Method for constructing HPLC characteristic spectra of Chuantong bark medicinal materials, decoction pieces and related preparations

Abstract

The present invention provides a method for constructing a high-performance liquid chromatography characteristic spectrum of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates, and their formulated granules. The present invention constructs a high-performance liquid chromatography characteristic spectrum detection method for Chuantong bark (Cassia truncatula), confirms nine characteristic peaks, specifies their relative retention times, establishes a control characteristic spectrum, fully demonstrates the chemical composition characteristics of Chuantong bark, and comprehensively reflects the quality information of Chuantong bark, thereby achieving comprehensive and effective quality control of Chuantong bark medicinal materials, decoction pieces, and related preparations.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to a method for constructing a high-performance liquid phase characteristic spectrum of Chuantong bark medicinal materials, decoction pieces and related preparations thereof. Background Art

[0002] The earliest record of Kalopanax septemlobus in the Materia Medica of Zhu Su was in the "Jiuhuang Bencao" (Compendium of Materia Medica for Relief of Famine), which is a traditional Chinese medicine in my country. The 1977 edition of the "Chinese Pharmacopoeia" states that this product is the dried bark of Kalopanax septemlobus (Thunb.) Koidz., a plant of the Araliaceae family. It is mild in nature, pungent in taste, and slightly bitter. It can dispel rheumatism, dredge meridians, and relieve pain. It is used for rheumatoid arthritis, pain in the waist and knees; and for external treatment of skin eczema. Modern research has shown that the saponin components in Kalopanax septemlobus have obvious pharmacological activities such as anti-inflammatory, anti-tumor, anti-rheumatoid, antioxidant, and anti-diabetic [5]. However, the basic research on Kalopanax septemlobus medicinal materials in my country is relatively weak, and there is no research on its processing in the literature. Through the literature review, there are also few studies on the quality standards of Kalopanax septemlobus, and the indicators are insufficient. [ The quality of Chuantong bark cannot be fully reflected, nor can the quality of medicinal materials and decoction pieces be effectively controlled. This has led to the long-standing phenomenon of uneven quality of medicinal materials and their finished pharmaceutical preparations, restricting the development and effective utilization of Chuantong bark medicinal resources. This study provides a basis for the quality evaluation and resource development and utilization of Chuantong bark medicinal materials, decoction pieces, standard decoctions, extracts, and formula granules.

[0003] Currently, there is little research on the quality of Chuantong bark (Cassia truncatula) and its compound preparations, with no reports on high-performance liquid chromatography (HPLC) characteristic spectrum analysis and detection. Consequently, there is a lack of an effective means for quality control. The present invention aims to develop a HPLC characteristic spectrum method for Chuantong bark (Cassia truncatula) to comprehensively reflect the quality level of Chuantong bark and its related preparations, providing guidance for the quality control of Chuantong bark (Cassia truncatula) medicinal materials, decoction pieces, and their related preparations. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing a high-performance liquid chromatography characteristic spectrum of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules. The high-performance liquid chromatography characteristic spectrum method of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules constructed by the present invention is stable and reliable, and can be used to control the quality of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules.

[0005] The present invention constructs a high-performance liquid chromatography characteristic spectrum detection method for Chuantong bark (Cassia truncatula), confirms that there are 9 characteristic peaks in total, stipulates their relative retention times, establishes a control characteristic spectrum, fully displays the chemical component characteristics of Chuantong bark, and comprehensively reflects the quality information of Chuantong bark, thereby achieving comprehensive and effective control of the quality of Chuantong bark medicinal materials, decoction pieces and related preparations.

[0006] The present invention provides a method for constructing a high-performance liquid chromatography characteristic spectrum of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and formula granules thereof, comprising the following steps:

[0007] S1) extracting the raw material of Chuantong bark with a solvent to obtain a test solution;

[0008] S2) measuring the test solution by high performance liquid chromatography to obtain a high performance liquid phase characteristic spectrum of the Chuantong bark raw material;

[0009] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid solution, and the elution is performed in a gradient manner.

[0010] The present invention provides a method for constructing a high-performance liquid chromatography characteristic spectrum of a Chuantong bark medicinal material, a decoction piece, a standard decoction, an intermediate, and a formula granule thereof. First, a Chuantong bark raw material is taken, solvent extraction is performed, and a test liquid is obtained; in a specific embodiment provided by the present invention, the solvent is preferably 70% methanol; the ratio of the mass g of the Chuantong bark raw material to the volume mL of the solvent is preferably (0.1-1): (10-50), more preferably (0.1-1): (10-30), further preferably (0.1-1): (10-20), and most preferably (0.1-1): 20.

[0011] In a specific embodiment provided by the present invention, the Chuantong bark raw materials are Chuantong bark raw medicinal materials, decoction pieces, standard decoctions, intermediates and formula granules thereof; wherein, the Chuantong bark intermediates are intermediates well known to those skilled in the art and are not particularly limited. In the present invention, the Chuantong bark intermediates include but are not limited to Chuantong bark extracts, Chuantong bark extracts, etc.

[0012] In a specific embodiment provided by the present invention, the preparation of the test solution of the Chuantong bark medicinal material / decoction piece is specifically as follows: the Chuantong bark medicinal material / decoction piece is decocted with water for 20 to 40 minutes, and then 70% methanol is added for ultrasonic extraction, cooled, shaken, and filtered to obtain; wherein, the Chuantong bark medicinal material / decoction piece is preferably sieved through a No. 4 sieve and then decocted with water; the mass ratio of the Chuantong bark medicinal material / decoction piece to water is preferably 1: (40 to 60), more preferably 1: (45 to 55), and further preferably 1: 50; after decocting with water, it is preferably centrifuged, the supernatant is evaporated to dryness, and then ultrasonically extracted with 70% methanol; the Chuantong bark medicinal material / decoction piece The ratio of mass g to volume mL of 70% methanol is preferably (0.1-1):(10-50), more preferably (0.1-1):(10-30), further preferably (0.5-1):(10-20), and most preferably 1:20; the power of the ultrasound is preferably 500-700W, more preferably 600W; the frequency of the ultrasound is preferably 30-50kHz, more preferably 40kHz; the extraction time is preferably 10-30min; in some embodiments provided by the present invention, the extraction time is specifically 10min, 20min or 30min.

[0013] In a specific embodiment provided by the present invention, the preparation of the test solution of the standard decoction, intermediate or formula granule of Chuantong Cortex is specifically as follows: the standard decoction, intermediate or formula granule of Chuantong Cortex is ultrasonically extracted with 70% methanol, cooled, shaken, and filtered to obtain the product; the ratio of the mass g of the standard decoction, intermediate or formula granule of Chuantong Cortex to the volume mL of 70% methanol is preferably (0.1-1): (10-50), more preferably (0.1-1): (10-30), further preferably (0.1-5): (10-20), and most preferably 0.1:20; the power of the ultrasound is preferably 500-700W, more preferably 600W; the frequency of the ultrasound is preferably 30-50kHz, more preferably 40kHz; the extraction time is preferably 10-30min; in some embodiments provided by the present invention, the extraction time is specifically 10min, 20min or 30min.

[0014] In a specific embodiment provided by the present invention, the present invention further comprises preparing a reference substance and a reference medicinal material reference solution:

[0015] Take protocatechuic acid reference substance, neochlorogenic acid reference substance and chlorogenic acid reference substance respectively, dissolve them in 70% methanol to obtain reference substance solutions;

[0016] The control medicinal material was decocted with water, and then ultrasonically extracted with 70% methanol to obtain a control medicinal material reference solution;

[0017] The reference substance and the reference medicinal material reference solution are determined by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference, respectively; and based on the chromatograms of the reference substance and the reference medicinal material reference, the components of the high performance liquid phase characteristic spectra of the Chuantong bark medicinal material, decoction pieces, standard decoction, intermediates and their formula granules are qualitatively determined.

[0018] In the present invention, the preparation of the control medicinal material reference solution is preferably the same as the preparation of the Chuantong bark medicinal material / decoction piece test solution.

[0019] In a specific embodiment provided by the present invention, the chromatographic column used in the high performance liquid chromatography method is a C18 column (octadecylsilane bonded silica gel); the C18 column is a C18 column well known to those skilled in the art without any special limitation, and can specifically be ZORBAX SB-AQ.

[0020] In a specific embodiment provided by the present invention, the specifications of the chromatographic column are preferably 1.6-1.8 μm, 2.1×100-2.1×150 mm; more preferably 1.8 μm, 2.1×100 mm.

[0021] In a specific embodiment provided by the present invention, the column temperature of the chromatographic column is preferably 20°C to 30°C, specifically 20°C.

[0022] The mobile phase A of the high performance liquid chromatography method of the present invention is acetonitrile, the mobile phase B is 0.1% phosphoric acid solution, and the gradient elution is performed. In a specific embodiment provided by the present invention, the gradient elution is specifically as follows, in terms of volume percentage:

[0023] 0-5 min, mobile phase A 3%→10%, mobile phase B 97%→90%;

[0024] 5-16 min, mobile phase A 10% → 17%, mobile phase B 90% → 83%;

[0025] 16-25 min, mobile phase A 17% → 20%, mobile phase B 83% → 80%;

[0026] 25-36 min, mobile phase A 20%→28%, mobile phase B 80%→72%.

[0027] In a specific embodiment provided by the present invention, the mobile phase flow rate is preferably 0.1-0.4 mL / min, more preferably 0.2-0.3 mL / min, and even more preferably 0.3 mL / min. The present invention has found that when the flow rate is 0.3 ml / min, the peak shape of each chromatographic peak is better, which is the preferred embodiment.

[0028] In a specific embodiment provided by the present invention, the detection wavelength is preferably 300 nm.

[0029] In a specific embodiment provided by the present invention, the injection volume is preferably 1 μL.

[0030] The present invention uses a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of the high-performance liquid phase characteristic spectra of the Pittosporum tobira bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules, and obtains a high-performance liquid phase standard characteristic spectrum of the Pittosporum tobira bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules consisting of 9 characteristic peaks, wherein peak 1: protocatechuic acid, peak 2: chlorogenic acid peak, peak 3: caffeic acid, peak 4: cryptochlorogenic acid, peak 5 (S): chlorogenic acid, peak 7: isochlorogenic acid B, peak 8: isochlorogenic acid A, and peak 9: isochlorogenic acid C.

[0031] Further specifically, in the HPLC standard characteristic spectra of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.87 (peak 3), 0.96 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9), respectively.

[0032] In a specific embodiment provided by the present invention, in the high performance liquid chromatography characteristic spectrum of the Chuantong bark medicinal material, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively, and the chlorogenic acid peak is used as the reference peak S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.88 (peak 3), 0.95 (peak 4), 1.50 (peak 6), 1.97 (peak 7), 2.07 (peak 8), and 2.22 (peak 9).

[0033] In a specific embodiment provided by the present invention, in the high performance liquid chromatography characteristic spectrum of the Chuantong bark slices, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively, and the chlorogenic acid peak is used as the reference peak S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.89 (peak 3), 0.97 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9).

[0034] In the HPLC characteristic spectrum of the standard decoction of Chuantong bark, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.87 (peak 3), 0.93 (peak 4), 1.58 (peak 6), 2.11 (peak 7), 2.22 (peak 8), and 2.37 (peak 9).

[0035] In the high performance liquid chromatography characteristic spectrum of the Chuantong bark intermediate, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.87 (peak 3), 0.96 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9).

[0036] In a specific embodiment provided by the present invention, in the HPLC characteristic spectrum of the Chuantong bark formula granules, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.87 (peak 3), 0.96 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9).

[0037] The present invention also provides a quality control method for Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and formula granules thereof, and the quality control is achieved by adopting the above-mentioned high performance liquid phase characteristic spectrum construction method.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) The present invention establishes for the first time a high performance liquid chromatography characteristic spectrum method for the detection of Chuantong bark (Caltropis chinensis) medicinal materials, decoction pieces, standard decoctions, extracts, formula granules and related preparations;

[0040] (2) In the process of establishing the characteristic spectrum of Chuantong bark (Cassia truncatula), the present invention confirmed 9 common characteristic peaks, identified 8 components, and studied their relative retention times, ensuring the stability of its chemical composition and safety of use;

[0041] (3) The method of the present invention has good stability, high precision, good reproducibility, convenience and easy to master. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Examine the chromatogram for the extraction solvent;

[0043] Figure 2 Examining chromatograms for extraction methods;

[0044] Figure 3 This is the result diagram of the extraction time investigation;

[0045] Figure 4 This is the result diagram of the investigation on the amount of solvent added;

[0046] Figure 5 This is the chromatographic peak identification diagram of Chuantong bark formula granules;

[0047] Figure 6 Examine the chromatogram for intermediate precision;

[0048] Figure 7 This is the result diagram of the chromatographic column durability investigation;

[0049] Figure 8 This is the durability result diagram of chromatographic columns of the same model but different batches;

[0050] Figure 9 This is the characteristic spectrum of the medicinal material Chuantong bark (Calotaxus spinulosa);

[0051] Figure 10 This is the characteristic spectrum of Chuantong Bark (Cathay tree) decoction pieces;

[0052] Figure 11 This is the characteristic spectrum of the standard decoction of Chuantong Bark (Cassia truncatula);

[0053] Figure 12 This is the characteristic spectrum of the intermediate of Chuantong bark (Calotaxus spinulosa);

[0054] Figure 13 Verification diagram of characteristic spectra of three batches of Chuantong bark formula granules;

[0055] Figure 14 This is the reference characteristic spectrum of the medicinal material Chuantong bark (Calotaxus spinulosa);

[0056] Figure 15 This is the reference characteristic spectrum of Chuantongpi (Caltropis spinulosa) slices;

[0057] Figure 16 This is the reference characteristic spectrum of Chuantongpi (Caltropis chinensis) decoction;

[0058] Figure 17 This is the reference characteristic spectrum of the intermediate of Chuantong bark (Calotaxus spinulosa);

[0059] Figure 18 This is the comparative characteristic spectrum of Chuantong bark (Calotaxus spinulosa) formula granules. DETAILED DESCRIPTION

[0060] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0061] To further illustrate the present invention, the following describes in detail a method for constructing a high-performance liquid chromatography characteristic spectrum of a Chuantong bark medicinal material, decoction pieces, standard decoction, intermediate and formula granules thereof provided by the present invention in combination with examples.

[0062] The reagents used in the following examples are all commercially available.

[0063] Experimental instruments and materials

[0064] High performance liquid chromatograph: Agilent 1290 ultra-high performance liquid chromatograph, Waters H-Class Plus ultra-high performance liquid chromatograph;

[0065] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0066] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0067] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0068] Chromatographic column: ZORBAX SB-AQ, 2.1 mm × 100 mm, 1.8 μm;

[0069] Acetonitrile, phosphoric acid, formic acid, and acetic acid were of chromatographic grade, water was ultrapure water, and the remaining reagents were of analytical grade.

[0070] Chlorogenic acid (China Food and Drug Control Institutes, batch number: 110753-202119, purity: 96.3%);

[0071] Neochlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDX001504, purity: 98.80%);

[0072] Cryptochlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DST220104-035, purity: 99.30%);

[0073] Protocatechuic acid (China Food and Drug Inspection Institute, batch number: 110809-202207, purity: 97.5%);

[0074] Caffeic acid (China Food and Drug Administration, batch number: 110885-201703, purity: 99.7%);

[0075] Isochlorogenic acid A (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDY054703, purity: 99.36%);

[0076] Isochlorogenic acid B (Chengdu Desite Biotechnology Co., Ltd., batch number: DST210823-037, purity: 99.05%);

[0077] Isochlorogenic acid C (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDY003804, purity: 99.39%);

[0078] Chuantong bark ( Catalpa asiatica ) control medicinal material (Chengdu Desite Biotechnology Co., Ltd., batch number: DST230406-135);

[0079] Chuantong bark (Cathay tree bark) batch numbers: 2023001, 2023002, 2023003, 2023004, 2023005, 2023006, 2023007, 2023008, 2023009, 2023010, 2023011, 2023012, 2023013, 2023014, 2023015, 2013016, 2023017;

[0080] Batch numbers of Chuantong bark (Calotaxus spinulosa) slices: YP001, YP002, YP003, YP004, YP005, YP006, YP007, YP008, YP009, YP010, YP011, YP012, YP013, YP014, YP015, YP016, YP017;

[0081] Batch numbers of standard decoction of Chuantong bark (Cathay tree bark) (provided by New Green Technology Development Co., Ltd.): BT001, BT002, BT003, BT004, BT005, BT006, BT007, BT008, BT009, BT010, BT011, BT012, BT013, BT014, BT015, BT016, BT017;

[0082] Chuantong bark (catalpa asiatica) intermediate (extract) batch numbers (provided by New Green Technology Development Co., Ltd.): TQW001, TQW002, TQW003;

[0083] Batch numbers of Chuantong bark (Calotaxus spinulosa) formula granules (provided by New Green Technology Development Co., Ltd.): KL001, KL002, KL003.

[0084] Example 1: Chromatographic conditions and system suitability test

[0085] 1.1 Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the packing (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed as specified in the table below. The flow rate was 0.30 ml / min; the column temperature was 20°C; and the detection wavelength was 300 nm. The number of theoretical plates, calculated based on the chlorogenic acid peak, should be no less than 5000.

[0086]

[0087] 1.2 Preparation of reference solution

[0088] Take 1.0g of Chuantong bark (Cathay tree bark) as a control medicinal material, add 50mL of water and boil for 30 minutes. Remove, centrifuge, take the supernatant, evaporate to dryness, dissolve the residue in 10mL of 70% methanol, plug, and treat with ultrasound (power 600W, frequency 40kHz) for 20 minutes. Let cool, shake well, filter, and take the filtrate as the control medicinal material reference solution. Separately, take appropriate amounts of protocatechuic acid reference substance, neochlorogenic acid reference substance, and chlorogenic acid reference substance, accurately weigh them, and add 70% methanol to make a mixed solution containing 20μg of each per 1mL, which is used as the control reference solution.

[0089] Preparation of test solution

[0090] Medicinal material test solution: Take 1.0 g of the product powder (passed through No. 4 sieve), place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, remove it, centrifuge it, take the supernatant, evaporate it to dryness, add 10 mL of 70% methanol to the residue, stopper it, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 20 minutes. Cool it, shake it well, filter it, and take the filtrate as the test solution.

[0091] Decoction piece test solution: Take 1.0 g of the powder of this product (passed through No. 4 sieve), place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, take it out, centrifuge, take the supernatant, evaporate to dryness, add 10 mL of 70% methanol to the residue, stopper it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, and take the filtrate as the test solution.

[0092] Standard decoction test solution: Take 0.1 g of this product, place it in a stoppered conical flask, add 20 mL of 70% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 20 minutes. Let it cool, shake it well, filter it, and take the filtrate as the test solution.

[0093] Intermediate test solution: Take an appropriate amount of this product, grind it into powder, take 0.1 g, place it in a stoppered conical flask, add 20 mL of 70% methanol, stopper it tightly, treat it with ultrasound (power 600 W, frequency 40 kHz) for 20 minutes, cool it, shake it well, filter it, and take the filtrate as the test solution.

[0094] Formula granule test solution: Take an appropriate amount of the product, grind it into powder, take 0.1 g, place it in a stoppered conical flask, add 20 mL of 70% methanol, stopper it tightly, treat it with ultrasound (power 600 W, frequency 40 kHz) for 20 minutes, let it cool, shake it well, filter it, and take the filtrate as the test solution.

[0095] Assay

[0096] Accurately pipette 1 μL of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0097] 1.3 Establishment of feature map method

[0098] The characteristic spectrum method was established based on the above chromatographic conditions.

[0099] 1.3.1 Preparation of test solution

[0100] 1.3.1.1 Extraction solvent investigation

[0101] Take an appropriate amount of this product (formulated granules), grind it into powder, take 0.1g, place it in a stoppered conical flask, add 20mL each of water, 30% methanol, 70% methanol, methanol, 30% ethanol, 70% ethanol, and ethanol, stopper it tightly, and ultrasonically treat it (power 600W, frequency 40kHz) for 20 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 1 . Figure 1 Examine the chromatogram for the extraction solvent.

[0102] The results showed that when 70% methanol was used as the extraction solvent, the chromatographic peak information was large and the chromatogram peak shape was good, so the extraction solvent for the test sample was determined to be 70% methanol.

[0103] 1.3.1.2 Investigation of extraction methods

[0104] Take an appropriate amount of this product (formulated granules), grind it into powder, take 0.1g, place it in a stoppered conical flask, add 20mL of 70% methanol, stopper it tightly, ultrasonicate it (power 600W, frequency 40kHz), heat it under reflux for 20 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the product. Figure 2 . Figure 2 Examine the chromatograms for the extraction method.

[0105] The results showed that ultrasonic extraction and reflux extraction had the same effect on the test sample. Because ultrasonic extraction is more convenient, ultrasonic extraction was determined to be the extraction method for the test sample.

[0106] 1.3.1.3 Extraction time investigation

[0107] Take an appropriate amount of this product (formulated granules), grind it into powder, take 0.1g, place it in a stoppered conical flask, add 20mL of 70% methanol, stopper it tightly, and ultrasonically treat it (power 600W, frequency 40kHz) for 10 minutes, 20 minutes, and 30 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 3 . Figure 3 This is the result diagram of the extraction time investigation.

[0108] The results showed that there was no significant difference in chromatographic information when the extraction time was 10 minutes, 20 minutes, and 30 minutes. To ensure sufficient extraction, the extraction time of the test sample was determined to be 20 minutes.

[0109] 1.3.1.4 Investigation of solvent addition amount

[0110] Take an appropriate amount of this product (formulated granules), grind it into powder, take 0.1g, add 10mL, 20mL, and 50mL of 70% methanol respectively, plug it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 20 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 4 . Figure 4 This is the result of investigating the amount of solvent added.

[0111] The results showed that when the amount of solvent added was 20 mL, the peak shape and separation of each chromatographic peak were better, so the amount of solvent added for the test sample was determined to be 20 mL.

[0112] In summary, the preparation method of the test solution of the characteristic spectrum of the Chuantong bark formula granules is determined as follows: take an appropriate amount of the product, grind it into powder, take 0.1 g, place it in a stoppered conical flask, add 20 mL of 70% methanol, stopper it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 20 minutes, cool it, shake it evenly, filter it, and take the filtrate to obtain it.

[0113] 1.3.1.5 Feature Mapping Method

[0114] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 0.30 mL / min; the column temperature was 20°C; and the detection wavelength was 300 nm. The number of theoretical plates, calculated based on the chlorogenic acid peak, should be no less than 5000.

[0115]

[0116] Preparation of reference solution: Take 1.0g of Chuantong bark (Caltropis chinensis) as the reference medicinal material, add 50mL of water and boil for 30 minutes. Remove, centrifuge, take the supernatant, evaporate to dryness, dissolve the residue in 10mL of 70% methanol, plug, and treat with ultrasound (power 600W, frequency 40kHz) for 20 minutes. Let cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Separately, take appropriate amounts of protocatechuic acid reference substance, neochlorogenic acid reference substance, and chlorogenic acid reference substance, accurately weigh them, and add 70% methanol to make a mixed solution containing 20μg of each per 1mL. This is the reference solution.

[0117] Preparation of test solution: Take an appropriate amount of this product, grind it into powder, take 0.1 g, place it in a stoppered conical flask, add 20 mL of 70% methanol, stopper it tightly, treat it with ultrasound (power 600 W, frequency 40 kHz) for 20 minutes, let it cool, shake it well, filter it, and take the filtrate as the test solution.

[0118] Determination method: Accurately pipette 1 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0119] 1.3.2 Methodological Investigation

[0120] 1.3.2.1 Chromatographic peak identification

[0121] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Chuantong bark formula granules.

[0122] Preparation of reference solution: Take 1g of Chuantong bark (Cathay tree bark) as a reference medicinal material and place it in a stoppered conical flask. Add 50ml of water and boil for 30 minutes. Remove and centrifuge. Take the supernatant and evaporate to dryness. Dissolve the residue in 10ml of 70% methanol. Seal the flask tightly and sonicate (power 600W, frequency 40kHz) for 20 minutes. Let cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Take appropriate amounts of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, protocatechuic acid, caffeic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C as reference substances, accurately weigh them, and add 70% methanol to make a solution containing 20μg of each per 1mL. This is the reference substance solution.

[0123] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking the Chuantong bark formula granules was prepared.

[0124] The characteristic spectrum peaks of the Chuantong bark formula granules were located, see Figure 5 . Figure 5 This is the chromatographic peak identification diagram of the Chuantong bark formula granules. Figure 5It can be seen that there are 9 peaks in the test solution, peak 1 is protocatechuic acid; peak 2 is neochlorogenic acid; peak 3 is caffeic acid; peak 4 is cryptochlorogenic acid; peak 5 is chlorogenic acid; peak 7 is isochlorogenic acid B; peak 8 is isochlorogenic acid A; peak 9 is isochlorogenic acid C.

[0125] 1.3.2.2 Precision test

[0126] Prepare one portion of the test solution according to the proposed experimental method, inject the sample six times continuously, and calculate the retention time of each characteristic peak. See Table 1.

[0127] Table 1 Precision investigation-retention time

[0128]

[0129] The results showed that the RSD of the retention time of each characteristic peak in the precision investigation was 0.03% to 0.12%, and the method had good precision.

[0130] 1.3.2.3 Repeatability Study

[0131] According to the proposed experimental method, 6 test sample solutions were prepared, measured, and the relative retention time of each characteristic peak was calculated. See Table 2.

[0132] Table 2 Repeatability study - relative retention time ratio

[0133]

[0134]

[0135] The results showed that the RSDs of the characteristic peaks relative to the retention times were between 0.00% and 1.92%, indicating that the method had good repeatability.

[0136] 1.3.2.3 Intermediate precision study

[0137] Based on the experimental conditions proposed above, different personnel (A1, A2) accurately weighed 2 portions of Chuantongpi formula granules at different times (T1, T2) to prepare test solutions, which were then measured on different instruments (C1: Waters H-Class Plus, C2: Agilent 1290) for high performance liquid chromatography, see Figure 6 , Table 3. Figure 6 Chromatogram for intermediate precision investigation.

[0138] Table 3 Intermediate precision investigation - relative retention time ratio

[0139]

[0140] The results showed that when the test solutions were prepared by different personnel at different times and measured on different instruments, the RSD of the relative retention times of the characteristic peaks was 0.60% to 5.68%. Different instruments had a great influence on the relative retention times of Peak 1 and Peak 2. It was proposed to use reference substances to regulate Peak 1 and Peak 2. The intermediate precision of this method was good.

[0141] 1.3.2.5 Column durability assessment

[0142] Three different types of C18 columns were used to investigate the same sample: column 1 (HSS T3, 2.1 mm × 100 mm, 1.8 μm), column 2 ( C18, 2.1 mm × 100 mm, 1.6 μm), chromatographic column 3 (ZORBAX SB-AQ, 2.1 mm × 100 mm, 1.8 μm). See Table 4, Figure 7 . Figure 7 This is the result of the chromatographic column durability investigation.

[0143] Table 4 Column durability investigation - relative retention time

[0144]

[0145] As can be seen from the above figure, chromatographic columns 1 and 2 have poor separation effects on sample peaks 3 and 4, and the order of peak elution has changed. Therefore, this study recommends using chromatographic column 3, which has a good peak separation effect.

[0146] Based on the experimental conditions proposed above, three different batches of ZORBAX SB-AQ, 2.1mm×100mm, 1.8μm columns (Column 1: UPSK010, Column 2: UPSK020, Column 3: UPSK030) were investigated. The results are shown in Table 5. Figure 8 . Figure 8 This is the durability result chart of chromatographic columns of the same model but different batches.

[0147] Table 5 Column durability investigation - relative retention time

[0148]

[0149] The method is column-selective. Results showed that using the three ZORBAX SB-AQ, 2.1 mm × 100 mm, 1.8 μm columns, the relative retention time of peak 2 was significantly affected by instrumentation, by 5.21%. The RSDs for the relative retention times of the remaining characteristic peaks ranged from 2.11% to 3.94%, demonstrating the robustness of the columns.

[0150] 1.3.2.6 Stability

[0151] According to the experimental conditions proposed above, a sample solution was prepared and measured at 0h, 6h, 9h, 14h, 20h, and 24h respectively. The results are shown in Table 6.

[0152] Table 6 Stability Study-Retention Time

[0153]

[0154] The results showed that the RSD of the corresponding characteristic peak retention time was 0.39-1.70%, and the sample solution was stable within 24 hours.

[0155] 1.3.2.7 Determination of characteristic peaks and establishment of reference spectrum

[0156] Based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peak heights, a total of nine robust peaks were selected as characteristic peaks: Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, Peak 8, and Peak 9. Because the relative retention times of Peaks 1 and 2 were relatively short, the specified range was narrow, and the relative retention time (RSD) values ​​of Peaks 1 and 2 were large, reference substances were used to define the relative retention times of Peaks 1 and 2. The relative retention times of the remaining characteristic peaks were stable and within ±10% of the mean, with small RSD values. Therefore, only the specified values ​​of the relative retention times were considered, and the specified range for the relative retention times of each peak was tentatively set at ±10%.

[0157] 1.3.3 Sample characteristic spectrum verification

[0158] The proposed method was used to determine the characteristic spectra of 17 batches of Chuantong bark (Caltropis chinensis) medicinal materials, 17 batches of decoction pieces, 17 batches of standard decoctions, 3 batches of extracts, and 3 batches of formula granules, and the relative retention times were calculated. Figures 9-13 , Tables 7 to 11. Figure 9 This is the characteristic spectrum of the medicinal material of Chuantong Bark (Cathay tree) (S1-S17 are: 2023001, 2023002, 2023003, 2023004, 2023005, 2023006, 2023007, 2023008, 2023009, 2023010, 2023011, 2023012, 2023013, 2023014, 2023015, 2013016, 2023017); Figure 10 Characteristic spectra of Chuantong bark (Cassia truncatula) slices (S1-S17 are: YP001, YP002, YP003, YP004, YP005, YP006, YP007, YP008, YP009, YP010, YP011, YP012, YP013, YP014, YP015, YP016, YP017); Figure 11Characteristic spectra of standard decoction of Chuantong bark (catalpa asiatica) (S1 to S17 are: BT001, BT002, BT003, BT004, BT005, BT006, BT007, BT008, BT009, BT010, BT011, BT012, BT013, BT014, BT015, BT016, BT017); Figure 12 This is the characteristic spectrum of the intermediate of Chuantong bark (Calotaxus spinulosa); Figure 13 This is the verification diagram of the characteristic spectra of three batches of Chuantong bark formula granules.

[0159] Table 7 Relative retention time of characteristic spectrum of Chuantong bark (Cathay tree)

[0160]

[0161]

[0162] Table 8 Relative retention time of characteristic spectra of 17 batches of Chuantong bark (Cassia truncatula) slices

[0163]

[0164] Table 9 Relative retention time of 17 batches of standard decoction of Chuantong bark (Cassia truncatula)

[0165]

[0166]

[0167] Table 10 Relative retention times of three batches of Chuantong bark (Cassia truncatula) intermediates

[0168]

[0169]

[0170] Table 11 Relative retention time of three batches of Chuantong bark granules

[0171]

[0172] The results showed that the RSD of the relative retention times of 9 characteristic peaks of 17 batches of medicinal materials, 17 batches of decoction pieces, 17 batches of standard decoctions, 3 batches of extracts and 3 batches of formula granules of Chuantong bark (Caltropis spinulosa) were all less than 2.0%.

[0173] Final regulations: The test sample chromatogram should show nine characteristic peaks, and their retention times should correspond to those of the nine characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 2, and 5 should correspond to the retention times of the corresponding reference material peaks. The peak corresponding to the chlorogenic acid reference material is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.87 (peak 3), 0.96 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9).

[0174] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 17 batches of medicinal materials, 17 batches of decoction pieces, 17 batches of standard decoctions, 3 batches of extracts, and 3 batches of formula granules of Chuantong bark (Caltropis spinulosa), and the corresponding characteristic spectrum was established. Figures 14-18 . Figure 14 This is the reference characteristic spectrum of Chuantong bark (Caltropis spinulosa) medicinal material, peak 1: protocatechuic acid, peak 2: neochlorogenic acid, peak 3: caffeic acid, peak 4: cryptochlorogenic acid, peak 5 (S): chlorogenic acid, peak 7: isochlorogenic acid B, peak 8: isochlorogenic acid A, peak 9: isochlorogenic acid C; Figure 15 This is the reference characteristic spectrum of Chuantong bark (Cassia truncatula) slices, peak 1: protocatechuic acid, peak 2: neochlorogenic acid, peak 3: caffeic acid, peak 4: cryptochlorogenic acid, peak 5 (S): chlorogenic acid, peak 7: isochlorogenic acid B, peak 8: isochlorogenic acid A, peak 9: isochlorogenic acid C; Figure 16 This is the reference characteristic spectrum of Chuantongpi (Caltropis arborescens) standard decoction, peak 1: protocatechuic acid, peak 2: neochlorogenic acid, peak 3: caffeic acid, peak 4: cryptochlorogenic acid, peak 5 (S): chlorogenic acid, peak 7: isochlorogenic acid B, peak 8: isochlorogenic acid A, peak 9: isochlorogenic acid C; Figure 17 This is the reference characteristic spectrum of the intermediate of Chuantong bark (Cassia truncatula), peak 1: protocatechuic acid, peak 2: neochlorogenic acid, peak 3: caffeic acid, peak 4: cryptochlorogenic acid, peak 5 (S): chlorogenic acid, peak 7: isochlorogenic acid B, peak 8: isochlorogenic acid A, peak 9: isochlorogenic acid C; Figure 18 This is the reference characteristic spectrum of Chuantong bark (Aralia strychnifolia) formula granules, peak 1: protocatechuic acid, peak 2: neochlorogenic acid, peak 3: caffeic acid, peak 4: cryptochlorogenic acid, peak 5 (S): chlorogenic acid, peak 7: isochlorogenic acid B, peak 8: isochlorogenic acid A, peak 9: isochlorogenic acid C.

[0175] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing a high-performance liquid chromatography characteristic spectrum of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules, characterized in that: The following steps are involved: S1) extracting the Chuan Tong bark raw material with a solvent to obtain a test solution; the solvent is 70% methanol; the Chuan Tong bark raw material is from Castanopsis arvense; S2) measuring the test solution by high performance liquid chromatography to obtain a high performance liquid chromatography characteristic spectrum of the Chuantong bark raw material; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column with a specification of 1.8 μm and a diameter of 2.1×100 mm; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid solution, and the elution is performed with a gradient. In terms of volume percentage, the gradient elution is specifically as follows: 0-5 min, mobile phase A 3%→10%, mobile phase B 97%→90%; 5-16 min, mobile phase A 10%→17%, mobile phase B 90%→83%; 16-25 min, mobile phase A 17%→20%, mobile phase B 83%→80%; 25-36 min, mobile phase A 20%→28%, mobile phase B 80%→72%; The detection wavelength is 300 nm.

2. The construction method according to claim 1, characterized in that It also includes the preparation of reference substances and reference medicinal material solutions: Take protocatechuic acid reference substance, neochlorogenic acid reference substance and chlorogenic acid reference substance respectively, dissolve them in 70% methanol to obtain reference substance solutions; The control medicinal materials were decocted with water, and then ultrasonically extracted with 70% methanol to obtain the control medicinal material reference solution; The reference substance and the reference medicinal material reference solution are determined by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference, respectively; and based on the chromatograms of the reference substance and the reference medicinal material reference, the components of the high performance liquid phase characteristic spectra of the Chuantong bark medicinal material, decoction pieces, standard decoction, intermediates and their formula granules are qualitatively determined.

3. The construction method according to claim 1, characterized in that Column temperature: 20°C.

4. The construction method according to claim 3, characterized in that The flow rate of the mobile phase was 0.3 mL / min, and the injection volume was 1 μL.

5. The construction method according to claim 1, characterized in that The similarity of the HPLC characteristic spectra of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules was evaluated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules was obtained, which consisted of 9 characteristic peaks, including peak 1: protocatechuic acid, peak 2: chlorogenic acid peak, peak 3: caffeic acid, peak 4: cryptochlorogenic acid, peak 5: chlorogenic acid, peak 7: isochlorogenic acid B, peak 8: isochlorogenic acid A, and peak 9: isochlorogenic acid C.

6. The construction method according to claim 5, characterized in that: In the HPLC standard characteristic spectra of Chuantong bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules, Peak 1, Peak 2, and Peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.87 (peak 3), 0.96 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9), respectively.

7. The construction method according to claim 5, characterized in that: In the HPLC characteristic spectrum of the Chuantong bark medicinal material, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.88 (peak 3), 0.95 (peak 4), 1.50 (peak 6), 1.97 (peak 7), 2.07 (peak 8), and 2.22 (peak 9); In the HPLC characteristic spectrum of the Chuantong bark slices, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.89 (peak 3), 0.97 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9); In the HPLC characteristic spectrum of the standard decoction of Chuantong bark, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.87 (peak 3), 0.93 (peak 4), 1.58 (peak 6), 2.11 (peak 7), 2.22 (peak 8), and 2.37 (peak 9); In the HPLC characteristic spectrum of the Chuantong bark intermediate, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.87 (peak 3), 0.96 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9); In the HPLC characteristic spectrum of the Chuantong bark formula granules, peak 1, peak 2, and peak 5 correspond to the retention times of the corresponding reference substance peaks, respectively. The chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.87 (peak 3), 0.96 (peak 4), 1.51 (peak 6), 1.98 (peak 7), 2.08 (peak 8), and 2.22 (peak 9).

8. The construction method according to claim 1, wherein: Step S1) the Chuantong bark raw materials include Chuantong bark raw medicinal materials, decoction pieces, standard decoctions, intermediates and formula granules thereof; The preparation of the test solution of the Chuantong bark medicinal material / decoction piece is as follows: the Chuantong bark medicinal material / decoction piece is decocted with water for 20 to 40 minutes, and then 70% methanol is added for ultrasonic extraction, cooled, shaken, and filtered to obtain the solution; The preparation of the test solution of the standard decoction, intermediate or formula granule of Chuantong bark is specifically as follows: the standard decoction, intermediate or formula granule of Chuantong bark is ultrasonically extracted with 70% methanol, cooled, shaken and filtered to obtain the solution.

9. The construction method according to claim 8, characterized in that: The ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 10-30min; The ratio of the mass g of the Chuantong bark raw material to the volume mL of the solvent is (0.1~1):(10~50).

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