Method for detecting morroniside and loganin content in ten-tonifying granules

Abstract

The invention discloses a method for detecting the content of morroniside and loganin in ten-tonifying granules, and belongs to the technical field of traditional Chinese medicine preparation detection. The invention discloses a method for detecting morroniside and loganin content in ten-tonifying granules. The method comprises the following steps: (1) preparing a reference solution; (2) preparing a to-be-detected sample solution; (3) chromatographic conditions; and (4) content determination. According to the method, the contents of morroniside and loganin in the ten-tonifying granules are measured by using the high performance liquid chromatography, compared with the existing standard without content measurement item detection, the method can directly control the internal quality of the medicine by detecting the contents of morroniside and loganin, and in the measurement method, the separation effect of morroniside and loganin is good, and the detection result is accurate. The method is high in stability and accuracy and good in repeatability, so that the quality of the ten-tonifying granule product can be better controlled.

Description

Technical Field

[0001] The invention relates to a method for detecting the contents of morroniside and loganin in Shibu granules, belonging to the technical field of traditional Chinese medicine preparation detection. Background Art

[0002] The Chinese patent medicine Shibu Granule originated from the traditional prescription recorded in the second volume of "Le's Pills, Powders, Pastes and Pills" in the Qing Dynasty. It is a Chinese medicine preparation used to nourish yin, strengthen yang, and invigorate qi and blood. The prescription is composed of Chinese medicines such as Rehmannia glutinosa, stir-fried yam, and steamed cornus officinalis, and is mainly used to treat a series of symptoms caused by insufficient kidney yang and kidney yin deficiency.

[0003] In the prior art, Shibu Granules are widely used in the treatment of various diseases in the clinic, including chronic nephritis, kidney disease, chronic lumbar muscle strain, edema, etc. Its efficacy is similar to that of traditional famous prescriptions such as Guifu Dihuang Pills, both of which have the effect of nourishing kidney yang. In the prescription composition, Rehmannia root as the main drug has the effects of nourishing blood and yin, replenishing essence and benefiting marrow; salt Eucommia, wine cornus, stir-fried yam, etc. as the assistant drugs, together play the role of warming and nourishing kidney yang.

[0004] However, the prior art has the following deficiencies:

[0005] 1. The content determination item of Shibu Granules has not been established yet, and there is a lack of quantitative detection methods for its active ingredients;

[0006] 2. The existing technology lacks a complete quality control standard, making it difficult to ensure the stability and consistency of product quality;

[0007] 3. The lack of a standardized quality evaluation system is not conducive to the quality management of the drug and the safety assurance of its clinical application.

[0008] Therefore, it is urgent to develop a method for determining the content of the active ingredients in Shibu Granules and establish corresponding quality standards to ensure the quality controllability and clinical safety of this Chinese patent medicine. Summary of the invention

[0009] The technical problem to be solved by the present invention is to propose a method for determining the contents of morroniside and loganin in Shibu granules, so as to improve the quality standard of Shibu granules and better control the quality of the product.

[0010] To achieve the above objectives, the present invention adopts the following technical solutions.

[0011] A method for detecting the content of morroniside and loganin in Shibu granules, the method comprising the following steps:

[0012] (1) Preparation of reference solution: accurately weigh morroniside and loganin reference substances, add 30-90% methanol solution to prepare a mixed reference solution with a content of 20-60 μg per ml;

[0013] (2) Preparation of the sample solution to be tested: (2) Preparation of the sample solution to be tested: Grind the Shibubu granules, accurately weigh about 1 g, place in a stoppered conical flask, accurately add 15 to 50 ml of 30 to 90% methanol, weigh, ultrasonically treat for 15 to 45 minutes, cool and weigh, add 30 to 90% methanol to make up for the weight loss, shake evenly and filter, and take the filtrate to obtain;

[0014] (3) Chromatographic conditions:

[0015] Chromatographic column: bonded silica gel as filler;

[0016] Mobile phase: acetonitrile-0.05-0.2% phosphoric acid solution;

[0017] Detection wavelength: 236~244nm;

[0018] Flow rate: 0.9~1.1ml / min;

[0019] Column temperature: 30~40℃.

[0020] (4) Content determination: Accurately pipette 5-20 μl of the reference solution and the sample solution into a high performance liquid chromatograph, analyze under the above chromatographic conditions, record the peak areas, and calculate the contents of morroniside and loganin in the sample by the external standard method.

[0021] (1) Preparation of reference substance solution: accurately weigh morroniside and loganin reference substances, add 80% methanol solution, and prepare a mixed reference substance solution with a content of 50 μg per 1 ml.

[0022] (2) preparing the sample solution to be tested: grind the Shibubu granules into powder, accurately weigh about 1 g, place in a stoppered conical flask, accurately add 25 ml of 80% methanol, weigh, ultrasonically treat for 15 to 45 minutes, weigh after cooling, make up the weight loss with 80% methanol, shake evenly and filter, and take the filtrate.

[0023] The ultrasonic treatment time in step (2) is 30 minutes.

[0024] The ultrasonic treatment conditions in step (2) are: power 500W, frequency 40KHz.

[0025] The chromatographic conditions of (3) are:

[0026] Chromatographic column: bonded silica gel as filler;

[0027] Mobile phase: acetonitrile-0.1% phosphoric acid solution;

[0028] Detection wavelength: 240nm;

[0029] Flow rate: 1ml / min;

[0030] Column temperature: 35°C.

[0031] The chromatographic condition filler described in step (3) is octadecylsilane bonded silica gel with a specification of 5 μm and 4.6×250 mm.

[0032] The chromatographic conditions in step (3) are as follows: the mobile phase is acetonitrile-0.1% phosphoric acid solution = 10:90.

[0033] (4) Content determination: 10 μl of the reference solution and the sample solution to be tested were accurately pipetted into a high performance liquid chromatograph, analyzed under the above chromatographic conditions, the peak areas were recorded, and the contents of morroniside and loganin in the sample to be tested were calculated using the external standard method.

[0034] The present invention provides a high performance liquid chromatography method for detecting the content of morroniside and loganin in Shibu granules, which has the following characteristics:

[0035] Beneficial effects:

[0036] 1. Filling the gaps in existing technologies:

[0037] The present invention establishes for the first time a method for determining the contents of morroniside and loganin in Shibu granules, which solves the technical defect of having no content determination item in the existing standard and provides reliable technical support for the quality control of Shibu granules.

[0038] 2. Provides reliable quality control means:

[0039] By directly testing the content of morroniside and loganin, the intrinsic quality of the drug can be accurately reflected, providing a scientific basis for product quality control.

[0040] 3. Good analytical performance:

[0041] Good separation effect: Morroniside and loganin can be well separated to avoid mutual interference;

[0042] High stability: the measurement results are stable and reliable;

[0043] High accuracy: The analysis results are accurate and meet the requirements of the analysis method;

[0044] Good repeatability: The measurement results have good reproducibility.

[0045] 4. Strong practicality:

[0046] This method is simple to operate, and the required reagents and instruments are easily available, which is suitable for routine use in quality control laboratories and is conducive to the improvement of the quality standards of Shibu Granule products and their popularization and application.

[0047] The above beneficial effects indicate that the present invention provides a reliable detection method for the quality control of Shibu Granules, which has important practical significance for improving product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0049] Figure 1 Schematic diagram of the mixed reference solution of morroniside and loganin described in Example 2 of the present invention

[0050] Figure 2 Schematic diagram of the sample solution to be tested according to Example 2 of the present invention

[0051] Figure 3 Schematic diagram of the negative sample solution of cornus officinalis wine according to Example 2 of the present invention

[0052] Figure 4 Schematic diagram of the morroniside standard curve described in Example 2 of the present invention

[0053] Figure 5 Schematic diagram of the loganin standard curve described in Example 2 of the present invention DETAILED DESCRIPTION

[0054] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0055] The instruments and materials used in the following examples are as follows. Other materials are commercially available unless otherwise specified.

[0056] Instruments: electronic balance (AL204-IC, METTLER TOLEDO), electronic balance (MS303s, METTLER TOLEDO), ultrasonic cleaner (TH400-BQE, Jining Tianhua Ultrasonic Electronic Instrument Co., Ltd.), water purifier (Milli-Q, Merck Millipore), high performance liquid chromatograph (Arc, Waters, USA), high performance liquid chromatograph (1260, Agilent, USA);

[0057] Materials: Morroniside (batch number: 111998-202205, content 98.6%), loganin (batch number: 111640-202309, content 99.2%), both purchased from China Food and Drug Inspection Institutes; Shibu Granules (batch numbers: 900001, 900004, 900005, 900006), produced by Lerentang Pharmaceutical Factory of Jinyao Darentang Group;

[0058] Reagents: methanol (chromatographic grade, batch number: I1338907 416, Merck), acetonitrile (chromatographic grade, batch number: I1336830415, Merck);

[0059] Chromatographic column: Welch LP-C18 (specification 5 μm, 4.6×250 mm).

[0060] Example 1: Method for determining the content of morroniside and loganin in Shibu granules

[0061] 1. Preparation of reference solution

[0062] Take appropriate amount of morroniside and loganin reference substances, weigh accurately, add 80% methanol to make a solution containing 50 μg of each per 1 ml.

[0063] 2. Preparation of sample solution

[0064] Take an appropriate amount of this product, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of 80% methanol, weigh it, treat it with ultrasound (power 500W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0065] 3. Chromatographic conditions

[0066] Octadecylsilane bonded silica gel was used as filler; acetonitrile-0.1% phosphoric acid solution (10:90) was used as mobile phase; the detection wavelength was 240 nm; the flow rate was 1 ml / min; and the column temperature was 35°C.

[0067] 4. Determination of sample content

[0068] Accurately pipette 10 μl of the mixed reference solution of step (1) and the sample solution prepared in step (2), respectively, and inject them into a high performance liquid chromatograph. Determine and analyze according to the chromatographic conditions of step (3) to obtain the peak areas of morroniside, loganin reference and the sample to be tested. Calculate the contents of morroniside and loganin in the sample to be tested by the external standard method, and then calculate the contents of morroniside and loganin in the Shibu granules.

[0069] Example 2: Methodology Verification Experiment

[0070] In order to ensure the effect of the present invention, the applicant has carried out corresponding method verification on the content determination method of morroniside and loganin in Shibu granules.

[0071] 1. Exclusivity

[0072] Take Shibu Granules and wine-fed Cornus negative samples, and prepare the test solution and wine-fed Cornus negative sample solution by the method of "2. Preparation of the sample solution to be tested" in Example 1. Accurately pipette 10 μl of the mixed reference solution, the test solution, and the wine-fed Cornus negative sample solution, respectively, and inject them into the high performance liquid chromatograph. According to the chromatographic conditions of "3. Chromatographic conditions" in Example 1, the chromatogram is shown in the attached figure. Figures 1 to 3 As shown, in the test solution, the separation degree between morroniside and the impurity peak is 1.78, and the separation degree between loganin and the impurity peak is 1.90, which are well separated. The negative sample of Cornus officinalis has no interference at the chromatographic peaks of morroniside and loganin.

[0073] 2. Repeatability

[0074] Take an appropriate amount of Shibu Granule (batch number: 900001), grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical bottle, a total of 6 portions, and prepare the test solution in the same way as in Example 1 "2. Preparation of the sample solution to be tested". Accurately pipette 10μl of the mixed reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and measure and analyze them according to Example 1 "3. Chromatographic conditions", calculate the content of morroniside and loganin, and the result is that the content of morroniside is 1.202mg / g, RSD is 0.36%; the content of loganin is 0.6212mg / g, RSD is 0.37%. The results are shown in Tables 1 and 2.

[0075] Table 1 Results of the Morroniside Repeatability Test

[0076]

[0077] Table 2 Loganin repeatability test results

[0078]

[0079]

[0080] 3. Accuracy

[0081] Take an appropriate amount of Shibu Granules (batch number: 900001), grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical bottle, a total of 6 portions, accurately add 25ml of mixed reference solution prepared with 80% methanol (concentration of 24.56μg of morroniside and 30.06μg of loganin per 1ml), weigh it, ultrasonically treat it (power 500W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain it. According to the "3. Chromatographic conditions" of Example 1, the recovery rate was calculated. The average recovery rate of morroniside was 99.45%, and the RSD was 1.12%; the average recovery rate of loganin was 99.21%, and the RSD was 0.83%. The results are shown in Tables 3 and 4.

[0082] Table 3 Morroniside accuracy test results

[0083]

[0084] Table 4 Loganin accuracy test results

[0085]

[0086] 4. Intermediate precision

[0087] Take an appropriate amount of Shibu Granule (batch number: 900001), grind it into powder, take about 1g, weigh it accurately, put it into a stoppered conical bottle, a total of 6 portions, prepare the test solution in the same way as in Example 1 "2. Preparation of the sample solution to be tested", change the date, personnel and instruments, accurately pipette 10μl of the mixed reference solution and the test solution respectively, inject into the high performance liquid chromatograph, measure and analyze according to Example 1 "3. Chromatographic conditions", comprehensively analyze the repeatability results, calculate the content of morroniside and loganin, and the result is that the content of morroniside is 1.202mg / g, RSD is 0.33%; the content of loganin is 0.6178mg / g, RSD is 0.88%. The results are shown in Tables 5 and 6.

[0088] Table 5 Morroniside intermediate precision test results

[0089]

[0090] Table 6 Loganin intermediate precision test results

[0091]

[0092]

[0093] 5. Linear Relationship

[0094] Take an appropriate amount of morroniside reference substance, accurately weigh it, add 80% methanol to prepare reference substance solutions containing 217.5μg, 108.7μg, 52.19μg, 26.10μg, and 7.829μg of morroniside per 1ml, accurately pipette 10μl of each, inject into high performance liquid chromatograph, measure and analyze according to "3. Chromatographic conditions" of Example 1, make a standard curve with morroniside concentration as the horizontal axis (X) and peak area as the vertical axis (Y), and calculate the regression equation and correlation coefficient. The results are shown in the attached Figure 4 As shown, Y=17709.04X+6298.32 (r=1.000), and the linear relationship was good within the range of morroniside concentration of 7.829-217.5 μg / ml.

[0095] Take an appropriate amount of loganin reference substance, accurately weigh it, add 80% methanol to prepare reference substance solutions containing 189.2μg, 94.58μg, 50.44μg, 25.22μg, and 7.566μg of loganin per 1ml, accurately draw 10μl of each, inject into high performance liquid chromatograph, measure and analyze according to "3. Chromatographic conditions" of Example 1, make a standard curve with loganin concentration as the horizontal axis (X) and peak area as the vertical axis (Y), and calculate the regression equation and correlation coefficient. The results are shown in the attached Figure 5 As shown, Y=16622.41X-609.81 (r=1.000), and the linear relationship was good in the range of loganin concentration from 7.566 to 189.2 μg / ml.

[0096] 6. Durability-Solution Stability

[0097] Take an appropriate amount of Shibu Granules (batch number: 900001), grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, prepare the test solution in the same way as in Example 1 (2), prepare the test solution in the same way as in Example 1 "2. Preparation of the sample solution to be tested", and at regular intervals, measure and analyze according to Example 1 "3. Chromatographic conditions", calculate the relative standard deviation of the peak area, and the RSD of the peak area of ​​morroniside and loganin measured is less than 2%, indicating that the test solution has good stability within 26 hours. The results are shown in Tables 7 and 8.

[0098] Table 7 Morroniside test solution stability test results

[0099]

[0100]

[0101] Table 8 Loganin test solution stability test results

[0102]

[0103] 7. Durability - Chromatographic parameters

[0104] Take an appropriate amount of Shibu Granules (Batch No.: 900001), grind it into powder, take about 1 g, weigh it accurately, and put it into a stoppered conical flask, a total of 2 portions, and prepare the test solution in the same way as in Example 1 "2. Preparation of the sample solution to be tested". Accurately pipette 10 μl of the mixed reference solution and the test solution respectively, and inject them into the high performance liquid chromatograph. According to Example 1 "3. Chromatographic conditions", slightly change the detection wavelength, column temperature, flow rate and chromatographic column, respectively, and measure and analyze them respectively, and calculate the relative standard deviation of the measured content when different chromatographic parameters are used. The results are shown in Tables 9 and 10. The RSD of the measured morroniside and loganin contents when the chromatographic conditions are slightly changed is less than 2%, indicating that the method has good durability.

[0105] Table 9 Morroniside durability-chromatographic parameter test results

[0106]

[0107]

[0108] Table 10 Loganin durability-chromatographic parameter test results

[0109]

[0110] Example 3

[0111] Take 4 batches of Shibu Granules and prepare the test solution in the same manner as in Example 1 "2. Preparation of the sample solution to be tested". Accurately pipette 10 μl of the mixed reference solution and the test solution respectively and inject into the high performance liquid chromatograph. Determine and analyze according to "3. Chromatographic conditions" in Example 1 respectively. Calculate the contents of morroniside and loganin by the external standard method. The results are shown in Table 11.

[0112] Table 11 Test results of multiple batches of samples

[0113]

[0114] By measuring the content of 4 batches of Shibu Granule samples, the results showed that the content of morroniside was between 1.200-1.292 mg / g, and the content of loganin was between 0.6025-0.6147 mg / g. The contents of the two components in different batches of samples showed good batch-to-batch consistency, and the relative standard deviation was small. On the one hand, this shows that the production process of Shibu Granule is stable and controllable, the quality of the raw materials is well controlled, and the preparation process has good reproducibility; on the other hand, it also proves that the detection method provided by the present invention has good precision and reproducibility, and the detection effect is stable and reliable, which can be used as an analytical method for quality control of the product.

[0115] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Those skilled in the art can design many other modifications and implementation methods, which will fall within the scope and spirit of the principles disclosed in this application. More specifically, within the scope of the disclosure, drawings and claims of this application, various modifications and improvements can be made to the components and / or layout of the subject combination layout. In addition to the modifications and improvements made to the components and / or layout, other uses will also be obvious to those skilled in the art.

Claims

1. A method for detecting the content of morroniside and loganin in Shibu granules, characterized in that The method comprises the following steps: (1) Preparation of reference solution: accurately weigh morroniside and loganin reference substances, add 30-90% methanol solution to prepare a mixed reference solution with a content of 20-60 μg per ml; (2) Preparation of the sample solution to be tested: (2) Preparation of the sample solution to be tested: Grind the Shibubu granules, accurately weigh about 1 g, place in a stoppered conical flask, accurately add 15 to 50 ml of 30 to 90% methanol, weigh, ultrasonically treat for 15 to 45 minutes, cool and weigh, add 30 to 90% methanol to make up for the weight loss, shake evenly and filter, and take the filtrate to obtain; (3) Chromatographic conditions: Chromatographic column: bonded silica gel as filler; Mobile phase: acetonitrile-0.05-0.2% phosphoric acid solution; Detection wavelength: 236~244nm; Flow rate: 0.9~1.1ml / min; Column temperature: 30~40℃. (4) Content determination: Accurately pipette 5-20 μl of the reference solution and the sample solution into a high performance liquid chromatograph, analyze under the above chromatographic conditions, record the peak areas, and calculate the contents of morroniside and loganin in the sample by the external standard method.

2. A method for detecting the content of morroniside and loganin in Shibu granules according to claim 1, characterized in that: (1) Preparation of reference substance solution: accurately weigh morroniside and loganin reference substances, add 80% methanol solution, and prepare a mixed reference substance solution with a content of 50 μg per 1 ml.

3. A method for detecting the content of morroniside and loganin in Shibu granules according to claim 1, characterized in that: (2) preparing the sample solution to be tested: grind the Shibubu granules into powder, accurately weigh about 1 g, place in a stoppered conical flask, accurately add 25 ml of 80% methanol, weigh, ultrasonically treat for 15 to 45 minutes, weigh after cooling, make up the weight loss with 80% methanol, shake evenly and filter, and take the filtrate.

4. A method for detecting the content of morroniside and loganin in Shibu granules according to claim 3, characterized in that: The ultrasonic treatment time described in (2) is 30 minutes.

5. A method for detecting the content of morroniside and loganin in Shibu granules according to claim 3, characterized in that: The ultrasonic treatment conditions described in (2) are: power 500W, frequency 40KHz.

6. A method for detecting the content of morroniside and loganin in Shibu granules according to claim 1, characterized in that: The chromatographic conditions of (3) are: Chromatographic column: bonded silica gel as filler; Mobile phase: acetonitrile-0.1% phosphoric acid solution; Detection wavelength: 240nm; Flow rate: 1ml / min; Column temperature: 35°C.

7. A method for detecting the content of morroniside and loganin in Shibu granules according to claim 6, characterized in that: The chromatographic condition filler described in (3) is octadecylsilane bonded silica gel with a specification of 5 μm and 4.6×250 mm.

8. A method for detecting the content of morroniside and loganin in Shibu granules according to claim 6, characterized in that: The chromatographic conditions described in (3) above include a mobile phase of acetonitrile-0.1% phosphoric acid solution = 10:

90.

9. A method for detecting the content of morroniside and loganin in Shibu granules according to claim 1, characterized in that: (4) Content determination: 10 μl of the reference solution and the sample solution to be tested were respectively accurately aspirated and injected into a high performance liquid chromatograph, analyzed under the above chromatographic conditions, the peak areas were recorded, and the contents of morroniside and loganin in the sample to be tested were calculated using the external standard method.

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