Quality detection method of uterus-warming seven-ingredient pill

Abstract

The invention belongs to the field of quality control of traditional Chinese medicine preparations, and particularly relates to a quality detection method of uterus-warming seven-ingredient pills. According to the method, the UPLC fingerprint spectrum of the uterus-warming seven-ingredient pill is constructed on the basis of ultra-high performance liquid chromatography by optimizing liquid chromatography conditions, and the efficient and accurate uterus-warming seven-ingredient pill detection method is obtained. The detection method determines 23 common peaks, identifies 5 chemical components, has good precision, repeatability and stability, can be used for quality control and comprehensive evaluation of the uterus-warming seven-ingredient pill, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of quality control of traditional Chinese medicine preparations, and particularly relates to a quality detection method for Nuangong Qiwei Pills. Background Art

[0002] Nuangong Qiwei Pills (NQP) is a classic Mongolian prescription for treating gynecological diseases, and the prescription is from "The Joy of the Viewer". This product is a compound preparation composed of seven medicinal materials, namely Amomum kravanh Pierre ex Gagnep., Asparagus cochinchinensis (Lour.) Merr., Gymnadenia conopsea (L.) R. Br., Aquilaria sinensis (Lour.) Spreng., Myristica fragrans Houtt., Polygonatum sibiricum Redoute, and Eugenia caryophyllata Thunb., and has the effects of regulating menstruation and stopping bleeding, warming the uterus, and dispelling cold and relieving pain. It is mainly used for treating symptoms such as qi stagnation low back pain and irregular menstruation. NQP is currently included in the Mongolian medicine volume of the "Drug Standards of the Ministry of Health". The current quality standard only includes the description and inspection items, and there is no quantitative method for effectively controlling the quality of this product. At present, most of the literature reports focus on the pharmacological activities and action mechanisms of NQP, and there are few research literature reports on quality control, and most of them stay at the level of determining the content of a single chemical component.

[0003] The chemical components of traditional Chinese patent medicines are complex, and it is difficult for a single-index evaluation mode to comprehensively reflect the overall quality of the preparation. As a comprehensive and quantifiable chromatographic identification method, the fingerprint of traditional Chinese medicine can objectively reflect the overall information of the chemical component group in the drug, better reflect the complexity and correlation of the components, and is widely used to evaluate the quality of medicinal materials, cut crude drugs, and preparations. By using fingerprint technology combined with the determination of multi-component content and using chemometrics, such as cluster analysis, principal component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) and other analysis methods to integrate complex data, the quality differences of drugs between different enterprises and batches can be more intuitively reflected, and it has been widely used in the research and analysis of ready-made prescription preparations in recent years.

[0004] Therefore, establishing a fingerprint that can accurately and comprehensively reflect the chemical components of Nuangong Qiwei Pills is of great significance for the quality control of Nuangong Qiwei Pills. Summary of the Invention

[0005] In view of the problems of the prior art, the present invention provides a quality detection method for Nuangong Qiwei Pills.

[0006] The present invention provides a quality detection method for Nuangong Qiwei Pills, which comprises the following steps:

[0007] Step 1, extracting the active ingredients of Nuangong Qiwei Pills to obtain a test solution;

[0008] Step 2: Detect the test solution by UPLC to obtain the UPLC fingerprint of Warming the Palace Qiwei Pills;

[0009] Among them, the chromatographic conditions are as follows:

[0010] The chromatographic column is a C18 column;

[0011] Mobile phase: It consists of mobile phase A and mobile phase B. Among them, mobile phase A is a 0.1 - 0.3% phosphoric acid solution, and mobile phase B is acetonitrile.

[0012] Preferably, in step 1, the extraction steps include: dissolving the Warming the Palace Qiwei Pills powder in a solvent and performing ultrasonic extraction; the mass - to - volume ratio of the Warming the Palace Qiwei Pills powder to the solvent is 1 g: 20 - 100 ml; and / or, the extraction time is 30 - 90 min; the solvent is an aqueous methanol solution;

[0013] And / or, in step 1, the chromatographic column is a Shim - pack GIST C18 chromatographic column;

[0014] And / or, mobile phase A is a 0.2% phosphoric acid solution.

[0015] Preferably, the chromatographic conditions further include: detection wavelength: 215 - 235 nm;

[0016] Flow rate: 0.3 - 0.5 mL / min;

[0017] Column temperature: 20 - 40 °C;

[0018] Injection volume: 1 - 10 μL.

[0019] Preferably, the detection wavelength is 220 nm.

[0020] Preferably, the flow rate is 0.4 mL / min.

[0021] Preferably, the column temperature is 30 - 35 °C.

[0022] Preferably, the injection volume is 1 - 2 μL.

[0023] Preferably, the chromatographic conditions further include: the elution method is gradient elution, and the elution gradient is 0 - 4 min, 3% mobile phase B; 4 - 5 min, 3% - 10% mobile phase B; 5 - 11 min, 10% mobile phase B; 11 - 25 min, 10% - 23% mobile phase B; 25 - 35 min, 23% - 36% mobile phase B; 35 - 45 min, 36% mobile phase B; 45 - 52 min, 36% - 39% mobile phase B; 52 - 65 min, 39% mobile phase B; 65 - 66 min, 39% - 45% mobile phase B; 66 - 75 min, 45% mobile phase B.

[0024] Preferably, the UPLC fingerprint contains 23 common peaks, and the following chemical components are detected: agallochaol, eugenol, pseudoprotodioscin, myristicin, dehydrodiisoeugenol.

[0025] The present invention also provides the application of the UPLC fingerprint constructed by the above quality detection method in the quality control of Warming the Palace Seven Flavor Pills.

[0026] By optimizing the liquid chromatography conditions (such as detection wavelength, flow rate, column temperature, injection volume) of the present invention, on the basis of ultra-high performance liquid chromatography, the UPLC fingerprint of Warming the Palace Seven Flavor Pills is constructed to obtain an efficient and accurate detection method for Warming the Palace Seven Flavor Pills. This detection method determines 23 common peaks and identifies 5 chemical components (agallochaol, eugenol, pseudoprotodioscin, myristicin, dehydrodiisoeugenol). Its precision, repeatability and stability are good, and it can be used for the quality control and comprehensive evaluation of Warming the Palace Seven Flavor Pills, and has broad application prospects.

[0027] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0028] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0029] Figure 1 It is the UPLC fingerprint of 15 batches of samples of Warming the Palace Seven Flavor Pills.

[0030] Figure 2 It is the UPLC chromatogram of the reference substance (red line) and the test sample (blue line); among them, 4 is agallochaol, 9 is eugenol, 10 is pseudoprotodioscin, 15 is myristicin, and 22 is dehydrodiisoeugenol.

[0031] Figure 3 It is the PCA score chart of 15 batches of samples of Warming the Palace Seven Flavor Pills.

[0032] Figure 4 It is the PLS-DA score chart of the common peaks of 15 batches of samples of Warming the Palace Seven Flavor Pills.

[0033] Figure 5 It is the VIP chart of the common peaks of 15 batches of samples of Warming the Palace Seven Flavor Pills.

[0034] Figure 6 It is the cluster analysis chart of 15 batches of samples of Warming the Palace Seven Flavor Pills.

[0035] Figure 7 It is for the investigation of the chromatographic conditions (flow rate) in the UPLC fingerprint of Warming Gong Qiwei Pills.

[0036] Figure 8 It is for the investigation of the chromatographic conditions (column temperature) in the UPLC fingerprint of Warming Gong Qiwei Pills.

[0037] Figure 9 It is for the investigation of the chromatographic conditions (sample injection volume) in the UPLC fingerprint of Warming Gong Qiwei Pills.

[0038] Figure 10 It is for the investigation of the chromatographic conditions (detection wavelength) in the UPLC fingerprint of Warming Gong Qiwei Pills. Specific implementation mode

[0039] In the following examples and experimental examples, the reagents and raw materials not specifically described are all commercially available products.

[0040] 1. Instruments

[0041] Shimadzu LC-40B X3 type high performance liquid chromatograph, equipped with CTO-40C type DAD detector (Shimadzu Corporation, Japan); BSA124S analytical balance (one in one hundred thousand) (Sartorius Scientific Instruments Co., Ltd., Beijing); FA1004 analytical balance (one in ten thousand) (Shanghai Liangping Instrument Co., Ltd.); UPH-I ultrapure water preparation instrument (Sichuan Youpu Ultra-pure Technology Co., Ltd.); SB-8200DTS dual-frequency ultrasonic instrument (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.); H1650-W micro desktop high-speed centrifuge (Hunan Xiangyi Laboratory Development Co., Ltd.).

[0042] 2. Reagents and drugs

[0043] Phosphoric acid and acetonitrile are chromatographically pure (Thermo Fisher Scientific (China) Co., Ltd.); water is ultrapure water; other reagents are all analytically pure; agarotetrol (batch number: DSTDC004201, purity ≥ 98%); eugenol (batch number: DST230905-036, purity ≥ 98%); pseudoprotodioscin (batch number: DST231017-003, purity ≥ 98%); myristicin (batch number: DSTDR004301, purity ≥ 98%); dehydrodiisoeugenol (batch number: DST220326-055, purity ≥ 98%) reference substances are all purchased from Lemeitian Medicine / Dest Bio-Technology Co., Ltd.

[0044] A total of 15 batches of Nuangong Qiwei Pills were provided by Inner Mongolia Datang Pharmaceutical Co., Ltd., with batch numbers 23449001, 24449002, 23449004, 24449003, 24449001, 22449005, 21449002, 23449003, 21449005, 22449006, 22449003, 23449005, 24449006, 24449005, 24449004 respectively.

[0045] Example 1 Detection method for UPLC fingerprint of Nuangong Qiwei Pills

[0046] 1. Weigh appropriate amounts of each reference substance, dissolve them with methanol to obtain reference substance solutions (with mass concentrations of agarotetrol 12.05 μg / mL, eugenol 150.27 μg / mL, pseudoprotodioscin 85.51 μg / mL, myristicin 10.78 μg / mL, dehydrodiisoeugenol 3.66 μg / mL respectively).

[0047] 2. Weigh 1.0 g of Nuangong Qiwei Pills powder (passed through No. 3 sieve) accurately, place it in a stoppered conical flask, accurately add 50 mL of methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 min, let it cool, weigh it again, make up the lost mass with methanol, shake well, take the continuous filtrate, centrifuge it (13000 r / min) for 15 min, and take the supernatant as the test solution.

[0048] 3. Chromatographic conditions

[0049] Shim-pack GIST C18 chromatographic column (100 mm × 2.1 mm, 2 μm); using 0.2% phosphoric acid (A) - acetonitrile (B) as the mobile phase, gradient elution (0 - 4 min, 3% B; 4 - 5 min, 3% - 10% B; 5 - 11 min, 10% B; 11 - 25 min, 10% - 23% B; 25 - 35 min, 23% - 36% B; 35 - 45 min, 36% B; 45 - 52 min, 36% - 39% B; 52 - 65 min, 39% B; 65 - 66 min, 39% - 45% B; 66 - 75 min, 45% B); flow rate 0.4 mL / min; column temperature 30 °C; detection wavelength 220 nm; injection volume 2 μL.

[0050] 4. Construction of UPLC fingerprint

[0051] 4.1 Generation of fingerprint

[0052] For 15 batches of samples of Nuangong Qiwei Pills, the test solution was prepared according to Step 2 and injected for determination under the above chromatographic conditions. The relevant data was imported into the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)". The time window width was set to 0.1 min, and the multi-point calibration method was used for full-peak matching. A total of 23 common peaks were determined, as shown in Figure 1 .

[0053] 4.2 Identification of common peaks and selection of reference peaks

[0054] The reference substance solution and the test solution were separately injected for determination under the above chromatographic conditions. The results are shown in Figure 2 . By comparing the ultraviolet absorption values of the DAD detector, peak 4 was identified as aquilarol, peak 9 as eugenol, peak 10 as pseudoprotodioscin, peak 15 as myristicin, and peak 22 as dehydrodiisoeugenol. Then, peak 9 (eugenol), which has good separation from adjacent chromatographic peaks and stable retention time, was selected as the reference.

[0055] 4.3 Similarity analysis

[0056] The relevant data of 15 batches of samples was imported into the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 Edition). Using the reference chromatogram as the reference, the similarities were calculated, and the results were 0.996, 0.996, 0.997, 0.993, 0.997, 0.998, 0.995, 0.997, 0.991, 0.998, 0.991, 0.996, 0.995, 0.994, and 0.994 respectively, all of which were greater than 0.99, indicating good similarity among 15 batches of Nuangong Qiwei Pills samples.

[0057] 4.4 Chemical pattern recognition analysis

[0058] (1) Principal component analysis (PCA)

[0059] The common peak data was imported into SIMICA 13.0 software for unsupervised PCA analysis. The results are shown in Figure 3 . It can be seen from this that the 15 batches of samples can be divided into 2 categories, and the discrimination is relatively obvious.

[0060] (2) Partial least squares discriminant analysis (PLS-DA)

[0061] The common peak data was imported into SIMICA 13.0 software. On the basis of PCA analysis, a supervised PLS-DA discriminant analysis was established to obtain the PLS-DA score plot, VIP plot, and cluster analysis plot, as shown in Figures 4 - 6 . As can be seen from Figure 4 , the 20 batches of samples can be divided into 2 regions. As can be seen from Figure 5It can be seen that the peak No. 10 is pseudoprotodioscin with a contribution value greater than 1; the peak No. 11 comes from Aquilariae Lignum Resinatum, the peak No. 21 comes from Myristicae Semen, the peak No. 14 is a common peak of Aquilariae Lignum Resinatum, Eugeniae Flos, Myristicae Semen and Amomi Kravanh Fructus Rotundus, the peak No. 13 is a common peak of Aquilariae Lignum Resinatum and Myristicae Semen, the peak No. 2 comes from Gymnadeniae Conopseae Rhizoma, the peak No. 17 comes from Myristicae Semen, the peak No. 8 comes from Myristicae Semen, and the peak No. 18 comes from Myristicae Semen, all of which have a significant impact on classification and are thus quality markers of the main components. From Figure 6 It can be seen that the 15 batches of samples can be grouped into 2 major categories, which is consistent with Figure 4 that.

[0062] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0063] Experimental Example 1: Methodology investigation

[0064] 1. Precision test

[0065] Intraday precision: Take the same sample solution (batch number: 23449001) (prepared according to step 2 of Example 1), inject and determine 6 times under the chromatographic conditions of Example 1, with eugenol as the reference. The results show that the RSDs of the relative retention times of the common chromatographic peaks are 0.02 - 0.14%, and the RSDs of the relative peak areas are 0.57 - 0.97%, indicating good precision of the instrument.

[0066] Interday precision: Take the same sample solution (batch number: 23449001) (prepared according to step 2 of Example 1), inject and determine 6 times on the same day under the chromatographic conditions of Example 1, continuously determine for three days and analyze the data, with eugenol as the reference. The results show that the RSDs of the relative retention times of the common chromatographic peaks are 0.04 - 0.17%, and the RSDs of the relative peak areas are 0.65 - 2.40%, indicating good precision of the instrument.

[0067] 2. Repeatability test

[0068] Take the Warming the Palace Seven-Ingredient Pills of the same batch (batch number: 23449001), grind them finely, make 6 parallel portions, prepare the sample solution according to step 2 of Example 1, inject and determine under the chromatographic conditions of Example 1 and analyze the data, with eugenol as the reference peak. The results show that the RSDs of the relative retention times of the common chromatographic peaks are 0.03 - 0.25%, and the RSDs of the relative peak areas are 0.60 - 2.89%, indicating good repeatability of the method.

[0069] 3. Stability test

[0070] The Warming Gong Qiwei Pills samples of the same batch (batch number: 23449001) were ground finely, and the test solution was prepared according to the steps in Example 1. The samples were injected and analyzed at 0, 2, 4, 8, 12, 16, and 24 h at room temperature under the chromatographic conditions in Example 1, with eugenol as the reference. The results showed that the RSD of the relative retention time of each common chromatographic peak was 0 - 0.38%, and the RSD of the relative peak area was 0.02 - 2.86%, indicating that the test solution had good stability within 24 h after preparation.

[0071] Experimental Example 2 Liquid Chromatography Condition Screening Experiment

[0072] 1. According to the detection method in Example 1, the only difference was the flow rate in the chromatographic conditions, which was set to 0.3 ml / min, 0.4 ml / min, and 0.5 ml / min respectively. The chromatograms of the test samples obtained were as Figure 7 shown. Compared with the flow rates of 0.3 ml / min and 0.5 ml / min, at a flow rate of 0.4 mL / min, the resolution of the chromatographic peaks was better, the peak shapes were clearer, the intervals between the peaks were larger, and the shapes of the chromatographic peaks were also more symmetrical. Therefore, the flow rate should be selected as 0.4 ml / min.

[0073] 2. According to the detection method in Example 1, the only difference was the column temperature in the chromatographic conditions, which was set to 20°C, 25°C, 30°C, and 35°C respectively. The chromatograms of the test samples obtained were as Figure 8 shown. At a column temperature of 35°C or 30°C, the resolution of the chromatographic peaks was better, the peak shapes were clearer, and the intervals between the peaks were larger. Therefore, the column temperature was selected as 30°C - 35°C.

[0074] 3. According to the detection method in Example 1, the only difference was the injection volume in the chromatographic conditions, which was set to 1 μL, 2 μL, 5 μL, and 10 μL respectively. The chromatograms of the test samples obtained were as Figure 9 shown. At a relatively small injection volume (such as 1 μL and 2 μL), the shapes of the chromatographic peaks were more symmetrical and the resolution was better. As the injection volume increased, although the peak height increased, the peak shapes became less symmetrical. Therefore, the injection volume was selected as 1 - 2 μL.

[0075] 4. According to the detection method in Example 1, the only difference was the detection wavelength in the chromatographic conditions, which was set to 220 nm, 254 nm, and 274 nm respectively. The chromatograms of the test samples obtained were as Figure 10 shown. At a wavelength of 220 nm, the ultraviolet absorption of the peaks was large and relatively complete. Therefore, the detection wavelength was selected as 220 nm.

[0076] In summary, the present invention conducts screening experiments on liquid chromatography conditions, and respectively examines the effects of different flow rates (0.3 ml / min, 0.4 ml / min, 0.5 ml / min), different column temperatures (20 °C, 25 °C, 30 °C, 35 °C), different injection volumes (1 μL, 2 μL, 5 μL, 10 μL), and different detection wavelengths (220 nm, 254 nm, 274 nm) on the extraction of active ingredients in Warming the Palace and Nourishing the Seven Flavors Pills. By comprehensively considering the number of chromatographic peaks and response values, it is determined that the detection wavelength is 220 nm, the flow rate is 0.4 ml / min, and the column temperature is 30 - 35 °C; when the injection volume is 1 - 2 μL, the detection effect is the best; the UPLC fingerprint method for Warming the Palace and Nourishing the Seven Flavors Pills constructed by the present invention determines 23 common peaks and identifies 5 chemical components, and its precision, repeatability, and stability are good, which can be used for the quality control and comprehensive evaluation of Warming the Palace and Nourishing the Seven Flavors Pills, and has broad application prospects.

Claims

1. A quality inspection method for Warming the Uterus Seven-Ingredient Pills, characterized in that, It includes the following steps: Step 1: Extract the active ingredients of Warming the Palace Qiwei Pills to obtain a test solution. Step 2: Detect the test solution by UPLC to obtain the UPLC fingerprint of Warming the Palace Qiwei Pills. Among them, the chromatographic conditions are as follows: The chromatographic column is a C18 column. Mobile phase: It consists of mobile phase A and mobile phase B. Among them, mobile phase A is a 0.1 - 0.3% phosphoric acid solution, and mobile phase B is acetonitrile.

2. The quality inspection method according to claim 1, characterized in that, In Step 1, the extraction steps include: dissolving the powder of Warming the Palace Qiwei Pills in a solvent and performing ultrasonic extraction; the mass - volume ratio of the powder of Warming the Palace Qiwei Pills to the solvent is 1g:20 - 100ml; and / or, the extraction time is 30 - 90min; the solvent is an aqueous methanol solution. And / or, in Step 1, the chromatographic column is a Shim - pack GIST C18 chromatographic column. And / or, mobile phase A is a 0.2% phosphoric acid solution.

3. The quality inspection method according to claim 1, characterized in that The chromatographic conditions also include: Detection wavelength: 215 - 235nm. Flow rate: 0.3 - 0.5 mL / min. Column temperature: 20 - 40°C. Injection volume: 1 - 10 μL.

4. The quality inspection method according to claim 3, characterized in that The detection wavelength is 220nm.

5. The quality inspection method according to claim 3, characterized in that, The flow rate is 0.4 mL / min.

6. The quality inspection method according to claim 3, characterized in that, The column temperature is 30 - 35°C.

7. The quality inspection method according to claim 3, characterized in that The injection volume is 1 - 2 μL.

8. The quality inspection method according to claim 1, characterized in that, The chromatographic conditions also include: The elution method is gradient elution, and the elution gradient is 0 - 4min, 3% mobile phase B; 4 - 5min, 3% - 10% mobile phase B; 5 - 11min, 10% mobile phase B; 11 - 25min, 10% - 23% mobile phase B; 25 - 35min, 23% - 36% mobile phase B; 35 - 45min, 36% mobile phase B; 45 - 52min, 36% - 39% mobile phase B; 52 - 65min, 39% mobile phase B; 65 - 66min, 39% - 45% mobile phase B; 66 - 75min, 45% mobile phase B.

9. The quality inspection method according to claim 1, characterized in that, The UPLC fingerprint contains 23 common peaks, and the following chemical components are detected: Aquilarol, Eugenol, Pseudoprotodioscin, Myristicin, Dehydrodiisoeugenol.

10. Application of the UPLC fingerprint constructed by the quality detection method according to any one of claims 1 - 9 in the quality control of Warming the Palace Qiwei Pills.

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