Method for determining polysaccharide content in Yujin prescription capsule content

Abstract

The invention relates to the technical field of polysaccharide content detection, and provides a method for determining the polysaccharide content in Yujin prescription capsule contents, which comprises the following steps: taking Yujin prescription capsule contents, crushing, adding petroleum ether into powder, carrying out reflux extraction, and carrying out vacuum drying on filter residues to obtain the polysaccharide content in Yujin prescription capsule contents. Adding 1-3wt% of a compound enzyme system composed of cellulase, pectinase, papain, high-temperature-resistant-alpha-amylase and saccharifying enzyme into the degreased dry powder, adding water, uniformly mixing, and carrying out enzymolysis to obtain an enzymatic hydrolysate; carrying out enzyme deactivation, ultrasonic treatment and pressurized extraction, cooling to room temperature, carrying out suction filtration, and collecting final filtrate; preparing a crude polysaccharide test solution; measuring the content of crude polysaccharide in the crude polysaccharide test solution; preparing a reducing sugar test solution; determining the reducing sugar content in the reducing sugar test solution; and obtaining the polysaccharide content in the Yujin prescription capsule content based on the crude polysaccharide content and the reducing sugar content. According to the method provided by the invention, the detection accuracy of the polysaccharide content in the Yujin prescription capsule content is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharide content detection, and in particular to a method for determining the polysaccharide content in the contents of Yu Jin Fang capsules. BACKGROUND

[0002] Yu Jin Fang capsules are a compound preparation composed of multiple Chinese medicines. The contents of Yu Jin Fang capsules include ginseng, sea horse, prepared radix polygonati, rhizoma polygonati, actinidia, pig brain powder, and corn starch. Polysaccharides are one of the core active ingredients of most Chinese medicines, and have clear pharmacological effects such as immune regulation, antioxidant, anti-tumor, and hypoglycemic effects (for example, the tonifying effects of ginseng polysaccharides and radix polygonati polysaccharides in Yu Jin Fang, and the immune-enhancing effect of rubia polysaccharides). The polysaccharide content of Chinese medicines is directly related to the efficacy intensity. By determining the content, the potential efficacy of Chinese medicines can be predicted, and scientific basis can be provided for clinical dosage adjustment and compatibility optimization, so as to avoid poor efficacy caused by low polysaccharide content. The polysaccharide content of Chinese medicines is significantly affected by factors such as production place, harvesting time, processing method, and storage condition. Determining the polysaccharide content can establish a quantitative index, ensure the quality consistency of the same batch, different batches, and different sources of Chinese medicines (or compound preparations), and reduce the efficacy deviation caused by fluctuations in raw materials.

[0003] However, due to the complex composition of the contents of Yu Jin Fang capsules, including animal components, plant components, and excipients, the difficulty of extracting polysaccharides from various components is increased, and the difficulty of accurately determining the polysaccharide content is also increased. SUMMARY

[0004] To at least partially solve the above problems, the embodiments of the present application provide a method for determining the polysaccharide content in the contents of Yu Jin Fang capsules, comprising:

[0005] Step S1, taking the contents of Yu Jin Fang capsules, crushing to 120-150 meshes, adding 5-10 times the volume of petroleum ether to the powder, refluxing at 55-65℃ for 30-50 min, filtering and discarding the organic phase, placing the filter residue in a vacuum dryer at 45-55℃ for 20-40 min to obtain defatted dry powder, adding 1-3% by weight of a composite enzyme system composed of cellulase, pectinase, papain, high-temperature-resistant alpha-amylase, and saccharifying enzyme to the defatted dry powder, the mass ratio of cellulase: pectinase: papain: high-temperature-resistant alpha-amylase: saccharifying enzyme in the composite enzyme system being 1:1-2:0.3-0.5:1-2:1-2, mixing with water, adjusting the pH value to 5.5-6.5, and placing in a 55-60℃ enzyme solution to obtain an enzyme solution;

[0006] Step S2, heating the enzyme solution to boiling to inactivate the enzyme, cooling to room temperature, ultrasonic treating the sample suspension after inactivation of the enzyme, pressurized extracting the sample suspension after ultrasonic treatment, cooling to room temperature after extraction is completed, suction filtering, discarding the filter residue, and collecting the final filtrate;

[0007] Step S3, take part of the final filtrate, add anhydrous ethanol to make the system ethanol concentration of 75%~85%, 3~5℃ for 4h or more, 3500~4500r / min centrifugal 10~20min, discard the supernatant, the precipitate is washed with 75%~85% ethanol 2~5 times, each time centrifugal discard the supernatant, take the washed precipitate, dissolve and constant volume, obtain the crude polysaccharide test solution;

[0008] Step S4, determine the crude polysaccharide content in the crude polysaccharide test solution;

[0009] Step S5, take part of the final filtrate, add anhydrous ethanol to make the system ethanol concentration of 75%~85%, 3~5℃ for 4h or more, 3500~4500r / min centrifugal 10~20min, discard the supernatant; the precipitate is washed with 75%~85% ethanol 2~5 times, each time centrifugal discard the supernatant, take the washed precipitate, dissolve and constant volume, obtain the reducing sugar test solution;

[0010] Step S6, determine the reducing sugar content in the reducing sugar test solution;

[0011] Step S7, based on the crude polysaccharide content and the reducing sugar content, obtain the polysaccharide content in the Yujinfang capsule content.

[0012] Preferably, in step S1, 0.15~0.25mol / L phosphate buffer is used to adjust the pH value of the mixture to 5.5~6.5; after 0.5~2h of enzymolysis, iodine solution is added to the enzyme solution to test whether the starch excipients in the Yujinfang capsule content are completely hydrolyzed, and if not, the enzymolysis time is extended.

[0013] Preferably, in step S2, the ultrasonic treatment is at a frequency of 30~50Hz and a power of 400~600W for 0.5~2h; the pressure extraction is at a pressure of 0.08~0.15MPa and a constant temperature of 93~97℃ for 1~2h, with stirring every 15~25min; the filter membrane used for filtration has a pore size of 0.4~0.5μm.

[0014] Preferably, in step S4, the phenol-sulfuric acid method is used to determine the crude polysaccharide content in the crude polysaccharide test solution.

[0015] Preferably, step S4 includes: taking the crude polysaccharide test solution, adding 4~6% phenol solution after water replenishment, adding concentrated sulfuric acid at low temperature, boiling in a water bath for 8~12min, after cooling, using the corresponding reagent as blank, using ultraviolet spectrophotometer to scan at 400~550nm wavelength to determine the absorbance value.

[0016] Preferably, in step S4, the low temperature environment is 0~15℃, the volume ratio of concentrated sulfuric acid to crude polysaccharide test solution is 2.7:1~4.5:1, and the absorbance value is measured by scanning at a wavelength of 485nm±2nm using a UV-spectrophotometer.

[0017] Preferably, in step S6, the reducing sugar content in the reducing sugar test solution is determined using the 3,5-dinitrosalicylic acid colorimetric method.

[0018] Preferably, step S6 includes: taking the reducing sugar test solution, adding 3,5-dinitrosalicylic acid colorimetric reagent, mixing well, boiling in a water bath for 8-12 minutes, rapidly cooling to room temperature and adding water, using the corresponding reagent as a blank, and scanning at a wavelength of 400-800 nm using a UV-spectrum spectrophotometer to determine the absorbance value.

[0019] Preferably, the volume ratio of 3,5-dinitrosalicylic acid colorimetric reagent to reducing sugar test solution is 0.35:1 to 0.7:1, and the absorbance value is measured by scanning at a wavelength of 490 nm ± 2 nm using a UV-spectrum spectrophotometer.

[0020] Preferably, in step S7, the polysaccharide content of the contents of Yujinfang capsules = X 粗多糖 -X 还原糖 ;

[0021] X 粗多糖 X represents the crude polysaccharide content in the crude polysaccharide test solution. 还原糖 The reducing sugar content in the test sample solution.

[0022] The method for determining the polysaccharide content in the contents of Yujinfang capsules provided in the embodiments of this application detects the polysaccharide content of the traditional Chinese medicine components in the contents of Yujinfang capsules. Specifically, it removes lipids from the animal components in the capsule contents by petroleum ether reflux extraction, avoiding lipid substances covering the polysaccharide surface and reducing the polysaccharide extraction rate.

[0023] Furthermore, the complex enzyme system composed of cellulase, pectinase, papain, thermostable α-amylase, and saccharifying enzyme can rapidly break down the cell walls of plant components, promoting the release of polysaccharides from them. Simultaneously, it decomposes collagen and binding proteins in animal components (such as seahorse and pig brain powder), further promoting the release of polysaccharides from these components. In addition, it can completely decompose the starch excipients in the contents of Yujinfang capsules, avoiding any impact on the determination of polysaccharide content in the traditional Chinese medicine components. Existing complex enzymes are mostly mixed in equal proportions, without considering the content ratio of different components and the efficiency of enzyme action, resulting in the waste of some enzymes and the failure to achieve the desired effect due to insufficient enzymes. The formulation of this application is as follows: Plant components are adapted to: cellulase (1 part) + pectinase (1~2 parts), with the proportion of pectinase slightly higher or equal, because the decomposition of pectin in plant cell walls is more difficult than that of cellulose, requiring more pectinase to synergistically destroy the cell wall structure and ensure the full release of plant polysaccharides; Animal components are adapted to: papain (0.3~0.5 parts), with a lower proportion than other enzymes, because the content of animal components in the capsules is relatively less than that of plant components and excipients, and papain has high catalytic efficiency, so a small amount can decompose animal protein, avoiding excessive enzyme damage to the polysaccharide structure; Starch excipients are adapted to: heat-resistant α-amylase (1~2 parts) + saccharifying enzyme (1~2 parts), with the two enzymes working synergistically and in a proportion equal to that of cellulase, because the content of starch excipients is high, requiring stepwise hydrolysis by the two enzymes (α-amylase first decomposes starch into dextrin, and saccharifying enzyme then decomposes it into glucose), ensuring complete hydrolysis of starch and completely eliminating interference from excipients.

[0024] By subjecting the enzymatic hydrolysate to ultrasonic treatment and pressure extraction, the release of polysaccharide components from the plant and animal cells of traditional Chinese medicine is further promoted. This allows for the full extraction of polysaccharides with different solubilities (such as water-soluble polysaccharides in kiwifruit and acidic, insoluble polysaccharides in seahorse), while also preventing the degradation of heat-sensitive polysaccharides (such as kiwifruit polysaccharides and pig brain powder polysaccharides).

[0025] This invention improves the measurement of polysaccharide content by optimizing the selection of reagents and parameters in the above-mentioned multi-step process, especially by optimizing the use of the compound enzyme system and ultrasonic pressure. This makes the determination results of polysaccharide content in the sample closer to the true value, ensuring the consistency of quality of traditional Chinese medicine (or compound prescriptions) from the same batch, different batches, and different sources, and reducing the deviation in efficacy caused by fluctuations in raw materials.

[0026] In addition, polysaccharides may mechanically carry over small molecule impurities such as mono- and oligosaccharides during sedimentation, which can interfere with the color development of the phenol reaction. Therefore, in the preferred embodiment, by combining the phenol-sulfuric acid method with the 3,5-dinitrosalicylic acid colorimetric method, the crude polysaccharide content and reducing sugar content in the sample are determined separately, and the difference between the two is calculated. This can eliminate the influence of small molecule reducing sugars and non-sugar colorimetric substances on the determination results, and make the determination results of polysaccharide content in the sample closer to the true value. Attached Figure Description

[0027] Figure 1 This is the absorption spectrum of a sample obtained using the phenol-sulfuric acid method.

[0028] Figure 2 This is the sample absorption spectrum obtained by the 3,5-dinitrosalicylic acid colorimetric method. Figure 2 In the image, the upper curve represents the UV-spectrum photometry results of the reducing sugar test solution, and the lower curve represents the UV-spectrum photometry results of the DNS reagent.

[0029] Figure 3 It is the phenol-sulfuric acid method standard curve;

[0030] Figure 4 It is the standard curve of the 3,5-dinitrosalicylic acid colorimetric method. Detailed Implementation

[0031] The preferred embodiments of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection claimed in this application. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used is conventional equipment, and the equipment and materials used in each embodiment are the same.

[0032] Example

[0033] I. Polysaccharide Content Detection Method

[0034] 1. Instruments and reagents

[0035] 1.1 Instruments

[0036] Ultraviolet-Spectrophotometer, TU-1900, Beijing Purkinje General Instrument Co., Ltd.;

[0037] Ultrafine pulverizer, GM200, Beijing Greedman Machinery Co., Ltd.;

[0038] Vacuum drying oven, DZF-6050, Shanghai Yiheng Scientific Instruments Co., Ltd.;

[0039] Ultrasonic cleaner, KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.;

[0040] Pressure extraction tank, GS-1, Shanghai Shensheng Technology Co., Ltd.;

[0041] Centrifuge, TDZ4-WS, Changsha Xiangzhi Centrifuge Instrument Co., Ltd.;

[0042] Rotary mixer, AC100-240V, ABSON Scientific Instruments, USA;

[0043] Electric thermostatic water bath, XMTD-7000, Beijing Yongguangming Medical Instrument Co., Ltd.

[0044] 1.2 Test Drugs

[0045] Yujinfang Capsules, Luoyang Xinchundu Biopharmaceutical Co., Ltd., 250803, 251101;

[0046] Anhydrous ethanol, analytical grade, Yantai Shuangshuang Chemical Co., Ltd., 20241205;

[0047] Glucose, analytical grade, Tianjin Kemeo Chemical Reagent Co., Ltd., 20250116;

[0048] Phenol, analytical grade, Tianjin De'en Chemical Reagent Co., Ltd., 20161107;

[0049] Concentrated sulfuric acid, analytical grade, Luoyang Haohua Chemical Reagent Co., Ltd., 200116;

[0050] Disodium hydrogen phosphate (disodium hydrogen phosphate dodecahydrate), analytical grade, Tianjin Kemeo Chemical Reagent Co., Ltd., 20230224;

[0051] Sodium dihydrogen phosphate (sodium dihydrogen phosphate dihydrate), analytical grade, Yantai Shuangshuang Chemical Co., Ltd., 20220519;

[0052] Cellulase, Sigma-Aldrich, C1184-5KU;

[0053] Pectinase, Sigma-Aldrich, P2401-500UN;

[0054] Papain, Beijing Solarbio Technology Co., Ltd., P8160;

[0055] High-temperature resistant α-amylase, BR, Shanghai Maclean Biochemical Technology Co., Ltd., C14452933;

[0056] Glycoamylase, Tianjin Nuoxin Biotechnology Co., Ltd., G0010;

[0057] Iodine, analytical grade, Tianjin Kemeo Chemical Reagent Co., Ltd., 20130507;

[0058] Potassium iodide, analytical grade, Xilong Scientific Co., Ltd., B220224;

[0059] 3,5-Dinitrosalicylic acid (DNS) reagent, analytical grade, Ise-Hisa Biotechnology, 350646;

[0060] Petroleum ether, analytical grade, Yantai Shuangshuang Chemical Co., Ltd., 20241108;

[0061] The water used in the experiment was double-distilled water.

[0062] 2. Measurement Procedure

[0063] 2.1 Solution Preparation

[0064] 0.2 mol / L phosphate buffer: Mix 31.5 ml (0.2 mol / L) disodium hydrogen phosphate solution with 68.5 ml (0.2 mol / L) sodium dihydrogen phosphate thoroughly.

[0065] 5% phenol solution (W / V): Weigh 5.0g of purified phenol, dissolve it in water and bring the volume to 100ml (store at 4℃).

[0066] 80% ethanol solution (V / V): Add 80 ml of anhydrous ethanol to 20 ml of water and mix well.

[0067] 2.2 Preparation of reference solution

[0068] 2.2.1 Preparation of phenol-sulfuric acid method reference solution

[0069] Accurately weigh 0.5000g of dried, constant-weight analytical-grade glucose, dissolve it in water and bring the volume to 50ml. Dilute 100 times before use.

[0070] 2.2.2 Preparation of reference solution for the 3,5-dinitrosalicylic acid colorimetric method (DNS method)

[0071] Accurately weigh 0.2500g of dried, constant-weight analytical-pure glucose, dissolve it in water and bring the volume to 25ml. Dilute 20 times before use.

[0072] 2.3 Preparation of the test solution

[0073] 2.3.1 Sample Extraction

[0074] Take 1.5g of capsule contents, pulverize to 120-150 mesh, add 8 times the volume of petroleum ether to the powder, reflux at 60℃ for 40min, filter to discard the organic phase, and vacuum dry the filter residue at 50℃ for 30min to remove residual petroleum ether, obtaining defatted and dried powder. Place the defatted and dried powder in a 250ml Erlenmeyer flask, add 2% by weight of a compound enzyme system (the mass ratio of cellulase: pectinase: papain: thermostable α-amylase: saccharifying enzyme is 1:1:0.4:1:1), add 100ml of water, and adjust the pH to 6.0-6.5 with 0.2mol / L phosphate buffer. Stopper the flask and incubate at 55-60℃ for 1h for enzymatic hydrolysis (test for complete hydrolysis with iodine solution; if incomplete, extend the hydrolysis time until the enzymatic hydrolysate does not turn blue when iodine solution is added).

[0075] The enzymatic hydrolysate was heated to boiling in an electric furnace for 5 minutes to inactivate the enzyme, and then cooled to room temperature. The enzyme-inactivated sample suspension was sonicated at 500W and 40Hz for 1 hour. The sonicated suspension was then transferred to a pressure extraction vessel and extracted at 0.1MPa pressure and 95℃ for 1.5 hours, stirring every 20 minutes to ensure uniform extraction. After extraction, the solution was cooled to room temperature and filtered through a 0.45μm filter membrane. The filter residue was discarded, and the filtrate was collected and diluted to 200ml.

[0076] 2.3.2 Preparation of crude polysaccharide test solution

[0077] Accurately pipette 5.0 ml of the final filtrate from section 2.3.1 into a 50 ml centrifuge tube, add 20 ml of anhydrous ethanol, mix well, and incubate in a refrigerator for at least 4 hours. Centrifuge at 4000 rpm for 15 minutes, discard the supernatant, and wash the precipitate with several ml of 80% (v / v) ethanol. Centrifuge again and discard the supernatant. Repeat this process three times. Dissolve the precipitate in water and bring the volume to 25 ml for the determination of crude polysaccharide content.

[0078] 2.3.3 Preparation of reducing sugar test solution

[0079] Accurately pipette 20.0 ml of the final filtrate from section 2.3.1 into a 100 ml centrifuge tube, add 80 ml of anhydrous ethanol, mix well, and incubate in a refrigerator for at least 4 hours. Centrifuge at 4000 rpm for 15 minutes, discard the supernatant, and wash the residue with several ml of 80% (v / v) ethanol. Centrifuge again and discard the supernatant. Repeat this process three times. Dissolve the precipitate in water and bring the volume to 10 ml for the determination of reducing sugar content.

[0080] 2.4 Determination of detection wavelength

[0081] 2.4.1 Determination of the detection wavelength in the phenol-sulfuric acid method

[0082] Accurately pipette 1.0 ml of the crude polysaccharide test solution into a 25 ml colorimetric tube, add water to a final volume of 2.0 ml, add 1.0 ml of 5% phenol, and mix thoroughly using a vortex mixer. Under low temperature conditions, add 10.0 ml of concentrated sulfuric acid, mix thoroughly again using a vortex mixer, and incubate in a boiling water bath for 10 min. Remove from the water and rapidly cool to room temperature. Using the corresponding reagent as a blank, scan with a UV-spectrum spectrophotometer at a wavelength of 400–550 nm.

[0083] 2.4.2 Determination of the detection wavelength for the DNS method

[0084] Accurately pipette 2.0 ml of the reducing sugar test solution into a 25 ml graduated colorimetric tube, add 1.5 ml of DNS reagent, mix thoroughly with a vortex mixer, incubate in a boiling water bath for 10 min, remove, and rapidly cool to room temperature. Add water to a final volume of 15 ml, and mix thoroughly with a vortex mixer. Using the corresponding reagent as a blank, scan with a UV-spectrum spectrophotometer at a wavelength of 400–800 nm.

[0085] 2.5 Optimization of color development conditions

[0086] 2.5.1 Optimization of colorimetric conditions for the phenol-sulfuric acid method

[0087] (1) Investigation on the dosage of concentrated sulfuric acid

[0088] Accurately measure 1.0 ml of the test solution, add water to a final volume of 2.0 ml, add 1.0 ml of 5% phenol, and mix thoroughly using a vortex mixer. Then, under low temperature conditions, add 5.0, 7.0, and 10.0 ml of concentrated sulfuric acid respectively, mix thoroughly using a vortex mixer, and incubate in a boiling water bath for 10 minutes. Remove from the water and rapidly cool to room temperature. Using the corresponding reagent as a blank, measure the absorbance value at the optimal detection wavelength determined in section 2.4.1 using a UV-spectrum spectrophotometer.

[0089] (2) Temperature investigation when concentrated sulfuric acid is added

[0090] Accurately measure 1.0 ml of the crude polysaccharide test solution, add water to 2.0 ml, add 1.0 ml of 5% phenol, and mix with a rotary mixer. Add the amount of concentrated sulfuric acid determined in section 2.5.1(1) under low temperature, room temperature, and high temperature conditions, respectively, mix with a rotary mixer, boil in a water bath for 10 min, remove, and quickly cool to room temperature. Using the corresponding reagent as a blank, measure the absorbance value at the optimal detection wavelength determined in section 2.4.1 using a UV-spectrum spectrophotometer.

[0091] (3) Examination of color development time

[0092] Accurately measure 1.0 ml of the crude polysaccharide test solution, add water to 2.0 ml, add 1.0 ml of 5% phenol, mix with a rotary mixer, add the amount of concentrated sulfuric acid determined in section 2.5.1(1) at the temperature specified in section 2.5.1(2), mix with a rotary mixer, and boil in a water bath for 10, 20, and 30 min respectively. Remove and quickly cool to room temperature. Using the corresponding reagent as a blank, measure the absorbance value at the optimal detection wavelength determined in section 2.4.1 using a UV-spectrum spectrophotometer.

[0093] 2.5.2 Optimization of DNS-based color rendering conditions

[0094] (1) Examination of color development time

[0095] Accurately measure 2.0 ml of the reducing sugar test solution and place it in a 25 ml graduated colorimetric tube. Add 1.5 ml of DNS colorimetric reagent, mix thoroughly with a vortex mixer, and incubate in a boiling water bath for 7.5, 10, 12.5, and 15 min respectively. Remove the tube, cool rapidly to room temperature, add water to a final volume of 15 ml, and mix thoroughly with a vortex mixer. Using the corresponding reagent as a blank, measure the absorbance value at the optimal detection wavelength determined in section 2.4.2 using a UV-spectrum spectrophotometer.

[0096] (2) Investigation on the amount of color developer

[0097] Accurately measure 2.0 ml of the reducing sugar test solution and place it in a 25 ml graduated colorimetric tube. Add 1.0, 1.5, 2.0, and 2.5 ml of DNS colorimetric reagent, respectively. Mix thoroughly by rotating the mixer. After boiling in a water bath for the duration determined in section 2.5.2(1), remove the tube and cool it rapidly to room temperature. Add water to a final volume of 15 ml and mix thoroughly by rotating the mixer. Using the corresponding reagent as a blank, measure the absorbance value at the optimal detection wavelength determined in section 2.4.2 using a UV spectrophotometer.

[0098] 2.6 Methodological Examination

[0099] 2.6.1 Methodological Investigation of Phenol-Sulfuric Acid Process

[0100] (1) Examination of linear relationships

[0101] Accurately measure 0, 0.10, 0.20, 0.40, 0.60, 0.80, and 1.00 ml of the reference solution from section 2.2.1 into separate 25 ml colorimetric tubes. Add water to a final volume of 2.0 ml, then add 1.0 ml of 5% phenol solution. Mix thoroughly using a vortex mixer. Develop the colorimetric solution according to the optimized conditions described in section 2.5.1. Remove the tube and allow it to cool rapidly to room temperature. Using a UV-Vis spectrophotometer at the optimal detection wavelength determined in section 2.4.1, with the reagent blank solution as a reference, measure the absorbance value using a 1 cm cuvette. Plot a standard curve with glucose mass (mg) on ​​the x-axis and absorbance values ​​on the y-axis.

[0102] (2) Precision test

[0103] Take the reference solution under section 2.2.1 and measure it 6 times consecutively according to the method for investigating linearity in section 2.6.1 (1), and calculate the RSD value.

[0104] (3) Stability test

[0105] Take the same crude polysaccharide test solution and measure the absorbance at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 and 150 min respectively according to the method of linear relationship investigation in 2.6.1 (1), and calculate the RSD value.

[0106] (4) Repeatability test

[0107] Weigh out 6 portions of the contents of Yujinfang capsules and prepare them according to the preparation method in section 2.3. Measure the absorbance value according to the linear relationship investigation method in section 2.6.1(1) and calculate the crude polysaccharide content and RSD value of each sample.

[0108] 2.6.2 Methodological Examination of DNS Law

[0109] (1) Drawing the standard curve

[0110] Accurately measure 0, 0.50, 0.75, 1.00, 1.25, and 2.0 ml of the reference solution from section 2.2.2 into 25 ml graduated colorimetric tubes. Develop the colorimetric solution according to the optimized conditions described in section 2.5.2. Remove the tubes, rapidly cool them to room temperature, and measure the absorbance using a UV-spectrum spectrophotometer at the optimal detection wavelength determined in section 2.4.2, using the reagent blank solution as a reference, in a 1 cm cuvette. Plot a standard curve with glucose mass (mg) on ​​the x-axis and absorbance values ​​on the y-axis.

[0111] (2) Precision test

[0112] Take the reference solution under section 2.2.2 and measure it 6 times consecutively according to the method for investigating linearity in section 2.6.2 (1), and calculate the RSD value.

[0113] (3) Stability test

[0114] Take the same reducing sugar test solution and measure the absorbance at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 and 150 min respectively according to the method for investigating linear relationship in 2.6.2 (1), and calculate the RSD value.

[0115] (4) Repeatability test

[0116] Weigh out 6 portions of the contents of Yujinfang capsules and prepare them according to the preparation method in section 2.3. Measure the absorbance value according to the linear relationship investigation method in section 2.6.2(1) and calculate the reducing sugar content and RSD value of each sample.

[0117] 2.7 Sample Content Determination and Calculation

[0118] Weigh out 4 portions of the contents of Yujinfang capsules and prepare them according to the preparation method in section 2.3. Determine the absorbance value according to the linear relationship investigation method in section 2.6.2 (1). Calculate the content of crude polysaccharide, reducing sugar and polysaccharide in each sample, and calculate the average value and RSD value.

[0119] Calculation formula:

[0120] X — Crude polysaccharide or reducing sugar content in the sample (calculated as glucose) (mg / g);

[0121] m1 — Mass of glucose in the sample solution (mg);

[0122] m2 — Sample mass (g).

[0123] Yujinfang capsule polysaccharide content = X 粗多糖 -X 还原糖 .

[0124] 3. Specificity test of phenol-sulfuric acid method

[0125] Specific spectral scanning includes specific reagent spectral scanning, specific excipient spectral scanning, specific sample spectral scanning, and specific standard spectral scanning.

[0126] 3.1 Preparation of reference solution

[0127] Take 0.6 ml of the glucose reference solution under section 2.2.1 and prepare it as a reference solution according to the method.

[0128] 3.2 Preparation of the test solution

[0129] Weigh 1.5g of the contents of Yujinfang capsules and prepare it according to the method to serve as the test solution.

[0130] 3.3 Preparation of negative sample solution

[0131] For negative samples lacking ginseng, seahorse, processed Polygonum multiflorum, Polygonatum sibiricum, kiwifruit, and pig brain powder, prepare negative sample solutions according to the prescription under the quality standard.

[0132] 3.4 Preparation of blank reagent solution

[0133] Take 2.0 ml of water and place it in a 25 ml colorimetric tube. Add 1.0 ml of 5% phenol solution and mix with a vortex mixer. Quickly add 7 ml of concentrated sulfuric acid and mix with a vortex mixer. Heat in a boiling water bath for 10 minutes and quickly cool to room temperature to obtain the final product.

[0134] 3.5 Methods for Specific Spectral Scanning

[0135] The UV-spectral spectrophotometer was used to perform spectral scanning at wavelengths of 400–550 nm using a blank reagent solution as a reference and a 1 cm cuvette.

[0136] 4. Robustness Study of the Phenol-Sulfuric Acid Process

[0137] The robustness of the crude polysaccharide detection method was investigated by examining the impact of minor changes in key steps during sample processing on the determination of crude polysaccharide content. Robustness tests were conducted on the established experimental method, including experiments with different amounts of anhydrous ethanol, different centrifugation times, and different amounts of 5% phenol solution added (see Table 1).

[0138] Table 1 Durability Test Conditions

[0139]

[0140] II. Results Analysis

[0141] 1. Results of color development condition optimization

[0142] 1.1 Optimal colorimetric conditions for the phenol-sulfuric acid method

[0143] Experimental results show that the amount of concentrated sulfuric acid, water bath time, and reaction temperature all have varying degrees of influence on the absorbance value (see Tables 2-4). When adjusting the amount of concentrated sulfuric acid added, it was found that the reaction was incomplete when 5.0 ml was added, and the absorbance value was lower when 10.0 ml was added than when 7.0 ml was added; therefore, the optimal choice is to add 7.0 ml of concentrated sulfuric acid. When adjusting the ambient temperature at which the concentrated sulfuric acid is added, it was found that the reaction was incomplete at room temperature and high temperatures, while the reaction was best when added at low temperatures. When adjusting the boiling water bath time, it was found that the duration of the boiling water bath had little effect on the results; the reaction was complete after 10 minutes. In summary, the optimal color development conditions are: low temperature, 7 ml of concentrated sulfuric acid, and a boiling water bath for 10 minutes.

[0144] Table 2. Optimized colorimetric conditions for the phenol-sulfuric acid method - Results of experiments with different amounts of concentrated sulfuric acid added.

[0145]

[0146] Table 3. Optimized colorimetric conditions for the phenol-sulfuric acid method - experimental results at different reaction temperatures.

[0147]

[0148] Table 4. Results of Optimized Colorimetric Conditions for Phenol-Sulfuric Acid Method with Different Water Bath Times

[0149]

[0150] 1.2 Optimal color rendering conditions for DNS method

[0151] Experimental results showed that factors such as color development time and the amount of colorimetric reagent had varying degrees of influence on the absorbance value (see Tables 5 and 6). Adjusting the color development time had a relatively small impact on the results, but the absorbance value was highest at 10 minutes; therefore, the optimal color development time was 10 minutes. Adjusting the amount of colorimetric reagent resulted in the best color development results when 1.0 ml was added. In summary, the optimal color development conditions for using the DNS method to investigate the reducing sugar content in Yujinfang capsules were 1.0 ml of colorimetric reagent and 10 minutes of color development.

[0152] Table 5. Results of experiments on DNS-optimized color development conditions with different color development times.

[0153]

[0154] Table 6. Optimized colorimetric conditions for the DNS method - Results of experiments with different amounts of colorimetric reagent.

[0155]

[0156] 2. Determination of detection wavelength

[0157] 2.1 Determination of the detection wavelength in the phenol-sulfuric acid method

[0158] According to the scanning results of the ultraviolet-spectrum spectrophotometer, the crude polysaccharide test solution of Yujinfang capsules has the maximum absorption in the range of 485nm±2nm. Figure 1 Therefore, the optimal detection wavelength for the phenol-sulfuric acid method was determined to be 485 nm.

[0159] 2.2 Determination of the detection wavelength for the DNS method

[0160] Since DNS reagent itself has ultraviolet absorption, to prevent it from interfering with the ultraviolet absorption after the reducing sugar color development, the DNS colorimetric reagent and the reducing sugar test solution were subjected to spectral scanning respectively. According to the scanning results of the ultraviolet-spectrum spectrophotometer, the reducing sugar test solution of Yujinfang capsules had the maximum absorption in the range of 490nm±2nm (…). Figure 2 Furthermore, it is not interfered with by the ultraviolet absorption of the DNS colorimetric reagent itself, therefore the optimal detection wavelength for the DNS method is determined to be 490 nm.

[0161] 3. Methodological Investigation Results

[0162] 3.1 Results of Methodological Investigation of Phenol-Sulfuric Acid Process

[0163] 3.1.1 Results of the linear relationship examination

[0164] Accurately pipette 0, 0.10, 0.20, 0.40, 0.60, 0.80, and 1.00 ml of glucose standard working solution (0.1004 mg / ml), and measure the absorbance values ​​according to the prescribed method. Plot a standard curve with glucose mass as the x-axis and absorbance values ​​as the y-axis. The equation of the standard curve is obtained as y = 7.1259x + 0.0051, R0. 2 = 0.9999, showing good linearity in the range of 10.04 ~ 100.4 μg (see... Figure 3 ).

[0165] 3.1.2 Precision Test Results

[0166] As shown in Table 7, the RSD value of the precision test results is 0.10%, indicating that the precision of the phenol-sulfuric acid method is good and meets the requirements.

[0167] Table 7 Precision test results of phenol-sulfuric acid method

[0168]

[0169] 3.1.3 Repeatability Test Results

[0170] As shown in Table 8, the average crude polysaccharide content of the test solution was 43.1734 mg / g, and the RSD value was 1.11%, indicating that the phenol-sulfuric acid method for determining the crude polysaccharide content of Yujinfang capsules has good repeatability and meets the requirements.

[0171] Table 8 Results of repeatability tests using the phenol-sulfuric acid method

[0172]

[0173] 3.1.4 Stability Test Results

[0174] As shown in Table 9, the RSD value of the stability test results is 1.28%, indicating that the stability of the crude polysaccharide content of Yujinfang capsules determined by the phenol-sulfuric acid method is good and meets the requirements.

[0175] Table 9 Results of stability tests using the phenol-sulfuric acid method

[0176]

[0177] 3.2 Results of the Methodological Examination of the DNS Approach

[0178] 3.2.1 Results of the linear relationship examination

[0179] Accurately pipette 0, 0.50, 0.75, 1.00, 1.25, and 2.00 ml of glucose standard working solution (0.5012 mg / ml), and measure the absorbance values ​​according to the prescribed method. Plot a standard curve with glucose mass as the x-axis and absorbance values ​​as the y-axis. The equation of the standard curve is obtained as y = 0.8423x - 0.0819, R0. 2 = 0.9993, showing good linearity in the range of 0.2506 ~ 1.0024 mg (see...). Figure 4 ).

[0180] 3.2.2 Precision Test

[0181] As shown in Table 10, the RSD value of the DNS method precision test results is 0.18%, indicating that the DNS method has good precision and meets the requirements.

[0182] Table 10 Precision test results of DNS method

[0183]

[0184] 3.2.3 Repeatability Test Results

[0185] As shown in Table 11, the average reducing sugar content of the test solution was 9.5145 mg / g, and the RSD value was 1.73%, indicating that the DNS method for determining the reducing sugar content of Yujinfang capsules has good repeatability and meets the requirements.

[0186] Table 11 Results of DNS Method Repeatability Tests

[0187]

[0188] 3.3.4 Stability Test Results

[0189] As shown in Table 12, the RSD value of the stability test results is 1.05%, indicating that the DNS method for determining the reducing sugar content of Yujinfang capsules has good stability and meets the requirements.

[0190] Table 12 Results of DNS Method Stability Test

[0191]

[0192] 4. Content Calculation Results

[0193] Four portions of 1.5g of the contents of Yujinfang capsules were weighed, prepared according to the method, and their absorbance values ​​were measured. The calculation results show that the average crude polysaccharide content in Yujinfang capsules was 43.1684 mg / g, the average reducing sugar content was 10.5168 mg / g, and the average polysaccharide content was 33.1015 mg / g. The calculated RSD value was 1.52% (see Table 13).

[0194] Table 13 Calculation results of polysaccharide content in Yujinfang capsules

[0195]

[0196] 5. Specificity test results of phenol-sulfuric acid method

[0197] Negative samples were prepared according to the established method. Spectroscopic scanning was performed according to the method, including specific reagents (using a blank reagent as a reference), specific excipients, specific samples, and specific standards.

[0198] The blank reagent solution and negative sample solution showed no maximum absorption in the range of 485nm±2nm, while the test solution and reference solution showed maximum absorption in the same range. The detection of these four solutions indicates that the blank reagent and blank excipient did not interfere with the determination results of crude polysaccharides, and the specificity of this method meets the requirements.

[0199] 6. Results of the phenol-sulfuric acid method durability test

[0200] The established method was subjected to a robustness test, with conditions including different amounts of anhydrous ethanol, different centrifugation times, and different amounts of 5% phenol solution added.

[0201] During the sample processing, a comparative experiment was conducted on different amounts of anhydrous ethanol added, with 18 ml, 20 ml, and 22 ml added respectively. All other steps were performed in accordance with the procedure. The results are shown in Table 14. There was no significant difference in the test results of the sample solution under different amounts of anhydrous ethanol added.

[0202] During the sample processing, a comparative experiment was conducted at different centrifugation times, namely 4 min, 5 min, and 6 min. All other steps were performed in accordance with the same procedure. The results are shown in Table 15. There was no significant difference in the test results of the sample solution under different centrifugation time conditions.

[0203] During the sample processing, a comparative experiment was conducted on different amounts of 5% phenol solution added, namely 0.9 ml, 1.0 ml, and 1.1 ml. All other steps were performed in accordance with the procedure. The results are shown in Table 16. There was no significant difference in the test results of the sample solution under different amounts of 5% phenol solution added.

[0204] In summary, different amounts of anhydrous ethanol, different centrifugation times, and different amounts of 5% phenol solution had minimal impact on the method, with no significant differences in the results. This indicates that minor changes in the key steps have no effect on the determination of crude polysaccharide content, and the robustness of the method meets the requirements.

[0205] Table 14 Experimental results with different amounts of anhydrous ethanol added

[0206]

[0207] Table 15 Experimental results at different centrifugation times

[0208]

[0209] Table 16 Results of different addition amounts of 5% phenol solution

[0210]

[0211] Based on the various embodiments of this application described above, in the absence of explicit denial or conflict, the technical features of one embodiment may be advantageously combined with one or more other embodiments.

[0212] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application. The scope of this application is defined by the appended claims.

Claims

1. A method for determining the polysaccharide content in the contents of Yujinfang capsules, characterized in that, include: Step S1: Take the contents of Yujinfang capsules, pulverize to 120-150 mesh, add 5-10 times the volume of petroleum ether to the powder, reflux at 55-65℃ for 30-50 min, filter to discard the organic phase, and place the filter residue in a vacuum dryer at 45-55℃ for 20-40 min to obtain defatted dry powder. Add 1-3% by weight of a complex enzyme system composed of cellulase, pectinase, papain, thermostable α-amylase, and saccharifying enzyme to the defatted dry powder. In the complex enzyme system, the mass ratio of cellulase: pectinase: papain: thermostable α-amylase: saccharifying enzyme is 1:1-2:0.3-0.5:1-2:1-2. Add water and mix well, adjust the pH to 5.5-6.5, and place in a 55-60℃ environment for enzymatic hydrolysis to obtain the enzymatic hydrolysate. Step S2: Heat the enzyme hydrolysate to boiling to inactivate the enzyme, cool it to room temperature, sonicate the enzyme-inactivated sample suspension, extract the sonicated sample suspension under pressure, cool it to room temperature after extraction, filter it, discard the filter residue, and collect the final filtrate. Step S3: Take a portion of the final filtrate, add anhydrous ethanol to make the ethanol concentration of the system reach 75%~85%, let it stand at 3~5℃ for more than 4 hours, centrifuge at 3500~4500r / min for 10~20min, discard the supernatant, wash the precipitate with 75%~85% ethanol 2~5 times, discard the supernatant after each centrifugation, take the washed precipitate, dissolve and make up to volume to obtain the crude polysaccharide test solution; Step S4: Determine the crude polysaccharide content in the crude polysaccharide test solution; Step S5: Take a portion of the final filtrate, add anhydrous ethanol to make the ethanol concentration of the system reach 75%~85%, let it stand at 3~5℃ for more than 4 hours, centrifuge at 3500~4500r / min for 10~20min, and discard the supernatant; wash the precipitate with 75%~85% ethanol 2~5 times, discard the supernatant after each centrifugation, take the washed precipitate, dissolve and make up to volume to obtain the reducing sugar test solution; Step S6: Determine the reducing sugar content in the reducing sugar test solution; Step S7: Obtain the polysaccharide content in the contents of Yujinfang capsules based on the crude polysaccharide content and the reducing sugar content.

2. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 1, characterized in that, In step S1, the pH of the mixture liquid is adjusted to 5.5-6.5 using a 0.15-0.25 mol / L phosphate buffer solution. After enzymatic hydrolysis for 0.5-2 hours, iodine solution is added to the hydrolysate to check whether the starch excipient in the contents of Yujinfang capsules has been completely hydrolyzed. If not, the hydrolysis time is extended.

3. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 1, characterized in that, In step S2, during ultrasonic treatment, the frequency is 30~50Hz, the power is 400~600W, and the time is 0.5~2h; during pressure extraction, the pressure is 0.08~0.15MPa, the temperature is 93~97℃, and the extraction is carried out for 1~2h, with stirring once every 15~25min during the process. When using vacuum filtration, the filter membrane used has a pore size of 0.4~0.5μm.

4. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 1, characterized in that, In step S4, the crude polysaccharide content in the crude polysaccharide test solution is determined using the phenol-sulfuric acid method.

5. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 4, characterized in that, Step S4 includes: taking the crude polysaccharide test solution, adding water and then adding 4-6% phenol solution, adding concentrated sulfuric acid at low temperature, boiling water bath for 8-12 minutes, cooling, using the corresponding reagent as a blank, and scanning at a wavelength of 400-550 nm using a UV-spectrum spectrophotometer to determine the absorbance value.

6. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 5, characterized in that, In step S4, the low temperature environment is 0~15℃, the volume ratio of concentrated sulfuric acid to crude polysaccharide test solution is 2.7:1~4.5:1, and the absorbance value is measured by scanning at a wavelength of 485nm±2nm using a UV-spectrophotometer.

7. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 1, characterized in that, In step S6, the reducing sugar content in the reducing sugar test solution is determined using the 3,5-dinitrosalicylic acid colorimetric method.

8. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 7, characterized in that, Step S6 includes: taking the reducing sugar test solution, adding 3,5-dinitrosalicylic acid colorimetric reagent, mixing well, boiling in a water bath for 8-12 minutes, rapidly cooling to room temperature and adding water, using the corresponding reagent as a blank, and scanning at a wavelength of 400-800 nm using a UV-spectrum spectrophotometer to determine the absorbance value.

9. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 8, characterized in that, The volume ratio of 3,5-dinitrosalicylic acid colorimetric reagent to reducing sugar test solution was 0.35:1 to 0.7:

1. The absorbance value was measured using a UV-spectrum spectrophotometer at a wavelength of 490 nm ± 2 nm.

10. The method for determining the polysaccharide content in the contents of Yujinfang capsules according to claim 1, characterized in that, In step S7, The polysaccharide content of Yujinfang capsules = X 粗多糖 -X 还原糖 ; X 粗多糖 X represents the crude polysaccharide content in the crude polysaccharide test solution. 还原糖 The reducing sugar content in the test solution.

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