Method for establishing fingerprint of xiaozhengzhitong traditional chinese medicine composition and standard fingerprint

Abstract

The application belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition for relieving rash and pain and a standard fingerprint spectrum.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a fingerprint spectrum establishing method and standard fingerprint spectrum of a Xiaozheshuti Tong traditional Chinese medicine composition. BACKGROUND

[0002] The Xiaozheshuti Tong gel is a Chinese invention patent applied by the applicant on March 23, 2017, with the patent number CN201710178327.1. The traditional Chinese medicine composition involved is composed of six traditional Chinese medicines, i.e., earthworm, Corydalis, Bai Zhi, Forsythia, purple flower ground ivy and licorice, and has the effects of removing rashes and relieving pain and is used for the treatment of herpes zoster. Although the preparation method of the traditional Chinese medicine composition is provided in the above-mentioned invention, the product prepared by the method needs to be controlled in quality to ensure the stability and controllability of the product quality, and due to the particularity of traditional Chinese medicine products, the quality control method is also required to be specific. Only the content of one or two components in the traditional Chinese medicine composition is selected to indicate the internal quality of the product, which is insufficient to represent the overall quality and has certain limitations.

[0003] The fingerprint spectrum can reflect the quality of traditional Chinese medicine preparation as a whole, and the fingerprint spectrum / characteristic spectrum quality control method of some varieties has been collected in the Chinese Pharmacopoeia. Therefore, the fingerprint spectrum similarity calculation method can be used to evaluate the consistency of different batches of traditional Chinese medicine preparations.

[0004] Nowadays, there are also some studies on the establishment of fingerprint spectrum for traditional Chinese medicine prescriptions. For example, the patent document with the publication number CN106404974B discloses a method for establishing the fingerprint spectrum of women's menstrual cramps granules, takes tetrahydropalmatine and salvianolic acid B as the reference substances, establishes the fingerprint spectrum by high performance liquid chromatography, and adopts gradient elution, so that the characteristic peaks of each medicinal material in the women's menstrual cramps granule prescription can be effectively detected, which is beneficial to monitoring the internal quality of the product, thereby ensuring the safety and effectiveness in treatment. However, the quality control method is not universal for different medicinal materials and different traditional Chinese medicine prescriptions. SUMMARY

[0005] To solve the above problems, the application provides a fingerprint spectrum establishing method and standard fingerprint spectrum of a Xiaozheshuti Tong traditional Chinese medicine composition.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0007] A quality detection method of a Xiaozheshuti Tong traditional Chinese medicine composition, which comprises a content determination method and the establishment of a fingerprint spectrum, adopts tetrahydropalmatine as the reference substance for content determination.

[0008] The fingerprint spectrum is evaluated by reverse high performance liquid chromatography, and a total of 5 common peaks are calibrated, with a similarity of more than 0.99.

[0009] 1. Chromatographic conditions and system suitability test:

[0010] Take octadecylsilane-bonded silica gel as the filler; take acetonitrile-1.2% triethylamine solution (45:55) as the mobile phase; and the detection wavelength is 282 nm. The theoretical plate number should not be less than 5000 calculated according to the peak of fumarolic acid B.

[0011] (1) Preparation of the control solution: take fumarolic acid B control substance in an appropriate amount, accurately weigh, and add methanol to prepare a solution containing 25 μg per 1 mL, and thus obtain.

[0012] (2) Preparation of the test solution: accurately weigh 2 g of the Xiaozheng pain-relieving gel, place it in a conical flask with a plug, accurately add 10 mL of 70% ethanol, tightly plug, weigh, ultrasonically treat for 10 min under the condition of 250 W and 40 kHz, make up the weight loss with 70% ethanol, shake well, and filter, and thus obtain.

[0013] (3) Determination: accurately pipette 10 μL of the control solution and the test solution respectively, inject into the liquid chromatograph, and determine, and thus obtain.

[0014] 2. The method for establishing the fingerprint spectrum comprises the following steps:

[0015] (1) Preparation of the reference solution: take fumarolic acid B control substance in an appropriate amount, accurately weigh, and add methanol to prepare a solution containing 25 μg per 1 mL, and thus obtain.

[0016] (2) Preparation of the test solution: accurately weigh 2 g of the Xiaozheng pain-relieving gel, add 10 mL of methanol, tightly plug, ultrasonically treat for 10 min under the condition of 250 W and 40 kHz, filter, and take the filtrate, and thus obtain.

[0017] (3) Chromatographic identification: accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, perform gradient elution with acetonitrile as the mobile phase A and 0.4% phosphoric acid-water as the mobile phase B, determine, the detection wavelength is 290 nm, record the chromatogram, determine the common peaks, and thus obtain the characteristic spectrum of the Xiaozheng pain-relieving traditional Chinese medicine composition.

[0018] The gradient elution conditions are as follows:

[0019] 0-6 min, 5%→10% A, 95%→90% B;

[0020] 6-35 min, 10%→16% A, 90%→84% B;

[0021] 35-55 min, 16%→19% A, 84%→81% B;

[0022] 55-65 min, 19%→23% A, 81%→77% B;

[0023] 65-85min, 23%→30% A, 77%→70% B;

[0024] 85-86min, 30%→66% A, 70%→34% B;

[0025] 86-100min, 66%→75% A, 34%→25% B.

[0026] The fingerprint spectrum identifies five components: tetrahydropalmatine, forsythoside A, glycyrrhizin, forsythoside and imperatorin.

[0027] It should be noted that the anti-eczema and analgesic traditional Chinese medicine composition involved in the application is a traditional Chinese medicine composition involved in patent No.

[0028] The preparation method of the gel dosage form is as follows: the prescription amount of 6 pieces of decoction pieces is crushed into coarse powder, 60% ethanol is used as a solvent, and after immersion for 24 hours, the percolation extraction is carried out at a speed of 4mL / min, the percolation liquid is collected, and the supernatant is separated after the concentrated solution is concentrated under reduced pressure to a relative density of 1.01-1.05 (60℃) and refrigerated overnight; the supernatant is concentrated under reduced pressure twice to a relative density of about 1.20-1.30 (60℃) of the extract; 12g of poloxamer is dissolved in 170mL of purified water, and the above-mentioned precipitate is added, filtered, and the precipitate dissolved liquid is obtained; a proper amount of purified water is added, 100g of glycerol is stirred, then 20g of carbomer is added and swelled, and then 3g of hydroxyphenyl ethyl ester dissolved in ethanol is added, stirred uniformly, and then the above-mentioned extract and precipitate dissolved liquid are added, stirred uniformly, and then the pH is adjusted to 6.0-6.5 with triethanolamine, and then purified water is added to 1000g, stirred uniformly, and then filled, to obtain the gel dosage form.

[0029] In summary, the beneficial effects of the present application are that the present application can control the quality from multiple angles, perform value transmission, effectively evaluate the quality of traditional Chinese medicine preparations, control the quality from the source of medicinal materials, realize the uniformity of quality of multiple batches of medicines, and more accurately and clearly control the quality of medicines.

[0030] I. Content determination research

[0031] 1. Methodology verification

[0032] 1.1 Specificity test

[0033] Precisely take 10μL of the control sample solution, the test sample solution and the negative sample solution, inject into the liquid chromatograph, and determine the results as shown in Figure 1 .

[0034] The experimental results show that under the chromatographic conditions, the test sample and the control sample have the same peak time, and no chromatographic peak is found at the same retention time in the negative sample, indicating that there is no interference in the determination of tetrahydropalmatine. The purity of the tetrahydropalmatine peak is analyzed by the DAD detector, and the purity factor is 999, which is good in specificity.

[0035] 1.2 System suitability test

[0036] 10 μL of the control solution and the test sample solution were precisely taken, 6 injections of the control solution were continuously made, the peak area RSD (%) was calculated, and the injection precision test results are shown in Table 1; another injection of the test sample solution was made, and the theoretical plate number, separation degree and tailing factor were recorded.

[0037] Table 1 Injection precision test results

[0038]

[0039] The experimental results show that the theoretical plate number of the tetrahydropalmatine chromatographic peak of the test sample solution is 13556, the separation degree is 3.07, and the tailing factor is 1.2. The RSD of the peak area of the injection precision test is 1.4%, which meets the requirements of system suitability.

[0040] 1.3 Linear relationship investigation

[0041] 10.81 mg of tetrahydropalmatine control was precisely weighed into a 10 mL capacity bottle, dissolved and diluted to the mark with methanol, shaken well, and served as a control stock solution. The control stock solution was precisely removed and diluted with methanol to concentrations of 5.4, 10.8, 27.0, 54.0, 108.0 and 216.0 μg / mL, respectively. 10 μL was precisely taken and injected into the liquid chromatograph for determination. The standard curve was plotted with the concentration of tetrahydropalmatine control as the abscissa and the peak area as the ordinate, and the regression equation y = 8.6728x - 18.664 was obtained, and the correlation coefficient r = 0.9998, as shown in Figure 2 The experimental results show that tetrahydropalmatine has a good linear relationship in the range of 5.4 μg / mL to 216.0 μg / mL.

[0042] 1.4 Stability test

[0043] The test sample solution was prepared according to the method in (2) of the chromatographic conditions and system suitability test, and was placed at room temperature. 10 μL was precisely taken at 0, 2, 4, 6, 8, 10, 12, 18 and 24 h, and 1 injection was made at each time point, and the peak area was recorded, as shown in Table 2. The experimental results show that the RSD of the tetrahydropalmatine peak area at 9 different time points is 1.5%, indicating that the solution has good stability within 24 h.

[0044] Table 2 Stability test results

[0045]

[0046] 1.5 Repeatability test

[0047] The test solution was prepared according to the method (2) in 1. Chromatographic Conditions and System Suitability Test. Six replicates were prepared, and 10 μL of each sample was injected. The content of tetrahydropalmatine was calculated. The results are shown in Table 3. The test results showed that the average content of tetrahydropalmatine in the rash-relieving and analgesic gel was 0.13 mg / g, with an RSD of 1.7%, indicating good repeatability of the method.

[0048] Table 3 Repeatability test results

[0049]

[0050] 1.6 Intermediate precision test

[0051] Six sample solutions were prepared according to the method described in (2) of 1. Chromatographic Conditions and System Suitability Test. Samples were injected and measured at different times using different instruments. The content of tetrahydropalmatine was calculated, and the RSD of the 12 content data was calculated together with the 6 results from the repeatability test. The results showed that the RSD of the content of the 12 samples was 1.8%, indicating good intermediate precision. The results are shown in Table 4.

[0052] Table 4 Intermediate precision test results

[0053]

[0054] 1.7 Recovery test

[0055] 1 g of the rash-relieving analgesic gel was accurately weighed and placed in a stoppered conical flask. 9 replicates were prepared. 4 mL, 5 mL, and 6 mL of a 26.42 μg / mL reference solution were accurately measured in 3 replicates. 70% ethanol was accurately added to make 10 mL of each sample solution. The solution was sealed, weighed, and ultrasonicated for 10 minutes. 70% ethanol was added to compensate for the weight loss. The solution was shaken to obtain the product. The recovery rate (K) was calculated according to formula (1). The results are shown in Table 5.

[0056]

[0057] C1 represents the known amount of sample;

[0058] C2 represents the measured amount of tetrahydropalmatine in the sample;

[0059] C3 represents the amount of reference substance added.

[0060] Table 5 Recovery test results

[0061]

[0062] The results of the test showed that the average accuracy of the samples was 96.60% and the RSD was 0.72% in the recovery test of three different concentrations, indicating that the method was highly accurate.

[0063] 1.8 Durability test

[0064] The applicability of the system under different conditions was investigated by fine-tuning the proportion of the mobile phase, flow rate, column temperature, and changing different brands of chromatographic columns, as shown in Table 6. The results showed that the separation degree of tetrahydropalmatine was >1.5 and the theoretical plate number was >5000 under different conditions, meeting the requirements of system applicability.

[0065] Table 6 Results of durability test

[0066]

[0067]

[0068] 1.9 Determination of three batches of pilot samples

[0069] The sample solution was prepared according to the method in (2) of 1. Chromatographic conditions and system suitability test, and the content of tetrahydropalmatine in the sample was determined. According to the determination results of the content of three batches of Xiaozhen Alleviating Pain Gel, it was temporarily determined that each bottle of the product contained not less than 0.7 mg of tetrahydropalmatine (C21H25NO4) as tetrahydropalmatine. The results are shown in Table 7.

[0070] Table 7 Determination results of pilot sample

[0071]

[0072] 2 Fingerprint study

[0073] 2.1 Reproducibility test

[0074] The sample solution of Xiaozhen Alleviating Pain Gel was prepared according to the method in (2) of 2. Fingerprint establishment method, and 6 parallel samples were determined with peak 7 as the reference peak. The results showed that the RSD of relative retention time of the 6 reproducibility samples was <1.0% and the RSD of relative peak area was <10%, indicating that the method had good reproducibility. The results are shown in Table 8 and Table 9.

[0075] Table 8 Reproducibility test results (relative retention time)

[0076]

[0077]

[0078] Table 9 Reproducibility test results (relative peak area)

[0079]

[0080] 2.2 Stability test

[0081] The same batch of Xiaozheng Zhitong Gel was used to prepare the test solution, which was placed at room temperature. Samples were injected at 0, 2, 4, 8, 12, 18, and 24 h after preparation. Peak 7 was used as the reference peak. The results showed that the RSD of the relative peak area of each chromatographic peak was <15%, and the RSD of the relative retention time was <1% at the 7 time points. The Xiaozheng Zhitong Gel test solution was stable within 24 h. The results are shown in Tables 10 and 11.

[0082] Table 10 Stability test results (relative retention time)

[0083]

[0084]

[0085] Table 11 Stability test results (relative peak area)

[0086]

[0087] 3 Similarity evaluation

[0088] Ten batches of Xiaozheng Zhitong Gel were prepared, and 10 batches of test solution were prepared. The 10 batches of Xiaozheng Zhitong Gel were injected into the liquid chromatograph to obtain 10 batches of Xiaozheng Zhitong Gel characteristic chromatograms. The median method was used to generate the control chromatogram R and perform chromatographic peak matching to complete the similarity evaluation (see Figure 3 ). The results showed that the similarity of the 10 batches of samples compared with the control chromatogram was all ≥0.99. The results are shown in Table 12.

[0089] Table 12 Similarity results of 10 batches of Xiaozheng Zhitong Gel samples

[0090]

[0091]

[0092] A total of 8 common peaks were determined by analysis and evaluation. Through comparison with the control product chromatogram, peak 4 was glycyrrhizin, peak 5 was forsythoside A, peak 6 was fumarine, peak 7 was forsythoside, and peak 8 was imperatorin. See Figure 3 . Peak 2 was used as the reference peak to calculate the relative retention time and relative peak area of the common peaks. The results showed that the RSD of the relative retention time of the 10 batches of samples was <1%, and the RSD of the relative peak area was <2%. The results are shown in Tables 13 and 14. This indicated that the similarity of the 10 batches of samples was good. Through the identification of the characteristic chromatographic peaks, the composition of the chemical components in Xiaozheng Zhitong Gel was determined, which provided a reference for the subsequent improvement of the quality standard.

[0093] Table 13 Relative retention time of 10 batches of Xiaoshen Zhitong gel

[0094]

[0095] Table 14 Relative peak area of characteristic chromatogram of 10 batches of Xiaoshen Zhitong gel

[0096]

[0097] BRIEF DESCRIPTION OF DRAWINGS

[0098] Figure 1 HPLC chart for specificity test; wherein a is a negative sample, b is a control, c is a test sample, 1 is corydalis B.

[0099] Figure 2 Corydalis B linear relationship chart.

[0100] Figure 3 Characteristic chromatogram of 10 batches of Xiaoshen Zhitong gel; wherein S1-S10 is the characteristic chromatogram of 10 batches of Xiaoshen Zhitong gel, and R is a control chromatogram.

[0101] Figure 4 Characteristic chromatogram of Xiaoshen Zhitong gel; wherein a is a test sample, b is a mixed control, 4 is glycyrrhizin, 5 is forsythoside A, 6 is corydalis B, 7 is forsythoside, and 8 is imperatorin. DETAILED DESCRIPTION

[0102] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

[0103] Example 1

[0104] A quality detection method of a Xiaoshen Zhitong traditional Chinese medicine composition includes a content determination method and the establishment of a fingerprint, and corydalis B is used as a control for content determination.

[0105] The fingerprint is evaluated by reverse high performance liquid chromatography.

[0106] The fingerprint evaluation method includes the following steps:

[0107] (1) Preparation of reference solution: take corydalis B control substance, accurately weigh and determine, add methanol to prepare a solution containing 25 μg per 1 mL, and obtain;

[0108] (2) Preparation of test solution: about 2g of the test sample was precisely weighed, added with 10mL of methanol, tightly capped, ultrasonically treated for 10min under the condition of 250W and 40kHz, filtered, and the filtrate was obtained, namely the test solution;

[0109] (3) Chromatographic identification: 10ul of the reference solution and the test solution were precisely pipetted and injected into the liquid chromatograph, gradient elution was carried out with acetonitrile as the mobile phase A and 0.4% phosphoric acid-water as the mobile phase B.

[0110] Further, the gradient elution conditions are as follows:

[0111] 0-6min, 5%→10% A, 95%→90% B;

[0112] 6-35min, 10%→16% A, 90%→84% B;

[0113] 35-55min, 16%→19% A, 84%→81% B;

[0114] 55-65min, 19%→23% A, 81%→77% B;

[0115] 65-85min, 23%→30% A, 77%→70% B;

[0116] 85-86min, 30%→66% A, 70%→34% B;

[0117] 86-100min, 66%→75% A, 34%→25% B.

[0118] The fingerprint spectrum identifies five components, namely, tetrahydropalmatine, forsythoside A, glycyrrhizin, forsythoside and imperatorin, and five common peaks are determined, and the similarity is above 0.99.

[0119] The antiphlogistic and analgesic traditional Chinese medicine composition involved in the application is a traditional Chinese medicine composition involved in patent No.CN201710178327.1, which is composed of 6 traditional Chinese medicines, i.e., earthworm, corydalis, radix angelicae dahuricae, forsythia, viola philippica and licorice, and is prepared into a gelatin dosage form;

[0120] The preparation method of the gel dosage form is specifically as follows: prescription amount of 6 medicinal materials is crushed into coarse powder, 60% ethanol is used as a solvent, and then the medicinal materials are soaked for 24 hours; the percolation extraction is performed at a speed of 4 mL / min; the percolation liquid is collected; the percolation liquid is concentrated under reduced pressure to a relative density of 1.01-1.05 (60 DEG C); the supernatant is separated after the concentrated liquid is refrigerated overnight; the precipitate is reserved; 12g of poloxamer is dissolved in 170 mL of purified water; the precipitate is added; filtration is performed; the precipitate dissolving liquid is obtained; a proper amount of purified water is added; 100g of glycerol is stirred uniformly; 20g of carbomer is added and is allowed to swell sufficiently; 3g of hydroxyphenyl ethyl ester dissolved in ethanol is added; the above-mentioned extract and the precipitate dissolving liquid are added in sequence; the pH is adjusted to 6.0-6.5 by using triethanolamine; the purified water is added to 1000g; the mixture is stirred uniformly; and then the mixture is filled into bottles to obtain the gel dosage form.

Claims

1. A method for establishing a fingerprint of a Chinese medicine composition for relieving rash and relieving pain, characterized in that: The following steps are involved: (1) Preparation of reference solution: Take an appropriate amount of tetrahydropalmatine reference substance, accurately weigh it, and add methanol to make a solution containing 25 μg per 1 mL. (2) Prepare the test solution: remove 2 g of the rash analgesic gel, accurately weigh it, add 10 mL of methanol, plug it tightly, sonicate it at 250 W and 40 kHz for 10 min, filter it, and take the filtrate. The rash-relieving and analgesic Chinese medicine composition is composed of six Chinese herbs: earthworm, Corydalis yanhusuo, Angelica dahurica, Forsythia suspensa, Viola yedoensis, and Licorice, and is prepared into a gel; (3) Chromatographic identification: Accurately pipette 10 μL of the reference solution and the test solution into a liquid chromatograph, use octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, and phosphoric acid-water as the mobile phase B for gradient elution. The detection wavelength is 290 nm, and the chromatogram is recorded to determine the common peaks and obtain the characteristic spectrum of the rash-relieving and analgesic Chinese medicine composition. The gradient elution conditions are: 0-6min, 5%→10%A, 95%→90%B; 6-35min, 10%→16%A, 90%→84%B; 35-55min, 16%→19%A, 84%→81%B; 55-65min, 19%→23%A, 81%→77%B; 65-85min, 23%→30%A, 77%→70%B; 85-86min, 30%→66%A, 70%→34%B; 86-100min, 66%→75%A, 34%→25%B; The fingerprint spectrum identified five components: tetrahydropalmatine, forsythiaside A, liquiritin, forsythiaside, and imperatorin.

2. The method for establishing a fingerprint spectrum of a rash-relieving and analgesic Chinese medicine composition according to claim 1, characterized in that: The fingerprint establishment method is specifically the following steps: (1) Preparation of reference solution: Take an appropriate amount of tetrahydropalmatine reference substance, accurately weigh it, and add methanol to make a solution containing 25 μg per 1 mL. (2) Prepare the test solution: remove 2 g of the rash analgesic gel, accurately weigh it, add 10 mL of methanol, plug it tightly, sonicate it at 250 W and 40 kHz for 10 min, filter it, and take the filtrate. (3) Chromatographic identification: Accurately pipette 10 μL of the reference solution and the test solution into a liquid chromatograph, use acetonitrile as mobile phase A and 0.4% phosphoric acid-water as mobile phase B for gradient elution, measure, record the chromatogram, determine the common peaks, and obtain the characteristic spectrum of the rash-relieving and analgesic Chinese medicine composition; The gradient elution conditions are: 0-6min, 5%→10%A, 95%→90%B; 6-35min, 10%→16%A, 90%→84%B; 35-55min, 16%→19%A, 84%→81%B; 55-65min, 19%→23%A, 81%→77%B; 65-85min, 23%→30%A, 77%→70%B; 85-86min, 30%→66%A, 70%→34%B; 86-100min, 66%→75%A, 34%→25%B.

3. A method for establishing a fingerprint of a rash-relieving and analgesic Chinese medicine composition according to any one of claims 1 to 2, characterized in that: The standard fingerprint spectrum is obtained according to the fingerprint spectrum establishment method of the rash-relieving and analgesic Chinese medicine composition according to any one of claims 1-2.

4. A method for establishing a fingerprint of a rash-relieving and analgesic Chinese medicine composition according to any one of claims 1 to 2, characterized in that: The preparation method of the rash-relieving and analgesic gel is as follows: taking a prescribed amount of 6 medicinal pieces, crushing them into the coarsest powder, using 60% ethanol as a solvent, soaking for 24 hours, extracting by percolation at a speed of 4 mL / min, collecting the percolation liquid, and concentrating under reduced pressure until the relative density at 60° C. is 1.01-1.

05. After refrigerating overnight, separating the supernatant, and precipitating for later use; concentrating the supernatant under reduced pressure a second time until the relative density at 60° C. is 1.20-1.30, thereby obtaining an extract; taking 12 g of poloxamer and adding 170 mL of purified water to dissolve it, adding the above-mentioned precipitate, filtering, and obtaining a precipitate solution; taking an appropriate amount of purified water, adding 100 g of glycerol, stirring evenly, adding 20 g of carbomer and waiting for it to swell fully, then adding 3 g of ethyl hydroxybenzoate dissolved in ethanol, stirring evenly, adding the above-mentioned extract and precipitate solution in sequence, stirring evenly, adjusting the pH to 6.0-6.5 with triethanolamine, adding purified water to 1000 g, stirring evenly, and filling to obtain the product.

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