Method for detecting 10 components in rehmannia and processed product thereof

Abstract

The invention discloses a method for detecting 10 components in rehmannia and processed products thereof, and belongs to the technical field of traditional Chinese medicine component detection. The invention particularly discloses a method for detecting catalpol, rehmannia D, mannose, fructose, glucose, cane sugar, melibiose, raffinose, manninotriose and stachyose in rehmannia and processed products by using a high performance liquid reversed phase chromatography. The elution gradient and flow velocity are 0-30 min, 30% A, 70% B and 0.3 mL / min; 30-40 min, 30% of A and 70% of B are adopted, and 0.4 mL / min is adopted; 40-60 min, 35% of A and 65% of B are adopted, and 1 mL / min is adopted; according to the detection method disclosed by the invention, the detection limit of catalpol and the detection limit of rehmannia D can be equivalent to the detection limit of a variable-wavelength ultraviolet-visible spectrophotometric detector; meanwhile, according to the detection method disclosed by the invention, each detection substance can meet quantitative requirements; and a foundation is laid for research on synchronous detection of various components in rehmannia, accurate control of medicinal material quality, clarification of a processing mechanism and guarantee of clinical curative effect.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine component detection, specifically a method for detecting 10 components in Rehmannia glutinosa and its processed products. Background Technology

[0002] Rehmannia ( Rehmannia glutinosa As a traditional Chinese medicine, Rehmannia glutinosa has a wide range of clinical applications, and its processed products (such as stewed Rehmannia glutinosa) are also commonly used medicinal slices. Rehmannia glutinosa contains a variety of active ingredients, more than 300 of which have been isolated and identified. Different processed Rehmannia glutinosa products exhibit significant differences in medicinal properties and chemical composition, thus being used in different clinical directions in traditional Chinese medicine, such as clearing heat and promoting body fluid production, cooling blood and stopping bleeding, and nourishing yin and replenishing blood. Fresh Rehmannia glutinosa is rich in iridoid glycosides (such as catalpol) and various small-molecule oligosaccharides (such as stachyose and raffinose), which have strong activity but are unstable. After processing into raw Rehmannia glutinosa, the content of some iridoid glycosides such as catalpol decreases, while sugar components may partially decompose or transform. Further processing into stewed Rehmannia glutinosa, during the long steaming process, further reduces heat-sensitive components such as catalpol. Simultaneously, processes such as the Maillard reaction cause some sugars to combine with amino acids, changing the medicinal properties from cold to warm, and enhancing the effects of nourishing yin and replenishing blood. These differences in chemical composition are directly related to the efficacy and clinical application of different processed Rehmannia glutinosa products. Therefore, establishing an analytical method that can comprehensively and synchronously detect multiple key components in Rehmannia glutinosa and its processed products is of great significance for accurately controlling the quality of medicinal materials, elucidating the processing mechanism, and ensuring clinical efficacy.

[0003] Currently, there is considerable research on methods for detecting active ingredients in Rehmannia glutinosa and its processed products. Existing technologies mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and thin-layer chromatography (TLC).

[0004] Among these methods, high-performance liquid chromatography (HPLC) is the most widely used due to its simplicity and good repeatability, but it still has many limitations in practical implementation. Existing patent CN109085256A discloses a method for simultaneously detecting active components in Rehmannia glutinosa using HPLC, capable of simultaneously detecting catalpol, gallic acid, aucubin, rehmannia glycoside D, rehmannia glycoside A, verbascoside, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether. However, this method does not cover small molecule sugar components. Patent CN114796347B provides a method for detecting components in processed Rehmannia glutinosa, capable of simultaneously detecting five glycoside components—catalpol, rehmannia glycoside D, leonurin, verbascoside, and isoruascoside—using HPLC. It can also detect polysaccharides in processed Rehmannia glutinosa, but polysaccharide detection requires further extraction of polysaccharides from processed Rehmannia glutinosa, followed by ultraviolet spectrophotometry, and only the total polysaccharide content can be detected. In addition, patent CN108254470B reports an HPLC-ELSD method for detecting sugar components in Rehmannia glutinosa. This method can determine stachyose, raffinose, etc., but it cannot achieve simultaneous analysis with components such as catalpol and rehmannia glycoside D under the same chromatographic conditions.

[0005] Liquid chromatography-mass spectrometry (LC-MS) combines the excellent separation capabilities of liquid chromatography with the high sensitivity and selectivity of mass spectrometry, enabling accurate detection of trace components in complex matrices. However, its ability to quantify small-molecule sugars is limited. Furthermore, some methods lack systematic validation of linear range, precision, and accuracy, making it difficult to guarantee the reliability of component quantification results in different samples, from fresh to processed Rehmannia glutinosa. Thin-layer chromatography (TLC) is simple to operate, inexpensive, and can simultaneously analyze multiple samples without requiring complex equipment, but its separation capability is limited and its quantitative accuracy is poor. Currently, patent CN118483363B provides a method for simultaneously identifying components of both raw and processed Rehmannia glutinosa using TLC, but it can only simultaneously detect three polysaccharides: raffinose, mannotriose, and stachyose, and cannot simultaneously detect glycosides such as catalpol and rehmannia glycoside D.

[0006] In summary, although existing detection methods each have their advantages, none can simultaneously and quantitatively analyze catalpol, rehmannia glycoside D, and various small-molecule sugars in Rehmannia glutinosa and its different processed products while ensuring high sensitivity and accuracy. In particular, there is a lack of an efficient analytical method that can achieve both high sensitivity detection limits for catalpol and rehmannia glycoside D using ultraviolet detectors and accurate quantification of multiple small-molecule sugars. Therefore, there is an urgent need to develop a simple, cost-effective method suitable for the simultaneous detection of multiple components to systematically evaluate the quality of Rehmannia glutinosa raw materials and processed products, and to scientifically elucidate the transformation patterns of chemical components during processing. Summary of the Invention

[0007] To address or partially address the problems existing in related technologies, this invention provides a method for detecting 10 components in Rehmannia glutinosa and its processed products. This method employs high-performance liquid reverse-phase chromatography, which can rapidly, effectively, and accurately detect 10 components in Rehmannia glutinosa and its processed products. This lays the foundation for research on the simultaneous detection of multiple components in Rehmannia glutinosa, precise control of medicinal material quality, elucidation of processing mechanisms, and assurance of clinical efficacy.

[0008] The method of the present invention includes the following steps: (1) Preparation of test solution: Mix Rehmannia powder with methanol (there is no specific requirement for the solid-liquid ratio of Rehmannia powder and methanol solution, as long as the Rehmannia powder is fully mixed in methanol), sonicate, and filter to obtain Rehmannia test solution.

[0009] (2) Preparation of reference solutions: Take ziziphus alcohol, rehmannia glycoside D, mannose, fructose, glucose, sucrose, melibiose, raffinose, mannitol, and stachyose respectively, and add methanol to prepare reference solutions.

[0010] (3) Take the test solution, reference solution and blank solvent methanol respectively, and inject them into the liquid chromatograph for detection.

[0011] Preferably, in step (1) of the present invention, the Rehmannia glutinosa powder is filtered using a No. 3 sieve.

[0012] Preferably, the volume percentage of methanol in step (1) of the present invention is 25%.

[0013] Preferably, the ultrasonic treatment conditions in step (1) of the present invention are 500W power and 40kHz frequency.

[0014] Preferably, the concentration of the reference solution in step (2) of the present invention is 0.1 mg / mL.

[0015] Preferably, in step (3) of the present invention, the chromatographic column of the liquid chromatograph is COSMOISL Sugar-D (4.6mm×150mm, 5μm) with secondary amine / tertiary amine bonded silica as the packing material.

[0016] Preferably, the detector used in the liquid chromatograph in step (3) of the present invention is an evaporative light scattering detector (ELSD), and the parameters used are evaporator temperature 50-90℃, nebulizer temperature 50-90℃, and carrier gas flow rate 0.5-2.0SLM.

[0017] As a further preferred embodiment of the present invention, the ELSD parameters of the present invention are: evaporator temperature 80°C, atomizer temperature 70°C, and carrier gas flow rate 1.0 SLM.

[0018] Preferably, in step (3) of the present invention, the mobile phase A used in the liquid chromatograph is water, and the mobile phase B is acetonitrile. The elution gradient and flow rate are as follows: 0-30 min, 30% A, 70% B, 0.3 mL / min; 30-40 min, 30% A, 70% B, 0.4 mL / min; 40-60 min, 35% A, 65% B, 1 mL / min.

[0019] Preferably, the column temperature of the liquid chromatograph in step (3) of the present invention is 30℃-35℃.

[0020] Preferably, the injection volume of the liquid chromatograph in step (3) of the present invention is 3-5 μL.

[0021] Compared with the prior art, the present invention provides a method for detecting 10 components in Rehmannia glutinosa and its processed products, which has the following beneficial effects: (1) The present invention can simultaneously detect catalpol, rehmannia glycoside D, mannose, fructose, glucose, sucrose, melibiose, raffinose, mannitol and stachyose.

[0022] (2) The detection limits of catalpol and rehmannia glycoside D in this invention are comparable to the detection limits of variable wavelength ultraviolet-visible spectrophotometers. On the basis of realizing the simultaneous detection of multiple components, it takes into account both the high sensitivity of key active ingredients and the practicality of the detection method.

[0023] (3) The method described in this invention can achieve quantitative requirements for each detection substance. Attached Figure Description

[0024] Figure 1 This is the chromatogram of the mixed reference standard shown in Example 1 of the present invention.

[0025] Figure 2 This is the yellow spectrum of Rehmannia glutinosa shown in Embodiment 1 of the present invention.

[0026] Figure 3 This is a chromatogram of the processed Rehmannia glutinosa product (braised Rehmannia glutinosa) shown in Example 1 of the present invention.

[0027] Figure 4 This is the linear graph of catalpol shown in Embodiment 1 of the present invention.

[0028] Figure 5 This is the linear graph of rehmannia glycoside D shown in Example 1 of the present invention.

[0029] Figure 6 This is the mannose linear graph shown in Embodiment 1 of the present invention.

[0030] Figure 7 This is the fructose linear graph shown in Embodiment 1 of the present invention.

[0031] Figure 8 This is the glucose linear graph shown in Embodiment 1 of the present invention.

[0032] Figure 9 This is the sucrose linear graph shown in Embodiment 1 of the present invention.

[0033] Figure 10 This is a linear graph of melibiose shown in Embodiment 1 of the present invention.

[0034] Figure 11 This is a linear graph of raffinose shown in Embodiment 1 of the present invention.

[0035] Figure 12 This is a linear graph of mannotriose shown in Embodiment 1 of the present invention.

[0036] Figure 13 This is a linear graph of stachyose shown in Embodiment 1 of the present invention. Detailed Implementation

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0038] The information on the test sample and control sample used in the embodiments of the detection method of the present invention is as follows: Test sample: Rehmannia glutinosa: It is produced in Jiaozuo City, Henan Province, and has been identified as Rehmannia glutinosa by Senior Engineer Dai Shaoshan of Jianchangbang Pharmaceutical Co., Ltd. Rehmannia glutinosa (Jianchangbang Pharmaceutical Co., Ltd., batch number: R2506085): a commercially available product; Excipients for stewing Rehmannia glutinosa: Amomum villosum and Citrus reticulata peel (from Jianchangbang Pharmaceutical Co., Ltd., batch numbers: Y074-240601 and Y126-240601 respectively), both of which are commercially available products; Reference standard: Zizitan (batch number: 110808-202112, content calculated as 98.8%), China National Institutes for Food and Drug Control; Rehmannia glutinosa D (batch number: 112063-202304, content calculated as 97.0%), China National Institutes for Food and Drug Control; Mannose (batch number: 140651-202206, content calculated as 99.8%), China National Institutes for Food and Drug Control; Fructose (batch number: 100231-202309, content calculated as 99.9%), China National Institutes for Food and Drug Control; Glucose (batch number: 130809-202501, content calculated as 99.8%), China National Institutes for Food and Drug Control; Sucrose (batch number: 111507-202406, content calculated at 100.0%), China National Institutes for Food and Drug Control; Melibiose (batch number: DSTD01901, content calculated as 98.0%), Lemeitian Pharmaceutical Technology Co., Ltd. Raffinose (batch number: 190225-201901, calculated at 100.0%), China National Institutes for Food and Drug Control; Mannotriose (batch number: 112148-202401, content calculated as 95.4%), China National Institutes for Food and Drug Control; Stachyose (112031-202203, content calculated as 94.9%), China National Institutes for Food and Drug Control.

[0039] Example 1 (1) Preparation of test solutions: Take 0.5 g of raw Rehmannia glutinosa powder and simmered Rehmannia glutinosa powder (details in Table 1), add them to 50 mL of 25% methanol, weigh them, sonicate (500 W power, 40 kHz frequency) for 30 minutes, cool, weigh them again, replenish the lost weight with 25% methanol, shake well, filter, and take the filtrate to obtain the raw Rehmannia glutinosa test solution and simmered Rehmannia glutinosa test solution. Unless otherwise specified, all reagents used are commercially available analytical grade, and all raw materials can be purchased through conventional commercial channels.

[0040] Table 1 Information on test samples and reference samples (2) Preparation of reference solution: Accurately weigh 0.2g of catalpol, rehmannia glycoside D, mannose, fructose, glucose, sucrose, melibiose, raffinose, mannitolose, and stachyose (see Table 1 for details), add 25% methanol to a 20mL volumetric flask and dilute to volume to prepare a reference solution with a concentration of 0.1mg / mL. See Table 2 for detailed reference concentrations.

[0041] Table 2 Concentration of Reference Standards (3) Accurately pipette the reference solution, the test solution, and 10µL of 25% methanol into the liquid chromatograph.

[0042] The parameters of the ELSD detector for liquid chromatography are: evaporator temperature 80℃, nebulizer temperature 70℃, and carrier gas flow rate 1.0 SLM; Mobile phase A is water, and mobile phase B is acetonitrile; The elution gradient and flow rate are shown in Table 3: Table 3 Elution gradient and flow rate of mobile phase in liquid chromatography The column temperature is 33℃.

[0043] The injection volume was 5 μL.

[0044] Analyze and record the chromatograms ( Figures 1-3 As can be seen from the chromatogram, under the chromatographic conditions described in this study, the peaks of 10 compounds, including catalpol, rehmannia glycoside D, mannose, fructose, glucose, sucrose, melibiose, raffinose, mannotriose, and stachyose, all reached baseline separation, with no interference from the blank solvent, and the theoretical plate number calculated based on the mannotriose peak was above 5000.

[0045] The reference solutions from Example 1 were subjected to a current investigation. 10 μL of each reference solution was accurately pipetted into the liquid chromatograph and analyzed. A standard curve was plotted with the logarithm of the reference concentration on the x-axis and the logarithm of the peak area on the y-axis. The regression equations for each reference were calculated as follows: Zichun: y = 1.942x + 9.5137, (R 2 =0.9994); Rehmannia glycoside D: y = 1.314x + 9.6966, (R 2 =0.9995); Mannose: y = 1.0149x + 8.793, (R 2 =0.9998); Fructose: y = 1.2357x + 9.6348, (R 2 =0.9996); Glucose: y = 1.2821x + 9.34, (R 2 =0.9999); Sucrose: y = 1.6802x + 9.7487, (R 2 =0.9999); Melibiose: y = 1.185x + 9.0616, (R 2 =0.9991). Raffinose: y = 1.2418x + 8.2685, (R 2 =0.9993); Mannotriose: y = 1.1718x + 8.7642, (R 2 =0.9997); Stachyose: y = 0.9675x + 8.6233, (R 2 =0.9992).

[0046] In the above regression equation, x is the logarithmic value of concentration and y is the logarithmic value of peak area. By substituting the peak area of ​​the target substance detected in the liquid chromatograph into the regression equation, the concentration of the target substance in the solution can be obtained, thus achieving the quantitative requirement of each detected substance.

[0047] The results of the linear range studies for each reference solution are shown in Table 4-13.

[0048] Table 4 Results of linear range investigation for catalpol Table 5 Results of linear range investigation of rehmannia glycoside D Table 6 Results of linear range investigation for mannose Table 7 Results of the fructose linear range investigation Table 8 Results of the investigation into the linear range of glucose Table 9 Results of the linear range investigation for sucrose Table 10 Results of linear range investigation for melibiose Table 11 Results of linear range investigation for raffinose Table 12 Results of linear range investigation for mannotriose Table 13 Results of linear range investigation for stachyose Example 2 The difference between this embodiment and embodiment 1 is that the liquid chromatography detection parameters in step (3) have been modified, while the other steps are the same as in embodiment 1.

[0049] The parameters of the ELSD detector in liquid chromatography are: Evaporator temperature 50℃, atomizer temperature 50℃, carrier gas flow rate 0.5SLM; Mobile phase A is water, and mobile phase B is acetonitrile; The mobile phase elution gradient and flow rate were as follows: 0-30 min, 30% A, 70% B, 0.3 mL / min; 30-40 min, 30% A, 70% B, 0.4 mL / min; 40-60 min, 35% A, 65% B, 1 mL / min. The column temperature is 30℃; The injection volume was 3 μL.

[0050] The peak separation effect of liquid chromatography was slightly weakened, but the analytical results were not much different from those of Example 1.

[0051] Example 3 The difference between this embodiment and embodiment 1 is that the liquid chromatography detection parameters in step (3) have been modified, while the other steps are the same as in embodiment 1.

[0052] The parameters of the ELSD detector in liquid chromatography are: Evaporator temperature 90℃, atomizer temperature 90℃, carrier gas flow rate 2.0SLM; Mobile phase A is water, and mobile phase B is acetonitrile; The mobile phase elution gradient and flow rate were as follows: 0-30 min, 30% A, 70% B, 0.3 mL / min; 30-40 min, 30% A, 70% B, 0.4 mL / min; 40-60 min, 35% A, 65% B, 1 mL / min. The column temperature is 35℃; The injection volume was 4 μL.

[0053] The peak separation effect of liquid chromatography was slightly weakened, but the analytical results were not much different from those of Example 1.

[0054] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting 10 components in Rehmannia glutinosa and its processed products, characterized in that, Includes the following steps: (1) Preparation of test solution: Mix Rehmannia powder with methanol, sonicate, and filter to obtain Rehmannia test solution; (2) Preparation of reference solutions: Take citronellol, rehmannia glycoside D, mannose, fructose, glucose, sucrose, melibiose, raffinose, mannitol, and stachyose respectively, and add methanol to prepare reference solutions; (3) Take the test solution, reference solution and blank solvent methanol respectively, and inject them into the liquid chromatograph for detection.

2. The method according to claim 1, characterized in that: In step (3), the chromatographic column in the liquid chromatography detector is filled with secondary / tertiary amine bonded silica gel, COSMOISL Sugar-D.

3. The method according to claim 1, characterized in that: In step (3), the liquid chromatography detector is an evaporative light scattering detector. The parameters of the evaporative light scattering detector are: evaporation tube temperature 50-90℃, nebulizer temperature 50-90℃, and carrier gas flow rate 0.5-2.0 SLM.

4. The method according to claim 1, characterized in that: The elution gradient and flow rate of the mobile phase in step (3) are as follows: 0-30 min, 30% A, 70% B, 0.3 mL / min; 30-40 min, 30% A, 70% B, 0.4 mL / min; 40-60 min, 35% A, 65% B, 1 mL / min.

5. The method according to claim 1, characterized in that: In step (3), the column temperature of the liquid chromatograph is 30℃-35℃.

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