Method for determining contents of seven components in moutan bark and preparation thereof by adopting quantitative analysis of multi-components by single marker

Abstract

The invention discloses a method for determining the contents of seven components in moutan bark and a preparation thereof by adopting a quantitative analysis of multi-components by single marker. Paeonol is used as an internal standard for quantitative analysis of multiple components by single marker, and relative correction factors between the paeonol and gallic acid, oxypaeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose and benzoylpaeoniflorin are established. And calculating the contents of gallic acid, oxypaeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose and benzoylpaeoniflorin in the moutan bark and the preparation thereof through the relative correction factors. The result obtained by the method has no obvious difference from that obtained by an external standard method, has high repeatability, stability and credibility, and can provide a reference basis for quality evaluation of moutan bark and a preparation thereof.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and specifically relates to a method for determining the contents of seven components in Moutan Cortex and its preparations by using a one-measurement-multiple-evaluation method. The method is used to detect the contents of seven chemical components in Moutan Cortex and its preparations. Background Art

[0002] Paeonia suffruticosa was first recorded in Shennong's Herbal Classic. It is the dried root bark of Paeonia suffruticosa Andr., a plant of the Ranunculaceae family. It has the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis, and clearing away heat. The main bioactive ingredients in Paeonia suffruticosa include phenols (paeonol), terpenes (such as paeoniflorin, oxidized paeoniflorin, benzoylpeoniflorin, etc.), tannins (such as pentagalloyl glucose) and organic acids (such as gallic acid, methyl gallate, etc.). However, the current Chinese Pharmacopoeia only sets a lower quality limit for paeonol, and the single component evaluation method is not comprehensive, and the quantitative determination of multiple indicators is limited by the difficulty and high cost of obtaining reference materials. For this reason, the one-test-multiple-evaluation method has been adopted by the Pharmacopoeia and widely used in the quality control of traditional Chinese medicine because of its advantage of using easily available and inexpensive reference materials to achieve simultaneous determination of multiple components. There are reports on the determination of multiple components in Paeonia suffruticosa, but most of them are external standard methods, which leads to increased costs.

[0003] Quantitative analysis of multiple components by single marker (QAMS) is a new model for simultaneous quality control of multiple components in the field of traditional Chinese medicine. It refers to the use of the intrinsic functional relationship and proportional relationship of the effective ingredients of traditional Chinese medicine to achieve the simultaneous determination of multiple components (reference materials are difficult to obtain or supply) by only determining one of the stable, cheap and easily available components. It can effectively solve the problems of excessive use of reference materials, high cost and difficulty in supply in the process of multi-indicator quality control of traditional Chinese medicine. This method will be the development direction of simultaneous quantification of multiple components in traditional Chinese medicine.

[0004] The currently reported evaluation methods for the quality of peony bark and its preparations have problems such as few test components, many standard product varieties, complicated operation steps, long analysis cycle, and high cost. Therefore, this patent provides a new idea for establishing a stable, controllable, low-cost, accurate and efficient quality control method for peony bark and its preparations. Summary of the invention

[0005] The object of the present invention is to provide a method for determining the contents of seven components in peony bark and its preparations by using a one-test-multiple-evaluation method. The detection method can objectively, comprehensively and accurately detect the contents of gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, benzoylpeoniflorin and paeonol in peony bark and its preparations, and has the characteristics of good repeatability, high stability and strong credibility, which greatly saves resources and reduces detection costs.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is:

[0007] In the first aspect, the present invention claims protection for a method for determining the contents of seven components in peony bark and its preparations using a one-measurement, multiple-evaluation method, the method comprising: using paeonol as an internal standard for one-measurement, multiple-evaluation, establishing relative correction factors between paeonol and gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, and benzoylpeoniflorin, and calculating the contents of gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, and benzoylpeoniflorin in peony bark and its preparations using the relative correction factors.

[0008] Furthermore, the method specifically comprises the following steps:

[0009] (1) Preparing reference solution and establishing relative correction factors between internal standard and other components to be measured: Using paeonol as internal standard, the process of establishing relative correction factors between paeonol and gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, and benzoyl paeoniflorin is as follows: gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, benzoyl paeoniflorin and paeonol are dissolved in solvents respectively; different series of concentrations of reference substances and reference substances of components to be measured are taken to prepare reference substance and reference substance solutions of components to be measured, which are injected into high performance liquid chromatography for determination, and the concentration is linearly regressed against the peak area using the least squares method (the origin should be brought in), and the relative correction factor is calculated according to the following formula:

[0010]

[0011] Among them, f sx K is the relative slope ratio of the reference object s to the component to be measured x, s is the slope of the standard curve of the reference substance s, K s is the slope of the standard curve of the component to be tested x, and the relative correction factors f of other components are calculated by taking paeonol in the reference substance as the reference. sx ;

[0012] (2) preparing a test solution: mixing the peony bark and the solvent, dissolving them by ultrasonic treatment, cooling, fixing the volume, mixing, filtering, and taking the filtrate to obtain a test solution;

[0013] (3) Chromatographic conditions:

[0014] Mobile phase A: acetonitrile; Mobile phase B: formic acid water

[0015] Elution conditions:

[0016] From 0 to 30 minutes, the volume fraction of mobile phase A increased from 5% to 15% to 20% to 30%;

[0017] From 30 to 50 minutes, the volume fraction of mobile phase A increases from 20% to 30% to 35% to 55%;

[0018] 50min~55min, the volume fraction of mobile phase A is reduced from 35%~55% to 5%~15%;

[0019] 55min~60min, the volume fraction of mobile phase A is 5%~15%;

[0020] Detection wavelength: 250nm~260nm; flow rate: 0.8~1.2mL / min; column temperature: 25~40℃;

[0021] Chromatographic column: C18 column;

[0022] (4) Determination: Inject the sample solution into a high performance liquid chromatograph and calculate the contents of gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose and benzoylpeoniflorin by the internal standard method through the relative correction factor with paeonol.

[0023] Furthermore, the solvent in step (1) and step (2) is methanol or a methanol aqueous solution, preferably a 70-80% (v / v) methanol aqueous solution.

[0024] Furthermore, in step (1), the concentration of the reference solution is 0.400-0.700 mg / mL, preferably 0.436-0.667 mg / mL.

[0025] Furthermore, in step (2), the ultrasonic time is 30-50 minutes, the power is 100-500 W, and the frequency is 40-60 KHz; preferably, the ultrasonic time is 45 minutes, the power is 300 W, and the frequency is 50 KHz.

[0026] Furthermore, in step (3), the detection wavelength is 254 nm, the flow rate is 1.0 mL / min, and the column temperature is 30°C.

[0027] Further, in step (3), the conditions for gradient elution are:

[0028] From 0 to 30 min, the volume fraction of mobile phase A increased from 10% to 25%;

[0029] From 30 to 50 min, the volume fraction of mobile phase A increased from 25% to 45%;

[0030] From 50 to 55 minutes, the volume fraction of mobile phase A was reduced from 45% to 10%;

[0031] 55min~60min, the volume fraction of mobile phase A is 10%;

[0032] Furthermore, in step (3), the mobile phase B is a 0.01-1% (v / v) formic acid aqueous solution; preferably, a 0.2% (v / v) formic acid aqueous solution.

[0033] Furthermore, in step (3), the C18 column is selected from at least one of ZORBX Extend-C18, Aglient ZORBAX SB-C18 column, and Aglient TC-C18 (2) column; preferably, the C18 column is a ZORBX Extend-C18 chromatographic column. The specifications of the chromatographic column are: 4.6 mm×250 mm, 5 μm.

[0034] In the second aspect, the present invention seeks to protect the application of the above method in detecting the contents of paeonol, gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose and benzoylpeoniflorin in peony bark and its preparations.

[0035] The method of the present invention calculates the content of the index components by relative correction factors and chromatographic peak positioning, and can simultaneously and effectively detect the contents of seven components in the peony bark and its preparations, including gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose, benzoylpeoniflorin, and paeonol, can save costs, simplify operations, improve efficiency, and has high detection sensitivity, good stability, and accurate and reliable measurement results, which is of great significance to the quality control and clinical efficacy of the peony bark and its preparations

[0036] The beneficial effects of the present invention are:

[0037] The present invention provides a one-measurement-multiple-evaluation method for simultaneously determining the contents of gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, benzoylpeoniflorin and paeonol in moutan bark and its preparations. Through methodological verification, relative correction factor confirmation, repeatability investigation, chromatographic peak positioning and accuracy evaluation, the one-measurement-multiple-evaluation method has no significant difference in content determination results with the external standard method. The present invention has high repeatability, stability and credibility, and can provide a reference basis for the quality evaluation of moutan bark and its preparations. DETAILED DESCRIPTION

[0038] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0039] The instruments and reagents used in the embodiments of the present invention are as follows:

[0040] Instruments: Agilent 1260 high performance liquid chromatograph (Agilent (USA) Technology Co., Ltd.); Shimadzu LC-20AT high performance liquid chromatograph (Shimadzu Co., Ltd., Japan); 1 / 100,000 electronic analytical balance (Mettler Toledo Technology Co., Ltd., model: CP225D); 1 / 10,000 electronic balance (Mettler Toledo Technology (China) Co., Ltd., model: YTJ-05-001); desktop CNC ultrasonic cleaner (Dongguan Keqiao Ultrasonic Equipment Co., Ltd., model: KQ-300DE).

[0041] Test drugs: The sources of Paeonia suffruticosa samples are shown in Table 1; gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose, benzoylpeoniflorin, and paeonol were purchased from the China Food and Drug Inspection Institutes; acetonitrile (chromatographic grade); formic acid (analytical grade); and ultrapure water.

[0042] Table 116 Origin and Batch Number of Paeonia Suffruticosa Andrews

[0043] serial number Origin batch number serial number Origin batch number 1 Guizhou 181108 9 Kaifeng, Henan 130106 2 Longnan, Gansu 170401 10 Kaifeng, Henan 140901 3 Xiangyang, Hubei 130901 11 Jilin 190607 4 Heze, Shandong 110209 12 Jilin 181202 5 Heze, Shandong 180201 13 Anhui Tongling 20210819 6 Qiaodong, Anhui 120211 14 Anhui Tongling 20221218 7 Anhui Wei Gang 101003 15 Chongqing Dianjiang 20180302 8 Anhui Bozhou 23090075 16 Chongqing Dianjiang 191203

[0044] Example 1 One-step multiple-evaluation content determination method for seven chemical components in Paeonia suffruticosa and its preparations

[0045] 1. Specific steps of the determination method:

[0046] (1) Preparation of reference solution: Accurately weigh appropriate amounts of gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose, benzoylpeoniflorin, and paeonol reference substances, accurately weigh, and add 70% (v / v) methanol to prepare 0.522 mg / mL gallic acid reference solution, 0.530 mg / mL oxidized paeoniflorin reference solution, 0.448 mg / mL methyl gallate reference solution, 0.455 mg / mL paeoniflorin reference solution, 0.667 mg / mL pentagalloylglucose reference solution, 0.436 mg / mL benzoylpeoniflorin reference solution, and 0.524 mg / mL paeonol reference solution, respectively.

[0047] (2) Preparation of the test solution of medicinal materials: Take 1 g of peony bark (MDP) powder, weigh it accurately, put it in a conical flask, accurately add 50 mL of 80% (v / v) methanol, weigh it, and then ultrasonically treat it (power 300 W, frequency 50 kHz) for 45 min. After cooling at room temperature (25±5°C), weigh it again, and make up the weight loss with the extraction solvent. Shake well, filter, take the filtrate, and filter it through a 0.45 μm microporous membrane to obtain the test solution.

[0048] (3) Accurately pipette 10 μL of the reference solution of step (1) and 16 batches of test solution of step (2) respectively, and inject them into a liquid chromatograph, using octadecylsilane bonded silica gel as a filler; using acetonitrile (A)-0.2% formic acid water (B) as a mobile phase, gradient elution, see Table 2; detection wavelength is 254 nm; column temperature is 30° C., flow rate is 1 mL / min;

[0049] Table 2 Gradient elution conditions

[0050] Time (min) Acetonitrile (v / v,%) 0.2% formic acid water (v / v,%) 0 10 90 30 25 75 50 45 55 55 10 90 60 10 90

[0051] 2. Methodological Investigation

[0052] 1. Linear relationship investigation

[0053] Take the mixed reference solution, accurately transfer 1, 2, 4, 6, 8, 10 ml to 10 ml volumetric flasks respectively, and dilute to the scale with 70% methanol solution to obtain the mixed reference solution of reference 1-reference 6. Perform the determination under the same chromatographic conditions. Take the concentration of the reference solution as the X-axis and the chromatographic peak area as the Y-axis for linear regression. The results of the linear relationship investigation are shown in Table 3.

[0054] Table 3 Regression equations, linear ranges, and correlation coefficients of the seven components

[0055] Element Linear equations Linear range (μg / mL) <![CDATA[R 2 ]]> gallic acid y=17766x+45874 16.31～522.00 0.9999 Oxidized paeoniflorin y=14872x-26625 16.55～529.50 1.0000 Methyl Gallate y=18303x-5053.6 14.00～448.00 1.0000 Paeoniflorin y=1248.5x-4267.3 14.22～455.00 0.9998 Pentagalloylglucose y=9353.4x-27669 20.84～667.00 1.0000 Benzoylpaeoniflorin y=1897.8x-7578.7 13.63～436.00 0.9998 Paeonol y=11236x-1842.2 16.36～523.50 0.9998

[0056] 2. Precision test

[0057] Accurately pipette 10 μl of the mixed reference solution, inject 6 times continuously, and calculate the RSD of the peak areas of the seven components. The results show that the peak area RSD of gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose, benzoylpeoniflorin, and paeonol is less than 4%, indicating that the instrument has good precision.

[0058] 3. Stability test

[0059] Take the same sample solution, let it stand at room temperature for 24 hours, and perform liquid phase analysis at 6 different time points (0h, 2h, 4h, 8h, 12h, 24h) according to the above chromatographic conditions to examine the stability. The results show that the RSD value of each peak area measured under the chromatographic conditions is less than 4%, indicating good stability within 24 hours.

[0060] 4. Repeatability test

[0061] Six portions of the same batch of test solution were taken and measured under the above chromatographic conditions to investigate the repeatability of the method. The results showed that the RSD value of each peak area measured under the chromatographic conditions was less than 4%, indicating that the method had good repeatability.

[0062] 5. Determination of relative correction factor

[0063] The specific method is to take different series of concentrations of reference substances and reference substances of the components to be tested, prepare reference substance and reference substance solutions of the components to be tested, inject them into the high performance liquid chromatograph, use the least square method to perform linear regression of the peak area with concentration (the origin should be brought in), and calculate the relative correction factor according to the following formula, where f sx K is the relative slope ratio of the reference object s to the component to be measured x, s is the slope of the standard curve of the reference substance s, K x is the slope of the standard curve of the component to be tested x, and the relative correction factors f of other components are calculated by taking paeonol in the reference substance as the reference. sx , relative correction factor f sx As shown in Table 4.

[0064]

[0065] Table 4 Relative correction factors

[0066]

[0067] III. Evaluation of the durability of the relative correction factor f (I) The influence of different HPLC instruments and chromatographic columns on the relative correction factor.

[0068] The effects of different HPLC instruments (Shimadzu, Agilent 1260Ⅱ) and three different batches of different chromatographic columns on the correction factor were investigated. The results showed that f had good durability when using different instruments and different chromatographic columns, as shown in Table 5 for details.

[0069] Table 5 Effects of different HPLC instruments and chromatographic columns on relative correction factors

[0070]

[0071] (II) Effects of different flow rates and column temperatures on fk / m values

[0072] The mixed reference solution was accurately pipetted and measured under the same chromatographic conditions. The effects of different flow rates (0.9, 1.0, 1.1 mL / min) and column temperatures (28°C, 30°C, 32°C) on f were investigated. The results showed that f had good durability at different flow rates and column temperatures. See Tables 6 and 7 for details.

[0073] Table 6 Effect of different flow rates on f

[0074]

[0075] Table 7 Effect of different column temperatures on f

[0076]

[0077]

[0078] (III) Chromatographic peak positioning of the component to be measured

[0079] The relative retention time was used to locate the chromatographic peak of the component to be measured, that is, the ratio of the retention time of each component to be measured to that of paeonol, and the reproducibility of the relative retention value on high performance liquid chromatographs of different brands and chromatographic columns of different models, as well as the reproducibility of the relative retention value at different flow rates and column temperatures were investigated. The results showed that the reproducibility was good, as shown in Table 8.

[0080] Table 8 Effect of different HPLC instruments on t

[0081]

[0082] (IV) Sample content determination and validation of QAMS method

[0083] Take the MDP (Paeonia suffruticosa) sample solution, inject and measure it according to the above chromatographic conditions, record the peak area, and calculate the contents of the seven components in the sample by external standard method and QAMS method respectively.

[0084] Table 9 Comparison of the contents of seven components in Paeonia suffruticosa by ESM and QAMS (with paeonol as internal reference)

[0085]

[0086]

[0087] The relative correction factors of the external standard one-point method and the slope correction method were used to calculate the content of each component as shown in Table 9. By calculation, the average relative error of each component to be tested was 3.041% for gallic acid, 1.804% for oxidized paeoniflorin, 0.097% for methyl gallate, 3.623% for paeoniflorin, 1.688% for pentagalloyl glucose, and 4.458% for benzoylpeoniflorin, which was less than 5%. It can be seen that there was no significant difference in the content determination results, so the experiment can prove that the established method has good accuracy.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the contents of seven components in Paeonia suffruticosa and its preparations using a one-measurement-multiple-evaluation method, characterized in that: The method is as follows: paeonol is used as an internal standard for one-test-multiple-evaluations, and relative correction factors between paeonol and gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, and benzoylpeoniflorin are established; the contents of gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, and benzoylpeoniflorin in peony bark and its preparations are calculated by the relative correction factors.

2. The method according to claim 1, characterized in that The method specifically comprises the following steps: (1) Preparing reference solution and establishing relative correction factors between internal standard and other components to be tested: Using paeonol as internal standard, the process of establishing relative correction factors between paeonol and gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, and benzoylpeoniflorin is as follows: gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloyl glucose, benzoylpeoniflorin and paeonol are dissolved in solvents respectively; different series of concentrations of reference substances and reference substances of components to be tested are taken to prepare reference substance and reference substance solutions of components to be tested, which are injected into high performance liquid chromatography for determination, and the concentration is linearly regressed against the peak area using the least squares method, and the relative correction factor is calculated according to the following formula: Among them, f sx K is the relative slope ratio of the reference object s to the component to be measured x, s is the slope of the standard curve of the reference substance s, K x is the slope of the standard curve of the component to be tested x, and the relative correction factors f of other components are calculated by taking paeonol in the reference substance as the reference. sx ; (2) preparing the test solution: mixing the peony bark and the solvent, dissolving them by ultrasonic treatment, cooling, fixing the volume, mixing, filtering, and taking the filtrate to obtain the test solution; (3) Chromatographic conditions: Mobile phase A: acetonitrile; Mobile phase B: formic acid water Elution conditions: From 0 to 30 minutes, the volume fraction of mobile phase A increased from 5% to 15% to 20% to 30%; From 30 to 50 minutes, the volume fraction of mobile phase A increases from 20% to 30% to 35% to 55%; 50min~55min, the volume fraction of mobile phase A is reduced from 35%~55% to 5%~15%; 55min~60min, the volume fraction of mobile phase A is 5%~15%; Detection wavelength: 250nm~260nm; flow rate: 0.8~1.2mL / min; column temperature: 25~40℃; Chromatographic column: C18 column; (4) Determination: Inject the sample solution into a high performance liquid chromatograph and calculate the contents of gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose and benzoylpeoniflorin by the internal standard method through the relative correction factor with paeonol.

3. The method according to claim 2, characterized in that The solvent in step (1) and step (2) is methanol or methanol aqueous solution, preferably 70-80% methanol aqueous solution.

4. The method according to claim 2, characterized in that: In step (1), the concentration of the reference solution is 0.400-0.700 mg / mL, preferably 0.436-0.667 mg / mL.

5. The method according to claim 2, characterized in that: In step (2), the ultrasonic time is 30-50 minutes, the power is 100-500W, and the frequency is 40-60KHz; preferably, the ultrasonic time is 45 minutes, the power is 300W, and the frequency is 50KHz.

6. The method according to claim 2, characterized in that The detection wavelength in step (3) is 254 nm, the flow rate is 1.0 mL / min, and the column temperature is 30°C.

7. The method according to claim 1, characterized in that In step (3), the conditions for gradient elution are: From 0 to 30 min, the volume fraction of mobile phase A increased from 10% to 25%; From 30 to 50 min, the volume fraction of mobile phase A increased from 25% to 45%; From 50 to 55 minutes, the volume fraction of mobile phase A was reduced from 45% to 10%; 55min~60min, the volume fraction of mobile phase A is 10%.

8. The method according to claim 2, characterized in that: In step (3), the mobile phase B is a 0.01-1% formic acid aqueous solution, preferably a 0.2% formic acid aqueous solution.

9. The method according to claim 2, characterized in that: In step (3), the C18 column is selected from at least one of ZORBX Extend-C18, Aglient ZORBAX SB-C18 column, and Aglient TC-C18 (2) column; preferably, it is a ZORBX Extend-C18 column.

10. Use of the method according to any one of claims 1 to 9 in detecting the contents of paeonol, gallic acid, oxidized paeoniflorin, methyl gallate, paeoniflorin, pentagalloylglucose and benzoylpeoniflorin in peony bark and its preparations.

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