A method for processing, extracting, and detecting Polygonatum sibiricum.

Abstract

This invention relates to the field of traditional Chinese medicine processing and testing technology, specifically to a method for processing, extracting, and detecting Polygonatum sibiricum. The method includes the following steps: taking raw Polygonatum sibiricum, adding 20% ​​of its weight in rice wine, mixing well, allowing it to soak, steaming it thoroughly in a steamer, removing it, letting it cool slightly, mixing it back into the steaming liquid, letting it cool slightly again, and then drying it. This process is repeated 9 times. The processed, steamed Polygonatum sibiricum is then taken, decocted with 10-12 times its weight in water, and the decoction is centrifuged. The resulting centrifuged liquid is concentrated and then centrifuged again to obtain the final product. This application provides a detection method suitable for Polygonatum sibiricum extract, through which a Polygonatum sibiricum extract with a high content of effective components is obtained.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine processing and testing technology, specifically to a method for processing, extracting, and testing Polygonatum sibiricum. Background Technology

[0002] Polygonatum is the dried rhizome of *Polygonatum kingianum* Coll. et Hemsl., *Polygonatum sibiricum* Red., or *Polygonatum cyrtonema* Hua, belonging to the Liliaceae family. It possesses the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. Polygonatum is not suitable for direct clinical application. Although it is not toxic, it has a numbing taste, and direct consumption can lead to side effects such as sore throat, dry tongue, itchy skin, and dizziness. The Chinese Pharmacopoeia specifies that Polygonatum should be thoroughly stewed or steamed using wine-based methods, then dried, sliced, and dried again. It stipulates that 20 kg of rice wine should be used for every 100 kg of Polygonatum. This wine-processing eliminates the irritation and improves the unpleasant taste of Polygonatum. Compared to the processed wine-processed Polygonatum, the extract obtained from the wine-processed Polygonatum does not require decoction and can be taken directly, making it more convenient and faster to use. The Pharmacopoeia does not disclose the specific processing and extraction techniques and parameters for the Polygonatum extract. Based on this, it is necessary to propose a method for processing and extracting Polygonatum sibiricum to prepare Polygonatum sibiricum extract, and to detect the effective components in the prepared Polygonatum sibiricum extract to determine the optimal process and process parameters for Polygonatum sibiricum processing and extraction. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for processing, extracting, and detecting Polygonatum sibiricum. It clarifies the processing and extraction techniques and parameters for Polygonatum sibiricum extract and establishes a method for detecting the content of key polysaccharides and flavonoids in the extract. The optimized method yields Polygonatum sibiricum extract with a high content of effective components.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The purpose of this invention is to provide a method for processing and extracting Polygonatum sibiricum, comprising the following steps:

[0006] (1) Take raw Polygonatum, add 20% of the weight of Polygonatum in rice wine, mix well and let it soak, steam it thoroughly in a steamer, take it out, let it cool slightly, mix it back into the steaming liquid, let it cool slightly and then dry it.

[0007] (2) Repeat step (1) 9 times;

[0008] (3) Take the wine-steamed Polygonatum sibiricum processed in step (2), add 10-12 times the amount of water and decoct, then centrifuge the decoction;

[0009] (4) Concentrate the centrifuged liquid obtained in step (3) and then centrifuge it to obtain the final product.

[0010] The processing of Polygonatum sibiricum using the alcohol-steaming method can be traced back to the Tang Dynasty in my country. This method effectively eliminates the irritation of Polygonatum sibiricum, improves its unpleasant taste, and combines it with the blood-activating properties of rice wine for enhanced tonic effects. This application, based on the existing alcohol-steaming process, further extracts and concentrates the extracted Polygonatum sibiricum to obtain an extract that can be taken directly without decoction and is easily absorbed. This application optimizes the processing and extraction process and parameters, and uses water as the extraction solvent to determine the extraction process and parameters. The proposed method has a good extraction effect on polysaccharides and flavonoids in Polygonatum sibiricum. The prepared Polygonatum sibiricum extract has a high content of effective components. The extraction process is simple and easy to operate, and no harmful waste liquid is generated during the extraction process, making it highly valuable for industrial application.

[0011] Furthermore, step (1) also includes slicing the raw Polygonatum sibiricum into slices with a slice thickness of 3-4 mm.

[0012] Furthermore, in step (1), high-pressure humidification is used, with a humidification pressure of 0.12 MPa and a humidification time of 4 hours. High-pressure humidification can effectively shorten the humidification time, and the prepared Polygonatum extract has a high content of effective components.

[0013] Furthermore, a butterfly centrifuge is used in step (3), and a tube centrifuge is used in step (4). The butterfly centrifuge has the advantages of large single-pass processing capacity and good clarification effect, while the tube centrifuge can further separate the tiny particles in the concentrate. This application uses a method of first separating the decoction with a butterfly centrifuge and then separating the concentrate with a tube centrifuge, which can greatly improve the overall separation efficiency. Moreover, the prepared Polygonatum extract is free of residue and has good clarity.

[0014] Furthermore, in step (3), the amount of water used for decocting is 10 times, the decocting time is 40-60 minutes, and the decocting is performed twice.

[0015] The Chinese Pharmacopoeia specifies that the content of Polygonatum polysaccharides is expressed as glucose content and describes the method for determining the glucose content in Polygonatum using ultraviolet-visible spectrophotometry. Existing research indicates that the main active components of Polygonatum include Polygonatum polysaccharides, Polygonatum flavonoids, and Polygonatum saponins. Therefore, characterizing only the sugar content in Polygonatum is insufficient to comprehensively evaluate the effective components in Polygonatum extract. Polygonatum saponins, belonging to the steroidal saponin class, are present in extremely low amounts in Polygonatum extract. This application proposes a method for detecting Polygonatum polysaccharides and Polygonatum flavonoids in Polygonatum extract using high-performance liquid chromatography (HPLC). By optimizing the chromatographic conditions, this method can simultaneously determine flavonoids and sugars, with Polygonatum polysaccharides expressed as glucose and Polygonatum flavonoids expressed as vitexin.

[0016] Specifically, the present invention provides a method for detecting processed extracts of Polygonatum sibiricum, the method being used for Polygonatum sibiricum extracts processed by the above method, the detection method comprising:

[0017] S1: Accurately measure the extract of Polygonatum sibiricum to be tested, dilute with water, shake well, filter, and take the filtrate as the test solution;

[0018] S2: Take glucose and vitexin as reference standards, weigh them accurately, dissolve them in water, dilute to the mark, shake well, filter, and take the filtrate as the reference solution;

[0019] S3: The test solution and the reference solution were detected by high performance liquid chromatography (HPLC). The HPLC method used an evaporative light scattering detector, a Lichrospher NH2 column (4.6 mm × 250 mm, 5 μm), and a mobile phase of 0.1% formic acid-water-methanol.

[0020] Furthermore, high performance liquid chromatography was performed using 0.1% formic acid water-methanol at a volume ratio of 80:20 for isocratic elution.

[0021] Furthermore, in the high-performance liquid chromatography described in S3, the column temperature is 25℃, the detector drift tube temperature is 50℃, the carrier gas is air, the flow rate is 1.2L / min, and the injection volume is 10μL.

[0022] Further, the preparation method of the test solution in S1 is as follows: accurately measure 1 ml of the extract of Polygonatum sibiricum to be tested, dilute with water to 50 ml, shake well, filter, and take the filtrate to obtain the solution;

[0023] Further, the preparation method of the reference solution described in S2 is as follows: accurately weigh about 8 mg of glucose and about 1.5 mg of vitexin, place them in a 10 ml volumetric flask, add water to dissolve, dilute to the mark, shake well, filter, and take the filtrate to obtain the solution.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. The processing and extraction method of Polygonatum sibiricum provided in this application clarifies the specific processing and extraction process and parameters for preparing Polygonatum sibiricum extract, resulting in a high content of polysaccharides and flavonoids in the prepared extract. Furthermore, this application optimizes the processing technology and parameters of Polygonatum sibiricum extract, significantly shortening the product processing cycle and effectively improving product processing efficiency while ensuring product quality.

[0026] 2. This application proposes a high-performance liquid chromatography (HPLC) method for detecting polysaccharides and flavonoids in Polygonatum extract. The method uses ELSD for detection and optimizes the chromatographic conditions. The method has good response to both glucose and vitexin, good linear range, and high detection accuracy. Attached Figure Description

[0027] Figure 1 The chromatogram of the test solution under the chromatographic conditions of this application is shown.

[0028] Figure 2 The chromatogram of the reference solution under the chromatographic conditions of this application is shown.

[0029] Figure 3 The chromatogram of the test solution using mobile phase ① is shown below.

[0030] Figure 4 The chromatogram of the test solution using mobile phase ② is shown below.

[0031] Figure 5 For glucose standard curve;

[0032] Figure 6 This is a standard curve diagram of vitexin. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise specified, all methods described herein are conventional methods, and all raw materials described herein are available from publicly available commercial sources.

[0035] Example 1: Processing and Extraction Technology of Polygonatum

[0036] (1) Take raw Polygonatum, cut it into 3-4 mm slices, add 20% of the weight of raw Polygonatum in rice wine, mix well, let it soak for 20 hours, steam it thoroughly in a steamer, take it out, let it cool slightly, mix it back into the steaming liquid, let it cool slightly, and then dry it at 45℃ for 20 hours.

[0037] (2) Repeat step (1) 9 times;

[0038] (3) Take the processed Polygonatum sibiricum, add 10 times the amount of water and decoct twice, 60 minutes each time. Centrifuge the decoction in a butterfly manner.

[0039] (4) The centrifuged liquid is concentrated to the weight of Polygonatum in step (3), and then centrifuged in a tube to obtain the Polygonatum extract. The Polygonatum extract obtained by the above method is dark brown in appearance and has no obvious impurities or precipitates.

[0040] Example 2: Establishment of a detection method for Polygonatum extract

[0041] 1. Instruments and reagents

[0042] 1.1 Instruments: Shimadzu LC-20AD high performance liquid chromatograph, METTLER MS205DU electronic balance, Alltech ELSD 2000ES evaporative light scattering detector.

[0043] 1.2 Reagents: Glucose (analytical grade, Sinopharm (Shanghai) Reagent Co., Ltd.), Vitexin (China National Institutes for Food and Drug Control), purified water (Hangzhou Wahaha Group Co., Ltd.), methanol (chromatographic grade, Sinopharm (Shanghai) Reagent Co., Ltd.), acetonitrile (chromatographic grade, Sinopharm (Shanghai) Reagent Co., Ltd.), formic acid (analytical grade, Sinopharm (Shanghai) Reagent Co., Ltd.).

[0044] 2. Solution preparation

[0045] Accurately measure 1 ml of the extract of Polygonatum sibiricum to be tested, dilute with water to 50 ml, shake well, filter, and take the filtrate as the test solution;

[0046] Weigh approximately 8 mg of glucose and 1.5 mg of vitexin accurately, place them in a 10 ml volumetric flask, add water to dissolve, dilute to the mark, shake well, filter, and take the filtrate as the reference solution.

[0047] Weigh approximately 8 mg of glucose and 1.5 mg of vitexin accurately, place them in 10 ml volumetric flasks, add water to dissolve, dilute to the mark, shake well, filter, and take the filtrate as a single reference solution for glucose and vitexin.

[0048] 3. Chromatographic conditions:

[0049] A Lichrospher NH2 column (4.6 mm × 250 mm, 5 μm) was used; the column temperature was 25 °C; the detector was ELSD; the drift tube temperature was 50 °C; the carrier gas was air, with a flow rate of 1.2 L / min; and the injection volume was 10 μL. The mobile phase consisted of 0.1% formic acid in water and methanol at a volume ratio of 80:20, with isocratic elution.

[0050] 4. Sample Determination

[0051] The Polygonatum extract prepared in Example 1 was used to prepare a test solution according to the method under "2. Solution Preparation". The chromatogram was determined according to the method under "3. Chromatographic Conditions", and the chromatogram was recorded. The chromatogram of the test solution is shown in [reference needed]. Figure 1 As shown. A mixed reference solution of glucose and vitexin was injected and analyzed using the same method. The chromatogram of the reference solution is shown below. Figure 2 As shown, peak 1 is vitexin and peak 2 is glucose. The identification of peaks 1 and 2 was determined by injection and detection of glucose and vitexin single reference solutions under the same chromatographic conditions.

[0052] The Polygonatum extract prepared in Example 1 was used to prepare a test solution according to "2. Solution Preparation". The chromatographic conditions were determined according to the method described in "3. Chromatographic Conditions". The difference was the use of two different mobile phases: mobile phase ①: water-acetonitrile (volume ratio 40:60); mobile phase ②: water-methanol (volume ratio 80:20). The chromatogram of mobile phase ① is shown below. Figure 3 As shown, the chromatogram of mobile phase ② is as follows. Figure 4 As shown: From Figure 3 It can be seen that vitexin cannot be completely separated from adjacent peaks. From Figure 4 It can be seen that vitexin can be separated from adjacent peaks, but most peak responses are relatively different. Figure 1 Lower.

[0053] 5. Methodological Examination

[0054] 5.1 Examination of Linear Relationships

[0055] Accurately pipette 1, 2, 4, 6, 8, and 10 ml of the single reference solution from section "2. Solution Preparation" into separate 10 ml volumetric flasks. Dilute each flask to the mark with water to prepare a series of concentration solutions of glucose and vitexin. Inject the solutions and perform the determination according to the method described in section "3. Chromatographic Conditions," recording the chromatograms. Perform linear regression analysis with the logarithm of peak area as the ordinate (y) and the logarithm of injection volume as the abscissa (x). The glucose standard curve is shown below. Figure 5 As shown, the regression equation is y = 0.5281x + 5.8742, with a correlation coefficient of 0.9996, indicating a good linear relationship between glucose concentration and peak area in the range of 0.08–0.8 mg / ml; the standard curve of vitexin is shown below. Figure 6 As shown, the regression equation is y=0.6829x+5.6694, and the correlation coefficient is 0.9999, indicating that the concentration of vitexin in the range of 0.015 to 0.15 mg / ml has a good linear relationship with the peak area.

[0056] 5.2 Precision Test

[0057] Accurately pipette 10 μL each of glucose and vitexin reference solutions and inject them 6 times consecutively. Perform the determination according to the method under "3. Chromatographic Conditions" and record the peak area. The results show that the peak area RSDs of glucose and vitexin are 1.3% and 2.4%, respectively, indicating that the method has good precision.

[0058] 5.3 Stability Test

[0059] Accurately pipette 10 μL of each test solution and perform determination at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h according to the method under "3. Chromatographic Conditions". The results show that the peak area RSDs of glucose and vitexin are 0.9% and 2.6%, respectively, indicating that the method has good stability.

[0060] 5.4 Repeatability Test

[0061] Accurately pipette 10 μL of each of the test solutions, for a total of 6 portions, and determine them according to the method under "3. Chromatographic Conditions". The results show that the peak area RSDs of glucose and vitexin are 1.4% and 2.5%, respectively, indicating that the method has good repeatability.

[0062] 5.5 Recovery Rate Test

[0063] Accurately pipette 1 ml of each of six extracts of Polygonatum sibiricum (0.8 mg / ml glucose and 0.16 mg / ml vitexin) into 50 ml volumetric flasks. Add reference standards according to the glucose and vitexin contents, respectively. Dilute with water to the mark and determine according to the method under "3. Chromatographic Conditions". The average recoveries of glucose and vitexin were calculated to be 95.3% and 92.4%, respectively, with RSDs of 1.8% and 2.9%, respectively, indicating that the method has good accuracy.

[0064] Example 3: Optimization of Extraction Process

[0065] 3.1 Optimization of water volume for decoction

[0066] The method of Example 1 was used to optimize the amount of water for decoction. The difference was that the amount of water for decoction in step (3) was 5 times, 8 times, and 12 times, which were respectively used as treatment groups 2#, 3#, and 4#. Example 1 was treatment group 1#.

[0067] 3.2 Optimization of simmering time

[0068] The decoction time was optimized according to the method of Example 1, except that the decoction time in step (3) was 20 min, 40 min, and 90 min, which were respectively used as treatment groups 5#, 6#, and 7#.

[0069] 3.3 Optimization of the number of decoctions

[0070] The number of decoctions was optimized according to the method of Example 1, except that the number of decoctions in step (3) was 1 time and 3 times, respectively, and they were used as treatment groups 8# and 9#.

[0071] The extracts of Polygonatum sibiricum obtained by the above method were all dark brown in appearance, without obvious impurities or precipitates. The test solution was prepared according to the method under "2. Solution Preparation" in Example 2, and the glucose and vitexin contents in the sample were determined according to the method under "3. Chromatographic Conditions". The results are shown in the table below:

[0072] Table 1: Content of glucose and vitexin

[0073]

[0074] As shown above, regarding the amount of water used for decoction, the content of vitexin decreases significantly when the amount of water used is 5 times that of the original volume. The content of vitexin increases accordingly with increasing the amount of water used, with the highest content observed when the amount of water used is 10-12 times that of the original volume. The glucose content does not differ significantly between the amounts of water used and the original volume, therefore, a water volume of 10-12 times is preferable.

[0075] Based on the decoction time, a decoction time of 40-60 minutes results in higher contents of glucose and vitexin. Extending the decoction time does not significantly change the content of vitexin, but the content of glucose actually decreases. If the decoction time is too short, glucose and vitexin cannot be effectively extracted. Therefore, the decoction time is determined to be 40-60 minutes.

[0076] From the perspective of the number of decoctions, decoctions exceeding two times do not significantly improve the extraction efficiency of glucose and vitexin from Polygonatum sibiricum. Therefore, the optimal number of decoctions is determined to be two.

[0077] In summary, treatment group 6# was determined to be the optimal extraction process, with a decoction volume of 10 times, a decoction time of 40 minutes, and a decoction frequency of 2 times. Further optimization experiments will be conducted.

[0078] Example 4: Optimization of processing techniques

[0079] 4.1 Optimization of slice thickness

[0080] The method of processing group 6 according to Example 3 is different in that the thickness of the raw Polygonatum slices in step (1) is 1-2mm and 5-6mm respectively, as processing groups 10# and 11#;

[0081] 4.2 Optimization of drying process

[0082] The method of processing group 6 according to Example 3 is different in that step (1) uses vacuum drying at 45°C for 2 hours and 3 hours respectively, and vacuum drying at 30°C for 5.5 hours, which are respectively used as processing groups 12#, 13# and 14#.

[0083] 4.4 Optimization of dampness

[0084] The method of processing group 6 according to Example 3 is different in that the humidification in step (1) is 0.12MPa for 2h, 0.12MPa for 4h, 0.15MPa for 2h, and 0.15MPa for 3h, respectively, as processing groups 15#, 16#, 17# and 18#;

[0085] The Polygonatum extract obtained by the above method was dark brown in appearance and free of obvious impurities and precipitates. The test solution was prepared according to the method described in "2. Solution Preparation" of Example 2, and the glucose and vitexin contents in the sample were determined according to the method described in "3. Chromatographic Conditions". The results are shown in the table below:

[0086] Table 2: Content of glucose and vitexin

[0087]

[0088] As shown in the table above, the section thickness should not be too thin. The overall section thickness should be 3-6 mm, with 3-4 mm being the best.

[0089] From the perspective of processing and drying technology, changing the drying process to vacuum drying significantly affects the extraction of effective components from Polygonatum extract. Vacuum drying can significantly shorten the drying time, but it requires strict control of drying conditions. Vacuum drying at 45℃ for 2-3 hours will lead to a significant decrease in the content of glucose and vitexin. Lowering the vacuum drying temperature and extending the drying time can reduce the loss of glucose and vitexin, but the levels are still lower than those in treatment group 6.

[0090] Conventional soaking time is relatively long. Treatment groups 15#-18# optimized the soaking pressure and time. The results showed that high-pressure soaking can promote the soaking effect. The best effect was achieved by soaking for 4 hours at 0.12MPa. This not only effectively shortened the soaking time, but also resulted in a glucose content of 4.4126% and a vitexin content of 0.8753% in the prepared Polygonatum extract.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features, all of which should be covered within the scope of the technical solutions claimed in this application.

Claims

1. A method for detecting the extract of processed Polygonatum sibiricum, characterized in that, The preparation method of Polygonatum sibiricum processed extract includes the following steps: (1) Take raw Polygonatum, add 20% of the weight of Polygonatum in rice wine, mix well and let it soak, steam it thoroughly in a steamer, take it out, let it cool slightly, mix it back into the steaming liquid, let it cool slightly and then dry it. (2) Repeat step (1) 9 times; (3) Take the wine-steamed Polygonatum sibiricum processed in step (2), add 10-12 times the amount of water and decoct, then centrifuge the decoction; (4) Concentrate the centrifuged liquid obtained in step (3) and then centrifuge it to obtain the final product; The detection method includes: S1: Accurately measure the extract of Polygonatum sibiricum to be tested, dilute with water, shake well, filter, and take the filtrate as the test solution; S2: Take glucose and vitexin as reference standards, weigh them accurately, dissolve them in water, dilute to the mark, shake well, filter, and take the filtrate as the reference solution; S3: The test solution and the reference solution were detected by high performance liquid chromatography (HPLC). The HPLC method used an evaporative light scattering detector, a Lichrospher NH2 column (4.6 mm × 250 mm, 5 μm), and a mobile phase of 0.1% formic acid-water-methanol. High performance liquid chromatography was performed using isocratic elution with 0.1% formic acid in water-methanol at a volume ratio of 80:

20. The high-performance liquid chromatography method described in S3 uses a column temperature of 25°C, a detector drift tube temperature of 50°C, air as the carrier gas, a flow rate of 1.2 mL / min, and an injection volume of 10 μL.

2. The method for detecting processed extracts of Polygonatum sibiricum according to claim 1, characterized in that, The preparation method of the test solution described in S1 is as follows: accurately measure 1 ml of the extract of Polygonatum sibiricum to be tested, dilute with water to 50 ml, shake well, filter, and take the filtrate to obtain the solution.

3. The method for detecting processed extracts of Polygonatum sibiricum according to claim 1, characterized in that, The preparation method of the reference solution described in S2 is as follows: Weigh approximately 8 mg of glucose and approximately 1.5 mg of vitexin accurately, place them in a 10 ml volumetric flask, add water to dissolve, dilute to the mark, shake well, filter, and take the filtrate to obtain the solution.

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