A method for determining the components of a zhusun capsule

Abstract

The application provides a method for determining components in Zhichun capsules, and simultaneously determines the contents of four components, i.e., isopsoralen, psoralen, icariin and cimicifugan, in Zhichun capsules by using high performance liquid chromatography-ultraviolet detection. By reasonably selecting chromatographic conditions and detection conditions, the method can realize rapid extraction and detection of the four components in Zhichun capsules, and has the advantages of simplicity, high sensitivity, fast analysis speed and the like, and can be used for quality control of Zhichun capsules.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine component determination technology, and in particular relates to a method for determining the components in Zhuchun capsules. Background Technology

[0002] Zhuchun Capsules are a traditional Chinese medicine preparation developed by Luoyang Orthopedic Hospital in Henan Province. It is mainly composed of 14 Chinese herbs, including Psoralea corylifolia, Cnidium monnieri, Epimedium, Lycium barbarum, Cistanche deserticola, stir-fried hawthorn, Shenqu (medicated leaven), oyster shell, stir-fried jujube seed, and Salvia miltiorrhiza. It has the effects of tonifying the liver and kidneys, strengthening the spleen, and enhancing bone quality. It is mainly used for lower back and leg pain, weakness, and heaviness caused by osteoporosis, and degenerative osteoarthritis caused by bone degeneration. It has been used clinically for many years with definite efficacy. Clinical studies have shown that Zhuchun Capsules can increase the levels of serum calcium and osteocalcin in patients' serum, and decrease the level of serum tartrate phosphatase 5b, thereby increasing the activity of osteoblasts and decreasing the activity of osteoclasts, and improving bone metabolism.

[0003] In prescriptions, psoralea corylifolia has anti-osteoporosis effects, with its representative components being isopsoralen and psoralen. Studies have reported that isopsoralen and psoralen can promote osteoblast proliferation and differentiation, and enhance bone metabolism. Epimedium, also known as Xianlingpi, has been shown in modern pharmacological studies to tonify kidney yang, strengthen tendons and bones, and dispel wind and dampness. Icariin, the main component of epimedium, can promote the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells, accelerate fracture healing, and promote new bone formation. Cnidium monnieri has aphrodisiac and debilitating effects. Osthol, the most abundant coumarin compound in Cnidium monnieri, is considered in traditional Chinese medicine to have warming kidney yang, dispelling wind and dampness effects. Zhuchun Capsules are composed of 14 Chinese herbs, with complex ingredients. Based on its formulation, indications, and biological indicators, the components that significantly influence its efficacy should be isopsoralen and psoralen from Psoralea corylifolia, icariin from Epimedium brevicornu, and osthol from Cnidium monnieri. However, there are very few reports on the quality control of Zhuchun Capsules; currently, only the content of icariin as a single component has been measured, and there are no reports on the simultaneous measurement of the content of multiple components.

[0004] Chinese patent application CN200710028079.9 establishes a high-performance liquid chromatography (HPLC) method for the determination of isopsoralen and psoralen. Chinese patent applications 202211224747.6 and 202210783992.4 establish HPLC methods for the determination of icariin. Chinese patent application CN202011629098.9 establishes an HPLC method for the determination of osthol. However, although these existing technologies all employ HPLC, there are significant differences between the methods. Using different methods to determine the four main components is not only cumbersome but also results in inconsistent detection limits and slow analytical speed, which is detrimental to rapid quality control in clinical practice. Summary of the Invention

[0005] To address the inconsistencies in the aforementioned analytical methods, the purpose of this invention is to establish a high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the simultaneous determination of four components in Zhuchun capsules: isopsoralen, psoralen, icariin, and osthol.

[0006] To achieve the above objectives, the present invention provides a method for determining the components in Zhuchun capsules, comprising:

[0007] (1) Chromatographic conditions: A C18 column was selected, and a methanol-water system was used as the mobile phase for gradient elution. The detection wavelength was 254 nm, and the flow rate was 1.0 mL / min. -1 ;

[0008] (2) Preparation of reference solution: Accurately weigh isopsoralen, icariin, psoralen and osthol reference standards into 10 mL volumetric flasks, dissolve and dilute with methanol to prepare a mixed reference standard stock solution.

[0009] (3) Preparation of test solution: Accurately weigh about 1g of Zhuchun capsule powder and put it into an Erlenmeyer flask. Add the extraction solvent, seal tightly, weigh the mass, sonicate at room temperature for 30min, cool to room temperature, replenish the lost mass with the extraction solvent, filter through a 0.22μm microporous membrane, and put the filtrate into a sample bottle for later use.

[0010] (4) Content determination: Take an appropriate amount of mixed reference standard and test solution, and perform high performance liquid chromatography analysis under the chromatographic conditions to obtain the chromatographic peak area of ​​the test solution and determine the content of isopsoralen, icariin, psoralen and osthol in the sample to be tested.

[0011] In a preferred embodiment, gradient elution is performed using a methanol-water system with an initial methanol volume fraction of 40%, 30%, or 10%, preferably using a methanol-water system with an initial methanol volume fraction of 10%.

[0012] In a preferred embodiment, elution is performed according to the following gradient:

[0013]

[0014] In a preferred embodiment, the detection wavelength, injection volume: 5 μL, column temperature: 30°C

[0015] In a preferred embodiment, the C18 column is an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm).

[0016] In a preferred embodiment, the extraction solvent for the test sample is water, methanol, or ethanol, preferably methanol. More preferably, the solvent for the reference standard is the same as that for the test sample.

[0017] In a preferred embodiment, the retention time of isopsoralen is 35.0 min, the retention time of psoralen is 36.5 min, the retention time of icariin is 49.8 min, and the retention time of osthol is 64.1 min.

[0018] The advantages of this invention are: the established HPLC method simultaneously determines the contents of isopsoralen, psoralen, icariin, and osthol in Zhuchun capsules; this method is stable, has high resolution, and good repeatability. This invention achieves rapid extraction and detection of these four components in Zhuchun capsules, offering advantages such as simplicity, high sensitivity, and fast analysis speed, and can be used for the quality control of Zhuchun capsules. Attached Figure Description

[0019] Figure 1 This is the HPLC chromatogram of the mixed reference standard (A) in Example 1 of this invention;

[0020] Figure 2 This is the HPLC chromatogram of the test sample (B) in Example 1 of the present invention.

[0021] In the attached diagram, 1. isopsoralen, 2. psoralen, 3. icariin, and 4. osthol. Detailed Implementation

[0022] In a specific embodiment, the present invention provides a method for determining the components in Zhuchun capsules, comprising:

[0023] (1) Chromatographic conditions: C18 column was selected, methanol-water system was selected as the mobile phase for gradient elution, detection wavelength was 254 nm, and volume flow rate was 1.0 mL·min-1;

[0024] (2) Preparation of reference solution: Accurately weigh isopsoralen, icariin, psoralen and osthol reference standards into 10 mL volumetric flasks, dissolve and dilute with methanol to prepare a mixed reference standard stock solution.

[0025] (3) Preparation of test solution: Accurately weigh about 1g of Zhuchun capsule powder and put it into an Erlenmeyer flask. Add the extraction solvent, seal tightly, weigh the mass, sonicate at room temperature for 30min, cool to room temperature, replenish the lost mass with the extraction solvent, filter through a 0.22μm microporous membrane, and put the filtrate into a sample bottle for later use.

[0026] (4) Content determination: Take an appropriate amount of mixed reference standard and test solution, and perform high performance liquid chromatography analysis under the chromatographic conditions to obtain the chromatographic peak area of ​​the test solution and determine the content of isopsoralen, icariin, psoralen and osthol in the sample to be tested.

[0027] Regarding the selection of mobile phase: This application investigated isocratic or gradient elution with acetonitrile-water and methanol-water, and also investigated the effect of the initial volume fraction of methanol (40%, 30%, 10%) on the separation effect. The results showed that the separation effect of isocratic or gradient elution with acetonitrile-water was not good. Only when the initial volume fraction of methanol was 10% did the chromatographic peak shape and resolution of isopsoralen, psoralen, icariin and osthol were better.

[0028] Regarding the selection of detection wavelength: This application investigated the response of each chromatographic peak at three wavelengths: 254 nm, 270 nm, and 324 nm. The results showed that at 254 nm, the peak area was larger, the resolution was better, and the sensitivity was higher, meeting the analytical requirements. Therefore, the content of four components—icariin, osthol, psoralen, and isopsoralen—in Zhuchun capsules was determined at a wavelength of 254 nm.

[0029] Regarding the selection of the test solution preparation method: This application investigated different extraction solvents, water, methanol, and ethanol. The results showed that methanol was the optimal extraction solvent due to the presence and size of the most chromatographic peaks. Ultrasonic and reflux extraction methods were also investigated, and the results indicated that ultrasonic extraction for 30 minutes resulted in the highest extraction rates for each component.

[0030] To provide a more complete understanding of the implementation of this invention, the following description uses typical embodiments to further illustrate the invention. Unless otherwise defined, the technical or scientific terms used in this patent application specification and claims should have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains.

[0031] Example 1:

[0032] Instruments: Agilent 1260 high-performance liquid chromatograph, equipped with a DADWR detector; AL204 electronic balance (Shanghai Mettler Toledo Instruments Co., Ltd.); KQ-250DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)

[0033] Reagents: Psoralen (batch number B20123-20mg, purity ≥98%) reference standard, isopsoralen (batch number: B21515-20mg, purity ≥98%) reference standard, icariin (batch number B21576-20mg, purity ≥98%) reference standard, osthol (batch number B21152-20mg, purity ≥98%) reference standard (Shanghai Yuanye Biotechnology Co., Ltd.); chromatographic grade methanol (Merck, Germany); Zhuchun capsules were produced by the preparation room of Luoyang Orthopedic Hospital, Henan Province.

[0034] Chromatographic conditions: Column: Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm); mobile phase: methanol-water (10:90); gradient elution; detection wavelength: 254 nm; flow rate: 1.0 mL·min⁻¹; injection volume: 5 μL; column temperature: 30 °C. Elution conditions are shown in Table 1.

[0035] Table 1 Gradient elution program

[0036]

[0037] Solution preparation:

[0038] Preparation of reference solutions: Accurately weigh 5.91 mg, 3.15 mg, 4.94 mg, and 4.83 mg of isopsoralen, icariin, psoralen, and osthol reference standards into 10 mL volumetric flasks, dissolve them in methanol and dilute to volume to prepare a mixed reference stock solution.

[0039] Preparation of the test solution: Take several Zhuchun capsules, remove the shells and take the powder to obtain Zhuchun capsule sample powder. Accurately weigh about 1g of Zhuchun capsule powder and put it into an Erlenmeyer flask. Add 50mL of methanol, seal tightly, weigh it, sonicate at room temperature for 30min, cool to room temperature, replenish the lost mass with methanol, filter through a 0.22μm microporous membrane, and put the filtrate into a sample bottle for later use.

[0040] Methodological examination:

[0041] System suitability test

[0042] Take appropriate amounts of the mixed reference standard and test solution, and perform high-performance liquid chromatography analysis under the above chromatographic conditions. The results are shown in the figure. Figure 1 , 2 .analyze Figure 1 , 2 It can be seen that under the chromatographic conditions, the separation degree of the four components, iridoidin, osthol, psoralen and isopsoralen, is greater than 1.5, indicating that the method has good system applicability.

[0043] Examining linear relationships

[0044] Take an appropriate volume of the mixed reference solution and perform high-performance liquid chromatography (HPLC) analysis under the above chromatographic conditions. Linear regression was performed with the reference concentration as the abscissa (X) and the peak area as the ordinate (Y). The linear regression equation was obtained, and the results are shown in Table 2. It can be seen that each component has a good linear relationship within its respective range.

[0045] Table 2 Linear Relationships of Each Component

[0046]

[0047] Precision test

[0048] Take 5 μL of the mixed reference solution and inject it 6 times consecutively under the above chromatographic conditions. The peak area RSDs of isopsoralen, psoralen, icariin and osthol were 2.76%, 0.37%, 0.43% and 2.40%, respectively, indicating that the instrument has good precision.

[0049] Stability test

[0050] Take 5 μL of the test solution and place it at room temperature for 0, 2, 4, 6, 8, and 12 hours. Then, determine the peak areas RSD of isopsoralen, psoralen, icariin, and osthol under the above chromatographic conditions. The RSDs are 0.46%, 0.21%, 0.29%, and 2.83%, respectively, indicating that the test solution has good stability within 12 hours.

[0051] Repeatability test

[0052] Six parallel solutions of the same batch of Zhuchun capsules (batch number: 211201) were prepared according to the above method. The solutions were measured under the above chromatographic conditions, and the peak areas were recorded. The RSDs of the peak areas of isopsoralen, psoralen, icariin, and osthol were 2.87%, 1.43%, 0.71%, and 0.28%, respectively, indicating good repeatability of the method.

[0053] Recovery test

[0054] Six portions of Zhuchun Capsules (batch number: 211201) with known content, each approximately 0.5 g, were accurately weighed. Equal amounts of reference standards were accurately added to each portion, and test solutions were prepared according to the above method. The solutions were then determined under the above chromatographic conditions. The recoveries of isopsoralen, psoralen, icariin, and osthol were calculated to be 98.97%, 99.90%, 102.80%, and 101.80%, respectively. The results are shown in Table 3.

[0055] Table 3. Results of the spiking recovery rate test for four components of Zhuchun Capsules (n=6)

[0056]

[0057] Determination of content of test sample

[0058] Take 6 portions of Zhuchun capsule sample powder, prepare test solutions according to the above method, and determine them under the above chromatographic conditions. Record the chromatographic peak area and calculate the content. The results are shown in Table 4.

[0059] Table 4. Results of component content determination (mg / g)

[0060]

[0061] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for determining the components in Zhuchun capsules, comprising: (1) Chromatographic conditions: A C18 column was selected, and a methanol-water system was used as the mobile phase for gradient elution. The detection wavelength was 254 nm, and the flow rate was 1.0 mL / min. -1 ; The gradient elution is as follows: (2) Preparation of reference solution: Accurately weigh isopsoralen, icariin, psoralen and osthol reference standards into 10 mL volumetric flasks, dissolve them in solvent and dilute to volume to prepare a mixed reference standard mother solution; (3) Preparation of test solution: Accurately weigh 1 g of Zhuchun capsule powder, place it in an Erlenmeyer flask, add extraction solvent, seal tightly, weigh it, sonicate at room temperature for 30 min, cool to room temperature, replenish the lost mass with extraction solvent, filter through a 0.22 μm microporous membrane, and place the filtrate in a sample bottle for later use; the extraction solvent is methanol; (4) Content determination: Take an appropriate amount of mixed reference standard and test solution, perform high performance liquid chromatography analysis under the chromatographic conditions, obtain the chromatographic peak area of ​​the test solution, and determine the content of isopsoralen, icariin, psoralen and osthol in the sample to be tested.

2. The method according to claim 1, wherein, Injection volume: 5 μL, column temperature: 30 ℃.

3. The method according to claim 1, wherein, The C18 column is an Agilent ZORBAX SB-C18 column.

4. The method according to claim 1, wherein, The reference standard and the test sample use the same solvent.

5. The method according to claim 1, wherein, In step (2), 5.91 mg, 3.15 mg, 4.94 mg, and 4.83 mg of isopsoralen, icariin, psoralen, and osthol reference standards were accurately weighed respectively.

6. The method according to claim 1, wherein, The retention times of isopsoralen were 35.0 min, psoralen was 36.5 min, icariin was 49.8 min, and osthol was 64.1 min.

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