Method for determining content of 4-coumaric acid in bidens pilosa and application thereof

Abstract

The application provides a determination method of 4-coumaric acid content in bidens pilosa and application thereof, S1, the bidens pilosa is mixed with a solvent, ultrasonic treatment is carried out, and a test sample solution is obtained; S2, 4-coumaric acid control samples and methanol are mixed, and a control sample solution is obtained; S3, under the same detection conditions, the chromatograms of chemical components in the test sample solution and the control sample solution are obtained by adopting high performance liquid chromatography; a C18 column is used as a chromatographic column; mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and gradient elution is used; S4, according to the concentration of the control sample solution, the peak area of the control sample solution in the chromatogram and the peak area of the component corresponding to the control sample solution in the chromatogram in the bidens pilosa, and based on the detection conditions of S3, the content of 4-coumaric acid is calculated by an external standard method. The application firstly establishes a high performance liquid content determination method taking 4-coumaric acid as an index component for the detection of the bidens pilosa, and the method has good stability, high precision and good reproducibility.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to a determination method of 4-coumaric acid content in bidens pilosa and application thereof. BACKGROUND

[0002] Bidens pilosa is an annual herbaceous plant of the family Asteraceae, also known as Bidens pilosa, sticky grass, and three-leaf Bidens pilosa. It is a commonly used medicine in China and has the effects of clearing heat and resolving toxins, promoting blood circulation and removing blood stasis. It is often used for upper respiratory tract infection, sore throat, malaria, acute jaundice hepatitis, etc. Bidens pilosa contains complex and diverse components, and the main active ingredient is flavonoids.

[0003] At present, there are few quality researches on Bidens pilosa and its compound preparations, and there is no relevant report on high-performance liquid analysis and content determination. Therefore, there is a lack of an effective means for quality control. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a determination method of 4-coumaric acid content in Bidens pilosa and application thereof. The determination method takes 4-coumaric acid as an index component to establish a content determination method, which provides a more scientific identification basis for the quality of Bidens pilosa medicinal materials, decoction pieces, standard decoction and formula granules.

[0005] The present application provides a determination method of 4-coumaric acid content in Bidens pilosa, comprising the following steps:

[0006] S1, mixing Bidens pilosa and a solvent, and ultrasonic treatment to obtain a test solution;

[0007] S2, mixing 4-coumaric acid reference substance and methanol to obtain a 4-coumaric acid solution of 10 μg / mL;

[0008] S3, under the same detection conditions, obtaining chromatograms of chemical components in the test solution in step S1 and the reference solution in step S2 by high-performance liquid chromatography, and the specific chromatographic parameters are as follows: a C18 column is used as the chromatographic column; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid solution, and gradient elution is adopted;

[0009] S4, calculating the content of 4-coumaric acid in Bidens pilosa by external standard method based on the detection conditions in step S3, according to the concentration of the reference solution, the peak area of the reference solution in the chromatogram, and the peak area of the component corresponding to the reference solution in the chromatogram in Bidens pilosa.

[0010] Preferably, the Bidens pilosa is at least one selected from the group consisting of Bidens pilosa medicinal materials, Bidens pilosa decoction pieces, Bidens pilosa formula granules, and Bidens pilosa standard decoction;

[0011] If the Bidens pilosa L. is selected from Bidens pilosa L. medicinal materials and / or Bidens pilosa L. prepared slices, the solvent is water; the mass of the Bidens pilosa L. to the volume of water is 1 g: (24-26) mL;

[0012] When the Bidens pilosa L. is selected from the Bidens pilosa L. formula granules or the Bidens pilosa L. standard decoction, the solvent is 70% methanol; the volume ratio of the Bidens pilosa L. mass to 70% methanol is 1 g: (24-26) mL.

[0013] Preferably, the power of the ultrasonic treatment is 580-620 W, the frequency of the ultrasonic treatment is 35-45 kHz, and the time of the ultrasonic treatment is 20-30 min.

[0014] Preferably, the gradient elution comprises:

[0015] 0-10 min, phase A: 10-17%, phase B: 90-83%;

[0016] 10-20 min, phase A: 17%, phase B 83%;

[0017] 20-25min, phase A: 17-28%, phase B 83-72%.

[0018] Preferably, the injection concentration of the 4-coumaric acid solution is 0.0068 to 0.0888 μg / mL, and the regression equation of 4-coumaric acid is y=48690.4286x-6386.0, R 2 =0.9999.

[0019] Preferably, the specifications of the chromatographic column are ZORBAX SB C18 4.6×250 mm;

[0020] The column temperature is 30-35°C.

[0021] Preferably, the detection wavelength is 309 nm;

[0022] The flow rate of the mobile phase is 0.8-1.2 ml / min;

[0023] The number of theoretical plates calculated based on the 4-coumaric acid peak is not less than 5000.

[0024] Preferably, the injection volume of the test solution and 4-coumaric acid solution is 4 to 5 μL.

[0025] The present invention provides a method for identifying Bidens pilosa L., which adopts the determination method described in the above technical solution to perform detection and analyze the detection results.

[0026] The invention provides a method for determining the content of 4-coumaric acid in Bidens pilosa L. The method comprises the following steps: S1, mixing Bidens pilosa L. and a solvent, and performing ultrasonic treatment to obtain a test solution; S2, mixing a 4-coumaric acid reference substance and methanol to obtain a 10 μg / mL 4-coumaric acid solution; S3, using high performance liquid chromatography to respectively obtain chromatograms of chemical components in the test solution in step S1 and the reference substance solution in step S2 under the same detection conditions, wherein specific chromatographic parameters are as follows: a chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is a 0.1% phosphoric acid solution, and gradient elution is performed; S4, calculating the content of 4-coumaric acid in Bidens pilosa L. by an external standard method according to the concentration of the reference substance solution, the peak area of ​​the reference substance solution in the chromatogram, and the peak area of ​​a component in Bidens pilosa L. corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step S3. This invention establishes for the first time a high-performance liquid chromatography (HPLC) content determination method for Bidens pilosa medicinal materials, decoction pieces, standard decoctions, formulated granules, and other related preparations. Using 4-coumaric acid as an indicator component, this method can comprehensively and macroscopically control the intrinsic quality of Bidens pilosa and its related preparations, ensuring the efficacy of the drugs and enabling more standardized quality control of the medicinal materials and their related preparations. This method offers excellent stability, high precision, good reproducibility, convenience, and ease of use. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the absorption spectrum of 4-coumaric acid;

[0028] Figure 2 Chromatograms at different column temperatures;

[0029] Figure 3 The chromatograms are at different flow rates;

[0030] Figure 4 To investigate the specificity of Bidens pilosa granules;

[0031] Figure 5 is the standard curve graph;

[0032] Figure 6 Survey results for different people, at different times, and with different instruments;

[0033] Figure 7 The results of the durability test of different chromatographic columns are shown in Figure 2. DETAILED DESCRIPTION

[0034] The present invention provides a method for determining the content of 4-coumaric acid in Bidens pilosa L., comprising the following steps:

[0035] S1. Mix Bidens pilosa L. and a solvent, and perform ultrasonic treatment to obtain a test solution;

[0036] S2. Mix 4-coumaric acid reference substance with methanol to obtain a 10 μg / mL 4-coumaric acid solution.

[0037] S3. Using high performance liquid chromatography, obtain chromatograms of the chemical components in the test solution described in step S1 and the reference solution described in step S2 under the same detection conditions. The specific chromatographic parameters are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid solution, and the gradient elution is performed;

[0038] S4. According to the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the component corresponding to the reference solution in Bidens pilosa L. in the chromatogram, and based on the detection conditions in step S3, the content of 4-coumaric acid in Bidens pilosa L. is calculated by the external standard method.

[0039] The present invention mixes Bidens pilosa and methanol, and performs ultrasonic treatment to obtain a test solution.

[0040] The Bidens pilosa is selected from at least one of Bidens pilosa herb materials, Bidens pilosa herb slices, Bidens pilosa herb formula granules or Bidens pilosa herb standard decoction; if Bidens pilosa herb is selected from Bidens pilosa herb materials and / or Bidens pilosa herb slices, the solvent is water; the volume ratio of the Bidens pilosa herb to water is 1g:(24~26)mL; when Bidens pilosa herb is selected from the solvent Bidens pilosa herb formula granules or Bidens pilosa herb standard decoction, the solvent is 70% methanol; the volume ratio of the Bidens pilosa herb to 70% methanol is 1g:(24~26)mL; in a specific embodiment, the volume ratio of the Bidens pilosa herb to the solvent is 1g:25mL.

[0041] The ultrasonic treatment power of the present invention is 580-620W, the ultrasonic treatment frequency is 35-45kHz, and the ultrasonic treatment time is 20-30min. In a specific embodiment, the ultrasonic treatment power is 600W, the ultrasonic treatment frequency is 40kHz, and the ultrasonic treatment time is 20min or 30min.

[0042] In a specific embodiment of the present invention, the test solution was prepared according to the following method:

[0043] Take about 1 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper it, weigh it, and treat it at an ultrasonic power of 600 W and a frequency of 40 kHz for 20 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0044] The present invention mixes a 4-coumaric acid reference substance with methanol to obtain a 10 μg / mL 4-coumaric acid solution, wherein the methanol in the reference substance solution is 70% methanol.

[0045] After obtaining the test solution and the reference solution, the present invention uses high performance liquid chromatography to obtain chromatograms of the chemical components in the test solution in step S1 and the reference solution in step S2 under the same detection conditions. The specific chromatographic parameters are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid solution, and the gradient elution is performed.

[0046] Gradient elution in the present invention comprises:

[0047] 0-10 min, phase A: 10-17%, phase B: 90-83%;

[0048] 10-20 min, phase A: 17%, phase B 83%;

[0049] 20-25min, phase A: 17-28%, phase B 83-72%.

[0050] The injection volume of the test solution and the reference solution in the present invention is preferably 4-5 μL; the chromatographic column used in the present invention has a specification of ZORBAX SB C18 4.6×250 mm; and the column temperature is 30-35°C. In a specific embodiment, the injection volume of the test solution and the reference solution is 5 μL; and the column temperature is 35°C or 30°C.

[0051] In the present invention, the detection wavelength is 309 nm; the flow rate of the mobile phase is 0.8 to 1.2 ml / min, preferably 1.0 to 1.2 ml / min; and the theoretical plate number is not less than 5000 calculated based on the 4-coumaric acid peak.

[0052] The present invention calculates the content of 4-coumaric acid in Bidens pilosa L. by an external standard method based on the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the component in Bidens pilosa L. corresponding to the reference solution in the chromatogram, and based on the detection conditions in step S3.

[0053] The present invention uses the peak area as the ordinate and the injection volume as the abscissa to calculate the regression equation and correlation coefficient of the standard curve. In the present invention, the injection concentration of the 4-coumaric acid solution is 0.0068-0.0888 μg / mL, and the regression equation of 4-coumaric acid is y=48690.4286x-6386.0, R 2 =0.9999.

[0054] The determination method of the present invention has good intermediate precision, good repeatability, high accuracy and good stability.

[0055] The present invention also provides a method for identifying Bidens pilosa L., which uses the determination method described in the above technical solution to perform detection and analyze the detection results.

[0056] This identification method uses 4-coumaric acid as the indicator component, and the determination method used is consistent with the above.

[0057] To further illustrate the present invention, the method for determining the content of 4-coumaric acid in Bidens pilosa L. and its application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Example 1 Establishment of a method for determining the content of Bidens pilosa formula granules

[0059] 1.1 Instruments and reagents

[0060] High-performance liquid chromatographs: Instrument-1 is Agilent 1260, Instrument-2 is Shimadzu LC-20AD;

[0061] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0062] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0063] Ultrasonic cleaner: KQ5200DB (600 W, 40 kHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0064] Phosphoric acid and acetonitrile were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade.

[0065] 4-Coumaric acid (China Food and Drug Inspection Institute, batch number: 112037-202102, content is 99.7%)

[0066] Bidens pilosa medicinal materials and decoction pieces batch number: YC-1~16, YP-1~16;

[0067] Bidens pilosa L. standard decoction batch number: BT-1~16;

[0068] Bidens pilosa formula granules batch numbers: KL-1, KL-2, KL-3;

[0069] The proposed chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 1 below; a flow rate of 1.0 ml per minute; a column temperature of 35°C; and a detection wavelength of 309 nm.

[0070] Table 1

[0071]

[0072] Preparation of reference solution: Take an appropriate amount of 4-coumaric acid reference substance, accurately weigh it, and add 70% methanol to make a mixed solution containing 10 μg per 1 ml.

[0073] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1 g, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 20 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0074] Determination method: Accurately aspirate 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0075] 1.2 Selection of chromatographic conditions and investigation of system suitability

[0076] Based on the experimental conditions proposed above, a diode array detector was used to perform full-band scanning of 4-coumaric acid to determine the content of 4-coumaric acid in Bidens pilosa granules at a wavelength of 309 nm. Figure 1 .

[0077] 1.2.1 Column temperature investigation

[0078] Based on the experimental conditions proposed above, the column temperature was investigated at 25℃, 30℃ and 35℃ respectively, and the separation degree, theoretical plate number and tailing factor of 4-coumaric acid chromatographic peak were used as evaluation indicators. Figure 2 , Table 2:

[0079] Table 2 Analysis results at different column temperatures

[0080]

[0081] The results showed that when the column temperature was 35℃, the separation was better than that at 25℃ and 30℃, so 35℃ was selected as the optimal column temperature.

[0082] 1.2.2 Flow rate investigation

[0083] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated, with the separation degree of 4-coumaric acid, the number of theoretical plates, and the tailing factor as evaluation indicators. Figure 3 and Table 3:

[0084] Table 3 Analysis results of different flow rates

[0085]

[0086] The results showed that 4-coumaric acid could be well separated under various flow rate conditions, and the flow rate of 1.0 mL / min was selected after comprehensive consideration.

[0087] 1.3 Preparation of test solution

[0088] 1.3.1 Investigation of extraction solvent

[0089] Take an appropriate amount of this product (batch number: KL-1), grind it finely, and take approximately 1g, accurately weigh it, and place it in a stoppered conical flask. Accurately add 25ml each of water, 10% methanol, 30% methanol, 70% methanol, methanol, and 30% ethanol. Stopper the flask tightly, weigh it, and sonicate it (power 600W, frequency 40kHz) for 20 minutes. Remove it, let it cool, and weigh it again. Make up the lost weight with the appropriate solvent, shake it well, filter it, and collect the filtrate to obtain the product. See Table 4:

[0090] Table 4 Extraction solvent investigation results

[0091]

[0092] The results showed that the contents of 4-coumaric acid extracted by water, 10% methanol and 70% methanol were similar. After comprehensive consideration, 70% methanol was selected as the extraction solvent.

[0093] 1.3.2 Investigation of extraction methods

[0094] Take an appropriate amount of this product (batch number: KL-1), grind it finely, and take approximately 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper it tightly, and weigh it. Heat it under reflux and ultrasonicate it (power 600W, frequency 40kHz) for 20 minutes respectively. Remove it, let it cool, and weigh it again. Make up the loss with 70% methanol, shake it well, filter it, and collect the filtrate to obtain the product. See Table 5:

[0095] Table 5 Results of the extraction method investigation

[0096]

[0097]

[0098] The results showed that the two extraction methods had similar extraction effects, and ultrasonic extraction was simpler to operate, so ultrasonic extraction was selected as the extraction method.

[0099] 1.3.3 Extraction time investigation

[0100] Take the product (batch number: KL-1) appropriate amount, grind, take about 1g, precision weighing, placed in a conical flask with a plug, precision add 70% methanol 25ml, sealed, weighed, ultrasonic treatment (power 600W, frequency 40kHz) 20 minutes, 40 minutes, 60 minutes, take out, cool, re-weighed, with 70% methanol to make up for the weight loss, shake, filter, take the filtrate, that is. See table 6.

[0101] Table 6 extraction time investigation results

[0102]

[0103] The results show that the test sample is extracted completely in 20 minutes, so 20 minutes is selected as the extraction time.

[0104] 1.3.4 extraction solvent addition amount investigation

[0105] Take the product (batch number: KL-1) appropriate amount, grind, take about 1g, precision weighing, placed in a conical flask with a plug, respectively precision add 70% methanol 25ml, 50ml, 100ml, sealed, weighed, ultrasonic treatment (power 600W, frequency 40kHz) 20 minutes, take out, cool, re-weighed, with 70% methanol to make up for the weight loss, shake, filter, take the filtrate, that is. See table 7.

[0106] Table 7 extraction solvent addition amount investigation results

[0107]

[0108]

[0109] The results show that the extraction effect of different solvent addition amount is not much different, and 25ml is selected as the solvent addition amount.

[0110] 1.3.5 determination method of cocklebur formula granules content

[0111] Chromatographic conditions and system suitability test with octadecylsilane bonded silica gel as filler; with acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in table 1 above; flow rate of 1.0ml per minute; column temperature is 35℃; detection wavelength is 309nm. The theoretical plate number should not be less than 5000 calculated by 4-coumaric acid peak.

[0112] Preparation of reference solution take 4-coumaric acid reference substance appropriate amount, precision weighing, add 70% methanol to make 10ug per 1ml mixed solution, that is.

[0113] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1 g, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 20 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0114] Determination method: Accurately aspirate 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0115] 1.4 Methodological Investigation

[0116] 1.4.1 Specificity Investigation

[0117] Prepare reference solution, test solution and negative control solution according to the proposed method and conduct the test. Figure 4 : The results showed that the chromatogram of negative solution had no interference with the determination of the peak to be tested, indicating that the method had good specificity.

[0118] 4.4.2 Precision investigation

[0119] The reference solution was injected 5 times continuously, the peak area of ​​4-coumaric acid was recorded, and the RSD value was calculated. The results are shown in Table 8.

[0120] Table 8 Precision inspection results

[0121]

[0122]

[0123] The results showed that the RSD of 4-coumaric acid peak area in the precision study was less than 1%, indicating that the instrument had good injection precision.

[0124] 1.4.3 Linear Relationship

[0125] Accurately weigh an appropriate amount of 4-coumaric acid reference substance and add 70% methanol to prepare a solution with a concentration of 6.8334380μg / ml. Accurately pipette 1μL, 3μL, 5μL, 7μL, 9μL, 11μL, and 13μL into the HPLC instrument, using the peak area as the ordinate and the injection volume (μl) as the abscissa to calculate the regression equation and correlation coefficient of the standard curve. The results are shown in Table 9. Figure 5 .

[0126] Table 9 Standard curve analysis results

[0127]

[0128] The results show that the linear relationship is y=48690.4286x-6386.0, R 2=0.9999, and the linear relationship was good when the concentration of 4-coumaric acid was 0.0068-0.0888 μg.

[0129] 1.4.4 Repeatability

[0130] Take an appropriate amount of this product (batch number: KL-1), grind it into powder, take about 1g, accurately weigh 6 portions, and prepare the test solution by the same operator according to the determined method. Calculate the content of 4-coumaric acid in the 6 portions of the test solution. The results are shown in Table 10.

[0131] Table 10 Repeatability test results

[0132]

[0133] The results showed that the RSD value of 4-coumaric acid content was 2.4%, indicating that the method had good repeatability.

[0134] 1.4.5 Intermediate precision

[0135] Based on the experimental conditions proposed above, different personnel (A and B) took about 1g of this product at different times and used different instruments, and each accurately weighed 6 portions to prepare test samples for determination. The calculated total 4-coumaric acid content is shown in Table 11. Figure 6 .

[0136] Table 11 Results of investigations of different personnel and time

[0137]

[0138] The results showed that the RSD value of the 4-coumaric acid content determination results was 1.7% when different personnel tested at different times, and the intermediate precision of this method was good.

[0139] 1.4.6 Sample recovery

[0140] Take 6 portions of a test sample with a known content (4-coumaric acid content 0.21 mg / g), accurately weigh them, and accurately add a certain amount of 4-coumaric acid reference substance to each portion. Prepare and measure the test sample solution according to the proposed method, and calculate the recovery rate. The results are shown in Table 12. The calculation formula is as follows:

[0141]

[0142] Table 12 Sample recovery test results

[0143]

[0144] The results showed that the average recovery rate of 4-coumaric acid was 98%, and the accuracy of this method was good.

[0145] 1.4.7 Durability inspection

[0146] Based on the experimental conditions proposed above, the chromatographic columns were: chromatographic column-1 (ZORBAX SBC18 4.6×250mm, 5μm), chromatographic column-2 (Atlantis T3 4.6×250mm, 5μm), chromatographic column-3 (Agilent5TC-C18 4.6×250mm, 5μm)

[0147] The results are shown in Table 13. Figure 7 .

[0148] Table 13 Durability test results

[0149]

[0150] The results showed that the results were similar when different chromatographic columns were used for analysis, and the chromatographic column of this method had good durability.

[0151] 1.4.8 Stability test

[0152] Take about 1g of the granules of this product, accurately weigh a portion, prepare the test solution, and measure the peak area of ​​4-coumaric acid at 0h, 4h, 8h, 16h, and 24h respectively. The results are shown in Table 14.

[0153] Table 14 Stability test results

[0154]

[0155] The results showed that under the experimental conditions, the RSD value of the 4-coumaric acid peak area was 1.2%, and the test solution had good stability within 24 hours.

[0156] 1.5 Sample content determination verification

[0157] The Bidens pilosa formula granules were tested using the determined method, and the results are shown in Table 15.

[0158] Table 15 Test results of the content of three batches of Bidens pilosa formula granules

[0159]

[0160] Conclusion: This method can effectively detect Bidens pilosa L. granules. Combined with the methodological test results, this method can be used to detect Bidens pilosa L. granules.

[0161] Example 2 Establishment of content determination of Bidens pilosa medicinal materials and decoction pieces

[0162] The content determination method of Bidens pilosa L. medicinal materials and decoction pieces was established based on the content determination method of Bidens pilosa L. formula granules, as follows:

[0163] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 1 above; a flow rate of 1.0 ml / min; a column temperature of 30°C; and a detection wavelength of 309 nm. The number of theoretical plates, calculated based on the 4-coumaric acid peak, should be no less than 5000.

[0164] Preparation of reference solution: Take an appropriate amount of 4-coumaric acid reference substance, accurately weigh it, and add 70% methanol to make a mixed solution containing 10 μg per 1 ml.

[0165] Preparation of test solution: Take about 1.0 g of the product powder (passed through No. 3 sieve), accurately weigh it, place it in a conical flask, accurately add 25 ml of water, plug it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it.

[0166] Determination method: Accurately aspirate 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0167] 16 batches of medicinal materials and 16 batches of decoction pieces were tested for content:

[0168] Table 16 4-Coumaric acid content of 16 batches of Bidens pilosa L.

[0169]

[0170]

[0171] Table 17 Content results of 16 batches of Bidens pilosa slices

[0172]

[0173]

[0174] Example 3 Establishment of a method for determining the content of Bidens pilosa L. standard decoction

[0175] The content determination method of Bidens pilosa L. standard decoction was established based on the content determination method of Bidens pilosa L. formula granules, as follows:

[0176] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 1 above; flow rate, 1.0 ml / min; column temperature, 35°C; detection wavelength, 309 nm. The number of theoretical plates, calculated based on the 4-coumaric acid peak, should be no less than 5000.

[0177] Preparation of reference solution: Take an appropriate amount of 4-coumaric acid reference substance, accurately weigh it, and prepare a solution containing 10 μg per 1 ml of 70% methanol solution.

[0178] Preparation of test solution: Take an appropriate amount of the product, take about 1.0g, accurately weigh, place in a conical flask, accurately add 25ml of 70% methanol solution, stopper, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes, let cool, weigh the weight again, make up the lost weight with 70% methanol solution, shake well, filter, and obtain.

[0179] Determination method: Accurately aspirate 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0180] Verification of content determination of 16 batches of Bidens pilosa standard decoction:

[0181] Table 18 4-Coumaric acid content of 16 batches of Bidens pilosa standard decoction

[0182]

[0183]

[0184] As can be seen from the above examples, the present invention provides a method for determining the content of 4-coumaric acid in Bidens pilosa L., comprising the following steps: S1, mixing Bidens pilosa L. and a solvent, and ultrasonically treating the mixture to obtain a test solution;

[0185] S2. Mix a 4-coumaric acid reference substance and methanol to obtain a 10 μg / mL 4-coumaric acid solution; S3. Use high performance liquid chromatography to obtain chromatograms of the chemical components in the test solution in step S1 and the reference solution in step S2 under the same detection conditions. The specific chromatographic parameters are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, and the mobile phase B is a 0.1% phosphoric acid solution, and the gradient elution; S4. According to the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the component in Bidens pilosa corresponding to the reference solution in the chromatogram, and based on the detection conditions in step S3, the content of 4-coumaric acid in Bidens pilosa was calculated by the external standard method. This invention establishes for the first time a high-performance liquid chromatography (HPLC) content determination method for Bidens pilosa medicinal materials, decoction pieces, standard decoctions, formulated granules, and other related preparations. Using 4-coumaric acid as an indicator component, this method can comprehensively and macroscopically control the intrinsic quality of Bidens pilosa and its related preparations, ensuring the efficacy of the drugs and enabling more standardized quality control of the medicinal materials and their related preparations. This method offers excellent stability, high precision, good reproducibility, convenience, and ease of use.

[0186] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for determining the content of 4-coumaric acid in Bidens pilosa L., comprising the following steps: S1. Mix Bidens pilosa L. and a solvent, and perform ultrasonic treatment to obtain a test solution; The Bidens pilosa is selected from at least one of Bidens pilosa herb, Bidens pilosa herb slices, Bidens pilosa herb formula granules or Bidens pilosa herb standard decoction; if the Bidens pilosa herb is selected from Bidens pilosa herb and / or Bidens pilosa herb slices, the solvent is water; if the Bidens pilosa herb is selected from the Bidens pilosa herb formula granules or Bidens pilosa herb standard decoction, the solvent is 70% methanol; S2. Mix 4-coumaric acid reference substance with methanol to obtain a 10 μg / mL 4-coumaric acid solution. S3. Using high performance liquid chromatography, obtain chromatograms of the chemical components in the test solution described in step S1 and the reference solution described in step S2 under the same detection conditions. The chromatographic parameters are as follows: chromatographic column: C18 column; detection wavelength: 309 nm; mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid solution, gradient elution; Described gradient elution comprises: 0-10 min, phase A: 10-17%, phase B: 90-83%; 10-20 min, phase A: 17%, phase B 83%; 20-25 min, phase A: 17-28%, phase B 83-72%; S4. According to the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the component corresponding to the reference solution in Bidens pilosa L. in the chromatogram, and based on the detection conditions in step S3, the content of 4-coumaric acid in Bidens pilosa L. is calculated by the external standard method.

2. The measuring method according to claim 1, wherein The mass of the Bidens pilosa L. to the volume ratio of water is 1 g: (24-26) mL; The volume ratio of the Bidens pilosa L. mass to 70% methanol is 1 g: (24-26) mL.

3. The measuring method according to claim 1, wherein The power of the ultrasonic treatment is 580-620 W, the frequency of the ultrasonic treatment is 35-45 kHz, and the time of the ultrasonic treatment is 20-30 min.

4. The measuring method according to claim 1, wherein The injection concentration of 4-coumaric acid solution was 0.0068~0.0888μg / mL, and the regression equation of 4-coumaric acid was y=48690.4286x-6386.0, with R²=0.9999.

5. The measuring method according to claim 1, wherein The specifications of the chromatographic column were ZORBAX SB C18 4.6 × 250 mm, 5 μm; The column temperature is 30~35℃.

6. The measuring method according to claim 1, wherein The flow rate of the mobile phase is 0.8~1.2ml / min; The number of theoretical plates calculated based on the 4-coumaric acid peak is not less than 5000.

7. The measuring method according to claim 1, wherein The injection volume of the test solution and 4-coumaric acid solution is 4~5μL.

8. A method for identifying Bidens pilosa L., characterized in that: The method according to any one of claims 1 to 7 is used for detection, and the detection results are analyzed.

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