Rhizoma polygonati processing extraction method and detection method

Abstract

The invention relates to the technical field of traditional Chinese medicinal material processing and detection, in particular to a rhizoma polygonati processing extraction method and a detection method. The rhizoma polygonati processing and extracting method comprises the following steps: taking raw rhizoma polygonati, adding yellow wine accounting for 20% of the weight of the rhizoma polygonati, uniformly stirring, moistening, thoroughly steaming in a steamer over water, taking out, slightly airing, stirring back with steaming liquid, slightly airing and then drying, and repeating for 9 times; taking the treated rhizoma polygonati steamed with wine, adding 10-12 times of water to decoct, and centrifuging the decoction; and concentrating the obtained centrifugate, and centrifuging to obtain the product. The invention provides a detection method suitable for a polygonatum kingianum extracting solution. The polygonatum kingianum extracting solution with high effective component content is obtained through optimization of the detection method.

Description

Technical Field

[0001] The invention relates to the technical field of Chinese medicinal material processing and detection, and in particular to a polygonatum processing and extraction method and a detection method. Background Art

[0002] Polygonatum sibiricum is the dried rhizome of Polygonatum kingianum Coll.et Hemsl., Polygonatum sibiricum Red. or Polygonatum cyrtonema Hua, which are plants of the Liliaceae family. It has the effects of nourishing qi and yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. Polygonatum sibiricum is not suitable for direct clinical application. Although Polygonatum sibiricum is non-toxic, it has a numb taste. Direct consumption of Polygonatum sibiricum can cause side effects such as sore throat, dry tongue, itchy skin, and dizziness. Polygonatum sibiricum included in the Chinese Pharmacopoeia requires that it be stewed or steamed thoroughly by wine stewing or wine steaming, then dried and sliced, and dried. It is stipulated that 20kg of yellow wine is used for every 100kg of Polygonatum sibiricum. After being processed into wine Polygonatum sibiricum by wine processing, the irritation can be eliminated and the bad taste of Polygonatum sibiricum can be improved. Compared with the processed products of wine Polygonatum sibiricum, the Polygonatum sibiricum extract obtained by extracting wine Polygonatum sibiricum does not need to be decocted when used, and can be taken directly, which is more convenient and quick when used. The pharmacopoeia does not disclose the specific processing and extraction process and parameters of the Polygonatum sibiricum extract. Based on this, it is very necessary to propose a processing and extraction method for polygonatum to prepare polygonatum extract, and to detect the effective ingredients in the prepared polygonatum extract, so as to determine the optimal process and process parameters for the processing and extraction method of polygonatum. Summary of the invention

[0003] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a processing and extraction method and a detection method for Polygonatum sibiricum, clarify the processing and extraction process and parameters of the Polygonatum sibiricum extract, and establish a method for detecting the content of key polysaccharides and flavonoids in the Polygonatum sibiricum extract. The Polygonatum sibiricum extract obtained by the optimized processing and extraction method of Polygonatum sibiricum has a high content of effective ingredients.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: The object of the present invention is to provide a method for processing and extracting polygonatum, comprising the following steps: (1) Take raw polygonatum, add 20% of the weight of yellow wine to mix well, steam thoroughly in a steamer, take out, let it dry for a while, mix it back with the steaming liquid, let it dry for a while, and then dry it; (2) Repeat step (1) 9 times; (3) taking the wine-steamed polygonatum sibiricum processed in step (2), adding 10-12 times water and boiling, and centrifuging the decoction; (4) The centrifuge liquid obtained in step (3) is concentrated and then centrifuged to obtain the product.

[0005] The use of wine steaming method to process Polygonatum sibiricum can be traced back to the Tang Dynasty in my country. The wine steaming method can effectively eliminate the irritation of Polygonatum sibiricum, improve the bad taste of Polygonatum sibiricum, and integrate the blood-activating function of rice wine to play a better tonic effect. Based on the original wine steaming process, this application further extracts and concentrates the wine-steamed Polygonatum sibiricum to obtain a Polygonatum sibiricum extract. It does not need to be decocted when used, and can be taken directly, which is easy to absorb. This application optimizes the processing extraction process and parameters, and determines the extraction process and parameters with water as the extraction solvent. The above-mentioned method has a good extraction effect on polysaccharides and flavonoids in Polygonatum sibiricum. The prepared Polygonatum sibiricum extract has a high content of effective ingredients. The extraction process involved is simple and easy to operate. No harmful waste liquid is generated during the extraction process, and it has a high industrial promotion value.

[0006] Furthermore, step (1) also includes slicing the raw Polygonatum sibiricum, with the slice thickness being 3-4 mm.

[0007] Furthermore, in step (1), high-pressure steaming is used, the steaming pressure is 0.12 MPa, and the steaming time is 4 hours. The high-pressure steaming can effectively shorten the steaming time, and the prepared polygonatum sibiricum extract has a high content of effective ingredients.

[0008] Furthermore, butterfly centrifugation is used in step (3), and tubular centrifugation is used in step (4). The butterfly centrifugation has the advantages of large single-time processing volume and good clarification effect, and the tubular centrifugation can further separate the tiny particles in the concentrated liquid. The present application uses a method of first separating the decoction liquid by butterfly centrifugation and then separating the concentrated liquid by tubular centrifugation, which can greatly improve the overall separation efficiency, and the prepared polygonatum sibiricum extract is free of residue and has good clarity.

[0009] Furthermore, in step (3), the amount of decoction water is 10 times, the decoction time is 40-60 minutes, and the decoction times are 2 times.

[0010] The Chinese Pharmacopoeia stipulates that the content of polygonatum polysaccharide is measured in terms of glucose content, and records the method for determining the glucose content in polygonatum by UV-visible spectrophotometry. Existing studies have pointed out that the main effective ingredients of polygonatum include substances such as polygonatum polysaccharide, polygonatum flavonoids and polygonatum saponins. Therefore, it is difficult to comprehensively evaluate the effective ingredients in the polygonatum extract by only characterizing the sugar content in polygonatum. Polygonatum saponins belong to steroidal saponins and are present in very low levels in the polygonatum extract. The present application proposes a method for detecting polygonatum polysaccharide and polygonatum flavonoids in polygonatum extract using high performance liquid chromatography. By optimizing the chromatographic conditions, the detection method can simultaneously determine flavonoids and sugar components, wherein polygonatum polysaccharide is measured in terms of glucose and polygonatum flavonoids is measured in terms of vitexin.

[0011] Specifically, the present invention provides a detection method for a processed extract of polygonatum sibiricum, the method being used for the polygonatum sibiricum extract processed by the above method, the detection method comprising: S1: Accurately measure the Polygonatum sibiricum extract to be tested, dilute it with water, shake it well, filter it, and take the filtrate as the test solution; S2: Take glucose and vitexin as reference substances, accurately weigh, dissolve in water, dilute to scale, shake well, filter, and take the filtrate as the reference substance solution; S3: The test solution and the reference solution were detected by high performance liquid chromatography, wherein the high performance liquid chromatography used an evaporative light scattering detector, a Lichrospher NH2 column (4.6 mm×250 mm, 5μm), and a mobile phase of 0.1% formic acid water-methanol.

[0012] Furthermore, the high performance liquid chromatography method was isocratic elution using 0.1% formic acid water-methanol at a volume ratio of 80:20.

[0013] Furthermore, the column temperature of the HPLC method described in S3 is 25° C., the detector drift tube temperature is 50° C., the carrier gas is air, the flow rate is 1.2 L / min; and the injection volume is 10 μL.

[0014] Furthermore, the preparation method of the test solution described in S1 is: accurately measure 1 ml of the polygonatum sibiricum extract to be tested, dilute it to 50 ml with water, shake well, filter, and take the filtrate to obtain; Furthermore, the preparation method of the reference solution described in S2 is: take about 8 mg of glucose and about 1.5 mg of vitexin, accurately weigh, place in a 10 ml volumetric flask, add water to dissolve, dilute to the scale, shake well, filter, and take the filtrate to obtain.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The processing and extraction method of polygonatum provided in this application clarifies the specific processing and extraction process and parameters of the polygonatum extract, and the prepared polygonatum extract has high content of polysaccharides and flavonoids. In addition, this application optimizes the processing process and parameters of the polygonatum extract, while ensuring product quality, significantly shortening the product processing cycle and effectively improving product processing efficiency.

[0016] 2. This application proposes a high performance liquid chromatography detection method suitable for detecting polygonatum polysaccharides and flavonoids in polygonatum extract. The detection method adopts ELSD for detection and optimizes the chromatographic conditions. The method has a good response to both glucose and vitexin. The detection method has a good linear range and high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The chromatogram of the test sample solution under the chromatographic conditions of this application; Figure 2 The chromatogram of the reference substance solution under the chromatographic conditions of this application; Figure 3 This is the chromatogram of the test solution using mobile phase ①; Figure 4 This is the chromatogram of the test solution using mobile phase ②; Figure 5 is the glucose standard curve; Figure 6 This is the standard curve of vitexin. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0020] Embodiment 1: Polygonatum sibiricum processing and extraction process (1) Take raw polygonatum, cut into 3-4 mm slices, add 20% of the weight of raw polygonatum rice wine, mix well, and simmer for 20 hours. Steam thoroughly in a steamer, take out, let it dry for a while, mix back into the steaming liquid, let it dry for a while, and then dry it at 45°C for 20 hours. (2) Repeat step (1) 9 times; (3) Take the processed Polygonatum sibiricum, add 10 times the amount of water and boil twice, each time for 60 minutes, and perform butterfly centrifugation on the decoction; (4) The centrifuged liquid is concentrated to the weight of the polygonatum sibiricum obtained in step (3), and then subjected to tubular centrifugation to obtain the polygonatum sibiricum extract. The polygonatum sibiricum extract extracted by the above method has a dark brown appearance and is free of obvious impurities and precipitation.

[0021] Example 2: Establishment of a method for detecting polygonatum extract 1. Instruments and Reagents 1.1 Instruments: Shimadzu LC-20AD high performance liquid chromatograph, METTLER MS205DU electronic balance, Alltech ELSD 2000ES evaporative light scattering detector.

[0022] 1.2 Reagents: Glucose (analytical grade, Sinopharm Group (Shanghai) Reagent Company), vitexin (China Food and Drug Control Institutes), purified water (Hangzhou Wahaha Group Co., Ltd.), methanol (chromatographic grade, Sinopharm Group (Shanghai) Reagent Company), acetonitrile (chromatographic grade, Sinopharm Group (Shanghai) Reagent Company), formic acid (analytical grade, Sinopharm Group (Shanghai) Reagent Company).

[0023] 2. Solution Preparation Accurately measure 1 ml of the Polygonatum sibiricum extract to be tested, dilute it to 50 ml with water, shake well, filter, and take the filtrate as the test solution; Take about 8 mg of glucose and about 1.5 mg of vitexin, weigh accurately, put into a 10 ml volumetric flask, add water to dissolve, dilute to the scale, shake well, filter, and take the filtrate as the reference solution; Take about 8 mg of glucose and about 1.5 mg of vitexin, weigh them accurately, put them in 10 ml volumetric flasks, add water to dissolve, dilute to scale, shake well, filter, and take the filtrate as the single reference solution of glucose and vitexin; 3. Chromatographic conditions: Lichrospher NH2 chromatographic column (4.6 mm×250 mm, 5μm); column temperature was 25℃; detector was ELSD, drift tube temperature was 50℃; carrier gas was air, flow rate was 1.2L / min; injection volume was 10μL. The mobile phase was 0.1% formic acid water-methanol at a volume ratio of 80:20 for isocratic elution.

[0024] 4. Sample determination Take the polygonatum extract prepared in Example 1 and prepare the test solution according to the method under "2. Solution preparation", measure according to the method under "3. Chromatographic conditions", record the chromatogram, and the chromatogram of the test solution is shown in Figure 1 As shown. Take the mixed reference solution of glucose and vitexin and perform the injection test in the same way. The reference solution spectrum is shown in Figure 2 As shown, peak 1 is vitexin, peak 2 is glucose, and the identification of peak 1 and peak 2 is determined by injecting and testing single reference solutions of glucose and vitexin under the same chromatographic conditions.

[0025] The polygonatum extract prepared in Example 1 was used to prepare a test solution according to "2. Solution preparation", and the test was performed according to the method under "3. Chromatographic conditions", except that the following two mobile phases were used: 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 in Figure 3 As shown, the chromatogram of mobile phase ② is as follows Figure 4 Shown: From Figure 3 It can be seen that the vitexin and the adjacent peaks cannot be completely separated. Figure 4 It can be seen that vitexin can be separated from adjacent peaks, but most peak responses are Figure 1 Lower.

[0026] 5. Methodological considerations 5.1 Linear relationship investigation Accurately pipette 1, 2, 4, 6, 8, and 10 ml of the single reference solution under "2. Solution preparation" into 10 ml volumetric flasks, dilute to scale with water, prepare a series of glucose and vitexin solutions, inject, and measure according to the method under "3. Chromatographic conditions" and record the chromatogram. Perform linear regression analysis with the logarithm of the peak area as the ordinate (y) and the logarithm of the injection volume as the abscissa (x). The glucose standard curve is shown in the figure below. Figure 5 As shown in the figure, the regression equation is: y=0.5281x+5.8742, and the correlation coefficient is 0.9996, indicating that the glucose concentration has a good linear relationship with the peak area between 0.08 and 0.8 mg / ml; the standard curve of vitexin is shown in the figure 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 has a good linear relationship with the peak area between 0.015 and 0.15 mg / ml.

[0027] 5.2 Precision test Accurately pipette 10 μL of glucose and vitexin reference solution, inject 6 times continuously, measure according to the method under "3. Chromatographic conditions", record the peak area, and the results show that the RSD of the peak area of ​​glucose and vitexin are 1.3% and 2.4%, respectively, indicating that this method has good precision.

[0028] 5.3 Stability test Accurately pipette 10 μL of the test solution, and measure according to the method under "3. Chromatographic conditions" at 0h, 2h, 4h, 6h, 8h, 10h and 12h. The results showed that the RSDs of the peak areas of glucose and vitexin were 0.9% and 2.6%, respectively, indicating that this method has good stability.

[0029] 5.4 Repeatability test Accurately pipette 10 μL of each test solution, a total of 6 portions, and measure according to the method under "3. Chromatographic conditions". The results show that the RSDs of the peak areas of glucose and vitexin are 1.4% and 2.5%, respectively, indicating that this method has good repeatability.

[0030] 5.5 Recovery test Accurately pipette 1 ml of each of 6 portions of Polygonatum sibiricum extract (glucose content: 0.8 mg / ml, vitexin content: 0.16 mg / ml), put into a 50 ml volumetric flask, add reference substances according to the glucose and vitexin content, dilute to the scale with water, and measure 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, and the RSDs were 1.8% and 2.9%, respectively, indicating that this method has good accuracy.

[0031] Example 3: Extraction process optimization 3.1 Optimization of decoction water volume The decoction water volume was optimized according to the method of Example 1, except that the decoction water volume in step (3) was 5 times, 8 times, and 12 times, which were respectively used as treatment groups 2#, 3#, and 4#, while Example 1 was used as treatment group 1#; 3.2 Optimization of decoction time 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 used as treatment groups 5#, 6#, and 7#, respectively.

[0032] 3.3 Optimization of decoction times The decoction times were optimized according to the method of Example 1, except that the decoction times in step (3) were 1 time and 3 times, which were used as treatment groups 8# and 9#, respectively.

[0033] The appearance of the polygonatum extracts extracted by the above method was dark brown, without obvious impurities and precipitation. The test solution was prepared according to the method under "2. Solution preparation" in Example 2, and the glucose and vitexin contents in the samples were detected according to the method under "3. Chromatographic conditions". The results are shown in the following table: Table 1: Contents of glucose and vitexin

[0034] From the above, it can be seen that from the perspective of the amount of decoction water, when the amount of decoction water is 5 times, the content of vitexin is significantly reduced. When the amount of decoction water increases, the content of vitexin increases accordingly, and the content of vitexin is higher when the amount of decoction water is 10-12 times; the content of glucose does not differ much between 5-12 times, so the amount of decoction water is preferably 10-12 times.

[0035] From the perspective of decoction time, when the decoction time is 40-60min, the content of glucose and vitexin is relatively high. When the decoction time is extended, the content of vitexin has no obvious change, and the glucose content is reduced. If the decoction time is too short, the glucose and vitexin cannot be effectively extracted. Therefore, the decoction time is determined to be 40-60min.

[0036] Judging from the number of decoctions, decoctions exceeding 2 times cannot significantly improve the extraction effect of glucose and vitexin in Polygonatum sibiricum, so the optimal number of decoctions is determined to be 2 times.

[0037] In summary, treatment group 6# was determined to be the best extraction process, and the decoction water volume was selected to be 10 times, the decoction time was 40 minutes, and the decoction number was 2 times, and subsequent optimization experiments were carried out.

[0038] Example 4: Optimization of processing technology 4.1 Optimization of slice thickness The method of treating group 6 in Example 3 was followed, except that the thickness of the slices of raw polygonatum in step (1) were 1-2 mm and 5-6 mm, respectively, as treating groups 10# and 11#; 4.2 Optimization of drying process The method of treating group 6 in Example 3 was followed, except that step (1) adopted vacuum drying at 45°C for 2 h and 3 h, respectively, and vacuum drying at 30°C for 5.5 h, respectively, as treating groups 12#, 13#, and 14#.

[0039] 4.4 Optimization of Muffle The method of treating group 6 in Example 3 was followed, except that in step (1), the humidification was performed at 0.12 MPa for 2 h, 0.12 MPa for 4 h, 0.15 MPa for 2 h, and 0.15 MPa for 3 h, respectively, as treatment groups 15#, 16#, 17#, and 18#; The extracts of Polygonatum sibiricum extracted by the above method are dark brown in appearance, without obvious impurities and precipitation. The test solution was prepared according to the method under "2. Solution preparation" of Example 2, and the glucose and vitexin contents in the samples were detected according to the method under "3. Chromatographic conditions". The results are shown in the following table: Table 2: Contents of glucose and vitexin

[0040] From the above table, we can see that the slice thickness should not be too thin. The overall slice thickness should be 3-6mm, and 3-4mm is the best.

[0041] From the perspective of the processing and drying process, changing the drying process to vacuum drying during the processing process will significantly affect the extraction of effective ingredients in the polygonatum extract. Vacuum drying can significantly shorten the drying time, but the drying conditions need to be strictly controlled. Vacuum drying at 45°C for 2-3h will lead to a significant decrease in the content of glucose and vitexin. Reducing the vacuum drying temperature and extending the drying time can reduce the loss of glucose and vitexin, but it is lower than that of treatment group 6#.

[0042] The conventional steaming time is relatively long. The steaming pressure and time of treatment groups 15#-18# were optimized. The results showed that high-pressure steaming can promote the steaming effect. The best effect was achieved by steaming for 4 hours at 0.12MPa. It not only effectively shortened the steaming time, but also the glucose content in the prepared Polygonatum sibiricum extract reached 4.4126%, and the content of vitexin could reach 0.8753%.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present application can still be modified or some technical features can be replaced by equivalents, which should all be included in the scope of the technical solution requested for protection in this application.

Claims

1. A method for processing and extracting Polygonatum sibiricum, characterized in that: The following steps are involved: (1) Take raw polygonatum, add 20% of the weight of yellow wine to mix well, steam thoroughly in a steamer, take out, let it dry for a while, mix it back with the steaming liquid, let it dry for a while, and then dry it; (2) Repeat step (1) 9 times; (3) taking the wine-steamed polygonatum sibiricum processed in step (2), adding 10-12 times water and boiling, and centrifuging the decoction; (4) The centrifuge liquid obtained in step (3) is concentrated and then centrifuged to obtain the product.

2. The method for processing and extracting polygonatum according to claim 1, characterized in that: Step (1) also includes slicing the raw Polygonatum sibiricum, with the slice thickness being 3-4 mm.

3. The method for processing and extracting polygonatum according to claim 1, characterized in that: In step (1), high pressure humidification is adopted, the humidification pressure is 0.12 MPa, and the humidification time is 4 h.

4. The method for processing and extracting polygonatum according to claim 1, characterized in that: In step (3), butterfly centrifugation is used, and in step (4), tube centrifugation is used.

5. The method for processing and extracting polygonatum according to claim 1, characterized in that: In step (3), the amount of decoction water is 10 times, the decoction time is 40-60 minutes, and the decoction times are 2 times.

6. A detection method for a processed extract of Polygonatum sibiricum, characterized in that: The method is used for the polygonatum extract processed by the method according to any one of claims 1 to 5, and the detection method comprises: S1: Accurately measure the Polygonatum sibiricum extract to be tested, dilute it with water, shake it well, filter it, and take the filtrate as the test solution; S2: Take glucose and vitexin as reference substances, accurately weigh, dissolve in water, dilute to scale, shake well, filter, and take the filtrate as the reference substance solution; S3: The test solution and the reference solution were detected by high performance liquid chromatography, wherein the high performance liquid chromatography used an evaporative light scattering detector, a Lichrospher NH2 column (4.6 mm×250 mm, 5μm), and a mobile phase of 0.1% formic acid water-methanol.

7. A detection method for the processed extract of polygonatum sibiricum according to claim 6, characterized in that: High performance liquid chromatography was performed with isocratic elution using 0.1% formic acid in water-methanol at a volume ratio of 80:

20.

8. A detection method for the processed extract of polygonatum sibiricum according to claim 6, characterized in that: The column temperature of the HPLC method described in S3 is 25°C, the detector drift tube temperature is 50°C, the carrier gas is air, the flow rate is 1.2 L / min, and the injection volume is 10 μL.

9. A detection method for the processed extract of polygonatum sibiricum according to claim 6, characterized in that: The preparation method of the test solution described in S1 is: accurately measure 1 ml of the polygonatum sibiricum extract to be tested, dilute it to 50 ml with water, shake well, filter, and take the filtrate to obtain the solution.

10. A detection method for the processed extract of Polygonatum sibiricum according to claim 6, characterized in that: The preparation method of the reference solution S2 is as follows: take about 8 mg of glucose and about 1.5 mg of vitexin, accurately weigh them, place them in a 10 ml volumetric flask, add water to dissolve them, dilute to the scale, shake well, filter, and take the filtrate to obtain the reference solution.

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