A method for detecting duyiwei capsule, a method for constructing a control fingerprint spectrum of duyiwei capsule, a method for controlling the quality of duyiwei capsule, and a method for detecting characteristic components in duyiwei samples

Abstract

The present invention provides a detection method of a single-flavor capsule, a method for constructing a single-flavor capsule control fingerprint spectrum, a single-flavor capsule quality control method, and a method for detecting characteristic components in a single-flavor sample, and belongs to the technical field of drug detection. The chromatographic conditions of the method of the present invention are as follows: a chromatographic column: octadecylsilane bonded silica gel is used as a filler; a mobile phase: composed of phase A and phase B, wherein phase A is acetonitrile and phase B is 0.05%-0.2% phosphoric acid aqueous solution. The method of the present invention is simple and stable, has high precision, good repeatability and durability, can perform multi-component indications on single-flavor capsules and perform similarity evaluation, and can more fully and comprehensively reflect the component information in single-flavor capsules than the prior art, and increases the means for monitoring the quality control of single-flavor capsule intermediates and finished products, which is conducive to improving the control level of the overall quality of single-flavor capsules, ensuring the stability and uniformity of drug quality, and ensuring efficacy and drug safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection, and specifically relates to a method for detecting a Lamiophlomis capsule, a method for constructing a Lamiophlomis capsule control fingerprint spectrum, a method for controlling the quality of a Lamiophlomis capsule, and a method for detecting characteristic components in a Lamiophlomis sample. Background Art

[0002] The Special Provisions on Registration and Administration of Traditional Chinese Medicines point out that the development of new Chinese medicines should focus on reflecting the original thinking and holistic view of traditional Chinese medicine. The research and formulation of major registration standards should reflect the quality status of the products as much as possible, and pay attention to the relationship with the effectiveness and safety of traditional Chinese medicines. It also supports the use of new technologies and methods to explore the establishment of fingerprints or characteristic spectra for preparation quality control, biological effect detection, etc. The fingerprint of traditional Chinese medicine is a comprehensive identification method. It is based on the systematic study of the chemical components of traditional Chinese medicines and is mainly used to evaluate the authenticity, excellence and stability of the quality of Chinese medicinal materials and finished Chinese medicine preparations. Traditional Chinese medicines and their preparations are multi-component complex systems. Therefore, the evaluation of their quality should adopt a detection method that is suitable for them and can provide rich identification information. The establishment of a fingerprint of traditional Chinese medicine will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations, and then provide an overall description and evaluation of the quality of the drugs.

[0003] The fingerprint of traditional Chinese medicine can basically reflect the quality characteristics of traditional Chinese medicine and embody the characteristics of the varieties. The method should be scientific and feasible and take into account the requirements of simplicity, ease of use, economy and environmental protection. The fingerprint of traditional Chinese medicine is the same as the characteristic spectrum. It is of great significance for identifying the key quality attributes of traditional Chinese medicine preparations, studying the transfer of quantity and quality, evaluating the uniformity and stability of the quality of traditional Chinese medicine preparations, and improving the overall quality control level of traditional Chinese medicine preparations.

[0004] Duyiwei Capsule is an exclusive product of Kangxian Duyiwei Biopharmaceutical Co., Ltd. It is included in the 2020 edition of the Chinese Pharmacopoeia and its approval number is Z10970053. This drug variety is a single-ingredient preparation that has the effects of promoting blood circulation and relieving pain, removing blood stasis and stopping bleeding. It is used for incision pain and bleeding after various surgical operations, traumatic fractures, sprains of muscles and bones, rheumatic pain, metrorrhagia, dysmenorrhea, gingival swelling and pain, and bleeding. In addition, studies have shown that the herb has antibacterial, anti-inflammatory, immunomodulatory and anti-tumor effects (Li Jiwen, Bi Yingyan, Li Junjiang. Research progress on the chemical composition and pharmacological effects of herb and its quality marker prediction analysis [J]. Modern Chinese Medicine, 2024, 26(05):903-911.), and the various anti-inflammatory components in herb can also synergize (Kang Diandian, Zhang Rongrong, Zhan Hupo, et al. Exploration of the synergistic effect and compatibility structure of the anti-inflammatory active ingredients of herb based on network pharmacology and component structure theory [J]. Chinese Journal of Traditional Chinese Medicine, 2024, 49(13):3505-3514.); the production process of herb capsules has well inherited and retained the various active ingredients in herb. In order to improve the quality standard of herb capsules and ensure the controllable quality of drugs, it is urgent to establish a fingerprint map of herb capsules for the quality control of herb capsules.

[0005] The literature (HPLC characteristic spectrum combined with chemometrics to evaluate the consistency of Du Yiwei medicinal materials and Du Yiwei capsules, Natural Product Research and Development, Nat Prod Res Dev 2018, 30: 251-256) used 10 batches of Du Yiwei medicinal materials and 11 batches of Du Yiwei capsules to determine the fingerprint spectrum, and the chromatographic conditions used were: Kromasil100-5 C18 column (4.6mm × 250 mm, 5μm) as the chromatographic column; acetonitrile as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, gradient elution according to the provisions of Table 1; flow rate of 1ml per minute; column temperature of 35℃; detection wavelength of 235nm. The preparation method of the test solution is as follows: take about 1g of the fine powder of the medicinal material or capsule of Lamiophlomis odoratissimum, accurately weigh it, put it in a stoppered conical flask, accurately add 25mL of 30% methanol, weigh the weight, reflux for 60min, make up the lost weight after cooling, shake well, filter, and take the filtrate to pass through a 0.45μm microporous filter membrane.

[0006]

[0007] However, the method for constructing the fingerprint spectrum of Duyiwei capsule reported in the above literature has the following problems:

[0008] 1. According to the chromatographic conditions recorded in the literature, the experiment was repeated with Duyiwei Capsule as the test product, but no effective peak was obtained (chromatogram as shown in Figure 1 as shown).

[0009] 2. In the fingerprint spectrum of Duyiwei Capsule reported in the literature, chlorogenic acid, cryptochlorogenic acid and iso-verbascoside were not identified. Among these three ingredients, chlorogenic acid is an active ingredient in Duyiwei Capsule that stops bleeding, relieves pain and fights inflammation. Cryptochlorogenic acid has the pharmacological effects of promoting blood circulation and removing blood stasis, antibacterial and anti-inflammatory. Iso-verbascoside is the same as verbascoside, and both belong to the phenylethanol glycoside components in Duyiwei Capsule, which have anti-inflammatory, anti-tumor, anti-pathogenic microorganism and neuroprotective effects. The identification of these three chemical components plays an important role in the characterization of the component information of Duyiwei Capsule.

[0010] 3. The method reported in the literature for constructing the fingerprint spectrum of Duyiwei Capsule cannot fully separate the absorption peaks of jasminoides methyl ester, chlorogenic acid and caffeic acid.

[0011] 4. The method for constructing the fingerprint of Duyiwei Capsules reported in this literature has poor durability and specificity and cannot be used for quality control of Duyiwei Capsules.

[0012] 5. The Duyiwei Capsules used in this document do not completely include all the raw material origins of the Duyiwei Capsules produced by Kangxian Duyiwei Bio-Pharmaceutical Co., Ltd. It is not representative to use the fingerprint map of the Duyiwei Capsules reported in this document as a control fingerprint map to evaluate the Duyiwei Capsules of Kangxian Duyiwei Bio-Pharmaceutical Co., Ltd.

[0013] 6. The method for preparing the test solution reported in this document has the problems of difficult filtration and complicated operation. Summary of the invention

[0014] In order to overcome the above-mentioned problems existing in the prior art, the first purpose of the present invention is to provide a method for detecting Lamiaceae capsules, the second purpose is to provide a method for constructing a control fingerprint of Lamiaceae capsules, the third purpose is to provide a quality control method of Lamiaceae capsules, and the fourth purpose is to provide a method for detecting characteristic components in Lamiaceae samples.

[0015] The present invention provides a method for detecting a fingerprint spectrum of a Lamiaceae capsule, the method comprising the following steps:

[0016] (1) Preparation of test solution: Take the contents of the capsule of D. leucoderma and extract with solvent to obtain the test solution;

[0017] (2) Pipette the test solution, inject it into the liquid chromatograph, perform the test, and record the chromatogram; the chromatographic conditions for the test are as follows:

[0018] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0019] Mobile phase: It is composed of phase A and phase B, wherein phase A is acetonitrile and phase B is 0.05%-0.2% phosphoric acid aqueous solution.

[0020] Furthermore, in step (1), the solvent is methanol, methanol-water solution, ethanol or ethanol-water solution; the extraction method is heating reflux extraction or ultrasonic extraction, and the extraction time is 60-90 minutes.

[0021] Furthermore, in step (1), the solvent is 70% methanol aqueous solution.

[0022] Furthermore, in step (2), in the chromatographic conditions, the chromatographic column is Alletch Leader C18, Yuexu XBC18, Kromasil 100-5-C18, Zhongpuhong RD-C18, Pheromone Luna or Agilent SB C18;

[0023] Phase B is 0.1% phosphoric acid aqueous solution;

[0024] The detection wavelength was 230-240 nm, the column temperature was 30°C ± 5°C, and the flow rate was 0.9-1.1 ml / min.

[0025] Furthermore, in step (2), the chromatographic conditions are as follows: the detection wavelength is 235 nm, the column temperature is 30° C., and the flow rate is 1.0 ml / min.

[0026] Furthermore, the elution conditions of the mobile phase are:

[0027]

[0028] The present invention also provides a method for constructing a control fingerprint of Lamiaceae capsules, the method comprising the following steps: using the detection method described above to detect Lamiaceae capsules, and merging the recorded chromatograms to generate a control fingerprint of Lamiaceae capsules.

[0029] According to the "Establishment of Characteristic Spectra" in the "Technical Guiding Principles for the Study of Characteristic Spectra of Traditional Chinese Medicine Preparations" (Trial Implementation), "The characteristic spectrum should be sufficiently representative, should be able to reflect multi-component information, and characterize the quality characteristics of traditional Chinese medicine. Generally, it should be studied and formulated based on the measurement results of 15 batches or more of representative samples."

[0030] Furthermore, the batches of the Duyiwei capsules are more than 15 batches.

[0031] In the present invention, "15 batches or more" means 15 batches or more.

[0032] Furthermore, the Lamiophlomis capsule is a single-ingredient preparation prepared with Lamiophlomis as a single drug.

[0033] Furthermore, the Duyiwei capsule is produced by Kangxian Duyiwei Bio-Pharmaceutical Co., Ltd.

[0034] Furthermore, the control fingerprint of Duyiwei Capsule is generated by merging the recorded chromatograms using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system.

[0035] Furthermore, the method also includes the following steps: taking a reference substance of shanzhioside methyl ester and a reference substance of 8-O-acetylshanzhioside methyl ester, adding a solvent to dissolve them to obtain a reference solution; aspirating the reference solution, injecting it into a liquid chromatograph, and performing detection.

[0036] Furthermore, the solvent is an alcohol solvent, preferably methanol.

[0037] The present invention also provides a quality control method for Lamiaceae capsules, the method comprising the following steps: (i) taking Lamiaceae capsules, detecting them using the detection method described above, and recording a chromatogram;

[0038] (ii) evaluating the similarity between the recorded chromatogram and a reference fingerprint, the similarity being not less than 0.90; the reference fingerprint being constructed using the method described above.

[0039] Furthermore, in step (ii), the similarity evaluation is performed using a Chinese medicine chromatographic fingerprint similarity evaluation system.

[0040] Furthermore, in step (ii), the similarity evaluation is performed for an outflow time range of 5-80 minutes.

[0041] The present invention also provides another quality control method for Lamiaceae capsules, the method comprising the following steps: ① taking Lamiaceae capsules, detecting them using the detection method described above, and recording a chromatogram;

[0042] ② Take the shanzhioside methyl ester reference substance and the 8-O-acetylshanzhioside methyl ester reference substance, add a solvent to dissolve them, and obtain a reference solution; aspirate the reference solution, inject it into a liquid chromatograph, and detect it according to the chromatographic conditions described above;

[0043] ③ Perform chromatographic peak comparison. The chromatogram recorded in step ① should show chromatographic peaks corresponding to the retention times of the chromatographic peaks of the reference substances jasminoidin methyl ester and 8-O-acetyljasminoidin methyl ester.

[0044] The present invention also provides another quality control method for Lamiaceae capsules, the method comprising the following steps: (a) taking Lamiaceae capsules, detecting them using the detection method described above, and recording a chromatogram;

[0045] (b) taking a reference substance of shanzhioside methyl ester and a reference substance of 8-O-acetylshanzhioside methyl ester, dissolving them in a solvent to obtain a reference solution; taking the reference solution, injecting it into a liquid chromatograph, and detecting it according to the chromatographic conditions described above;

[0046] (c) The chromatogram recorded in step (a) is evaluated for similarity with the reference fingerprint, and the similarity shall not be less than 0.90, and the chromatogram recorded in step (a) shall present chromatographic peaks corresponding to the retention times of the chromatographic peaks of the reference substances jasminoides methyl ester and 8-O-acetyljasminoides methyl ester; the reference fingerprint is constructed using the method described above.

[0047] Furthermore, in step (c), the similarity evaluation is performed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system.

[0048] Furthermore, in step (c), the similarity evaluation is targeted at an outflow time range of 5-80 minutes.

[0049] In the present invention, "quality control method" refers to the use of this method to perform quality control on the sample. If the conditions are met, it meets the control requirements.

[0050] The present invention also provides a method for detecting characteristic components in a Lamiaceae sample, the method comprising the following steps:

[0051] (a) Preparation of test solution: taking a sample of Radix Angelicae Pubescentis and extracting it with a solvent to obtain a sample of Radix Angelicae Pubescentis; the sample of Radix Angelicae Pubescentis is Radix Angelicae Pubescentis medicinal material, Radix Angelicae Pubescentis capsule intermediate and / or Radix Angelicae Pubescentis capsule;

[0052] (b) Preparation of reference solution: taking a reference, adding a solvent to dissolve it, and obtaining a reference solution; the reference solution is a characteristic component, including one or more of chlorogenic acid, cryptochlorogenic acid, and isoverbascoside;

[0053] (c) The test solution and the reference solution are respectively aspirated and injected into a liquid chromatograph, and the characteristic components in the Lamiophlomis lappa sample are detected according to the chromatographic conditions described above.

[0054] In the present invention, "one or more" means one or more than one.

[0055] Furthermore, the characteristic components are scutellaria glycoside, jasmine glycoside methyl ester, chlorogenic acid, cryptochlorogenic acid, 8-O-acetyljasmine glycoside methyl ester, forsythiaside B, luteolin, verbascoside and isoverbascoside.

[0056] The present invention also provides the use of the method for detecting characteristic components in a Lamiophlomis sample in the quality control of Lamiophlomis medicinal materials, Lamiophlomis capsule intermediates and Lamiophlomis capsules.

[0057] In the present invention, the alcohol solvent includes alcohol and its aqueous solution.

[0058] In the present invention, phosphoric acid solution refers to phosphoric acid aqueous solution, methanol solution refers to methanol aqueous solution, and ethanol solution refers to ethanol aqueous solution. Unless otherwise specified, the concentration of the solution refers to the volume concentration.

[0059] The present invention has achieved the following beneficial effects:

[0060] The invention provides a method for detecting a Lamiaceae capsule, and the method can fully separate the components of the Lamiaceae capsule.

[0061] Using this detection method, a control fingerprint of Duyiwei Capsule can be further constructed, and multi-component indications and similarity evaluations of Duyiwei Capsule can be performed. Compared with the existing technical effects, it can more fully and comprehensively reflect the component information in Duyiwei Capsule.

[0062] Before the application date of the present invention, the registration standard of Duyiwei Capsule included 1 item of properties and thin layer chromatography identification, 2 items of content determination (i.e. UV method for determining the total flavonoid content and HPLC method for determining the total amount of shanzhioside methyl ester and 8-O-acetylshanzhioside methyl ester) in addition to the routine inspection items of preparations. In order to more fully and comprehensively reflect the component information in Duyiwei Capsule, the present invention provides a method for establishing the fingerprint of Duyiwei Capsule, which is conducive to improving the overall quality control level of the variety and ensuring the stability and uniformity of product quality. The present invention establishes a fingerprint construction method unique to Duyiwei Capsule for the first time, further improving and perfecting the quality control standards of drugs.

[0063] By using the detection method of the present invention, each batch of Duyiwei Capsules is detected under the same chromatographic conditions and compared with the control fingerprint spectrum. The quality consistency between batches is evaluated based on the similarity of the fingerprint spectrum, thereby further ensuring the quality controllability of the drugs and improving the overall quality control level of the drugs.

[0064] The fingerprint spectrum constructed by the method of the present invention has identified the peaks of each component compound of a unique medicinal material and its preparation as much as possible, effectively achieving the requirement that "the fingerprint spectrum of traditional Chinese medicine can more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and then provide an overall description and evaluation of the quality of the medicine."

[0065] By using the detection method of the present invention, starting from the finished preparation, a chromatographic method specifically for fingerprint detection of Duyiwei Capsules was established, which focuses on and embodies the holistic view of traditional Chinese medicine, and can reflect the quality characteristics of traditional Chinese medicine and embody the characteristics of the variety.

[0066] During the establishment of the fingerprint method of the present invention, mass spectrometry was used for research, and the chemical components in the Du Yiwei capsule were fully analyzed and identified. Compared with the existing technology, more compound components such as chlorogenic acid, cryptochlorogenic acid, and isoverbascoside were identified, which made quality control more accurate, further laid the foundation for the subsequent research on Du Yiwei medicinal materials and preparations, and can effectively reflect that the production process of Du Yiwei capsules effectively inherits and retains the effective ingredients and pharmacological effects of the medicinal materials.

[0067] The fingerprint spectrum constructed by the detection method of the present invention fully separates jasmine glycoside methyl ester, chlorogenic acid and caffeic acid.

[0068] The detection method of the present invention can also be used to detect characteristic components (including genus glycosides, jasmine glycoside methyl ester, chlorogenic acid, cryptochlorogenic acid, 8-O-acetyl jasmine glycoside methyl ester, forsythiaside B, luteolin, verbascoside and isoverbascoside) in the medicinal materials, preparation intermediates (extract powder, granules) and finished preparations of Lamiaceae, thereby increasing the means for monitoring the quality control of the intermediate products and finished products of Lamiaceae capsules.

[0069] In the detection method of the present invention, the amount of test sample used in the preparation method of the test solution is significantly reduced compared with the prior art, and solution filtration is simpler. The solution preparation method is the same as the "Senjaminoside Methyl Ester, 8-O-Acetylsenjaminoside Methyl Ester" determination item under the [Content Determination] item of the quality standard of Duyiwei Capsules in the 2020 edition of the "Chinese Pharmacopoeia", and can be shared, saving detection costs and further reducing the possibility of environmental pollution.

[0070] Compared with the methods in the prior art, the detection method of the present invention is simple to operate, has good precision and repeatability, and has better specificity, system applicability and method durability.

[0071] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0072] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 .According to the chromatographic conditions recorded in the literature (HPLC characteristic spectrum combined with chemometrics to evaluate the consistency of Duyiwei medicinal materials and Duyiwei capsules, Natural Product Research and Development, Nat Prod Res Dev 2018, 30: 251-256), the chromatogram obtained by repeating the experimental test with Duyiwei capsules as the test sample.

[0074] Figure 2 .Compare with fingerprint map.

[0075] Figure 3. The identification results of the 9 chromatographic peaks in the reference fingerprint (genistein, jasmine glycoside methyl ester, chlorogenic acid, cryptochlorogenic acid, 8-O-acetyljasmine glycoside methyl ester, forsythiaside B, luteolin, verbascoside, and isoverbascoside). From bottom to top are the mixed reference chromatogram and the Duyiwei capsule chromatogram.

[0076] Figure 4 .Chromatograms under different elution systems. The mobile phases used from bottom to top are: acetonitrile-0.1% phosphoric acid solution, acetonitrile-water, methanol-water, methanol-0.1% phosphoric acid solution.

[0077] Figure 5 . Chromatogram at 210nm wavelength.

[0078] Figure 6 . Chromatogram at 220nm wavelength.

[0079] Figure 7 . Chromatogram at 235nm wavelength.

[0080] Figure 8 . Chromatogram at 250nm wavelength.

[0081] Fig. 9 . Chromatogram at 270nm wavelength.

[0082] Fig.10 .Chromatogram at 300nm wavelength.

[0083] Fig.11 . Chromatogram at 330nm wavelength.

[0084] Fig.12 . Chromatogram at 360nm wavelength.

[0085] Fig.13 .Chromatograms under different extraction solvents. The extraction solvents used from bottom to top are methanol, 70% methanol, 50% methanol, and 70% ethanol.

[0086] Fig.14 .Chromatograms under different extraction methods and extraction times. The extraction method and extraction time used from bottom to top were reflux for 60 minutes, ultrasound for 60 minutes, and reflux for 90 minutes.

[0087] Fig.15 . Results of injection precision investigation.

[0088] Fig.16 . Repeatability test results.

[0089] Fig.17 . Results of stability test on test sample solution.

[0090] Fig.18 . Column temperature and flow rate durability test results. The column temperature and flow rate used from bottom to top are: 30℃, 1.0ml / min, 30℃, 0.9ml / min, 30℃, 1.1ml / min, 25℃, 1.0ml / min, 35℃, 1.0ml / min.

[0091] Fig.19 .Durability test chart of instruments of different brands. The liquid chromatographs used from bottom to top are Agilent1200-DAD, Waters2695-2489, and Shimadzu LC20A-SPD.

[0092] Fig. 20 . Durability inspection chart of chromatographic columns of different brands (Agilent instrument). The chromatographic columns used from bottom to top are: column ①Alletch Leader C18 4.6*250mm, 5μm; column ②Shiseido AQ C18 4.6*250mm, 5μm; column ③Yuexu XB C18 4.6*250mm, 5μm; column ⑤Kromasil 100-5-C18 4.6*250mm 5μm; column ⑥Agilent Extend C18 4.6*250mm, 5μm; column ⑦Zhongpuhong RD-C18 4.6*250mm, 5μm; column ⑧Phenomenon Luna 4.6*250mm, 5μm; column ⑨Agilent SB C18 4.6*250mm, 5μm.

[0093] Fig.21 .Durability inspection chart of chromatographic columns of different brands (Waters instrument). The chromatographic columns used from bottom to top are: Column ① Alletch Leader C18 4.6*250mm, 5μm; Column ⑩ Inertsil ODS-3 4.6*250mm, 5μm; Column ⑪ Zhongpu HD-3 4.6*250mm, 5μm; Column ⑤ Kromasil 100-5-C18 4.6*250mm 5μm.

[0094] Fig. 22 .Inspection chart of Alletch Leader chromatographic columns of different batches. The batches used from bottom to top are SN: FEDG1854 and SN: FEDG1823.

[0095] Fig.23 . Specificity investigation results. From bottom to top, they are the chromatograms of negative sample solution, reference solution, and Duyiwei capsule.

[0096] Fig.24 .Comparison chart of fingerprint spectra of batch A.

[0097] Fig.25 . Comparison chart of fingerprints of batch B.

[0098] Fig.26 . Comparison chart of fingerprint spectra of batch C.

[0099] Fig. 27 . Comparison chart of fingerprint spectra of batch D.

[0100] Fig.28 . Comparison chart of fingerprint spectra of batch E.

[0101] Fig.29 . Comparison chart of fingerprint spectra of batch F.

[0102] Fig.30 . Identification results of caffeic acid components. From bottom to top are the chromatograms of Duyiwei capsule and reference solution. DETAILED DESCRIPTION

[0103] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0104] The Duyiwei capsules used in the following embodiments and experimental examples are all commercially available products of Kangxian Duyiwei Bio-Pharmaceutical Co., Ltd.

[0105] Example 1: Method for constructing a comparative fingerprint of Duyiwei capsule

[0106] 1. Detection method of Duyiwei capsule

[0107] Chromatographic conditions: The chromatographic column is Alltech Leader C18 with octadecylsilane bonded silica as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 µm); acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the conditions specified in Table 2 below; the flow rate is 1 ml per minute; the column temperature is 30 °C; the detection wavelength is 235 nm. The theoretical plate number calculated based on the jasmine methyl ester peak should not be less than 10,000.

[0108]

[0109] Preparation of reference solution: Take appropriate amount of shanzhioside methyl ester reference substance and 8-O-acetylshanzhioside methyl ester reference substance, weigh accurately, add methanol to make a mixed solution containing 30 μg of each per 1 ml.

[0110] Preparation of test solution: Grind the contents of Du Yiwei capsules into powder, take out about 0.2 g, weigh accurately, put it in a conical flask, add 25 ml of 70% methanol accurately, weigh the weight, heat and reflux for 60 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0111] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, record the chromatogram, and obtain the result.

[0112] 2. Method for constructing the comparative fingerprint of Duyiwei capsule

[0113] 22 batches of Du Yiwei capsules were used as test samples, and 22 chromatograms were obtained according to the method in step 1. Then, the control fingerprint was generated by merging them using the similarity evaluation system of Chinese medicine chromatographic fingerprint. Figure 2 The reference substance chromatographic peak is confirmed based on the chromatogram of the reference substance solution.

[0114] According to the stability of chromatographic peaks in different batches of samples and the results of the chromatographic column durability test, 14 common peaks were selected on the control fingerprint, such as Figure 2 Peaks 1-14 are shown.

[0115] The chemical components in Duyiwei capsules were studied by UPLC-Q-TOF method, and compared with the reference chromatographic peaks. As a result, 9 chromatographic peaks in the reference fingerprint were identified, namely, scutellaria glycoside, scutellaria glycoside methyl ester, chlorogenic acid, cryptochlorogenic acid, 8-O-acetyl scutellaria glycoside methyl ester, forsythiaside B, luteolin, verbascoside, and isoverbascoside. Figure 3 shown.

[0116] 3. Quality Control Methods of Duyiwei Capsules

[0117] In the liquid chromatogram of Duyiwei Capsule, the retention time of Peak 6 and Peak 10 should correspond to the retention time of the reference peak.

[0118] According to the Chinese medicine chromatographic fingerprint similarity evaluation system, the chromatographic peaks from 5 to 80 minutes are calculated, and the similarity between the fingerprint of Duyiwei Capsule and the control fingerprint shall not be less than 0.88.

[0119] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0120] Experimental Example 1: Screening of Chromatographic Conditions

[0121] 1. Selection of elution system

[0122] With reference to the content determination and fingerprint methods in pharmacopoeia and relevant literature, four mobile phase compositions of acetonitrile-water, acetonitrile-0.1% phosphoric acid solution, methanol-water, and methanol-0.1% phosphoric acid solution were investigated, and different elution procedures were screened. The results showed that when acetonitrile-0.1% phosphoric acid solution was used for gradient elution, the peak shape and separation of each chromatographic peak in the fingerprint were the best, so it was finally selected as the mobile phase for determination (some typical spectra are shown in Figure 2). Figure 4After optimization, the final elution system was determined as follows: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution according to the elution conditions in Table 2.

[0123] 2. Selection of detection wavelength

[0124] The test solution was scanned in the wavelength range of 200nm to 400nm using a diode array detector. Finally, 235nm was selected as the measurement wavelength based on the principle of less baseline interference, the most information on the chromatographic peak, and the greater absorbance of the main characteristic peak. The chromatograms under each wavelength are as follows Figure 5-12 shown.

[0125] 3. Investigation of the preparation method of the test solution

[0126] Duyiwei Capsule is a variety included in the 2020 edition of the "Chinese Pharmacopoeia". The preparation method of the test solution of shanzhioside methyl ester and 8-O-acetylshanzhioside methyl ester under the item [Content Determination] is: take the content of this product under the item of filling amount difference, grind it, take about 0.2g, accurately weigh it, put it in a conical flask, accurately add 25ml of 70% methanol, weigh the weight, heat and reflux for 60 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate. The present invention refers to the pharmacopoeia, starting from saving the cost of organic reagents and optimizing the drug detection procedure, and examines the possibility of sharing the test solution with the detection item. On this basis, the present invention considers the effects of different extraction solvents and different extraction methods and times on the fingerprint spectrum.

[0127] (1) Investigation of extraction solvents: Four extraction solvents were investigated, namely methanol, 70% methanol, 50% methanol and 70% ethanol. According to the overall peak conditions of the fingerprint, 70% methanol was finally selected as the extraction solvent. The chromatograms of each extraction solvent are shown in Figure 2. Fig.13 shown.

[0128] (2) Investigation of extraction method and extraction time: Using 70% methanol as the extraction solvent, a comparative study was conducted on heating reflux for 60 minutes and 90 minutes, and ultrasonic extraction for 60 minutes. The results showed that the overall behavior of the chromatographic peaks of the three fingerprints was basically consistent, and the peak height of the ultrasonic extraction chromatographic peak was slightly lower than that of the reflux extraction. Since the extraction method of the test sample solution for one of the content determination items in the current standard of this product is reflux for 1 hour, considering that sharing the solution for two items can improve the inspection efficiency and save the use of organic solvents, which is beneficial to the environment, it was finally determined that heating reflux for 60 minutes would be used as the test sample extraction method for the fingerprint of Duyiwei Capsules, which is consistent with the content determination method. The comparison spectrum is as follows Fig.14 shown.

[0129] In summary, it is confirmed that the method for preparing the test solution is: take the contents of Du Yiwei capsules, grind them into powder, take about 0.2 g, accurately weigh it, put it in a conical flask, accurately add 25 ml of 70% methanol, weigh the weight, heat and reflux for 60 minutes, cool, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the solution.

[0130] Experimental Example 2: Methodological Investigation

[0131] (1) Investigation of injection precision

[0132] Take the same test solution, accurately measure 10 μl of the solution, and inject it 6 times continuously to compare the similarity of each fingerprint spectrum. The similarity of each fingerprint spectrum is greater than 0.998, and the RSD value is less than 0.05%, indicating that the instrument injection precision is good ( Fig.15 , Table 3).

[0133]

[0134] (2) Repeatability study

[0135] Take Du Yiwei capsules and prepare 6 test solutions in parallel according to the above test solution preparation method. Samples are injected separately, and the fingerprints are recorded and the similarity is calculated. The results show that the similarity of the fingerprints between the samples is 1, indicating that the method has good repeatability. The results and spectra are shown in Fig.16 And as shown in Table 4.

[0136]

[0137] (3) Stability study of test solution

[0138] The same test solution was sampled at 0, 4, 8, 12, 16, 20, 24, and 32 hours. The fingerprints at each time point showed no significant difference, and the similarity was 1.0, indicating that the test solution was stable within 32 hours. Fig.17 shown.

[0139] (4) Durability study of chromatographic method

[0140] (1) Investigation of flow rate and column temperature

[0141] Keep other conditions unchanged, only change the column temperature or flow rate to conduct single factor investigation, and record the chromatogram ( Fig.18), the similarity of the fingerprints was calculated, and the results showed that when the column temperature was 30℃±5℃ and the flow rate was 0.9~1.1ml / min, except for slight changes in the retention time of each peak, the chromatographic peak shape was relatively stable as a whole and the similarity was greater than 0.99, indicating that the method had good durability when the column temperature and flow rate changed slightly. However, in order to enable the determination method to maintain better reproducibility and stability in future applications, the column temperature was set to 30℃ and the flow rate to 1.0ml / min.

[0142] (2) Liquid chromatography inspection

[0143] Three brands of liquid chromatographs (①Agilent1200-DAD; ②Waters2695-2489; ③Shimadzu LC20A-SPD) were used to measure the fingerprint of the same sample. The similarity of the results was greater than 0.99, indicating that the instrument has good durability. Fig.19 .

[0144] (3) Durability test of liquid chromatography column

[0145] 1) Investigation of different chromatographic column brands

[0146] A total of 11 C18 columns of different brands or models were investigated in the experiment (① and ④AlletchLeader C18 4.6*250mm, 5μm (① and ④ differ only in the batch number); ②Shiseido AQ C18 4.6*250mm, 5μm; ③Yuexu XB C18 4.6*250mm, 5μm; ⑤Kromasil 100-5-C18 4.6*250mm 5μm; ⑥Agilent Extend C18 4.6*250mm, 5μm; ⑦Zhongpuhong RD-C18 4.6*250mm, 5μm; ⑧Phenomenon Luna 4.6*250mm, 5μm; ⑨Agilent SB C18 4.6*250mm, 5μm; ⑩Inertsil ODS-3 4.6*250mm, 5μm; ⑪ medium spectrum HD-3 4.6*250mm, 5μm. The results show that the retention time of each characteristic peak of the fingerprint spectrum obtained by using the above chromatographic columns for determination is slightly different, but the overall chromatographic behavior of the fingerprint spectrum is basically the same. Except for columns ②, ⑥, ⑩ and ⑪, the similarity of the remaining chromatographic columns is greater than 0.96, indicating that the determination method has good durability for most of the chromatographic column brands examined. Fig. 20 and Fig.21 .

[0147] 2) Inspection of different batches of Alletch Leader C18 columns

[0148] Since Alletch Leader C18 is the chromatographic column used for the establishment of the method and the generation of the reference spectrum in this fingerprint study, the durability of the chromatographic columns of different batches (SN: FEDG1823; SN: FEDG1854) of this model was also investigated. The results showed that the chromatographic peaks were completely consistent with a similarity of 1.0, indicating that the quality of the products of different batches of this chromatographic column is relatively stable and has good durability. Fig. 22 .

[0149] Comprehensive durability investigation results show that the brand of liquid chromatography has good durability for the fingerprint determination of this product, and the liquid phase brand is not fixed in the method; the brand of chromatographic column used in the study has an impact on the retention time of the chromatographic peak, but most of the chromatographic columns have good similarity, and the fingerprint detection method of this invention has good durability.

[0150] Experimental Example 3: Specificity Investigation

[0151] 1) Reference solution

[0152] Take appropriate amounts of shanzhioside methyl ester reference substance and 8-O-acetylshanzhioside methyl ester reference substance, weigh them accurately, and add methanol to prepare a mixed solution containing 30 μg of each per 1 ml.

[0153] 2) Negative sample solution

[0154] Since this product is a single-ingredient preparation, the excipients used in this product are regarded as negative samples lacking a single ingredient.

[0155] The negative sample of Lactobacillus sutchuenensis was prepared and determined according to the test solution preparation method. The negative chromatogram showed no interference, indicating that the fingerprint determination method had good specificity. Fig.23 shown.

[0156] Experimental Example 4: Correlation study of fingerprints of medicinal materials, intermediates and finished products

[0157] The correlation between the fingerprints of 6 batches of single herbal medicines and their corresponding intermediates and preparations from three main production areas was studied. The results showed that the 9 identified chromatographic peaks could be detected in all three. Except for a few small peaks in the fingerprint of the preparation, the remaining chromatographic peaks could be traced back to the intermediates and herbal medicines, indicating that the fingerprints of the three have a strong correlation. The corresponding relationship between the samples of herbal medicines, intermediates and preparations is shown in Table 5, and the chromatograms of each sample are shown in Figure 24 to Figure 29 .

[0158]

[0159] The above results show that the method of the present invention can be used to detect safflower glycoside, shanzhioside methyl ester, chlorogenic acid, cryptochlorogenic acid, 8-O-acetylshanzhioside methyl ester, forsythiaside B, luteolin, verbascoside and isoverbascoside in the medicinal materials, intermediates and preparations of Lamiophlomis lappa, to control the quality of the medicinal materials, intermediates and preparations of Lamiophlomis lappa, and to increase the means for monitoring the quality control of the intermediates and finished products of Lamiophlomis lappa capsules.

[0160] Experimental Example 5: Separation of characteristic peaks of jasminoidin methyl ester, chlorogenic acid and caffeic acid

[0161] The content of caffeic acid in Duyiwei capsules is relatively low. Referring to the preparation method of the reference solution in Example 1, a reference solution was prepared using a caffeic acid reference substance, and tested according to the chromatographic conditions of Example 1. Comparison revealed that the reference fingerprint constructed in Example 1 of the present invention can fully separate jasmine methyl ester, chlorogenic acid and caffeic acid. Fig.30 .

Claims

1. A method for constructing a comparative fingerprint of Duyiwei capsule, characterized in that: The method comprises the following steps: using the detection method to detect the Duyiwei capsule, merging the recorded chromatograms to generate a Duyiwei capsule control fingerprint spectrum; the Duyiwei capsule control fingerprint spectrum presents chromatographic peaks corresponding to the retention times of genus scutellariae glycoside, jasmine glycoside methyl ester, chlorogenic acid, cryptochlorogenic acid, 8-O-acetyl jasmine glycoside methyl ester, forsythiaside B, luteolin, verbascoside and isoverbascoside; the detection method comprises the following steps: (1) Preparation of the test solution: Take the contents of the Lamiophlomis capsule and extract with a solvent to obtain the test solution; the solvent is methanol, methanol-water solution, ethanol or ethanol-water solution; (2) Pipette the test solution, inject it into the liquid chromatograph, perform the test, and record the chromatogram; the chromatographic conditions for the test are as follows: The chromatographic column is one of the following chromatographic columns: Alletch Leader C18, size: 4.6*250mm, 5μm; Yuexu XBC18, size: 4.6*250mm, 5μm; Kromasil 100-5-C18, size: 4.6*250mm 5μm; Medium Spectrum Red RD-C18, size: 4.6*250mm, 5μm; Pheromone Luna, size: 4.6*250mm, 5μm; Agilent SB C18, size: 4.6*250mm, 5μm; The detection wavelength was 230-240 nm, the column temperature was 30°C ± 5°C, and the flow rate was 0.9-1.1 ml / min; Mobile phase: composed of phase A and phase B, wherein phase A is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution; The elution conditions of the mobile phase are: 。 2. The method according to claim 1, characterized in that In step (1), the extraction method is heating reflux extraction or ultrasonic extraction, and the extraction time is 60-90 minutes.

3. The method according to claim 2, characterized in that In step (1), the solvent is 70% methanol aqueous solution.

4. The method according to claim 1, characterized in that In step (2), the chromatographic conditions are as follows: the detection wavelength is 235 nm, the column temperature is 30° C., and the flow rate is 1.0 ml / min.

5. The method according to any one of claims 1 to 4, characterized in that: The batches of the Duyiwei capsules are more than 15 batches.

6. A quality control method for Lamiaceae capsules, characterized in that: The method comprises the following steps: (i) taking Du Yi Wei capsules, testing them using the detection method described in any one of claims 1 to 4, and recording a chromatogram; (ii) evaluating the similarity between the recorded chromatogram and a reference fingerprint, the similarity being not less than 0.88; the reference fingerprint being constructed by the method according to any one of claims 1 to 5.

7. A quality control method for Lamiaceae capsules, characterized in that: The method comprises the following steps: (a) taking Du Yi Wei capsules, detecting them using the detection method described in any one of claims 1 to 4, and recording a chromatogram; (b) taking a reference substance of shanzhioside methyl ester and a reference substance of 8-O-acetylshanzhioside methyl ester, dissolving them in a solvent to obtain a reference solution; aspirating the reference solution, injecting it into a liquid chromatograph, and detecting it according to the chromatographic conditions of any one of claims 1 to 4; (c) evaluating the similarity between the chromatogram recorded in step (a) and the reference fingerprint, the similarity shall not be less than 0.88, and the chromatogram recorded in step (a) shall present chromatographic peaks corresponding to the retention times of the chromatographic peaks of the reference substances jasmine methyl ester and 8-O-acetyljasmine methyl ester; the reference fingerprint is constructed by the method described in any one of claims 1 to 5.

8. A method for detecting characteristic components in a sample of Lamiophlomis lappa, characterized in that: The method comprises the following steps: (a) Preparation of test solution: taking a sample of Radix Angelicae Pubescentis and extracting it with a solvent to obtain a sample of Radix Angelicae Pubescentis; the sample of Radix Angelicae Pubescentis is Radix Angelicae Pubescentis medicinal material, Radix Angelicae Pubescentis capsule intermediate and / or Radix Angelicae Pubescentis capsule; (b) Preparation of reference solution: taking a reference, adding a solvent to dissolve it, and obtaining a solution; the reference is a characteristic component, which is safflower glycoside, shanzhioside methyl ester, chlorogenic acid, cryptochlorogenic acid, 8-O-acetylshanzhioside methyl ester, forsythiaside B, luteolin, verbascoside and isoverbascoside; (c) respectively taking the test solution and the reference solution, injecting them into a liquid chromatograph, and detecting characteristic components in the Lamiophlomis lappa sample according to the chromatographic conditions described in any one of claims 1 to 4.

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