Method for simultaneously determining contents of five chemical components in fermented soybean and fresh rehmannia root simultaneously-processed product

Abstract

According to the present invention, the high performance liquid chromatography is adopted, the five symbolic components in the fermented soybean fresh rehmannia root simultaneously-processed product are subjected to simultaneous quantitative analysis under the acetonitrile 0.1% phosphoric acid water system gradient elution condition, the method is simple, the catalpol, the rehmannia glutinosa libosch D, the leonuride, the daidzein and the genistein can be rapidly and simultaneously separated, and the peak pattern and the separation degree are good. The simultaneous determination method established by the invention is good in repeatability and accurate and reliable in result, can be used for determining the contents of catalpol, rehmannia glutinosa libosch D, leonurus glycoside, daidzein and genistein, can be used for research on a processing technology of a fermented soybean fresh-rehmannia root simultaneous processing process product, and can also provide a basis for quality control of the fermented soybean fresh-rehmannia root simultaneous processing product.

Description

Technical Field

[0001] The invention relates to a method for simultaneously determining the contents of five chemical components in a product of light fermented black beans, fresh rehmannia and processed rehmannia by using a high performance liquid chromatograph, and belongs to the field of traditional Chinese medicine component analysis. Background Art

[0002] Clinical preparation of Chinese medicines refers to the process by which a TCM pharmacist prepares a Chinese medicine into the required clinical preparation specifications based on its properties and therapeutic needs. This traditional pharmaceutical technique, developed to meet the needs of TCM's syndrome differentiation and treatment, is crucial for clinical syndrome differentiation and treatment, ensuring the quality of preparations, and passing on the unique processing techniques of Chinese medicine. Commonly used preparations in prescriptions include: stir-frying with water, wine, vinegar, bran, salt, ginger juice, earth, rice, charcoal, simmering, honey-roasting, stir-frying two herbs together, and beating two herbs together.

[0003] The "two-ingredient combined" method involves combining and crushing two different medicinal herbs, allowing them to interpenetrate and alter their properties and flavors to achieve the desired therapeutic effect. Physicians of the Jiangsu School of Medicine, inspired by the black plaster formula in the "Emergency Prescriptions for the Elbow" and incorporating clinical experience, prioritized the preparation of medicinal ingredients, using them to create medicine. This unique clinical preparation, "combined fermented black beans and fresh Rehmannia root," is documented in medical texts such as "Liu Baoyi's Medical Cases," "Yang Boliang's Medical Cases," and "Continuation of Ding Ganren's Medical Cases," and is commonly used clinically to treat blood-heat and hemorrhage. Even today, the renowned Chinese medicine master Zhang Jingren still routinely uses this combination of fermented black beans and fresh Rehmannia root to treat acute and feverish conditions.

[0004] Although the processed product of light fermented black beans and fresh raw rehmannia has significant therapeutic effects in clinical applications, there is currently no research on its ingredients, processing technology and mechanism of action. The present invention discloses a method for simultaneously determining the contents of five ingredients in the processed product of light fermented black beans and fresh raw rehmannia using HPLC. This method can achieve comprehensive evaluation and control of the quality of the processed product of light fermented black beans and fresh raw rehmannia, and can be used to study the processing technology of the processed product of light fermented black beans and fresh raw rehmannia, ensure the quality stability of the processed product and the safety of clinical medication, and provide important data reference for improving the quality standard of the processed product of light fermented black beans and fresh raw rehmannia. Summary of the Invention

[0005] In order to solve the above problems, the present invention adopts HPLC technology to establish a method for simultaneous content determination of five representative ingredients in a special prescription processed product of light fermented black beans and fresh Rehmannia glutinosa. The method is simple, stable and reproducible, and can be used for quality control of the special prescription processed product of light fermented black beans and fresh Rehmannia glutinosa.

[0006] The invention adopts the following technical solutions:

[0007] A method for simultaneously determining the contents of five chemical components in a product of light fermented black beans and fresh raw rehmannia, comprising the following steps:

[0008] (1) Preparation of test solution of light fermented black beans, fresh raw earth and processed products

[0009] Fourteen batches of fresh Rehmannia root were collected from different locations and were identified as fresh tubers of Rehmannia glutinosa Libosch. (Scrophulariaceae). Detailed information is shown in Table 1. Light fermented black beans are fermented from mature seeds of Glycine max (L.) Merr. (Batch No. 211001, Sichuan).

[0010] Table 1 Source information of 14 batches of fresh Rehmannia root

[0011]

[0012] After crushing light fermented black beans, add fresh Rehmannia into a mortar and pound together. After crushing evenly, let it stand at 25℃ for 5h to obtain the processed product of light fermented black beans and fresh Rehmannia. Weigh the processed product of light fermented black beans and fresh Rehmannia into a conical flask, add 70% methanol, weigh it, and extract it ultrasonically for 30min. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and accurately pipette the supernatant through a 0.22μm microporous filter membrane to obtain the test solution.

[0013] (2) Preparation of mixed reference solution

[0014] Weigh appropriate amounts of catalpol, rehmannia glutinosa D, leonurin, daidzein, and genistein reference substances into a 5mL volumetric flask. Dissolve in 70% methanol and sonicate. Cool to room temperature, then dilute to volume. Prepare a stock solution of each standard using methanol. Accurately pipette 1mL of each standard stock solution and dilute to 10mL to create a mixed standard solution.

[0015] (3) Determination method

[0016] Use HPLC method to accurately aspirate the mixed reference solution and test solution respectively, inject them into the analytical instrument, and measure to obtain the result.

[0017] The method for simultaneously determining the contents of five chemical components in the light fermented black beans, fresh raw rehmannia and processed products is as follows: the light fermented black beans are ground into 120 meshes, and the weight ratio of fresh raw rehmannia to light fermented black beans is 12:9.

[0018] The method for simultaneously determining the contents of five chemical components in the prepared product of light fermented black beans and fresh raw earth, wherein the HPLC chromatographic conditions in step (3) are: Kromasil 100-5-C 18Chromatographic column (4.6 mm × 250 mm, 5 μm, Nouryon, Sweden); mobile phase: 0.1% phosphoric acid water (A)-acetonitrile (B); gradient elution: 0-5 min, 2%-6% B; 5-10 min, 6%-10% B; 10-30 min, 10%-25% B; 30-40 min, 25%-35% B; 40-50 min, 35%-60% B; 50-52 min, 60%-50% B; 52-55 min, 50%-2% B; 55-60 min, 2% B; volume flow rate: 0.7 mL / min; detection wavelength: 203 nm; column temperature: 35°C; injection volume: 10 μL.

[0019] The method for simultaneously determining the contents of five chemical components in the product of light fermented black beans, fresh raw rehmannia and processed rehmannia comprises the following steps: step (3) comprises determining the linear relationship; accurately aspirating the mixed reference solution, diluting it step by step, and obtaining a series of concentration solutions with dilution multiples of 2 times, 4 times, 8 times, 16 times and 32 times.

[0020] The method for simultaneously determining the contents of five chemical components in the prepared product of fresh and processed Rehmannia glutinosa with light fermented black beans comprises, in step (3), precision determination; namely, accurately aspirating a mixed reference solution containing 1.50 mg / mL catalpol, 0.30 mg / mL rehmannia glutinosa D, 0.30 mg / mL leonurin, 0.25 mg / mL daidzein and 0.24 mg / mL genistein, injecting the sample six times in succession under the test conditions, and recording the area of each peak.

[0021] The method for simultaneously determining the contents of five chemical components in the processed product of fresh Rehmannia glutinosa with fermented black beans comprises step (3) of repeatable determination; that is, taking the same batch of processed product of fresh Rehmannia glutinosa with fermented black beans with special clinical prescription, preparing six test samples in parallel, determining them six times respectively under chromatographic conditions, and recording the area of each peak.

[0022] The method for simultaneously determining the contents of five chemical components in the prepared product of light fermented black beans and fresh raw rehmannia, step (3) includes stability determination; that is, taking the test solution, placing it at room temperature, injecting the sample at 0, 2, 4, 8, 12, and 24 hours, analyzing it under the test conditions, and recording the peak area.

[0023] The method for simultaneously determining the contents of five chemical components in the prepared product of fresh Rehmannia glutinosa and processed fermented black beans comprises a recovery test in step (3); 1.0 mL of a test sample of known content is precisely aspirated, and 6 portions are placed in 5 mL volumetric flasks; 1.0 mL of a mixed reference solution of a certain concentration, namely, catalpol, rehmannia glutinosa glycoside D, leonurin, daidzein and genistein, is precisely added respectively; 70% methanol solution is added, ultrasonicated for 30 minutes, cooled, diluted to the scale with 70% methanol solution, centrifuged at 12000 r / min for 10 minutes, the supernatant is aspirated, and the sample is analyzed under the test conditions; the peak area is recorded, and the sample recovery of the method is calculated.

[0024] The advantages of the present invention are:

[0025] 1. The present invention adopts high performance liquid chromatography to simultaneously quantitatively analyze the five characteristic components in the fresh and processed products of light fermented black beans under the conditions of acetonitrile-0.1% phosphoric acid water system gradient elution. The method is simple and can quickly and simultaneously separate catalpol, rehmannia glutinosa glycoside D, motherwort sideroside, daidzein and genistein with good peak shape and separation.

[0026] 2. The simultaneous determination method established in the present invention has good repeatability and accurate and reliable results. It can be used for the determination of the contents of catalpol, rehmannia glutinosa D, leonurin, daidzein and genistein. It can be used for the research on the preparation technology of the processed products of light fermented black beans and fresh raw rehmannia glutinosa, and can also provide a basis for the quality control of the processed products of light fermented black beans and fresh raw rehmannia glutinosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 HPLC chart of the mixed reference substance (A) of the present invention and the processed product of fresh Rehmannia root with fermented black beans (B) (1 to 5 are catalpol, rehmannia root glycoside D, motherwort sideroside, daidzein, and genistein, respectively) DETAILED DESCRIPTION

[0028] The above contents of the present invention are further described in detail below through examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0029] Example 1: A processing technology for preparing light fermented black beans and fresh rehmannia

[0030] Take fresh Rehmannia root produced in Wen County, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and pound them together, after crushing them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia pounded together.

[0031] Example 2: A processing technology for preparing light fermented black beans and fresh rehmannia

[0032] Take fresh Rehmannia root produced in Zhaoxian, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and beat them together, after breaking them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0033] Example 3: A processing technology for preparing light fermented black beans and fresh rehmannia

[0034] Take fresh Rehmannia root produced in Lingbao, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and beat them together, after breaking them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0035] Example 4: A processing technology for preparing light fermented black beans and fresh rehmannia

[0036] Take fresh Rehmannia root produced in Huixian, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and pound them together, after crushing them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0037] Example 5: A processing technology for preparing light fermented black beans and fresh rehmannia

[0038] Take fresh Rehmannia root produced in Nanzhao, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and beat them together, after breaking them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0039] Example 6: A processing technology for preparing light fermented black beans and fresh rehmannia

[0040] Take fresh Rehmannia root produced in Wuzhi, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and beat them together, after breaking them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0041] Example 7: A processing technology for preparing light fermented black beans and fresh rehmannia

[0042] Take fresh Rehmannia root produced in Boai, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and beat them together, after breaking them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0043] Example 8: A processing technology for preparing light fermented black beans and fresh rehmannia

[0044] Take fresh Rehmannia root produced in Qinyang, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and pound them together, after crushing them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia pounded together.

[0045] Example 9: A processing technology for preparing light fermented black beans and fresh rehmannia

[0046] Take fresh Rehmannia root produced in Neixiang, Henan Province, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and pound them together, after crushing them evenly, place them at 25°C for 5 hours, and you can get the product of light fermented black beans and fresh Rehmannia pounded together.

[0047] Example 10: A processing technology for preparing light fermented black beans and fresh rehmannia

[0048] Take fresh Rehmannia root produced in Lushan, Henan, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and pound them together, after crushing them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0049] Example 11: A processing technology for preparing light fermented black beans and fresh rehmannia

[0050] Take fresh Rehmannia root produced in Shandong, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and pound them together, after crushing them evenly, place them at 25℃ for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0051] Example 12: A processing technology for preparing light fermented black beans and fresh rehmannia

[0052] Take fresh Rehmannia root produced in Hebei, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and pound them together, after crushing them evenly, place them at 25°C for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0053] Example 13: A processing technology for preparing light fermented black beans and fresh rehmannia

[0054] Take fresh Rehmannia root produced in Shanxi, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and pound them together, after crushing them evenly, place them at 25℃ for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0055] Example 14: A processing technology for preparing light fermented black beans and fresh rehmannia

[0056] Take fresh Rehmannia root produced in Shaanxi, add light fermented black beans (fresh Rehmannia root: light fermented black beans = 12g: 9g) crushed with a mesh number of 120, put them into a mortar and beat them together, after breaking them evenly, place them at 25℃ for 5h, and you can get the product of light fermented black beans and fresh Rehmannia beaten together.

[0057] Example 15: Determination of the contents of five index components in the products of light fermented black beans and fresh raw earth and processed

[0058] 1. Instruments and test drugs

[0059] 1.1 Instrument

[0060] WT6002 electronic balance (Hangzhou Wante Weighing Instrument Co., Ltd.); AG-285 1 / 100,000 balance (Mettler Toledo, Switzerland); KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); DZF-6050 vacuum drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.); Waters e2695 high-performance liquid chromatograph (Waters, USA); QE-500 high-speed pulverizer (Zhejiang Yili Industry and Trade Co., Ltd.); Milli-Q ultrapure water machine (Millipore, USA).

[0061] 1.2 Decoction pieces and test drugs

[0062] Catalpol (Batch No. J07GB150957), rehmannoside D (Batch No. M12IB208721), leonurin (Batch No. J11GB154353), daidzein (Batch No. C11D11Y134057), and genistein (Batch No. H30A9Z69019) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity of ≥98%. Analytical-grade phosphoric acid was purchased from ACS Enke Chemical Reagent Co., Ltd., USA; chromatographic-grade acetonitrile and methanol were purchased from Anhui Tiandi High Purity Solvent Co., Ltd.; and ultrapure water was used. All other reagents were of analytical grade. Fresh Rehmannia glutinosa medicinal materials (originating from Wen County, Henan Province, etc.) were identified by Professor Li Juan of Henan Agricultural University as fresh tuberous roots of Rehmannia glutinosa Libosch., a plant of the genus Rehmannia in the family Scrophulariaceae; the light fermented black bean sample (batch number: 211001 Sichuan) was identified as a fermented product of mature seeds (black beans) of Glycine max (L.) Merr., a plant of the legume family.

[0063] 2 Methods and Results

[0064] 2.1 Preparation of test solution

[0065] Take 2.0 g of the fresh and processed products of light fermented black beans in Examples 1 to 14 respectively into a conical flask, accurately weigh, add 15 mL of 70% methanol, weigh, and ultrasonically extract for 30 minutes. Cool, weigh again, add 70% methanol to make up for the lost weight, shake well, filter, and accurately draw the supernatant through a 0.22 μm microporous filter membrane to obtain the test solution.

[0066] 2.2 Preparation of mixed reference solution

[0067] Accurately weigh appropriate amounts of catalpol, rehmannia glutinosa D, motherwort sideroside, daidzein and genistein reference substances and place them in a 5 mL volumetric flask. Add 70% methanol to dissolve and sonicate. Cool to room temperature and then dilute to the mark to prepare mixed reference substance solutions containing 1.50 mg / mL catalpol, 0.30 mg / mL rehmannia glutinosa D, 0.30 mg / mL motherwort sideroside, 0.25 mg / mL daidzein and 0.24 mg / mL genistein, respectively. Dilute with 70% methanol by 2 times, 4 times, 8 times, 16 times and 32 times, respectively, to obtain a series of mixed standard solutions.

[0068] 2.3 Chromatographic conditions

[0069] Column: Kromasil 100-5-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm, Nouryon, Sweden); mobile phase: 0.1% phosphoric acid (A)-acetonitrile (B); gradient elution: 0–5 min, 2%–6% B; 5–10 min, 6%–10% B; 10–30 min, 10%–25% B; 30–40 min, 25%–35% B; 40–50 min, 35%–60% B; 50–52 min, 60%–50% B; 52–55 min, 50%–2% B; 55–60 min, 2% B. Flow rate: 0.7 mL / min, detection wavelength: 203 nm, column temperature: 35°C, injection volume: 10 μL.

[0070] 2.4 System adaptability investigation

[0071] Accurately inject 10 μL of the mixed reference solution and test solution respectively. Figure 1 ,Under the above chromatographic conditions, the five components were well separated (R>1.5), indicating that the method has good system adaptability.

[0072] 2.5 Linear Relationship Investigation

[0073] The mixed standard solutions of a series of concentrations under item "2.2" were precisely pipetted and injected separately for determination. With concentration (X) as the abscissa and peak area (Y) as the ordinate, the regression equation, linear range and correlation coefficient of each component were calculated (Table 2). The linear range of each component was good between the corresponding mass concentrations.

[0074] Table 2 Regression equations, correlation coefficients and linear ranges of five chemical components

[0075]

[0076] 2.6 Precision Investigation

[0077] Accurately pipette 10 μL of the reference solution under item "2.2" and continuously inject and measure 6 times according to the chromatographic conditions under item "2.3". Record the peak area of each component and calculate the RSDs of the peak areas of catalpol, rehmannia glutinosa D, motherwort sideroside, daidzein and genistein, which are 0.60%, 0.49%, 0.38%, 0.18% and 0.16%, respectively, indicating that the precision of the instrument is good.

[0078] 2.7 Stability investigation

[0079] Take the same aliquot of the test sample solution and inject it for determination at 0, 2, 4, 8, 12 and 24 h after preparation according to the chromatographic conditions under "2.3". Record the peak area of each component and calculate the RSDs of the peak areas of catalpol, rehmannia glutinosa, motherwort sideroside, daidzein and genistein to be 1.81%, 2.21%, 0.72%, 0.88% and 1.01%, respectively, indicating that the test sample is stable within 24 h.

[0080] 2.8 Repeatability Study

[0081] Prepare 6 test sample solutions in parallel according to item "2.1", and inject and measure according to the chromatographic conditions under item "2.3". Record the peak area of each component, and calculate the RSDs of the contents of catalpol, rehmannia glutinosa D, motherwort sideroside, daidzein and genistein in the 6 test samples to be 1.92%, 1.48%, 1.71%, 1.20% and 2.03%, respectively, indicating that the method has good repeatability.

[0082] 2.9 Sample recovery rate investigation

[0083] Six portions of fresh Rehmannia root with known contents of fermented black beans and processed products were accurately weighed and the sample recovery method (1:1 addition) was used to prepare the test solution according to the method in "2.1". The solution was then assayed according to the chromatographic conditions in "2.3". The sample recovery was calculated and the results are shown in Table 3. The results show that the sample recovery met the requirements.

[0084] Table 3 Recovery rates of five chemical components

[0085]

[0086] 2.10 Sample content determination

[0087] Prepare multiple batches of test solutions of fresh Rehmannia glutinosa and processed products with light fermented black beans according to the method in “2.1”. Determine the solution according to the method in “2.3”, record the peak areas and calculate the compound contents. The determination results are shown in Table 4.

[0088] Table 4 Determination results of the content of fresh Rehmannia glutinosa and processed products in fermented black beans (n=3, )

[0089]

[0090] 3. Conclusion

[0091] The present invention adopts high performance liquid chromatography to simultaneously quantitatively analyze five characteristic components, namely catalpol, rehmannia glutinosa D, leonurin, daidzein and genistein, in a processed product of light fermented black beans and fresh raw rehmannia glutinosa under the conditions of a preferred acetonitrile-0.1% phosphoric acid aqueous system gradient elution. The chromatographic peak separation and peak shape are both good, and the method has the characteristics of simple operation, good precision and high repeatability. The method can comprehensively and more effectively control the quality of the processed product of light fermented black beans and fresh raw rehmannia glutinosa.

[0092] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for simultaneously determining the contents of five chemical components in fresh and processed Rehmannia root with fermented black beans, characterized in that: The following steps are involved: (1) Preparation of test solution of light fermented black beans, fresh raw earth and processed products After crushing the light fermented black beans, add fresh Rehmannia into a mortar and pound together. After crushing evenly, place at 15℃-25℃ for 3-5h to obtain the processed product of light fermented black beans and fresh Rehmannia. Weigh the processed product of light fermented black beans and fresh Rehmannia into a conical flask, add 70% methanol, weigh it, and extract it ultrasonically for 30min. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and accurately pipette the supernatant through a 0.22μm microporous filter membrane to obtain the test solution. (2) Preparation of mixed reference solution Weigh appropriate amounts of catalpol, rehmannia glutinosa D, leonurin, daidzein, and genistein reference substances into a 5 mL volumetric flask, dissolve in 70% methanol, and sonicate. Cool to room temperature and dilute to volume. Prepare a stock solution of each standard substance with methanol. Accurately pipette 1 mL of each standard substance stock solution and dilute to 10 mL to obtain a mixed standard solution. (3) Determination method Use HPLC method to accurately aspirate the mixed reference solution and test solution respectively, inject them into the analytical instrument, and measure to obtain the result.

2. The method for simultaneously determining the contents of five chemical components in the light fermented black beans and fresh raw rehmannia glutinosa products as claimed in claim 1, characterized in that: The crushing mesh number of the light fermented black beans is 80-120 meshes, and the weight ratio of fresh rehmannia to light fermented black beans is 12:

9.

3. The method for simultaneously determining the contents of five chemical components in the product of light fermented black beans, fresh raw rehmannia and processed rehmannia as claimed in claim 1, characterized in that: HPLC chromatographic conditions in step (3): Kromasil 100-5-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm, Nouryon, Sweden); mobile phase: 0.1% phosphoric acid in water (A)-acetonitrile (B); gradient elution: 0-5 min, 2%-6% B; 5-10 min, 6%-10% B; 10-30 min, 10%-25% B; 30-40 min, 25%-35% B; 40-50 min, 35%-60% B; 50-52 min, 60%-50% B; 52-55 min, 50%-2% B; 55-60 min, 2% B; flow rate: 0.7 mL / min; detection wavelength: 203 nm; Column temperature: 35°C; injection volume: 10 μL.

4. The method for simultaneously determining the contents of five chemical components in the product of light fermented black beans and fresh rehmannia glutinosa and processed products as claimed in claim 1, characterized in that: Step (3) includes determining the linear relationship; accurately aspirating the mixed reference solution and diluting it step by step to obtain a series of concentration solutions with dilution multiples of 2 times, 4 times, 8 times, 16 times, and 32 times.

5. The method for simultaneously determining the contents of five chemical components in the product of light fermented black beans and fresh raw rehmannia and processed rehmannia as claimed in claim 1, characterized in that: Step (3) includes the determination of precision; that is, accurately aspirating a mixed reference solution containing 1.50 mg / mL catalpol, 0.30 mg / mL rehmannia glutinosa D, 0.30 mg / mL leonurin, 0.25 mg / mL daidzein and 0.24 mg / mL genistein, injecting the sample six times continuously under the test conditions, and recording the area of each peak.

6. The method for simultaneously determining the contents of five chemical components in the product of light fermented black beans, fresh raw rehmannia and processed rehmannia as claimed in claim 1, characterized in that: Step (3) includes repeatability determination; that is, taking the same batch of light fermented black beans and fresh Rehmannia glutinosa and preparing the same special clinical recipe, 6 test samples are prepared in parallel, and the results are measured 6 times under chromatographic conditions, and the chromatographic peak area of each reference substance is calculated.

7. The method for simultaneously determining the contents of five chemical components in the product of light fermented black beans, fresh raw rehmannia and processed rehmannia as claimed in claim 1, characterized in that: Step (3) includes the determination of stability; that is, taking the test solution, placing it at room temperature, injecting it at 0, 2, 4, 8, 12, and 24 hours, analyzing it under the test conditions, and recording the peak area.

8. The method for simultaneously determining the contents of five chemical components in the product of light fermented black beans, fresh raw rehmannia and processed rehmannia as claimed in claim 1, characterized in that: Step (3) includes a recovery test; accurately draw 1.0 mL of the test sample of known content, parallel 6 portions, place in 5 mL volumetric flasks, accurately add 1.0 mL of a mixed reference solution of a certain concentration, respectively, catalpol, rehmannia glutinosa D, leonurin, daidzein and genistein, add 70% methanol solution, ultrasonicate for 15 minutes, cool, dilute to the scale with 7% methanol solution, centrifuge at 12000 r / min for 10 minutes, draw the supernatant, sample and analyze under the test conditions, record the peak area, and calculate the sample recovery of the method.

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