalus, resulting in more accurate results and facilitating quality comparison and monitoring. Hydrolysis is performed using trifluoroacetic acid, which is highly volatile, easily removed to eliminate interference, and less corrosive than concentrated sulfuric acid, avoiding the toxicity associated with phenol. This method is suitable for large-scale industrial production. The hydrolysis apparatus has been changed from high-temperature oven hydrolysis to temperature-controlled water bath hydrolysis, ensuring safety and ease of operation. Ultrasonic removal of air further reduces polysaccharide oxidation.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide determination technology, and particularly relates to a method for determining Astragalus polysaccharides. Background Technology

[0002] Polysaccharides are polymeric carbohydrates composed of at least 10 monosaccharides linked by glycosidic bonds. Polysaccharides composed of identical monosaccharides are called homopolysaccharides, such as starch, cellulose, and glycogen; polysaccharides composed of different monosaccharides are called heteropolysaccharides, such as gum arabic, which is composed of pentoses and galactose. Polysaccharides are not pure chemical substances, but rather mixtures of substances with varying degrees of polymerization. Polysaccharides are generally insoluble in water, have no sweet taste, cannot crystallize, and exhibit no reducing or mutarotation properties. Polysaccharides are also glycosides, and therefore can be hydrolyzed. During hydrolysis, a series of intermediate products are often produced, eventually leading to complete hydrolysis and the extraction of monosaccharides.

[0003] Astragalus polysaccharide is a water-soluble heteropolysaccharide extracted, concentrated, and purified from the dried roots of *Astragalus mongholicus* or *Astragalus membranaceus*, both belonging to the legume family. It is pale yellow, with a fine, uniform powder free of impurities, and is hygroscopic. Astragalus polysaccharide is composed of hexuronic acid, glucose, fructose, rhamnose, arabinose, galacturonic acid, and glucuronic acid, among other components. It can be used as an immune stimulant or regulator, and also possesses antiviral, antitumor, anti-aging, anti-radiation, anti-stress, and antioxidant effects.

[0004] The determination of polysaccharides currently mostly uses the phenol-sulfuric acid method. However, sulfuric acid is a controlled substance, which is inconvenient to purchase and difficult to conduct subsequent application research. Sulfuric acid is also highly corrosive, making its use in the laboratory very unsafe. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining Astragalus polysaccharides, which aims to solve the problems that the determination of polysaccharides currently mostly uses the phenol-sulfuric acid method, but sulfuric acid is a controlled substance, inconvenient to purchase, not easy to conduct subsequent application research, and has strong corrosiveness, making its use in the laboratory very unsafe.

[0006] The present invention is implemented as follows: a method for determining Astragalus polysaccharides, the method comprising the following steps:

[0007] Step 1: Accurately transfer multiple sets of glucose reference solutions into 2 mL volumetric flasks using a pipette. Place the diluted reference solutions into an ultrasonic instrument and sonicate for 30 min, then let stand for 60 min. Subsequently, use a Sinocare brand Safe and Code-Free Blood Glucose Meter to measure the blood glucose value. Plot a standard curve with the blood glucose value on the ordinate and the glucose concentration on the abscissa.

[0008] Step 2: Extract polysaccharides from the medicinal materials and place them in a 10mL threaded test tube. Add 2mol / L trifluoroacetic acid at a material-to-liquid ratio of 1:2 and seal the tube. Place the tube in an ultrasonic cleaner and sonicate for 10 minutes to remove air. Then place the tube in a beaker and put the beaker in an electric heating mantle. Set the temperature to 96℃ and the time to 1.7h for hydrolysis.

[0009] Step 3: After the hydrolysate has cooled, transfer it to a 100mL round-bottom flask and place it in a water bath to evaporate to dryness under reduced pressure.

[0010] Step 4: After cooling, add 3 mL of distilled water to a round-bottom flask, sonicate for 30 min and let stand for 60 min, then measure the glucose content with a blood glucose meter. Subtract interference from the sample blank and calculate the polysaccharide content in Astragalus membranaceus as glucose.

[0011] As a further aspect of the present invention: the extraction of polysaccharides from medicinal materials in step 2 includes the following steps:

[0012] Step 1: Weigh 100g of crude Astragalus membranaceus powder, add 300mL of 75% ethanol solution, desorb for 40min, add 12 times the volume of distilled water at 80℃, reflux extract at 80℃ under reduced pressure (vacuum degree is -0.0740MPa) for 7min, centrifuge the extract, and repeat the same method twice from the step of "adding 12 times the volume of distilled water at 80℃", and combine the filtrates;

[0013] Step 2: Concentrate the filtrate to 170 mL at 80 °C, add 3 times the amount of 80% ethanol solution while stirring continuously during the addition process, let it stand overnight, and filter the solution after standing overnight to obtain Astragalus polysaccharide.

[0014] As a further aspect of the present invention: the polysaccharide content in Astragalus membranaceus calculated as glucose in step 4 is determined by the following formula:

[0015]

[0016]

[0017] As a further embodiment of the present invention: in step 3, the product is evaporated to dryness under reduced pressure. During the evaporation process, methanol is added repeatedly to the round-bottom flask 4 times, 2 mL each time, to remove trifluoroacetyl groups, until there is no sour taste.

[0018] The method for determining Astragalus polysaccharides provided in this invention has the following beneficial effects:

[0019] The glucose content was measured using a code-free blood glucose meter, which is convenient to operate, highly specific, and has little interference. It does not require polysaccharide purification such as protein removal, thus reducing polysaccharide loss. Using the sample as a blank can effectively reduce the interference of glucose contained in Astragalus membranaceus itself, resulting in more accurate measurement results and facilitating the comparison and monitoring of the quality of medicinal materials.

[0020] Trifluoroacetic acid is used for hydrolysis. Its high volatility makes it easy to remove and eliminate interference, and its corrosiveness is weaker than concentrated sulfuric acid. This avoids the toxicity associated with phenol and allows for large-scale industrial production. The hydrolysis apparatus has been changed from high-temperature oven hydrolysis to temperature-controlled water bath hydrolysis, which is safer and easier to operate. Ultrasonic removal of air further reduces polysaccharide oxidation. Attached Figure Description

[0021] Figure 1 A contour plot showing the effects of temperature and time on the results;

[0022] Figure 2 Response surface 3D plot showing the effects of temperature and time on the results;

[0023] Figure 3 Contour plot showing the effects of temperature and polysaccharide dosage on the results;

[0024] Figure 4 Response surface 3D plot showing the effect of temperature and polysaccharide dosage on the results;

[0025] Figure 5 Contour plot showing the effects of time and polysaccharide dosage on the results;

[0026] Figure 6 Response surface 3D plot of the effects of time and polysaccharide dosage on the results;

[0027] Figure 7 The graph shows the effect of room temperature volume adjustment on the measurement results;

[0028] Figure 8 Figure showing the effect of volume fixation in a 36℃ water bath;

[0029] Figure 9 Figure showing the effect of volume fixation in an 80℃ water bath;

[0030] Figure 10 This is a graph showing the effect of ultrasound on the measurement results;

[0031] Figure 11 This is a standard curve plot of the linear relationship. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0034] like Figure 1 As shown in the embodiment of the present invention, a method for determining Astragalus polysaccharides includes the following steps:

[0035] Step 1: Accurately transfer multiple sets of glucose reference solutions into 2 mL volumetric flasks using a pipette. Place the diluted reference solutions into an ultrasonic instrument and sonicate for 30 min, then let stand for 60 min. Subsequently, measure the blood glucose value using an Anwen coded blood glucose meter. Plot a standard curve with the blood glucose value on the ordinate and the glucose concentration on the abscissa.

[0036] Step 2: Extract polysaccharides from the medicinal materials and place them in a 10mL threaded test tube. Add 2mol / L trifluoroacetic acid at a material-to-liquid ratio of 1:2 and seal the tube. Place the tube in an ultrasonic cleaner and sonicate for 10 minutes to remove air. Then place the tube in a beaker and put the beaker in an electric heating mantle. Set the temperature to 96℃ and the time to 1.7h for hydrolysis.

[0037] Step 3: After the hydrolysate has cooled, transfer it to a 100mL round-bottom flask and place it in a water bath to evaporate to dryness under reduced pressure.

[0038] Step 4: After cooling, add 3 mL of distilled water to a round-bottom flask, sonicate for 30 min and let stand for 60 min, then measure the glucose content with a blood glucose meter. Subtract interference from the sample blank and calculate the polysaccharide content in Astragalus membranaceus as glucose.

[0039] As a further aspect of the present invention: the extraction of polysaccharides from medicinal materials in step 2 includes the following steps:

[0040] Step 1: Weigh 100g of crude Astragalus membranaceus powder, add 300mL of 75% ethanol solution, desorb for 40min, add 12 times the volume of distilled water at 80℃, reflux extract at 80℃ under reduced pressure (vacuum degree is -0.0740MPa) for 7min, centrifuge the extract, repeat the same method twice from the point of "adding 12 times the volume of distilled water at 80℃", and combine the filtrates;

[0041] Step 2: Concentrate the filtrate to 170 mL at 80 °C, add 3 times the amount of 80% ethanol solution while stirring continuously during the addition process, let it stand overnight, and filter the solution after standing overnight to obtain Astragalus polysaccharide.

[0042] As a further aspect of the present invention: the polysaccharide content in Astragalus membranaceus calculated as glucose in step 4 is determined by the following formula:

[0043]

[0044]

[0045] As a further embodiment of the present invention: in step 3, the product is evaporated to dryness under reduced pressure. During the evaporation process, methanol is added repeatedly to the round-bottom flask 4 times, 2 mL each time, to remove trifluoroacetyl groups, until there is no sour taste.

[0046] Example 1:

[0047] Accurately weigh 132.7 mg of the reference standard using an analytical balance with a concentration of 0.01%, dilute to 25 mL with distilled water, and shake well to obtain a glucose reference solution with a concentration of 5.308 mg / mL.

[0048] Accurately pipette 0.14, 0.18, 0.205, 0.24, 0.40, 0.60, 0.80, 1.00, and 1.20 mL of the reference solution into 2 mL volumetric flasks. Place the diluted reference solution in an ultrasonic instrument and sonicate for 30 min. After standing for 60 min, measure the blood glucose value using an Anwen codeless blood glucose meter. Plot a standard curve with blood glucose value (B) on the ordinate and glucose concentration (X) on the abscissa.

[0049] The glucose reference standard concentration showed good linearity in the range of 0.5443–3.1848, with a linear correlation coefficient r = 0.9993.

[0050] Weigh a certain amount of polysaccharide extracted from the medicinal material and place it in a 10mL threaded test tube. Add 2mol / L trifluoroacetic acid at a material-to-liquid ratio of 1:2. Seal the tube and place it in an ultrasonic cleaner for 10 minutes to remove air. Then place it in a beaker filled with water and place the beaker in an electric heating mantle. Set the temperature to 96℃ and the time to 1.7h for hydrolysis.

[0051] After the hydrolysate has cooled, it is transferred to a 100 mL round-bottom flask and placed in a water bath to evaporate to dryness under reduced pressure. The process is repeated four times, with 2 mL of methanol added each time to remove trifluoroacetyl groups, until there is no sour taste.

[0052] After cooling, add 3 mL of distilled water to a round-bottom flask, sonicate for 30 min and let stand for 60 min, then measure the glucose content with a blood glucose meter. Subtract interference from the sample blank and calculate the polysaccharide content in Astragalus membranaceus as glucose.

[0053] Example 2:

[0054] Extraction of Astragalus polysaccharides:

[0055] Astragalus root was dried in the shade, pulverized, and 100g of crude powder was accurately weighed. 300mL of 75% ethanol solution was added, and the mixture was desorbed for 40min. 12 times the volume of distilled water at 80℃ was added, and the mixture was extracted under reduced pressure by reflux at 80℃ for 7min. The mixture was centrifuged twice in advance, starting from the step of adding 12 times the volume of distilled water at 80℃. The filtrates were combined. The mixture was concentrated to 170mL at 80℃, and 3 times the volume of 80% ethanol was added by slow and fast stirring. The mixture was allowed to stand overnight and then filtered to obtain 47.35g of Astragalus polysaccharide.

[0056] Example 3:

[0057] Investigation of hydrolysis time:

[0058] Five portions of Astragalus polysaccharide, each 100 mg, were accurately weighed and placed in 10 mL spiral test tubes. 15 mL of 2 mol / L trifluoroacetyl was added, and the tubes were sonicated for 10 min to remove air. The tubes were then placed in beakers filled with water and placed in a heating mantle. Hydrolysis was carried out at 100 °C for 0.5, 1, 1.5, 2, and 3 h, respectively. Then, the procedure was followed according to step 3 of the Astragalus polysaccharide determination method. After cooling, 3 mL of distilled water was added, and the tubes were sonicated for 30 min. After standing for 60 min, the blood glucose level was measured using a blood glucose meter, and the glucose and polysaccharide contents were calculated. The measurement results are shown in Table 3. As can be seen from the table, the amount of polysaccharide hydrolyzed into monosaccharides increases with time. The glucose percentage reaches its highest point at 1.5 h. Subsequently, the monosaccharide content decreases instead of increasing when the time is extended. Therefore, the optimal hydrolysis time is 1.5 h.

[0059] Table 3 Effect of hydrolysis time on glucose percentage content

[0060]

[0061] Investigation of hydrolysis temperature:

[0062] Five 100mg portions of Astragalus polysaccharide were accurately weighed and placed in separate 10mL spiral test tubes. 15mL of 2mol / L trifluoroacetyl was added, the tubes were sealed, and the mixture was sonicated for 10 minutes. The tubes were then placed in beakers filled with water and heated in a heating mantle at 80, 85, 90, 95, and 100℃ respectively. Following step 3 of the Astragalus polysaccharide determination method, "after the hydrolysate has cooled, transfer it to a 100mL round-bottom flask," the process was repeated. After cooling, 3mL of distilled water was added to the round-bottom flask, and the mixture was sonicated for 30 minutes. After standing for 60 minutes, blood glucose levels were measured using a glucometer, and the glucose and polysaccharide contents were calculated. Table 4 shows that the higher the temperature, the greater the amount of polysaccharide hydrolyzed into monosaccharides, reaching its peak at 95℃. After 95℃, the glucose content decreased slightly. This may be because the excessively high hydrolysis temperature led to the oxidation of glucose by oxygen in the air. Therefore, the optimal hydrolysis temperature is 95℃.

[0063] Table 4 Effect of hydrolysis temperature on glucose percentage content

[0064]

[0065] Investigation of the liquid-to-material ratio:

[0066] Accurately weigh five portions of Astragalus polysaccharides (45, 30, 15, 7.5, and 5 mg respectively) and place them in separate 10 mL spiral test tubes. Add 15 mL of 2 mol / L trifluoroacetyl to achieve a material-to-liquid ratio of 3:1, 2:1, 1:1, 1:2, and 1:3 (since the amount of trifluoroacetic acid remains constant at 15 mL, only the amount of polysaccharides needs to be changed). Seal the tubes, sonicate for 10 min, and place them in a beaker filled with water. Place the beaker on a heating mantle and hydrolyze at 100 °C for 1.5 h. Then, follow step 3 of the Astragalus polysaccharide determination method, "transfer the hydrolysate to a 100 mL round-bottom flask after cooling." After cooling, add 3 mL of distilled water to the round-bottom flask, sonicate for 30 min, and let stand for 60 min. Measure the blood glucose level using a glucometer and calculate the glucose and polysaccharide content. Table 5 shows that the material-to-liquid ratio has a significant impact on the hydrolysis of polysaccharides. The glucose content reaches its highest level when the material-to-liquid ratio is 1:2 (at which point the polysaccharide content is 7.5 mg), and then gradually decreases. This phenomenon may be due to the following reasons: too much polysaccharide may lead to incomplete hydrolysis, while too little may cause glucose to be carbonized. Therefore, 7.5 mg of polysaccharide is considered the optimal dosage, i.e., a material-to-liquid ratio of 1:2.

[0067] Table 5. Effect of feed-to-liquid ratio on glucose percentage.

[0068]

[0069]

[0070] Experiments on optimizing the hydrolysis process of Astragalus polysaccharides using response surface methodology:

[0071] Based on the single-factor experiments, using the Design-Expert software and the Box-Behnken central composite experimental design principle, the hydrolysis conditions of Astragalus polysaccharide were optimized with three factors as independent variables: hydrolysis temperature (A), hydrolysis time (B), and polysaccharide dosage (C), and the percentage of glucose extracted from Astragalus hydrolysis as the response value. The response surface experimental factors and levels are shown in Table 6.

[0072] Table 6. Response Surface Experiment Factor Levels

[0073]

[0074] Results of response surface methodology:

[0075] Based on the optimal hydrolysis conditions of coltsfoot flower polysaccharide obtained from single-factor experiments, a three-factor, three-level response surface design was carried out. The three levels (-1, 0, 1) of the three factors were used as independent variables, and the percentage of glucose content measured by the blood glucose meter was used as the dependent variable (response value). The response surface experimental design and results are shown in Table 7.

[0076] Table 7 Response Surface Experimental Design and Results

[0077]

[0078]

[0079] The experimental data in Table 7 were analyzed using response surface methodology software. A multiple regression model was established, and the quadratic multinomial regression equations between hydrolysis temperature (°C), hydrolysis time (h), polysaccharide content (mg), and glucose percentage were obtained as follows:

[0080] Y=33.39+3.79A+4.27B+2.04C-1.74AB-0.14AC-0.15BC-5.42A 2 -5.4 2 B 2 -8.90C 2

[0081] Where Y represents the percentage content.

[0082] Analysis of variance using response surface methodology:

[0083] An ANOVA analysis of the binomial model was performed on the model, and the results are shown in Table 7.

[0084] Table 8. Analysis of Variance of the Regression Model

[0085]

[0086] A brief summary of the ANOVA: Table 7 shows that the p-value for the model terms is <0.0500, indicating that the model terms are significant. The lack-of-fit term of the model is significant (p>0.05), therefore, using this model to infer the change in glucose percentage caused by changes in factors is reliable. Furthermore, the F-values ​​show that the order of influence of the three factors on the hydrolysis of Astragalus polysaccharides is: B (time) > A (temperature) > C (solid-liquid ratio).

[0087] Response surface analysis:

[0088] Response surface methodology was used to analyze the experimental results, and 3D plots and contour plots were generated. This method is more conducive to analyzing the effects of the three factors on the percentage glucose content obtained from the hydrolysis of Astragalus polysaccharides.

[0089] Depend on Figures 1 to 6It can be observed that the curve for time has the greatest curvature, indicating that the change in glucose percentage caused by changing time is the most significant. In contrast, the effects of temperature and polysaccharide dosage on glucose percentage are not very significant, but the effect of temperature is slightly greater than that of polysaccharide dosage. This conclusion is consistent with the trend shown in the analysis of variance.

[0090] Response surface optimization criteria:

[0091] The optimized scheme based on the response surface methodology (RSM) predictions designed using Design Expert 8.0 is as follows: hydrolysis time 1.673 h, hydrolysis temperature 96.466 °C, and feed-to-liquid ratio 1:2. Under these conditions, the model prediction value is 34.797%.

[0092] in conclusion:

[0093] The optimal conditions predicted by response surface methodology were: time 1.67 h, temperature 96.46 °C, and polysaccharide dosage 7.774 mg. Based on actual operation and combined with single-factor results, the hydrolysis conditions were finally modified to: hydrolysis time 1.7 h, hydrolysis temperature 96 °C, and material-to-liquid ratio 1:2.

[0094] This experiment utilizes a novel method of hydrolyzing Astragalus polysaccharides with trifluoroacetic acid. The method is highly operable, yields stable experimental data, and can be applied in practical applications. However, it was found that oxygen in the air oxidizes glucose during the experiment; therefore, the hydrolysate should be allowed to cool to room temperature before removal. Furthermore, during vacuum distillation to remove trifluoroacetic acid, temperature also affects the measurement results; the process should be carried out at 80℃.

[0095] Example 4:

[0096] The effect of room temperature volume adjustment on the measurement results:

[0097] Accurately weigh 11.6 mg of glucose reference standard, dilute to 5 mL with distilled water, shake well, and store at room temperature protected from light. Measurements were taken at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6 hours, with three consecutive measurements within each 0.5-hour period, and the average value was calculated. The results are shown in Table 1. The results indicate that blood glucose levels measured within the first 4.5 hours after storage at room temperature protected from light are unstable and fluctuate significantly. After 4.5 hours, the blood glucose levels tend to stabilize with smaller errors. The effects are discussed in [the table below]. Figure 1 .

[0098] Table 1. Effect of room temperature volume adjustment on the measurement results

[0099]

[0100] The effect of 36℃ water bath volume adjustment on the test results:

[0101] Accurately weigh 11.6 mg of glucose reference standard, dilute to 5 mL with distilled water, shake well, and heat in a 36℃ water bath for 30 min. Measurements were taken at 0.5, 1, 1.5, 2, 2.5, and 3 h, with three consecutive measurements every 0.5 h, and the average value was calculated. The results are shown in Table 2. The results indicate that blood glucose levels measured within the first 1.5 h after heating the glucose solution to 36℃ in a water bath are unstable and fluctuate significantly. Blood glucose levels tend to stabilize after 1.5 h, with a smaller error. The effects are discussed in [the table below]. Figure 2 .

[0102] Table 2. Effect of 36℃ water bath volume adjustment on the test results.

[0103]

[0104] The effect of 80℃ water bath for volume fixation:

[0105] Accurately weigh 11.6 mg of glucose reference standard and dilute to 5 mL with distilled water, then shake well. Heat in an 80℃ water bath for 30 min, then remove and measure at 0.5, 1, 1.5, 2, 2.5, and 3 h, three times consecutively every 0.5 h, and calculate the average value. The results are shown in Table 3. The results indicate that the blood glucose levels measured within the first 2 h after heating the glucose solution in an 80℃ water bath for 30 min are unstable and fluctuate significantly. After 2 h, the blood glucose levels tend to stabilize with smaller errors. The effects are discussed in [the table below]. Figure 3 .

[0106] Table 3 Effect of 80℃ water bath for volume fixation

[0107]

[0108] The effect of ultrasound on measurement results:

[0109] Accurately weigh 11.6 mg of glucose reference standard and dilute to 5 mL with distilled water, then shake well. Sonicate the solution in an ultrasonicator for 30 min, then remove and measure the blood glucose levels at 15, 30, 60, 90, 120, and 180 min, repeating three times consecutively. Calculate the average value. The results are shown in Table 4. It can be seen that after 60 min of sonication, the blood glucose levels measured within the first hour are unstable and fluctuate significantly. After one hour, the blood glucose levels tend to stabilize with smaller errors. The effects are discussed in [the table below]. Figure 4 .

[0110] Table 4. Effects of ultrasound on measurement results

[0111]

[0112] Example 5:

[0113] I. Stability Assessment:

[0114] Four 6.0 mg D-glucose reference solutions were accurately weighed and placed in beakers, then diluted to 5 mL with distilled water in volumetric flasks. Reference solution 1 was incubated at room temperature for 4.5 h; reference solution 2 was heated in a 36℃ water bath for 30 min and then incubated at room temperature for 1.5 h; reference solution 3 was heated in an 80℃ water bath for 30 min and then incubated at room temperature for 2 h; and reference solution 4 was sonicated for 30 min and then incubated at room temperature for 60 min. The stability of the glucose solutions after different pretreatment methods was investigated at 0, 2, 4, 6, 8, 10, 12, and 24 h. Blood glucose levels were measured and recorded using a Sinocare Safe Blood Glucose Meter (no code adjustment required). The results are shown in Table 5. The results showed that: RSD (room temperature) > RSD (80℃ water bath) > RSD (36℃ water bath) > RSD (sonication), indicating that the reference solution had the best stability after sonication.

[0115] Table 5. Stability assessment of glucose after different pretreatments

[0116]

[0117] Based on the above analysis, when using a blood glucose meter to measure glucose levels, the most stable blood glucose values ​​are obtained by pre-treating glucose with ultrasound for 30 minutes and then allowing it to stand for 60 minutes.

[0118] II. Detection Limit Assessment:

[0119] Accurately weigh 132.7 mg of glucose reference standard and dilute to 25 mL with distilled water. Mix well. Accurately pipette 0.14, 0.18, 0.205, 0.24, 0.40, 0.60, 0.80, 1.00, 1.20, 1.40, 1.60, and 1.80 mL of the reference solution into 2 mL volumetric flasks. Sonicate for 30 min, let stand for 60 min, and then measure blood glucose levels six times consecutively using a glucometer. The results are shown in Table 6. The experimental results indicate that the instrument's stable measurement range is 1.1 mmol / L to 17.0 mmol / L.

[0120] Table 6. Examination of the instrument's measurement range

[0121]

[0122] By gradually diluting the reference standard, it was found that the instrument could not detect it when the blood glucose level was below 1.1 mmol / L. Therefore, 1.1 mmol / L is the detection limit of the instrument.

[0123] Examining linear relationships:

[0124] Accurately weigh 132.7 mg of the reference standard using an analytical balance with a concentration of 0.01%, and dilute to 25 mL with distilled water. Shake well to obtain a glucose reference solution with a concentration of 5.308 mg / mL. Accurately pipette 0.14, 0.18, 0.205, 0.24, 0.40, 0.60, 0.80, 1.00, and 1.20 mL of the reference solution into 2 mL volumetric flasks. Sonicate the diluted reference solution for 30 min, let it stand for 60 min, and then measure the blood glucose level using a Sinocare Safe Blood Glucose Meter (no calibration required). The results are shown in Table 13. A standard curve is plotted with blood glucose level (B) on the ordinate and glucose concentration (X) on the abscissa. Within the instrument's measurement range, the glucose standard concentration showed good linearity in the range of 0.5443–3.1848, with a linear correlation coefficient r = 0.9993 and a linear equation B = 5.1803x + 0.3552. The results are shown in [Figure number missing]. Figure 11 .

[0125] Table 13 Results of Standard Curve Measurement

[0126]

[0127]

[0128] III. Precision Examination: Accurately transfer 0.6 mL of a 5.308 mg / mL glucose standard solution to a 2 mL volumetric flask, sonicate for 30 min, let stand for 60 min, and then measure the glucose level 6 times consecutively using a Sinocare Safe Blood Glucose Meter (no calibration required). The results are shown in Table 14. As can be seen from the table, the RSD was 1.64% (n=6), indicating that the instrument precision is good and meets the requirements.

[0129] Table 14 Precision Experiment Results

[0130]

[0131] IV. Repeatability of Examinations

[0132] Six portions of Astragalus polysaccharide, each approximately 7.5 mg, were accurately weighed and placed in separate 10 mL spiral test tubes. 15 mL of 2 mol / L TFA was added, and the tubes were ultrasonically cleaned for 10 min to remove air. The tubes were then placed in a water-filled beaker and placed in a heating mantle at 96 °C for 1.7 hours for hydrolysis. After cooling, the hydrolysate was transferred to a 100 mL round-bottom flask and evaporated to dryness under reduced pressure in a water bath. TFA was removed by adding 2 mL of methanol four times, each time, until no sour taste remained. After cooling, a certain amount of distilled water was added to the round-bottom flask, and the tubes were ultrasonically cleaned for 30 min. After standing for 60 min, blood glucose levels were measured using a glucometer. Three measurements were taken for each sample, and the average value was calculated. The RSD was 2.52% (n=6), indicating good repeatability of the method. The results are shown in Table 15.

[0133] Table 15 Results of Repeatability Experiments

[0134]

[0135]

[0136] V. Recovery Rate Assessment

[0137] Six portions of Astragalus polysaccharide, each approximately 7.5 mg, were accurately weighed and placed into separate 10 mL spiral test tubes. 15 mL of 2 mol / L TFA was added, and the tubes were ultrasonically cleaned for 10 min to remove air. The tubes were then placed in a water-filled beaker and placed in a heating mantle at 96 °C for 1.7 hours for hydrolysis. After cooling, the hydrolysate was transferred to a 100 mL round-bottom flask and evaporated to dryness under reduced pressure in a water bath. The TFA was removed by adding 2 mL of methanol four times, each time, until no sour taste remained. After cooling, 1.5 mL of a 1.5924 mg·mL⁻¹ glucose standard solution was added to each flask, along with 3 mL of distilled water. The flasks were ultrasonicated for 30 min and allowed to stand for 60 min. Blood glucose levels were measured using a glucometer. After subtracting the blank sample, the glucose content was calculated using a regression equation. The percentage glucose content was calculated, and the recovery rate was calculated. The average recovery rate of this method was 97.49%, with an RSD of 2.48% (n=6). This indicates good recovery of the method. The results are shown in Table 16.

[0138] Table 16 Results of Sample Recovery Rate Experiment

[0139]

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining Astragalus polysaccharides, characterized in that, The method for determining Astragalus polysaccharides includes the following steps: Step 1: Accurately transfer multiple sets of glucose reference solutions into 2 mL volumetric flasks using a pipette. Place the diluted reference solutions into an ultrasonic instrument and sonicate for 30 min, then let stand for 60 min. Subsequently, measure the blood glucose value using an Anwen coded blood glucose meter. Plot a standard curve with the blood glucose value on the ordinate and the glucose concentration on the abscissa. Step 2: Extract polysaccharides from the medicinal materials and place them in a 10mL threaded test tube. Add 2mol / L trifluoroacetic acid at a material-to-liquid ratio of 1:2 and seal the tube. Place the tube in an ultrasonic cleaner and sonicate for 10 minutes to remove air. Then place the tube in a beaker and put the beaker in an electric heating mantle. Set the temperature to 96℃ and the time to 1.7h for hydrolysis. Step 3: After the hydrolysate has cooled, transfer it to a 100mL round-bottom flask and evaporate it to dryness by vacuum distillation in a water bath. Step 4: After cooling, add 3 mL of distilled water to a round-bottom flask, sonicate for 30 min and let stand for 60 min, then measure the glucose content with a blood glucose meter, subtract interference from the sample blank, and calculate the polysaccharide content in Astragalus membranaceus as glucose. Step 2, which describes the extraction of polysaccharides from medicinal materials, includes the following steps: Step 1: Weigh 100g of crude Astragalus membranaceus powder, add 300mL of 75% ethanol solution, desorb for 40min, add 12 times the volume of distilled water at 80℃, reduce the pressure to -0.0740MPa at 80℃, and reflux for 7min. Centrifuge the extract. Repeat the extraction twice starting from the addition of 12 times the volume of distilled water at 80℃, and combine the filtrates. Step 2: Concentrate the filtrate to 170 mL at 80 °C, add 3 times the volume of 80% ethanol solution while stirring continuously during the addition, then let it stand overnight. After overnight standing, filter the solution to obtain Astragalus polysaccharide. The formula for calculating the polysaccharide content in Astragalus membranaceus as measured by glucose in step 4 is as follows: In step 3, the process involves vacuum distillation to remove trifluoroacetic acid. During the distillation process, methanol is added repeatedly (2 mL each time) to the round-bottom flask to remove the trifluoroacetic acid until no sour taste remains.

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