Method for determining contents of various flavone components in clematis tenuifolia medicinal material
Optimizing the detection of flavonoid components in clematis medicinal materials through high-performance liquid chromatography, solving the problem that the determination method in the prior art is not accurate and efficient, and achieving rapid and accurate flavonoid content analysis, simplifying the process and improving the Accuracy of medicinal quality evaluation.
Patent Information
- Application Number
- CN202411260638.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2024-12-27
AI Technical Summary
The existing method for measuring the content of flavonoids in fine leaf clematis medicinal materials is not accurate and efficient enough, and it is difficult to effectively reflect the quality standards of the medicinal materials. The analysis process is complicated and the detection time is long, and the results are not accurate enough.
High performance liquid chromatography was used to determine the content of quercetin, luteolin, yamphetol and apigenin in the medicinal materials of fine leaf clematis by optimizing the length of the chromatographic column, mobile phase composition and detection conditions, and ultrasonic extraction and acid Hydrolysis destroys glycosidic bonds and generates flavonoid aglycones for detection.
It realizes rapid and accurate measurement of flavonoid components in clematis medicinal materials, simplifies the analysis process, improves the scientificity and feasibility of detection, provides a more intuitive reflection of flavonoid content, and is suitable for medicinal quality evaluation and standards. promote.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining the content of flavonoid components in a Clematis tenuifolia medicinal material. Background Art
[0002] Clematis aethusifolia Turcz is a herbaceous vine of the genus Clematis in the family Ranunculaceae. It is also known as celery-leaf clematis. Its Mongolian name is "Temur-Aoriyanggu". It is harvested when the flowers are in full bloom in summer, dried in the sun or cut into sections and dried in the sun. It is pungent, slightly sweet, hot, light, sharp, dry, and rough; it is poisonous and has the effects of "breaking lumps, regulating temperature, drying, stopping corruption, reducing swelling, and stopping diarrhea". It is mainly used for cold lumps, indigestion, cold "Xie Ri Wu Su" disease, "Wu Ya Man" disease, impetigo, wounds, edema, and cold diarrhea.
[0003] Modern research shows that the chemical components isolated and identified from Clematis truncatula mainly include flavonoids, lignans, phenylpropionic acid derivatives, coumarins, and saponins. Among them, the active aglycones of flavonoids are mainly quercetin, luteolin, kaempferol and apigenin.
[0004] The present invention adopts high performance liquid chromatography to establish a method for determining the contents of flavonoid components quercetin, luteolin, kaempferol and apigenin in Clematis tenuifolia medicinal materials, so as to provide a reference for quality control of Clematis medicinal materials and improvement and perfection of the quality standards of Clematis tenuifolia medicinal materials. Summary of the invention
[0005] The purpose of the present invention is to provide a method for determining the content of flavonoid components in a Clematis tenuifolia medicinal material.
[0006] To achieve the above object, the technical solution of the present invention is as follows: The method for determining the content of flavonoid components in the Clematis chinensis medicinal material of the present invention adopts high performance liquid chromatography to simultaneously determine the content of the main flavonoid active components quercetin, luteolin, kaempferol, and apigenin in the Clematis chinensis medicinal material. The main active components determined by the present invention are all flavonoid components, which have biological activities such as anti-oxidation, anti-inflammatory, antibacterial, improving cardiovascular health, absorbing ultraviolet radiation, and anti-allergic effects.
[0007] The chromatographic conditions of the high performance liquid chromatography method of the present invention are: the chromatographic column is a C18 chromatographic column; the mobile phase is methanol-0.2% phosphoric acid aqueous solution (50:50, v / v); the flow rate is 1.0 mL / min; the detection wavelength is 360 nm; and the column temperature is 25°C.
[0008] The chromatographic column used in the high performance liquid chromatography method of the present invention is UItimate XB-C18 (4.6 mm×250 mm, 5 μm), which is a reverse phase chromatographic column of octadecylsilane bonded silica gel, whose silica gel particle diameter is 5 μm, column length is 250 mm, and column inner diameter is 4.6 mm. The present invention compares two C18 chromatographic columns of 150 mm and 250 mm in length, and the results show that when using the C18 chromatographic column of 150 mm in length, the separation degree of kaempferol and apigenin is less than 1.0, while using the C18 chromatographic column of 250 mm can make the separation degree of kaempferol and apigenin greater than 1.5.
[0009] The mobile phase of the chromatographic conditions of the present invention is methanol-0.2% phosphoric acid aqueous solution (50:50, v / v), wherein methanol is the organic phase and the phosphoric acid aqueous solution is the aqueous phase.
[0010] The present invention compares 0.05%, 0.1%, 0.2%, 0.3% and 0.4% phosphoric acid aqueous solutions as aqueous phases, and the results show that when 0.05% and 0.1% phosphoric acid aqueous solutions are used as aqueous phases, the tailing factors of quercetin and kaempferol chromatographic peaks are greater than 1.05; when 0.3% and 0.4% phosphoric acid aqueous solutions are used as aqueous phases, the separation of kaempferol and apigenin chromatographic peaks is less than 1.5; when 0.2% phosphoric acid aqueous solution is used as aqueous phase, the separation of quercetin, luteolin, kaempferol and apigenin chromatographic peaks is greater than 1.5, the tailing factors are all between 0.95 and 1.05, and the theoretical plate numbers are all greater than 3 000, so 0.2% phosphoric acid aqueous solution is selected as the aqueous phase.
[0011] The present invention compares methanol and 0.2% phosphoric acid aqueous solution according to volume ratios of 40:60, 45:55, 50:50, 55:45 and 60:40 as mobile phases. The results show that when methanol and 0.2% phosphoric acid aqueous solution are used as mobile phases in volume ratios of 40:60 and 45:55, the chromatographic peak separation of quercetin and kaempferol is less than 1.5, and the tailing factor is greater than 1.05; when methanol and 0.2% phosphoric acid aqueous solution are used as mobile phases in volume ratios of 55:45 and 60:40, the chromatographic peak separation of luteolin and apigenin is less than 1.5; when methanol and 0.2% phosphoric acid aqueous solution are used as mobile phases in volume ratios of 50:50, the chromatographic peak separation of quercetin, luteolin, kaempferol and apigenin are all greater than 1.5, and the tailing factors are all between 0.95 and 1.0. 1.05, and the theoretical plate numbers were all greater than 3000, so methanol and 0.2% phosphoric acid aqueous solution were selected as the mobile phase in a volume ratio of 50:50.
[0012] The methanol-0.5% phosphoric acid aqueous solution (50:50, v / v) described in the present invention is a solution prepared by mixing anhydrous methanol and a 1% phosphoric acid aqueous solution in a volume ratio of 50:50.
[0013] The methanol described in the present invention is a chromatographic grade methanol solution with a purity of ≥99.9%.
[0014] The preparation method of the test solution of the present invention is as follows: the sample of Clematis serrata is placed in a 40°C forced air drying oven for 24 hours, taken out, crushed into fine powder, 3 g of fine powder is accurately weighed, placed in a stoppered conical bottle, and methanol solution is added at a solid-liquid ratio of 1:20, ultrasonic extraction (temperature 50°C, power 250W, frequency 40kHz) is performed for 1.5 hours, cooled to room temperature, filtered under reduced pressure, the filtrate is concentrated to dryness under reduced pressure, and the residue is added with 25 mL of a methanol-25% hydrochloric acid solution (4:1, v / v) mixed solution, shaken, heated and refluxed in a 90°C water bath for 0.5 hours, cooled to room temperature, filtered, and the filtrate is placed in a 25 mL volumetric flask, methanol is added to the volume, shaken, and filtered through a 0.45 μm organic microporous filter membrane to obtain the test solution. Flavonoid compounds are usually combined with sugars in plants to form glycosides, and a small part exists in the form of free state (aglycones). The detection of flavonoid aglycones requires the destruction of the glycosidic bonds of flavonoid compounds by pre-column treatment to generate flavonoid aglycones. Therefore, the present invention selects to first extract flavonoids by ultrasonic method using organic solvent, and then acid hydrolyzes to destroy glycosidic bonds to generate flavonoid aglycones.
[0015] The high performance liquid chromatography method of the present invention also includes a method for preparing a reference substance solution, which comprises: accurately weighing reference substances quercetin, luteolin, kaempferol, and apigenin, respectively adding methanol to dissolve, and shaking well to prepare respective reference substance stock solutions; accurately aspirating the quercetin, luteolin, kaempferol, and apigenin stock solutions, placing them in the same volumetric flask, adding methanol to make up to volume, and preparing a mixed reference substance solution.
[0016] The high performance liquid chromatography method of the present invention also includes a method for preparing a blank solution. The blank solution method is: no Clematis medicinal material is added, and the remaining operations are the same as those of the test solution to prepare a blank solution.
[0017] Beneficial effects of the present invention: (1) The present invention establishes a high performance liquid chromatography method to simultaneously conduct quantitative determination of quercetin, luteolin, kaempferol, and apigenin in the medicinal material of Clematis tenuifolia, and the methodological verification is carried out. The results show that the method is scientific and feasible; (2) Compared with the analysis of flavonoid glycoside compounds, the present invention can more intuitively reflect the flavonoid content of the Clematis chinensis medicinal material by analyzing the main flavonoid aglycone components, effectively reducing the inability to effectively separate the spectrum caused by the complexity of the reference substance, shortening the detection time, and achieving accurate detection results, which is simple and easy to operate; (3) The linear range of the present invention is wide, the repeatability is good, the sample processing method is stable and feasible, and the obtained test solution has good stability at room temperature; (4) The method of the present invention is stable and accurate, and can be used for the quality evaluation of Clematis serrata, and can provide a reference for improving the quality standard of Clematis serrata. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 is the HPLC chart of blank solution; Figure 2 is the HPLC chart of the mixed control solution; Figure 3 HPLC chart of the test solution; Figure 4 is the linear relationship diagram of quercetin reference substance; Figure 5 is the linear relationship diagram of luteolin reference substance; Figure 6 is the linear relationship diagram of kaempferol reference substance; Figure 7 This is the linear relationship diagram of apigenin reference substance. DETAILED DESCRIPTION
[0019] The following experimental examples are used to further illustrate but not limit the present invention.
[0020] The Clematis tenuifolia medicinal materials used in the following specific embodiments are from Tibet Shenhou Pharmaceutical Co., Ltd., with batch numbers 20220314, 20220729, 20240322, and 20240464.
[0021] Rutin reference substance (batch number 100080-202012, purity 92.2%), luteolin reference substance (batch number 111520-202107, purity 96.3%), kaempferol reference substance (batch number 110861-202214, purity 97.4%), and quercetin reference substance (batch number 100081-201610, purity 99.1%) were all purchased from China Food and Drug Inspection Institutes; apigenin reference substance (batch number DSTDQ002602, purity 98.5%) was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.; methanol (batch number 20230418) was chromatographic grade and was purchased from Anhui Shilian Special Solvent Co., Ltd.; phosphoric acid (batch number 20210906) was optimized pure and was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). Hydrochloric acid (batch number P2141688) and anhydrous ethanol (batch number P1988182) were purchased from Shanghai Titan Technology Co., Ltd. Example
[0022] The method of simultaneously determining the contents of quercetin, luteolin, kaempferol and apigenin in Clematis tenuifolia medicinal materials by high performance liquid chromatography comprises the following steps: (1) Preparation of test solution The sample of Clematis slender-leaved was placed in a 40°C forced air drying oven for 24 hours, taken out, crushed into fine powder, 3 g of fine powder was accurately weighed, placed in a stoppered conical flask, and methanol solution was added at a solid-liquid ratio of 1:20. Ultrasonic extraction (temperature 50°C, power 250W, frequency 40kHz) was performed for 1.5 h, cooled to room temperature, filtered under reduced pressure, and the filtrate was concentrated to dryness under reduced pressure. 25 mL of a mixture of methanol-25% hydrochloric acid solution (4:1, v / v) was added to the residue, shaken well, heated under reflux at 90°C in a water bath for 0.5 h, cooled to room temperature, filtered, and the filtrate was placed in a 25 mL volumetric flask, methanol was added to the volume, shaken well, and filtered through a 0.45 μm organic microporous filter membrane to obtain the test solution.
[0023] (2) Preparation of mixed reference solution Accurately weigh the reference substances quercetin, luteolin, kaempferol, and apigenin, add methanol to dissolve them respectively, shake well, and prepare the reference substance stock solutions; aspirate appropriate amounts of quercetin, luteolin, kaempferol, and apigenin stock solutions, place them in the same volumetric flask, add methanol to make up to volume, and prepare mixed reference substance solutions with mass concentrations of quercetin, kaempferol, luteolin, and apigenin of 231.36 μg / mL, 40.08 μg / mL, 4.13 μg / mL, and 20.16 μg / mL, respectively.
[0024] (3) Preparation of blank solution Without adding Clematis chinensis, the remaining operations were the same as those for the test solution in (1) to prepare a blank solution. (4) Chromatographic conditions The chromatographic column was UItimate XB-C18 (4.6 mm×250 mm, 5 μm); the mobile phase was methanol-0.2% phosphoric acid aqueous solution (50:50, v / v); the flow rate was 1.0 mL / min; the detection wavelength was 360 nm; the column temperature was 25 ℃; and the injection volume was 20 μL.
[0025] (5) Methodological investigation System suitability test: Take blank solution, mixed reference solution, test solution, and inject according to chromatographic conditions, record the chromatogram, and the results are shown in Figure 1 , Figure 2 , Figure 3 The chromatographic separation of the reference substances quercetin, luteolin, kaempferol and apigenin was >1.5, the theoretical plate number was >3000, the tailing factor was in the range of 0.95-1.05, and the retention time of the mixed reference solution was consistent with that of the test solution, and there was no interference from the blank solution, indicating that the method had good specificity. The chromatographic system suitability test met the requirements for content determination.
[0026] Linear relationship investigation: Accurately pipette 0.25, 0.5, 1, 2, 3, 4, and 5 mL of the mixed reference solution into 5 mL volumetric flasks, dilute with methanol, shake well, and prepare mixed reference solutions of different mass concentrations. Inject and measure according to the chromatographic conditions under (4). Draw a standard curve with the peak area as the ordinate Y and the mass concentration (μg / mL) as the abscissa X and perform regression analysis. The regression equation is shown in Table 1, and the linear relationship diagram results are shown in Figure 4 , Figure 5 , Figure 6 , Figure 7 .
[0027] Table 1 Linear relationship of reference substances Element Regression equation Linear range (μg / mL) Quercetin =73.685+132.79 0.9997 11.57-231.36 Luteolin =81.895-9.7433 0.9993 0.21-4.13 Kaempferol =65.236+4.7068 0.9993 2.01-40.08 Apigenin =36.293+2.4679 0.9995 1.01-20.16 Precision test: The test solution was taken and injected according to the chromatographic conditions. The measurement was performed continuously for 6 times. The RSD (n=6) of the peak areas of quercetin, luteolin, kaempferol and apigenin were 1.23%, 1.77%, 1.02% and 0.88%, respectively, indicating that the instrument had good precision.
[0028] Repeatability test: Take the same batch of Clematis sample powder (batch number 20220314), and prepare 6 test solutions in parallel according to the test solution preparation method. The samples were injected and measured according to the chromatographic conditions. The RSDs (n=6) of the peak areas of quercetin, luteolin, kaempferol, and apigenin were 1.73%, 1.95%, 1.39%, and 1.41%, respectively, indicating that the method has good repeatability.
[0029] Stability test: A new portion of the test solution was prepared and injected at room temperature after 0, 2, 4, 8, 10, 12, and 24 h. The RSDs (n=7) of the peak areas of quercetin, luteolin, kaempferol, and apigenin were 1.29%, 2.13%, 0.69%, and 0.81%, respectively, indicating that the solution was stable within 24 h.
[0030] Sample addition recovery test: 6 portions of test samples with known content (3 g each) were accurately weighed and added with appropriate amount of quercetin, luteolin, kaempferol and apigenin reference sample stock solutions. The test sample solutions were prepared according to the method under (1). The samples were injected and measured respectively. The average recoveries of quercetin, luteolin, kaempferol and apigenin were 99.52%, 98.63%, 99.71% and 98.99%, respectively. The RSDs (n=6) were 0.93%, 1.32%, 1.24% and 1.13%, respectively.
[0031] (6) Determination of flavonoid content in Clematis medicinal materials Four batches of Clematis (batch numbers: 20220314, 20220729, 20240322, 20240464) were taken, and two samples were taken in parallel from each batch. The test solutions were prepared separately, and the samples were injected and measured according to the chromatographic conditions. The results are shown in Table 2.
[0032] Table 2 Determination results of the contents of four flavonoid aglycones in Clematis tenuifolia (n=2) batch number <![CDATA[Quercetin / μg·g -1 > <![CDATA[Luteolin / μg·g -1 > <![CDATA[Kaempferol / μg·g -1 > <![CDATA[Apigenin / μg·g -1 > 20220314 600.33 10.24 92.71 96.30 20220729 599.24 10.08 90.46 94.29 20240322 603.35 11.02 92.89 98.48 20240464 605.27 11,09 92.99 99.38 The present invention establishes a high performance liquid chromatography method to quantitatively determine the flavonoid index components quercetin, luteolin, kaempferol, and apigenin in the Clematis ulmoides medicinal material at the same time and validates the method. The results show that the method is scientific and feasible. Compared with the analysis of flavonoid glycoside compounds, the analysis of the main flavonoid aglycone components can more intuitively reflect the flavonoid content of the Clematis medicinal materials, effectively reduce the inability to effectively separate the spectrum caused by the complexity of the reference substance, the detection time is short, the detection result is accurate, and it is simple and easy. At the same time, the linear range of the present invention is wide, the repeatability is good, the sample processing method is stable and feasible, and the obtained test solution has good stability at room temperature. The method of the present invention is stable and accurate, can be used for the quality evaluation of Clematis ulmoides, and can provide a reference for the improvement of the quality standard of Clematis ulmoides.
[0033] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.
Claims
1. A method for determining the content of multiple flavonoid components in Clematis tenuifolia medicinal materials, characterized in that: The contents of four flavonoid aglycones, including quercetin, luteolin, kaempferol and apigenin, in Clematis tenuifolia were simultaneously determined by HPLC.
2. The method for determining the contents of various flavonoid components in the medicinal material of Clematis tenuifolia according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 chromatographic column; the mobile phase is methanol-0.2% phosphoric acid aqueous solution (50:50, v / v); the flow rate is 1.0 mL / min; the detection wavelength is 360 nm; and the column temperature is 25°C.
3. The method for determining the contents of various flavonoid components in the medicinal material of Clematis tenuifolia according to claim 2, characterized in that: The C18 chromatographic column is a reverse phase chromatographic column of octadecylsilane bonded silica gel, the silica gel particle size is 5 μm, the column length is 250 mm, and the column inner diameter is 4.6 mm.
4. The method for determining the contents of various flavonoid components in the Clematis tenuifolia medicinal material according to claim 1, characterized in that: The method for preparing the test solution in the high performance liquid chromatography method is as follows: the sample of Clematis slenderii is crushed into fine powder, 3 g of the fine powder is accurately weighed, placed in a stoppered conical flask, and a methanol solution is added at a solid-liquid ratio of 1:20, ultrasonic extraction (temperature 50°C, power 250W, frequency 40kHz) is performed for 1.5 h, cooled to room temperature, filtered under reduced pressure, and the filtrate is concentrated to dryness under reduced pressure, and 25 mL of a mixture of methanol-25% hydrochloric acid solution (4:1, v / v) is added to the residue, shaken, heated in a water bath at 90°C for reflux for 0.5 h, cooled to room temperature, filtered, and the filtrate is placed in a 25 mL volumetric flask, methanol is added to the volume, shaken, and filtered through a 0.45 μm organic microporous filter membrane to obtain the test solution.
5. The method for determining the contents of various flavonoid components in the Clematis tenuifolia medicinal material according to claim 1, characterized in that: The method for preparing the reference substance solution in the high performance liquid chromatography method is as follows: accurately weighing the reference substances quercetin, luteolin, kaempferol and apigenin, respectively adding methanol to dissolve, shaking well, and preparing the respective reference substance stock solutions; taking the quercetin, luteolin, kaempferol and apigenin stock solutions, placing them in the same volumetric flask, adding methanol to make up to volume, and obtaining the mixed reference substance solution.
6. The method for determining the contents of various flavonoid components in the Clematis tenuifolia medicinal material according to claim 1, characterized in that: The injection volume in the HPLC method was 20 μL.