A quality control method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules

Abstract

The present invention provides a quality control method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules, comprising the following steps: extracting Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules to prepare a test solution; extracting Baiyaozi control medicinal materials to prepare a control medicinal material reference solution; mixing cephalothin and methanol to prepare a reference substance reference solution; using high performance liquid chromatography to detect the test solution, the control medicinal material reference solution and the reference substance reference solution respectively, to obtain the characteristic spectrum of Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules and the content of cephalothin in Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules. The present invention establishes a unified characteristic spectrum determination method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules, which can simultaneously determine the content of cephalothin in the sample.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a quality control method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules. Background Art

[0002] Baiyaozi is the dried root of Stephania cepharantha Hayata, a plant of the Menispermaceae family. It is distributed in Hanzhong, Shaanxi, eastern and southeastern Sichuan, eastern and southern Guizhou, east to Zhejiang, Jiangsu and Taiwan, and south to Guangxi and Guangdong.

[0003] The name "Baiyaozi" was recorded in the Tang Dynasty's "New Compendium of Materia Medica". The Song Dynasty's "Illustrated Materia Medica" recorded that "the leaves of Baiyaozi from Jiangxi are like Chinese tallow trees, and the seeds are like mung beans. In August, the seeds turn red." This description is basically consistent with the Menispermum capitatum of the Menispermaceae family. In the "Compendium of Materia Medica", "the vines and roots are like earth melons, the leaves are like money, and the roots are like Stephania tetrandra" of Chen's Baiyao is also similar to the Menispermum capitatum. In the "Illustrated Record of Plant Names and Realities", it is recorded as the official name by its common alias "Golden Thread Turtle", and its description is also consistent with the Menispermum capitatum. Although Baiyaozi is not recorded in the current "Chinese Pharmacopoeia", the dried tubers of the Menispermaceae plant Menispermum capitatum have been recorded as the authentic Baiyaozi in the 1977 edition of the "Chinese Pharmacopoeia".

[0004] Baiyaozi is often used clinically in most provinces of my country. It is included in the "Beijing Traditional Chinese Medicine Preparation Specifications (2008 Edition)", "Guizhou Traditional Chinese Medicine and Ethnic Medicine Quality Standards (2003 Edition)" and "Hunan Traditional Chinese Medicine Preparation Specifications (2010 Edition)", but only simple properties and physical and chemical identification are available, and the specificity is not strong, which cannot effectively control the quality of Baiyaozi. Baiyaozi tastes pungent, warm, and non-toxic; it is mainly used to treat wounds and promote tissue regeneration, and its efficacy is relatively significant, so it is widely used in clinical Chinese and Mongolian medicine. There are also counterfeit products on the market that use the rhizomes of the lily family plant Polygonatum as Baiyaozi. Therefore, it is urgent to improve the current local quality control standards.

[0005] The chemical components contained in Bletilla striata are mainly alkaloids, which can be divided into 7 categories. Among them, the content of bis-benzylisoquinoline type is the highest and the pharmacological effects are the most abundant, including anti-cancer effect, analgesic and sedative effect, antibacterial and anti-inflammatory effect, immune system effect, anti-drug resistance effect, anti-arrhythmic effect, etc. As a bis-benzylisoquinoline alkaloid, cephalanthrin plays an important role in Bletilla striata. At present, some literature studies have reported the determination method and quality control method of cephalanthrin in Bletilla striata.

[0006] For example, Wang Yingbin et al. (Wang Yingbin, Mu Xianli. Determination of the content of cephalaenopsis in Bletilla striata by RP-HPLC [J]. Chinese Journal of Ethnic Medicine, 2010, 4(4): 44-45.) determined the content of cephalaenopsis in Bletilla striata by RP-HPLC with methanol-0.05% triethylamine solution (75:25) for isocratic elution, and determined that cephalaenopsis had a maximum absorption at 282nm. However, the chromatogram obtained by this method had a poor peak shape, and the chromatographic peak had an obvious tailing phenomenon. In addition, only the content of cephalaenopsis in Bletilla striata was determined, and its characteristic peak was not specified.

[0007] Zhang Zhitong et al. (Zhang Zhitong, Fu Xingsheng, Zhu Lin, et al. Study on the quality standard of Baiyaozi [J]. Pharmacy and Clinical Research, 2019, 27(5): 356-358.) controlled the quality of Baiyaozi medicinal materials by property identification, microscopic identification, thin layer identification, moisture, total ash, and extracts. They also used methanol-0.1% triethylamine (75:25) for isocratic elution to determine the content of cephalothin in Baiyaozi medicinal materials. The peak shape of the chromatogram obtained by this method was improved, but except for the chromatographic peak containing the measured indicator cephalothin, the separation of other chromatographic peaks was poor, and no regulations were made for the characteristic peaks. Therefore, this method cannot be used directly to detect the characteristic peaks of Baiyaozi medicinal materials.

[0008] The above studies are all control indicators established for Baiyaozi medicinal materials, but no reports have been seen on the quality control of Baiyaozi standard decoctions and formula granules. Baiyaozi formula granules produced using modern technology have lost their inherent form. Existing detection methods are difficult to deeply analyze the transfer of Baiyaozi's basic medicinal substances from raw materials to standard decoctions and then to finished formula granules. It is impossible to compare and analyze the differences and changes in samples at key links in the entire process. Therefore, it is difficult to comprehensively evaluate and control the intrinsic quality of the process from Baiyaozi raw materials to finished formula granules.

[0009] Therefore, how to provide an analytical method for Bletilla striata seeds so that it can comprehensively evaluate and control the intrinsic quality of the process from Bletilla striata seeds raw materials to formulated granule finished products has become a problem that needs to be solved urgently. Summary of the invention

[0010] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a quality control method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules. The present invention establishes a unified method for determining the characteristic spectrum of Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules. The method can simultaneously determine the content of cephalaenopsis in the sample, which is conducive to the overall evaluation of the scientificity and rationality of the relevant process of Baiyaozi, and can more effectively control the intrinsic quality of Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules. The detection method is accurate, fast and stable, which is of great significance to ensuring the stability, clinical effectiveness and safety of the product.

[0011] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] The present invention provides a quality control method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules, the quality control method comprising the following steps:

[0013] (1) Extract the Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules and prepare the test solution;

[0014] Extract the reference medicinal material of Bletilla striata to prepare a reference medicinal material solution;

[0015] The cephalaenopsis indole and methanol were mixed to prepare a reference substance solution;

[0016] (2) The test solution, control medicinal material reference solution and control substance reference solution obtained in step (1) are respectively detected by high performance liquid chromatography to obtain characteristic spectra of Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules and the content of cephalaenopsis in Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules.

[0017] The technical problem to be solved by the present invention is that there is no detection method currently reported that is suitable for analyzing the transfer of the basic substance of the efficacy of Baiyaozi from the raw material to the standard decoction and then to the finished formula granules, and it is impossible to compare and analyze the differences and changes in the sample spectra of the key links in the whole process, so it is difficult to evaluate and control the intrinsic quality of the process from the raw material to the finished formula granules of Baiyaozi as a whole. This method can accurately and quickly detect the content of cephalanthrin in Baiyaozi and its preparations, and can also be used as a detection method for its characteristic spectrum, further enriching its relevant information and improving the quality standards of the products.

[0018] In the present invention, in step (1), the preparation of the test solution specifically comprises the following steps:

[0019] Take Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules, mix them with methanol aqueous solution, perform ultrasound, and filter to obtain the test solution.

[0020] Preferably, the volume fraction of methanol in the methanol aqueous solution is 50%-80% (for example, it can be 50%, 53%, 56%, 59%, 63%, 66%, 69%, 73%, 76%, 79%, 80%, etc.).

[0021] Preferably, the solid-to-liquid ratio of the Baiyaozi medicinal material, decoction piece, standard decoction freeze-dried powder or formula granules to the methanol aqueous solution is 0.25:(15-50) g / mL (wherein "15-50" can be 15, 20, 25, 30, 35, 40, 45, 50, etc.).

[0022] Preferably, the power of the ultrasound is 240-260 W (for example, it can be 240 W, 243 W, 246 W, 249 W, 253 W, 256 W, 259 W, 260 W, etc.), the frequency of the ultrasound is 30-50 kHz (for example, it can be 30 kHz, 33 kHz, 35 kHz, 38 kHz, 40 kHz, 43 kHz, 46 kHz, 49 kHz, 50 kHz, etc.), and the time of the ultrasound is 20-50 min (for example, it can be 20 min, 23 min, 26 min, 29 min, 35 min, 40 min, 43 min, 47 min, 50 min, etc.).

[0023] In the present invention, in step (1), the preparation of the control medicinal material reference solution specifically comprises the following steps:

[0024] The control medicinal material of Bletilla striata is mixed with water and boiled to obtain the control medicinal material reference solution.

[0025] Preferably, the mass ratio of the Bletilla striata reference medicinal material and water is 1:(25-35) (wherein, "25-35" can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, etc.).

[0026] Preferably, the decocting time is 10-40 min (for example, it may be 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, etc.).

[0027] Preferably, in step (1), the preparation of the reference substance solution specifically comprises the following steps: mixing cepharanthin and methanol to obtain the reference substance solution.

[0028] Preferably, the solid-to-liquid ratio of the cephaladin and methanol is (20-30):1 μg / mL (wherein, "20-30" can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.).

[0029] In the present invention, the preparation method of the standard decoction freeze-dried powder comprises the following steps:

[0030] Extracting Baiyaozi from Baiyaozi decoction pieces by water decoction to obtain Baiyaozi standard decoction; performing reduced pressure concentration on the Baiyaozi standard decoction, freeze drying, and obtaining Baiyaozi standard decoction freeze-dried powder;

[0031] Wherein, the water decoction method includes a first decoction and a second decoction in sequence, the weight of water added in the first decoction is 5-8 times the weight of the Baiyaozi decoction slices (for example, it can be 5 times, 6 times, 7 times, 8 times, etc.), and the decoction time is 10-30min (for example, it can be 10min, 13min, 16min, 19min, 23min, 26min, 29min, 30min, etc.); soaking treatment is also carried out before the first decoction, and the soaking time is 20-40min (for example, it can be 20min, 23min, 26min, 29min, 33min, 36min, 39min, 40min, etc.); the weight of water added in the second decoction is 4-8 times the weight of the Baiyaozi decoction slices (for example, it can be 4 times, 5 times, 6 times, 7 times, 8 times, etc.), and the decoction time is 10-30min (for example, it can be 10min, 13min, 16min, 19min, 23min, 26min, 29min, 30min, etc.).

[0032] Preferably, the temperature of the reduced pressure concentration is 60-70°C (for example, 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, etc.).

[0033] In the present invention, the freeze drying includes 10 stages, and the specific process of the freeze drying is shown in the following table:

[0034]

[0035] In the present invention, in step (2), the filler used in the chromatographic column in the high performance liquid chromatography detection includes C18 bonded silica gel.

[0036] Preferably, in step (2), the chromatographic column used in the high performance liquid chromatography detection includes any one of Waters XBridge C18, Waters XBridge Shield RP18 or Agilent ZORBAX Extend C18.

[0037] Preferably, in step (2), the column temperature in the HPLC detection is 28-32°C (for example, it may be 28°C, 29°C, 30°C, 31°C, 32°C, etc.).

[0038] In the present invention, in step (2), the flow rate of the mobile phase in the HPLC detection is 0.9-1.1 mL / min (for example, it can be 0.9 mL / min, 0.93 mL / min, 0.96 mL / min, 0.99 mL / min, 1.03 mL / min, 1.06 mL / min, 1.09 mL / min, 1.1 mL / min, etc.).

[0039] In the present invention, in step (2), the detection wavelength in the high performance liquid chromatography detection is 282 nm.

[0040] Preferably, in step (2), the injection volume in the HPLC detection is 8-12 μL (for example, it can be 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, etc.).

[0041] In the present invention, in the HPLC detection, methanol is used as mobile phase A, and 0.05%-0.15% (for example, 0.05%, 0.07%, 0.09%, 0.11%, 0.13%, 0.15%, etc.) triethylamine aqueous solution is used as mobile phase B, and gradient elution is performed according to the following table:

[0042] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 40→47 60→53 10～18 47→58 53→42 18～24 58 42 24～30 58→75 42→25 30～40 75 25 40～60 75→90 25→10 .

[0043] In the present invention, the Baiyaozi medicinal material, decoction slices, standard decoction freeze-dried powder or formula granules have a total of 4 characteristic peaks, which are peak 1, peak 2, peak 3 and peak 4 in order from short to long in terms of peak elution time, with peak 4 being the S peak, and the relative retention times of peaks 1-3 are within ±10% of the specified values, and the specified values ​​are: 0.51, 0.80 and 0.93, respectively; wherein peak 4 is cephalothin.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This study established a unified characteristic spectrum determination method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules. This method can simultaneously determine the content of cephalothin in the sample, which is conducive to the overall evaluation of the scientificity and rationality of Baiyaozi related process, and can more effectively control the intrinsic quality of Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules. This detection method is accurate, fast and stable, which is of great significance to ensure the stability, clinical effectiveness and safety of the product, and can further supplement and improve the quality evaluation system of Baiyaozi and its related preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the characteristic spectrum of the reference medicinal material of Bletilla striata, among which peak 4 is cephalaenopsis indica.

[0047] Figure 2 This is the characteristic spectrum of the freeze-dried powder test sample of the standard decoction of Bletilla striata in Example 1, wherein peak 4 is cephalaenopsis indica.

[0048] Figure 3 This is the characteristic spectrum of the Baiyaozi formula granules test sample in Example 1, wherein peak 4 is cephalaenopsisin.

[0049] Figure 4 This is the delayed chromatogram of the freeze-dried powder of Baiyaozi standard decoction.

[0050] Figure 5 This is a comparison chart of the correlation study of the freeze-dried powder of Baiyaozi standard decoction, the reference material of Baiyaozi reference medicinal material and the reference material of Stephania tetrandra;

[0051] Among them, S1 is the freeze-dried powder of the standard decoction of Bletilla striata, S2 is the reference material of the control medicinal material of Bletilla striata, S3 is the reference material of the reference substance of cephalothin, and peak 4 is cephalothin.

[0052] Figure 6 Characteristic spectra of 15 batches of freeze-dried powder of Baiyaozi standard decoction;

[0053] Among them, S1-S15 are YP202106-10-1 to YP202106-10-15, R is the reference spectrum, and peak 4 is cephalothin.

[0054] Figure 7 These are the characteristic spectra of freeze-dried powder of standard decoction of Bletilla striata fruit extracted with different solvents, where peak 4 is cephalaenopsis indica.

[0055] Figure 8 This is the standard curve of cephalaenopsisin.

[0056] Fig. 9 Characteristic spectra of 15 batches of Bletilla striata seeds;

[0057] Among them, R is the reference spectrum, S1-S15 are 202106-10-1 to 202106-10-15 respectively, and peak 4 is cephalothin.

[0058] Fig.10 Characteristic spectra of three batches of Baiyaozi formula granules;

[0059] Among them, S1 is KL202106-10-1, S2 is KL 202106-10-9, S3 is KL202106-10-14, R is the reference spectrum, and peak 4 is cephalothin.

[0060] Fig.11 It is the production verification characteristic spectrum of Baiyaozi YP202106-10-1;

[0061] Among them, S1 is the reference spectrum of freeze-dried powder of Baiyaozi standard decoction, S2 is the spectrum of Baiyaozi medicinal materials, S3 is the spectrum of extract, S4 is the spectrum of concentrated solution, S5 is the spectrum of extract powder, S6 is the spectrum of Baiyaozi formula granules, and peak 4 is cephalothin.

[0062] Fig.12 It is the production verification characteristic spectrum of Baiyaozi YP202106-10-9;

[0063] Among them, S1 is the reference spectrum of freeze-dried powder of Baiyaozi standard decoction, S2 is the spectrum of Baiyaozi medicinal materials, S3 is the spectrum of extract, S4 is the spectrum of concentrated solution, S5 is the spectrum of extract powder, S6 is the spectrum of Baiyaozi formula granules, and peak 4 is cephalothin.

[0064] Fig.13 It is the production verification characteristic spectrum of Baiyaozi YP202106-10-14;

[0065] Among them, S1 is the reference spectrum of freeze-dried powder of Baiyaozi standard decoction, S2 is the spectrum of Baiyaozi medicinal materials, S3 is the spectrum of extract, S4 is the spectrum of concentrated solution, S5 is the spectrum of extract powder, S6 is the spectrum of Baiyaozi formula granules, and peak 4 is cephalothin. DETAILED DESCRIPTION

[0066] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0067] The sources of instruments, reagents and reagents in the following examples are as follows:

[0068] Test drug: Cephalotaxel (batch number: 111647-200301, purity 100%, China Food and Drug Administration)

[0069] Baiyaozi reference medicinal material: batch number: 202106-10-3;

[0070] Baiyaozi medicinal materials: batch number: 202106-10-1; 202106-10-2; 202106-10-3; 202106-10-4; 202106-10-5; 202106-10-6; 202106-10-7; 202106-10-8; 202106-10-9; 202106-10-10; 202106-10-11; 202106-10-12; 202106-10-13; 202106-10-14; 202106-10-15;

[0071] Baiyaozi decoction pieces: batch number: yp202106-10-1; yp 202106-10-2; yp 202106-10-3; yp202106-10-4; yp202106-10-5; yp202106-10-6; yp202106-10-7; yp202106-10-8; yp202106-10-9; yp202106-10-10; yp202106-10-11; yp202106-10-12; yp202106-10-13; yp202106-10-14; yp202106-10-15;

[0072] Baiyaozi standard decoction freeze-dried powder: batch number: YP202106-10-1; YP202106-10-2; YP202106-10-3; YP202106-10-4; YP202106-10-5; YP202106-10-6; YP202106-10-7; YP202106-10-8; YP202106-10-9; YP202106-10-10; YP202106-10-11; YP202106-10-12; YP202106-10-13; YP202106-10-14; YP202106-10-15;

[0073] Baiyaozi formula granules: batch number: KL202106-10-1; KL202106-10-9; KL202106-10-14.

[0074] Reagents: Methanol (Merck) was of chromatographic grade; triethylamine (chromatographic grade, Thermo Fisher); water was distilled water (Watsons); other reagents were of analytical grade.

[0075] Instrument: One hundredth of a day balance (model: JA1002, manufacturer: Shanghai Puchun Measuring Instrument Co., Ltd.);

[0076] One-tenth balance (model: ML204T, manufacturer: Mettler Toledo Instruments (Shanghai) Co., Ltd.);

[0077] Electronic balance (model: MSA6 6S-OCE-DM, manufacturer: Sartorius Scientific Instruments (Beijing) Co., Ltd.);

[0078] Ultra-fast liquid chromatography (model: Waters UPLC Arc (UV), manufacturer: Waters Technology (Shanghai) Co., Ltd.);

[0079] High performance liquid chromatography (model: Thermo Ultimate 3000, manufacturer: Thermo Fisher Scientific (China) Co., Ltd.);

[0080] CNC ultrasonic cleaner (model: KQ-100DE, manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.);

[0081] Constant temperature water bath oscillator (model: THZ-82, manufacturer: Jintan Jingda Instrument Manufacturing Co., Ltd.);

[0082] Freeze dryer (model: BL-4000FD, manufacturer: Shanghai Bilang Instrument Manufacturing Co., Ltd.);

[0083] Chromatographic column: Waters XBridge C18 (4.6×250 mm, 5 μm);

[0084] Waters XBridge Shield RP18 (4.6mm×250mm, 5μm);

[0085] Agilent ZORBAX Extend C18 (4.6×250mm, 5μm).

[0086] The retention times in the following examples are all in minutes (min).

[0087] Preparation Example 1

[0088] This preparation example provides a Baiyaozi standard decoction freeze-dried powder, and the preparation method of the Baiyaozi standard decoction freeze-dried powder comprises the following steps:

[0089] Take the Baiyaozi slices, place them in a casserole, soak for 30 minutes, add 7 times the amount of water for the first decoction, boil over high heat, simmer for 20 minutes, filter while hot, cool quickly, and set aside; add 6 times the amount of water for the second decoction, boil over high heat, simmer for 15 minutes, filter while hot, cool quickly, and set aside; combine the filtrate to obtain the standard decoction. The standard decoction is concentrated under reduced pressure at 65°C, and then freeze-dried to obtain the Baiyaozi standard decoction freeze-dried powder.

[0090] The specific steps of freeze drying are as follows:

[0091] stage Temperature setting (℃) Maintenance time (h) 1 -40 4 2 -30 4 3 -20 4 4 -10 6 5 -5 6 6 0 10 7 10 6 8 20 6 9 30 6 10 35 15 .

[0092] Example 1

[0093] This embodiment provides a quality control method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules, and the quality control method specifically includes the following steps:

[0094] Preparation of reference substance solution: Take an appropriate amount of cephalaenopsis reference substance, weigh it accurately, and add methanol to make a solution containing 25 μg per 1 mL, which is used as the reference substance solution.

[0095] Preparation of reference solution of control medicinal materials: Take about 1.0 g of Baiyaozi medicinal material (batch number: 202106-10-3), put it in a beaker, add 30 mL of water, boil it for 30 minutes, cool it, filter it, and take the filtrate as the reference solution of control medicinal materials.

[0096] Preparation of Baiyaozi medicinal material test solution: Take an appropriate amount of Baiyaozi medicinal material, grind it into powder, take about 0.25g, accurately weigh it, put it in a conical flask, accurately add 25mL of 70% methanol aqueous solution, plug it tightly, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 70% methanol aqueous solution, shake it well, filter it, take the filtrate to obtain the Baiyaozi medicinal material test solution.

[0097] Preparation of the test solution of Baiyaozi decoction pieces: The only difference from the test solution of Baiyaozi medicinal materials is that the Baiyaozi medicinal materials are replaced with Baiyaozi decoction pieces of the same quality.

[0098] Preparation of the test solution of Baiyaozi standard decoction freeze-dried powder: The only difference from the test solution of Baiyaozi medicinal materials is that the Baiyaozi medicinal materials are replaced with the Baiyaozi standard decoction freeze-dried powder of the same quality.

[0099] Preparation of the test solution of Baiyaozi formula granules: The only difference from the test solution of Baiyaozi medicinal materials is that the Baiyaozi medicinal materials are replaced with Baiyaozi formula granules of equal quality.

[0100] High performance liquid chromatography was used to test the reference substance solution, the reference medicinal material solution, the Baiyaozi medicinal material test solution, the Baiyaozi decoction piece test solution, the Baiyaozi standard decoction freeze-dried powder test solution and the Baiyaozi formula granule test solution;

[0101] Chromatographic conditions: Waters XBridge Shield RP18 (4.6mm×250mm, 5μm) chromatographic column; methanol as mobile phase A, 0.1% triethylamine as mobile phase B, gradient elution according to the following table; flow rate 1.0mL per minute; column temperature 30°C; detection wavelength 282nm; injection volume 10μL. The theoretical plate number should be no less than 3000 based on the cephalothin peak, and the elution gradient is as follows:

[0102] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 40→47 60→53 10～18 47→58 53→42 18～24 58 42 24～30 58→75 42→25 30～40 75 25 40～60 75→90 25→10

[0103] like Figure 1 , Figure 2 and Figure 3As shown, the test samples of Baiyaozi standard decoction freeze-dried powder (batch number YP202106-10-1) and Baiyaozi formula granules (batch number KL202106-10-1) present 4 characteristic peaks, which should correspond to the 4 characteristic peaks in the chromatographic peaks of Baiyaozi reference medicinal material reference, among which peak 4 has the same retention time as the reference substance (Chenacanthus chinensis), and peak 4 is taken as the S peak, and the relative retention time of characteristic peaks 1 to 3 is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are 0.51 (peak 1), 0.80 (peak 2), 0.93 (peak 3), and 1.00 (peak 4S).

[0104] (I) Construction of characteristic spectrum of freeze-dried powder of Baiyaozi standard decoction

[0105] Experimental Example 1 Delay Experiment

[0106] Take the test solution of the lyophilized powder of the standard decoction of Baiyaozi (provided in Example 1), perform high performance liquid chromatography according to the method provided in Example 1, and record the chromatogram of 2 times the elution time of the mobile phase. The test results are as follows: Figure 4 As shown, the delayed results show that no obvious hysteresis peak appears after 60 minutes, which indicates that the chromatographic method provided by the present invention meets the analysis requirements.

[0107] Experimental Example 2 System Applicability Experiment

[0108] According to the method provided in Example 1, the lyophilized powder test solution of the standard decoction of Baiyaozi (provided in Example 1) was subjected to high performance liquid chromatography, and the system suitability test was performed with the cephalaenopsis acetosine peak as a reference. The test results are shown in Table 1 below:

[0109] Table 1

[0110] Number of measurements 1 2 3 4 5 average value RSD(%) Theoretical plate number 152730 153863 156274 154883 155488 154648 0.9 Tailing Factor 1.1 1.1 1.1 1.1 1.1 1.1 0.0 Peak area 135283 135525 136263 134382 134832 135257 0.5

[0111] The system suitability results meet the relevant requirements of 0512 High Performance Liquid Chromatography of the 2020 edition of the Chinese Pharmacopoeia.

[0112] Experimental Example 3 Methodological Verification

[0113] 3.1 Exclusivity

[0114] According to the method provided in Example 1, the lyophilized powder of the standard decoction of Bletilla striata (batch number: YP202106-10-03), the Bletilla striata control medicinal material reference and the spectrum of cephalaenopsis indica were determined by high performance liquid chromatography.

[0115] The test results are as follows Figure 5 As shown in the figure, it can be seen that the spectra of the freeze-dried powder of the standard decoction of Bletilla striata, the reference medicinal material of Bletilla striata and the reference solution of Cephalaenopsis indica have no interference, indicating that the method provided by the present invention has good specificity.

[0116] 3.2 Precision

[0117] 3.2.1 Repeatability

[0118] Take 6 portions of Baiyaozi standard decoction freeze-dried powder (batch number: YP202106-10-3) and perform high performance liquid chromatography determination according to the method provided in Example 1 to obtain its characteristic spectrum. Taking peak No. 4 as the reference peak S, calculate the relative retention time and relative peak area of ​​characteristic peaks No. 1 to 3, and calculate the RSD value.

[0119] The results of repeatability retention time and relative retention time are shown in Table 2 below:

[0120] Table 2

[0121]

[0122] The repeatability peak area and relative peak area are shown in Table 3 below:

[0123] Table 3

[0124]

[0125] The results of repeatability investigation showed that the relative retention time RSD of each characteristic peak was in the range of 0.01% to 0.13%, and the relative peak area RSD of each characteristic peak was in the range of 1.55% to 2.66%, indicating that the characteristic spectrum method had good repeatability.

[0126] 3.2.2 Intermediate precision

[0127] The freeze-dried powder of Baiyaozi standard decoction (YP202106-10-3) was taken, and different analysts used another Thermo Fisher high-performance liquid chromatograph (model: Ultimate3000) for intermediate precision test at different times. The high-performance liquid chromatography was determined according to the method provided in Example 1 to obtain its characteristic spectrum. Peak No. 4 was used as the reference peak S, and the relative retention time and relative peak area of ​​characteristic peaks No. 1 to 3 were calculated. The results were combined with the results of the repeatability investigation to calculate the RSD values ​​of different instruments.

[0128] The retention time and relative retention time of the intermediate precision test are shown in Table 4 below:

[0129] Table 4

[0130]

[0131] The peak area and relative peak area of ​​the intermediate precision test are shown in Table 5 below:

[0132] Table 5

[0133]

[0134] The relative retention time RSD of each characteristic peak is in the range of 0.01% to 0.09%, and the relative retention time RSD range between different instruments is 0.20% to 0.58%, indicating that the experimental results of this analytical method are relatively stable; the RSD values ​​of the relative peak areas of each peak between different instruments are large, so the relative peak area range is not limited.

[0135] 3.3 Durability

[0136] 3.3.1 Stability study

[0137] Take the freeze-dried powder of Baiyaozi standard decoction (YP202106-10-3), prepare the test solution according to the method provided in Example 1, and measure it according to the method provided in Example 1 at 0, 2, 4, 6, 8, 10, 12, and 24 h, respectively, to obtain its characteristic spectrum, take peak No. 4 as the reference peak S, calculate the relative retention time and relative peak area of ​​characteristic peaks No. 1 to 3, and calculate the RSD value.

[0138] The retention time and relative retention time of stability study are shown in Table 6 below:

[0139] Table 6

[0140]

[0141] The peak area and relative peak area of ​​stability investigation are shown in Table 7 below:

[0142] Table 7

[0143]

[0144]

[0145] The results of the stability experiment show that the characteristic components in the sample solution are stable within 24 hours, the relative retention time RSD of each characteristic peak is in the range of 0.02% to 0.24%, and the relative peak area RSD is in the range of 2.60% to 3.89%.

[0146] 3.3.2 Investigation of different column temperatures

[0147] Take Baiyaozibiaojian lyophilized powder (batch number: YP202106-10-3) and prepare it into a test solution according to the test solution preparation method provided in Example 1. The characteristic spectrum was obtained by measuring it at different column temperatures (28°C, 30°C and 32°C) according to the chromatographic conditions provided in Example 1. Peak 4 was used as the reference peak S, and the relative retention times of characteristic peaks 1 to 3 were calculated, and the RSD value was calculated.

[0148] The retention time and relative retention time at different column temperatures are shown in Table 8 below:

[0149] Table 8

[0150]

[0151] Different column temperatures (28°C, 30°C and 32°C) were investigated respectively, and the relative retention time RSD of the characteristic peaks was in the range of 0.24% to 0.40%, indicating that this method has good durability to column temperature.

[0152] 3.3.3 Investigation of different flow rates

[0153] Take Baiyaozibiaojian lyophilized powder (batch number: YP202106-10-3) and prepare it into a test solution according to the test solution preparation method provided in Example 1. The test solution was measured at different flow rates (0.9 mL / min, 1.0 mL / min and 1.1 mL / min) according to the chromatographic conditions provided in Example 1 to obtain its characteristic spectrum. Peak 4 was used as the reference peak S, and the relative retention times of characteristic peaks 1 to 3 were calculated, and the RSD value was calculated.

[0154] The retention time and relative retention time at different flow rates are shown in Table 9 below:

[0155] Table 9

[0156]

[0157] The relative retention time RSD of the characteristic peaks at different flow rates (0.9 mL / min, 1.0 mL / min and 1.1 mL / min) was in the range of 0.51% to 1.86%, indicating that the method has good durability for different flow rates.

[0158] 3.3.4 Investigation of different brands and models of chromatographic columns

[0159] Take Baiyaozibiaojian lyophilized powder (batch number: YP202106-10-3) to prepare the test solution according to the method provided in Example 1, and use different types of chromatographic columns to measure according to the method provided in Example 1 to obtain its characteristic spectrum, take peak No. 4 as the reference peak S, calculate the relative retention time of characteristic peaks No. 1 to 3, and calculate the RSD value.

[0160] Chromatographic column 1: Waters XBridge C18 (4.6*250mm, 5.0μm);

[0161] Column 2: Waters XBridge Shield RP18 (4.6*250 mm, 5.0 μm);

[0162] Column 3: Agilent ZORBAX Extend C18 (4.6*250mm, 5.0μm);

[0163] The retention time and relative retention time of different chromatographic columns are shown in Table 10 below:

[0164] Table 10

[0165]

[0166] Different chromatographic columns were investigated respectively, and the relative retention time RSD of characteristic peaks was in the range of 2.20% to 3.53%, indicating that the method has good durability for different chromatographic columns.

[0167] Experimental Example 4: Sample Testing and Data Processing

[0168] The characteristic spectrum of 15 batches of Baiyaozi standard decoction samples was determined by using the established characteristic spectrum method (test method provided in Example 1), and the common peaks were marked using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System" (2012.1 version), and S1 was used as the reference spectrum, and the median was calculated to obtain the control spectrum, and the similarity between the spectrum of each batch of Baiyaozi standard decoction and the control spectrum was calculated to determine the acceptable range of similarity. According to the characteristics of each chromatographic peak and the situation of the active ingredients in Baiyaozi, peak No. 4 was used as the reference peak S, and the relative retention time and relative peak area of ​​characteristic peaks No. 1 to No. 3 were calculated, and the relative retention time and relative peak area of ​​each common peak were counted, and the acceptability of the relative retention time of each common peak was clarified.

[0169] The characteristic spectrum of Baiyaozi standard decoction freeze-dried powder (15 batches) is as follows Figure 6 As shown, the similarity of the characteristic spectra of 15 batches of Baiyaozi standard decoction freeze-dried powder is shown in Table 11 below:

[0170] Table 11

[0171]

[0172]

[0173] The characteristic spectrum retention time and relative retention time of 15 batches of Baiyaozi standard decoction freeze-dried powder are shown in Table 12 below:

[0174] Table 12

[0175]

[0176] The peak areas and relative peak areas of the characteristic spectra of 15 batches of Baiyaozi standard decoction freeze-dried powder are shown in Table 13 below:

[0177] Table 13

[0178]

[0179]

[0180] The characteristic spectrum of the freeze-dried powder of Baiyaozi standard decoction generated by the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System" (2012.1 version) has 4 common peaks, of which peak 4 is used as the reference peak S, and the relative retention time and relative peak area of ​​peaks 1 to 3 are calculated based on peak S. The relative retention time of each characteristic peak is slightly different, all within the range of ±10% of the mean, which meets the quality control requirements. The average value of the relative retention time is selected as the specified value: 0.51 (peak 1), 0.80 (peak 2), 0.93 (peak 3), and the allowable error is ±10%. However, the relative peak area of ​​the characteristic peaks between batches varies greatly, with RSD values ​​of 9.48% to 17.85%, which is not suitable as a quality control requirement, so the relative peak area is not included in the standard.

[0181] (II) Determination of cephalaenopsis in freeze-dried powder of standard decoction of Baiyaozi

[0182] Experimental Example 5 System Applicability Experiment

[0183] According to the method provided in Example 1, the lyophilized powder test solution of the standard decoction of Baiyaozi (provided in Example 1) was subjected to high performance liquid chromatography, and the system suitability test was performed with the cephalaenopsis peak as a reference. The test results are shown in Table 14 below:

[0184] Table 14

[0185] Number of measurements 1 2 3 4 5 average value RSD(%) Theoretical plate number 152730 153863 156274 154883 155488 154648 0.89 Tailing Factor 1.1 1.1 1.1 1.1 1.1 1.1 0.00 Repeatability 35243 35838 35993 35499 36494 35813 1.34

[0186] The system suitability results meet the relevant requirements of 0512 High Performance Liquid Chromatography of the 2020 edition of the Chinese Pharmacopoeia.

[0187] Experimental Example 6 Preparation of test solution

[0188] 6.1 Selection of extraction solvent

[0189] Take the freeze-dried powder of Baiyaozi standard decoction (batch number: YP202106-10-3) respectively, weigh 0.25g in parallel, accurately weigh, put in a stoppered conical flask, accurately add 25mL of appropriate solvent (methanol, 70% methanol, 50% methanol, 30% methanol, water), seal, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter as the test solution. Determine according to the chromatographic conditions provided in Example 1, calculate the content of cephalothin, the results are shown in Table 15 below.

[0190] Table 15

[0191]

[0192] The characteristic spectra of freeze-dried powder of Baiyaozi standard decoction obtained by using different extraction solvents are as follows Figure 7 As shown, peak 4 is cephalaenopsisin.

[0193] From the above results, it can be seen that different extraction solvents have different extraction abilities for cephalanthrin. Among them, the cephalanthrin content is relatively high when 50% methanol aqueous solution, 70% methanol aqueous solution and methanol are used as extraction solvents. At the same time, considering that the peak shapes and separation conditions of the characteristic peaks in the characteristic spectra of different extraction solvents are similar, the extraction solvent was finally determined to be 70% methanol aqueous solution.

[0194] 6.2 Investigation of extraction solvent dosage

[0195] Take the freeze-dried powder of Baiyaozi standard decoction (batch number: YP202106-10-3) respectively, weigh 0.25g in parallel, accurately weigh, put in a stoppered conical bottle, accurately add 15mL, 25mL, and 50mL of 70% methanol water solvent respectively, plug it tightly, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, and filter it as the test solution. Determine according to the chromatographic conditions in the text, calculate the content of cephalothin, and the test results are shown in Table 16 below:

[0196] Table 16

[0197]

[0198]

[0199] From the above results, it can be seen that different amounts of extraction solvent have different effects on the extraction capacity of cephalothin, among which the cephalothin content extracted with 25mL and 50mL is relatively high. At the same time, due to cost and operational considerations, the extraction solvent amount was selected as 25mL.

[0200] 6.3 Investigation of extraction methods

[0201] Take the freeze-dried powder of Baiyaozi standard decoction (batch number: YP202106-10-3) separately, weigh 0.25g in parallel, accurately weigh, place in a stoppered conical flask, accurately add an appropriate amount of 70% methanol aqueous solution 25mL to seal the flask, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz), reflux extraction for 30 minutes, take out, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and use as the test solution. Determine according to the chromatographic conditions provided in Example 1, and calculate the content of cephalothin. The results are shown in Table 17 below.

[0202] Table 17

[0203]

[0204] The above results show that the content of cephaladin in the test solution obtained by different extraction methods is not much different. Considering the simplicity of experimental operation, ultrasonic treatment was determined as the extraction method for preparing the test solution.

[0205] 6.4 Investigation of extraction time

[0206] Take the freeze-dried powder of Baiyaozi standard decoction (batch number: YP202106-10-3) respectively, weigh 0.25g in parallel, accurately weigh, put in a stoppered conical flask, accurately add 25mL of 70% methanol aqueous solution, plug, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively, take out, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, and filter as the test solution. According to the chromatographic conditions provided in Example 1, the content of cephalothin was calculated, and the results are shown in Table 18 below.

[0207] Table 18

[0208]

[0209] From the above results, it can be seen that different extraction times have slightly different effects on the extraction capacity of cephalothin. The cephalothin content is relatively high when extracted for 40 minutes, so the extraction time is selected as 40 minutes.

[0210] The preparation method of the test solution is as follows: take 0.25 g of the test sample (freeze-dried powder of standard decoction of Bailiaozi), accurately weigh it, put it in a conical flask, accurately add 25 mL of 70% methanol aqueous solution, seal it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 70% methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain it.

[0211] Experimental Example 7 Methodology Verification

[0212] 7.1 Linearity

[0213] Take an appropriate amount of cephalaenopsis reference substance, accurately weigh it, add 70% methanol aqueous solution to make a reference substance solution containing 60.10μg of cephalaenopsis per 1mL, as cephalaenopsis reference substance mother solution ⑤; then accurately pipette 1mL of cephalaenopsis reference substance solution ⑤ into a 25mL brown volumetric flask, add 70% methanol aqueous solution to dilute to the scale, as cephalaenopsis reference substance solution ①. Then accurately pipette 5mL of cephalaenopsis reference substance solution ⑤ into a 100mL brown volumetric flask, add 70% methanol aqueous solution to dilute to the scale, as cephalaenopsis reference substance solution ②. Then accurately pipette 10mL of cephalaenopsis reference substance solution ⑤ into a 100mL brown volumetric flask, add 70% methanol aqueous solution to dilute to the scale, as cephalaenopsis reference substance solution ③. Then accurately pipette 5mL of cephalaenopsis reference substance solution ⑤ into a 25mL brown volumetric flask, add 70% methanol aqueous solution to dilute to the scale, as cephalaenopsis reference substance solution ④. The above-mentioned cephalaenopsis reference solution ① to ⑤ was filtered respectively, and 10 μL of the above-mentioned filtrate was accurately taken and injected into a high performance liquid chromatograph. The peak area of ​​the cephalaenopsis chromatographic peak was determined according to the method provided in Example 1. The peak area of ​​the cephalaenopsis chromatographic peak was used as the ordinate and the concentration of cephalaenopsis was used as the abscissa to draw a standard curve. The least squares method was used for linear regression to obtain the cephalaenopsis regression equation as Y=5667.31X–1850.12, R 2 =1.000, and its linear range is 2.4040-60.1000 μg / mL. The results are shown in Table 19 below.

[0214] Table 19

[0215]

[0216] Figure 8 is the standard curve of cepharanthine, from Table 19 and Figure 8 It can be seen that by using the method provided by the present invention, the linear relationship of cephalaenopsis is good.

[0217] 7.2 Precision

[0218] 7.2.1 Repeatability

[0219] Take 6 portions of the freeze-dried powder of the standard decoction of Bletilla striata (batch number: YP202106-10-3) for testing, and measure according to the method provided in Example 1 to calculate the content of cephalaenopsis in the sample. The results are shown in Table 20 below.

[0220] Table 20

[0221]

[0222] The average content of cephalaenopsis measured in the repeatability test was 0.66 mg / g, and its RSD was 1.62%, which met the repeatability requirements of the methodology validation.

[0223] 7.2.2 Intermediate precision

[0224] Different analysts used another Thermo Fisher Ultimate 3000 high performance liquid chromatograph (TUV detector) to conduct intermediate precision tests at different times. Six portions of the test sample of the lyophilized powder of the standard decoction of Baiyaozi (batch number: YP202106-10-03) were taken and measured according to the method provided in Example 1. The results are shown in Table 21 below.

[0225] Table 21

[0226]

[0227] The average content of cephalaenopsis in the intermediate precision test was 0.70 mg / g, with an RSD value of 3.31%, which was 3.84% compared with the RSD of the repeatability test, meeting the precision requirements of methodological validation.

[0228] 7.3 Accuracy

[0229] Take an appropriate amount of cephalaenopsis reference substance, accurately weigh it, and add 70% methanol aqueous solution to make a solution containing 60.10 μg per 1 mL. Take 9 portions of the lyophilized powder test sample of the standard decoction of Baiyaozi (batch number: YP202106-10-03) with a known content under the repeatability item, each portion is about 1 g, accurately weighed, and accurately added with reference substance solutions containing 0.0751 mg, 0.1503 mg, and 0.2254 mg of cephalaenopsis, respectively. The remaining operations are the same as the method provided in Example 1. The recovery rate is calculated according to the following formula. The results are shown in Table 22 below.

[0230]

[0231] Table 22

[0232]

[0233] The recovery rate range measured by the recovery test was 95.50% to 103.37%, the average recovery rate was 99.1%, and the RSD value was 2.73%, which met the recovery rate requirements of the methodology validation, indicating that the results measured by this method are accurate.

[0234] 7.4 Durability

[0235] 7.4.1 Stability Study

[0236] Take an appropriate amount of Baiyaozi standard decoction freeze-dried powder test sample (batch number: YP202106-10-3), prepare the test solution according to the method provided in Example 1, and measure according to the method provided in Example 1 at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, and record the changes in the peak area of ​​cephalothin. The results are shown in Table 23 below.

[0237] Table 23

[0238]

[0239] From the above data, it can be seen that the peak area RSD value of cephalaenopsis within 24 hours is 3.93%, which meets the requirements of the system suitability test, indicating that the stability of cephalaenopsis in the sample within 24 hours is good.

[0240] 7.4.2 Investigation of different column temperatures

[0241] Take the test sample of the freeze-dried powder of the standard decoction of Baiyaozi (batch number: YP202106-10-3), prepare the test solution according to the test solution preparation method provided in Example 1, and measure it at different column temperatures (28°C, 30°C and 32°C) according to the method provided in Example 1 to examine the durability of the experimental method for column temperature. The results are shown in Table 24 below.

[0242] Table 24

[0243]

[0244] The RSD value of cepharanthin content measured at different column temperatures was 2.56%, which met the system suitability requirements, indicating that the method has good durability to column temperature.

[0245] 7.4.3 Investigation of different flow rates

[0246] Take the test sample of the lyophilized powder of the standard decoction of Baiyaozi (batch number: YP202106-10-3), prepare the test solution according to the preparation method of the test solution provided in Example 1, and use different flow rates (0.9 mL / min, 1.0 mL / min and 1.1 mL / min) to measure according to the method provided in Example 1 to investigate the durability of the experimental method for the flow rate. The results are shown in Table 25 below.

[0247] Table 25

[0248]

[0249] The RSD value of cepharanthin content measured at different flow rates was 4.80%, which met the system suitability requirements, indicating that the method has good flow rate durability.

[0250] 7.4.4 Investigation of different chromatographic columns

[0251] The test sample of the lyophilized powder of the standard decoction of Baiyaozi (batch number: YP202106-10-3) was prepared according to the method provided in Example 1, and the test solution was determined using different types of chromatographic columns according to the method provided in Example 1 to examine the durability of the experimental method for different chromatographic columns. The results are shown in Table 26 below.

[0252] Chromatographic column 1: Waters XBridge C18 (4.6*250mm, 5.0μm);

[0253] Column 2: Waters XBridge Shield RP18 (4.6*250 mm, 5.0 μm);

[0254] Column 3: Agilent ZORBAX Extend C18 (4.6*250mm, 5.0μm);

[0255] Table 26

[0256]

[0257]

[0258] The chromatograms obtained using different chromatographic columns showed that the theoretical plate number, tailing factor and separation degree of the cephalothin peak all met the system suitability requirements; the RSD value of the cephalothin content measured using different chromatographic columns was 2.25%, which met the system suitability requirements, indicating that this method has good durability for different chromatographic columns.

[0259] Experimental Example 8 Sample Content Determination

[0260] Fifteen batches of Baiyaozi standard decoction freeze-dried powder were taken respectively, and the content of cephalaenopsis in the standard decoction freeze-dried powder was determined according to the method provided in Example 1. The results are shown in Table 27 below.

[0261] Table 27

[0262] batch number Cephalaenopsis content (mg / g) YP202106-10-1 0.81 YP202106-10-2 0.64 YP202106-10-3 0.64 YP202106-10-4 0.64 YP202106-10-5 0.54 YP202106-10-6 0.66 YP202106-10-7 0.55 YP202106-10-8 0.77 YP202106-10-9 0.65 YP202106-10-10 0.76 YP202106-10-11 0.59 YP202106-10-12 0.66 YP202106-10-13 0.69 YP202106-10-14 0.64 YP202106-10-15 0.78

[0263] (III) Detection of characteristic spectrum of Baiyaozi medicinal material

[0264] The characteristic spectrum method established in Example 1 was used to measure the characteristic spectrum of 15 batches of Baiyaozi medicinal material samples, and the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System" (2012.1 version) was used to mark the common peaks, and S1 was used as the reference spectrum, and the median was calculated to obtain the control spectrum, and the similarity between the spectrum of each batch of Baiyaozi standard decoction and the control spectrum was calculated. Taking the No. 4 peak, cephalaenopsis as the reference peak S, the relative retention time and relative peak area of ​​the characteristic peaks 1 to 3 were calculated, the relative retention time of each common peak was statistically analyzed, and the acceptability of the relative retention time of each common peak was clarified.

[0265] The characteristic spectra of 15 batches of Baiyaozi medicinal materials are as follows Fig. 9 shown.

[0266] The similarity results of the characteristic spectra of 15 batches of Bletilla striata seeds are shown in Table 28 below.

[0267] Table 28

[0268]

[0269]

[0270] The relative retention times of the characteristic spectra of 15 batches of Bletilla striata seeds are shown in Table 29 below.

[0271] Table 29

[0272]

[0273] The characteristic spectrum of Baiyaozi medicinal material generated by the "Similarity Evaluation System for Chromatographic Characteristic Spectra of Traditional Chinese Medicine" (2012.1 version) has 4 common peaks. The similarities between 15 batches of Baiyaozi medicinal materials and the control medicinal materials are all above 95%. Among them, peak 4 is used as the reference peak S. The relative retention times of peaks 1 to 3 calculated based on peak S are slightly different, all within the range of ±10% of the specified value of the standard decoction. Therefore, the characteristic spectrum standard of Baiyaozi standard decoction can be used as the characteristic spectrum quality standard of Baiyaozi medicinal material.

[0274] (IV) Determination of the content of celastrin in Bletilla striata

[0275] 15 batches of Bletilla striata seed coarse powder were taken respectively, and the content of celastrin was determined according to the method provided in Example 1. The results are shown in Table 30 below:

[0276] Table 30

[0277]

[0278]

[0279] (V) Detection of characteristic spectra of Baiyaozi decoction pieces

[0280] Baiyaozi can be sliced ​​and diced on the spot while it is fresh, which is convenient for drying, processing and transportation. Since the processing specifications of various places describe that Baiyaozi medicinal materials are only purified and impurities are removed to obtain Baiyaozi decoction pieces, the chemical composition difference between the two is very small, and the characteristic spectra are almost the same, so I will not repeat them here.

[0281] (VI) Determination of celastrin content in Baiyaozi decoction pieces

[0282] Preparation method of medicinal pieces: clean and remove impurities.

[0283] 15 batches of Baiyaozi decoction pieces were taken respectively, and the content of cephalaenopsis was determined according to the method provided in Example 1. The results are shown in Table 31 below:

[0284] Table 31

[0285] batch number Cephalaenopsis content (mg / g) yp202106-10-1 1.9 yp202106-10-2 2.0 yp202106-10-3 2.3 yp202106-10-4 2.1 yp202106-10-5 1.9 yp202106-10-6 2.2 yp202106-10-7 2.1 yp202106-10-8 2.5 yp202106-10-9 1.7 yp202106-10-10 1.8 yp202106-10-11 2.2 yp202106-10-12 1.8 yp202106-10-13 2.3 yp202106-10-14 2.3 yp202106-10-15 2.1

[0286] (VII) Detection of characteristic spectrum of Baiyaozi formula granules

[0287] Take 3 batches of Baiyaozi formula granules respectively, obtain their liquid phase spectrum according to the method provided in Example 1, use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.1 version), use the S1 liquid phase spectrum as the reference spectrum, calculate according to the median, obtain the Baiyaozi granule reference spectrum, and identify the common peaks. Take peak No. 4 as the reference peak S, calculate the relative retention time and relative peak area of ​​characteristic peaks No. 1 to 3, perform statistics on the relative retention time and relative peak area of ​​each common peak, and clarify the acceptability of the relative retention time of each common peak, and verify the three batches of granules.

[0288] The characteristic spectra of 3 batches of Baiyaozi formula granules are as follows Fig.10 As shown, S1 is KL202106-10-1, S2 is KL202106-10-9, S3 is KL202106-10-14, and R is the control spectrum.

[0289] The retention time and relative retention time of three batches of Baiyaozi formula granules are shown in Table 32 below:

[0290] Table 32

[0291]

[0292] The relative retention times of the characteristic spectra of three batches of production verification of Baiyaozi formula granules are basically consistent with the relative retention times of the standard characteristic spectra, and the reproducibility of the characteristic spectra is good, all within the range of ±10% of the specified value of the standard decoction freeze-dried powder. Therefore, the characteristic spectrum standard of Baiyaozi standard decoction freeze-dried powder can be used as the characteristic spectrum quality standard of Baiyaozi formula granules.

[0293] (VIII) Determination of the content of celastrin in Baiyaozi formula granules

[0294] Three batches of Baiyaozi formula granules were taken respectively, and the content of cephalaenopsisin was determined according to the method provided in Example 1. The results are shown in Table 33 below:

[0295] Table 33

[0296] batch number Cephalaenopsis content (mg / g) KL202106-10-1 1.00 KL202106-10-9 0.90 KL202106-10-14 0.95

[0297] (IX) Transfer of characteristic peak components during the production of Baiyaozi formula granules

[0298] The quality control method used in this study can effectively monitor the transfer of each characteristic peak component during the production process of Baiyaozi formula granules, and can realize characteristic spectrum detection of samples from each production link of Baiyaozi production raw materials, extraction, concentration, drying, and preparation, and compare them with the characteristic spectrum of Baiyaozi standard decoction freeze-dried powder.

[0299] The preparation method of Baiyaozi formula granules comprises the following steps:

[0300] Take an appropriate amount of Baiyaozi slices, extract (100℃) twice, decoct for 1.5h each time, add 10 times the amount of water for the first decoction, and add 9 times the amount of water for the second decoction. Filter the liquid with a 150-mesh filter cloth while hot, combine the filtrate, and obtain the extract. Concentrate the extract under reduced pressure (85℃) to a relative density of 1.05-1.10 (60℃) to obtain a concentrated solution. Spray dry the concentrated solution with an inlet air temperature of 180±5℃, collect the spray-dried powder to obtain the extract powder. Add an appropriate amount of the extract powder to the auxiliary materials, mix well, and dry granulate to obtain the Baiyaozi formula granules.

[0301] Fig.11 This is the production verification characteristic spectrum of Baiyaozi YP202106-10-1. The comparison results of the production verification characteristic spectrum of Baiyaozi YP202106-10-1 are shown in Table 34 below:

[0302] Table 34

[0303]

[0304]

[0305] Fig.12 This is the production verification characteristic spectrum of Baiyaozi YP202106-10-9. The comparison results of the production verification characteristic spectrum of Baiyaozi YP202106-10-9 are shown in Table 35 below:

[0306] Table 35

[0307] serial number Sample name Control feature map S1 Standard soup comparison chart 1.000 S2 Medicinal Materials 0.946 S3 Extract 0.991 S4 Concentrate 0.991 S5 Extract powder 0.990 S6 Particles 0.990

[0308] Fig.13 This is the production verification characteristic spectrum of Baiyaozi YP202106-10-14. The comparison results of the production verification characteristic spectrum of Baiyaozi YP202106-10-14 are shown in Table 36 below:

[0309] Table 36

[0310] serial number Sample name Control feature map S1 Standard soup comparison chart 1.000 S2 Medicinal Materials 0.946 S3 Extract 0.989 S4 Concentrate 0.990 S5 Extract powder 0.989 S6 Particles 0.990

[0311] Depend on Figure 11-13The spectrum shows that the four characteristic peaks of the characteristic spectrum of the freeze-dried powder of the standard decoction of Baiyaozi can correspond to the characteristic peaks of the production process (extraction, concentration, drying, granulation) and the raw medicinal materials one by one, indicating that the four characteristic peaks are all stably transmitted; the similarity of the spectrum of the samples in each production link is higher than 95% compared with the control spectrum of the freeze-dried powder of the standard decoction of Baiyaozi, indicating that the basic substances of the samples in each link of the production process are consistent with the standard decoction of Baiyaozi. It can be seen that this study has established a unified characteristic spectrum determination method for Baiyaozi medicinal materials, decoction pieces, freeze-dried powder of standard decoctions and formula granules. This method can simultaneously determine the content of celastrol in the sample, which is conducive to the overall evaluation of the scientificity and rationality of the relevant process of Baiyaozi, and can more effectively control the intrinsic quality of Baiyaozi medicinal materials, decoction pieces, freeze-dried powder of standard decoctions and formula granules.

[0312] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A quality control method for Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder and formula granules, characterized in that: The quality control method comprises the following steps: (1) Extracting the medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules of Bletilla striata to prepare a test solution; extracting the control medicinal materials of Bletilla striata to prepare a control medicinal material reference solution; mixing cephalaenopsis indica with methanol to prepare a reference substance solution; The preparation of the test solution specifically includes the following steps: taking Baiyaozi medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules, mixing with methanol aqueous solution, ultrasonicating, filtering, and obtaining the test solution; the volume fraction of methanol in the methanol aqueous solution is 70%-76%; (2) The test solution, the control medicinal material reference solution and the control reference solution obtained in step (1) are respectively detected by high performance liquid chromatography to obtain characteristic spectra of the Baiyaozi medicinal material, decoction pieces, standard decoction freeze-dried powder or formula granules and the content of cephalaenopsis in the Baiyaozi medicinal material, decoction pieces, standard decoction freeze-dried powder or formula granules; The chromatographic column in the high performance liquid chromatography detection is a C18 bonded silica gel chromatographic column; The detection wavelength in the high performance liquid chromatography detection is 282nm; In the HPLC detection, methanol was used as mobile phase A, 0.05%-0.15% triethylamine aqueous solution was used as mobile phase B, and gradient elution was performed according to the following table:

2. The quality control method according to claim 1, characterized in that: In the preparation of the test solution in step (1), the solid-to-liquid ratio of the Bletilla striata seeds, decoction pieces, standard decoction freeze-dried powder or formula granules to the methanol aqueous solution is 0.25:(15-50) g / mL.

3. The quality control method according to claim 1, characterized in that: In the preparation of the test solution in step (1), the power of the ultrasound is 240-260 W, the frequency of the ultrasound is 30-50 kHz, and the time of the ultrasound is 20-50 min.

4. The quality control method according to claim 1, characterized in that: In step (1), the preparation of the control medicinal material reference solution specifically includes the following steps: mixing the Bletilla striata reference medicinal material and water, and decocting the mixture to obtain the control medicinal material reference solution.

5. The quality control method according to claim 4, characterized in that: The mass ratio of the Bletilla striata reference medicinal material to water is 1:(25-35).

6. The quality control method according to claim 4, characterized in that: The decocting time is 10-40min.

7. The quality control method according to claim 1, characterized in that: In step (1), the solid-liquid ratio of cephalaenopsis indole to methanol is (20-30):1 μg / mL.

8. The quality control method according to any one of claims 1 to 7, characterized in that: The preparation method of the standard decoction freeze-dried powder comprises the following steps: Extracting Baiyaozi from Baiyaozi decoction pieces by water decoction to obtain Baiyaozi standard decoction; performing reduced pressure concentration on the Baiyaozi standard decoction, freeze drying, and obtaining Baiyaozi standard decoction freeze-dried powder; Among them, the water decoction method includes the first decoction and the second decoction in sequence, the weight of water added in the first decoction is 5-8 times the weight of the Baiyaozi decoction pieces, and the decoction time is 10-30min; soaking treatment is also carried out before the first decoction, and the soaking time is 20-40min; the weight of water added in the second decoction is 4-8 times the weight of the Baiyaozi decoction pieces, and the decoction time is 10-30min.

9. The quality control method according to claim 8, characterized in that: The temperature of the reduced pressure concentration is 60-70°C.

10. The quality control method according to claim 8, characterized in that: The freeze drying process includes 10 stages, and the specific process of the freeze drying process is shown in the following table: 。 11. The quality control method according to claim 1, characterized in that: In step (2), the column temperature in the HPLC detection is 28-32°C.

12. The quality control method according to claim 1, characterized in that: In step (2), the flow rate of the mobile phase in the high performance liquid chromatography detection is 0.9-1.1 mL / min.

13. The quality control method according to claim 1, characterized in that: In step (2), the injection volume in the HPLC detection is 8-12 μL.

14. The quality control method according to claim 1, characterized in that: The Baiyaozi medicinal material, decoction slices, standard decoction freeze-dried powder or formula granules have a total of 4 characteristic peaks, which are peak 1, peak 2, peak 3 and peak 4 in order from short to long in terms of peak elution time, with peak 4 being the S peak. The relative retention times of peaks 1-3 are within ±10% of the specified values, and the specified values ​​are: 0.51, 0.80 and 0.93, respectively; wherein peak 4 is cephalothin.

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