Celosia cristata and preparation variable wavelength UPLC characteristic spectrum construction method and application thereof

Abstract

The invention discloses a construction method and application of a variable wavelength UPLC characteristic chromatogram of cockscomb and a preparation thereof, which adopts an ultra performance liquid chromatograph and a variable wavelength technology to measure the characteristic chromatograms of cockscomb and the preparation thereof, and realizes positioning of flavone, organic acid and amino acid components in cockscomb at the same time. The integrity and comprehensive effects of the effective components in the cockscomb and the preparation thereof can be more comprehensively reflected. According to the method, ethanol heating reflux and concentrated hydrochloric acid hydrolysis extraction technologies are adopted, so that the dissolution efficiency and types of effective components in a cockscomb sample are improved to a great extent. The optimal elution condition is constructed, so that the detection time of the cockscomb characteristic chromatogram is greatly shortened, the column washing effect of the chromatographic column is improved, and the service life of the chromatographic column is prolonged. The specific chromatogram obtained by the construction method disclosed by the invention has the advantages of good separation degree of each peak, strong stability and high precision, and the quality of the cockscomb and the preparation thereof can be controlled at the same time.

Description

Technical Field

[0001] The invention belongs to the field of identification of traditional Chinese medicine preparations, and specifically relates to a method for constructing a variable-wavelength UPLC characteristic spectrum of cockscomb and its preparations with high precision and good stability, and an application thereof. Background Art

[0002] Celosia cristata L. is a plant of the genus Celosia in the family Amaranthaceae. It is also known as cockscomb flower, cockscomb head, and old and young. It is native to tropical Africa and Asia and is now cultivated in all parts of my country. Its inflorescence is used as medicine, which has the functions of astringent hemostasis, leukorrhea and dysentery. Modern pharmacological research has fully proved that Celosia cristata can kill vaginal Trichomonas and improve human immunity. It is often used clinically to treat chronic pelvic inflammatory disease, leukorrhea, metrorrhagia, dysentery and other diseases. It is a Chinese medicine with high clinical value.

[0003] In clinical practice, the common medicinal forms of cockscomb include cockscomb slices, cockscomb formula granules, cockscomb standard decoction and some compound preparations containing cockscomb. The cockscomb standard decoction is made by boiling cockscomb slices with pure water, filtering while hot, combining the filtrate, and then concentrating under reduced pressure and vacuum drying at low temperature. The cockscomb formula granules are made by adding auxiliary materials to the cockscomb standard decoction after low-temperature vacuum drying and dry granulation.

[0004] At present, the Chinese Pharmacopoeia only defines the three items of properties, inspection and extracts of Cockscomb. There is little research on the active ingredients in Cockscomb and its preparations in the prior art. CN 110441445A discloses a method for constructing a common high-performance liquid phase characteristic spectrum of Cockscomb medicinal materials and the determination of flavonoid content. This method can effectively make up for the shortcomings of the quality detection method of Cockscomb medicinal materials in the Chinese Pharmacopoeia and has strong practicality. However, since the standard decoction, formula granules and other preparations of Cockscomb have lost the original appearance characteristics of the medicinal materials, their material basis must be different from the original medicinal materials. At present, there is no efficient detection method that is applicable to Cockscomb and its preparations at the same time. Therefore, constructing a characteristic spectrum identification method that effectively evaluates the integrity and comprehensive effects of the effective ingredients of Cockscomb has become the key to improving the quality control of Cockscomb and its preparations. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing and applying a variable wavelength UPLC characteristic spectrum of Celosia and its preparations, so as to overcome the complex problems in the prior art that the characteristic spectrum of Celosia and its preparations has low extraction efficiency, single detection component type, long detection time, poor repeatability, low stability, and easy clogging of the chromatographic column, resulting in reduced column efficiency of the chromatographic column. The present invention adopts ethanol heating reflux and concentrated hydrochloric acid hydrolysis extraction technology, which greatly accelerates the type of effective components dissolved from the test sample. The characteristic spectrum of Celosia and its preparations is constructed by ultra-high performance liquid chromatography and variable wavelength technology, which has the characteristics of short detection time, strong stability, high precision, etc., and can simultaneously achieve quality control of Celosia and its preparations.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparations, comprising the following steps:

[0008] (1) Preparation of test solution: Weigh 0.5 g to 1.0 g of the sample of Celosia cristata and its preparation to be tested, accurately add the extraction solvent, weigh, heat and reflux for 1.0 h to 1.5 h, cool, weigh again, and make up the weight with the extraction reagent to prepare the test solution;

[0009] (2) Using tryptophan, p-hydroxycinnamic acid, ferulic acid, quercetin, kaempferol, isorhamnetin, and kaempferol-4'-O-methyl ether as reference substances, the UPLC characteristic spectra of Celosia cristata and its preparations were obtained using ultra-high performance liquid chromatography and variable wavelength technology.

[0010] The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: using octadecylsilane bonded silica gel as filler, a column length of 100 mm, an inner diameter of 2.1 mm, and a silica gel particle size of 1.6 μm as a chromatographic column; using acetonitrile as mobile phase A, and using 0.1-0.3% phosphoric acid aqueous solution as mobile phase B for gradient elution, the flow rate is set to 0.28-0.32 mL / min, the detection wavelength is set to 286 nm and 365 nm, and the column temperature is 35° C.;

[0011] The gradient elution conditions are as follows: 0-2.0 min, the volume ratio of mobile phase A to mobile phase B is 5-8%:95-92%; 2.0-4.0 min, the volume ratio of mobile phase A to mobile phase B is 8-10%:92-90%; 4.0-7.0 min, the volume ratio of mobile phase A to mobile phase B is 10-21%:90-79%; 7.0-9.0 min, the volume ratio of mobile phase A to mobile phase B is 21-24%:79-76%; 9.0-10.0 min, the volume ratio of mobile phase A to mobile phase B is 24-26%:76-74%; 10.0-15.0 min, the volume ratio of mobile phase A to mobile phase B is 26-30%: 74-70%; 15.0-16.0min, the volume ratio of mobile phase A to mobile phase B is 30%:70%; 16.0-17.0min, the volume ratio of mobile phase A to mobile phase B is 30-38%:70-62%; 17.0-22.0min, the volume ratio of mobile phase A to mobile phase B is 38%:62%; 22.0-22.1min, the volume ratio of mobile phase A to mobile phase B is 38-100%:74-70%; 22.1-25.0min, the volume ratio of mobile phase A to mobile phase B is 100%:0%; 25.0-25.1min, the volume ratio of mobile phase A to mobile phase B is 100-5%:0-95%.

[0012] The preparation method of the reference substance is as follows: taking appropriate amounts of tryptophan, p-hydroxycinnamic acid, ferulic acid, quercetin, kaempferol, isorhamnetin, and kaempferol-4'-O-methyl ether reference substances, accurately weighing them, and dissolving them in methanol to prepare a mixed reference substance solution.

[0013] The preparation method of the control medicinal material reference solution is as follows: take an appropriate amount of cockscomb control medicinal material in a stoppered conical flask, add a certain volume of 70% ethanol solution or 50% ethanol solution or 95% ethanol solution and concentrated hydrochloric acid solution, heat and reflux for 1-1.5 hours, cool, filter, and take the filtrate as the control medicinal material reference solution.

[0014] Furthermore, the preparation method of the control medicinal material reference solution specifically includes taking 1.0 g of cockscomb control medicinal material, accurately weighing, placing in a stoppered conical flask, accurately adding 50 mL of 70% ethanol and 10 mL of concentrated hydrochloric acid solution, weighing, heating and reflux at 95°C for 1.5 hours, cooling, re-weighting, filtering, and taking the filtrate as the control medicinal material reference solution.

[0015] The preparation method of the test solution is as follows: take an appropriate amount of cockscomb and its preparation samples into a stoppered conical flask, add a certain volume of 70% ethanol solution or 50% ethanol solution or 95% ethanol solution and concentrated hydrochloric acid solution, weigh, heat and reflux at 95°C for 1-1.5h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution.

[0016] Furthermore, the preparation method of the test solution specifically includes taking an appropriate amount of cockscomb and its preparation to be tested, grinding, accurately adding 50 mL of 70% ethanol and 10 mL of concentrated hydrochloric acid solution, weighing, heating and reflux at 95°C for 1.5 hours, cooling, reweighing, filtering, and taking the filtrate as a control medicinal material reference solution.

[0017] The cockscomb and its preparations include cockscomb medicinal materials, cockscomb decoction pieces, cockscomb standard decoction dry products, and cockscomb formula granules.

[0018] The method for constructing a variable wavelength UPLC characteristic spectrum of cockscomb and its preparation is characterized in that: the characteristic spectrum of cockscomb and its preparation includes 15 characteristic peaks, with peak No. 6 (p-hydroxycinnamic acid) as reference peak S1, and peak No. 11 (kaempferol) as reference peak S2. The relative retention time limit values ​​of peaks 1 to 9 and S1 are respectively: 0.252 (peak 1), 0.336 (peak 2), 0.432 (peak 3), 0.546 (peak 4), 0.698 (peak 5), 1.054 (peak 7), 1.093 (peak 8), 1.113 (peak 9); the relative retention time limit values ​​of peaks 10 to 15 and S2 are respectively: 0.825 (peak 10), 1.041 (peak 12), 1.098 (peak 13), 1.217 (peak 14), 1.413 (peak 15). The relative retention time of each peak was within ±10% of the specified value. The remaining peaks were located as follows: peak 4 was tryptophan, peak 8 was ferulic acid, peak 10 was quercetin, peak 12 was isorhamnetin, and peak 15 was kaempferol-4'-O-methyl ether.

[0019] The reflux temperature is 90-98° C., and the concentration of the test solution is 8.0-16.0 mg / mL.

[0020] The concentrations of the reference substance solution are tryptophan 10 μg / mL, p-hydroxycinnamic acid 10 μg / mL, ferulic acid 10 μg / mL, quercetin 20 μg / mL, kaempferol 20 μg / mL, isorhamnetin 5 μg / mL, and kaempferol-4'-O-methyl ether 5 μg / mL.

[0021] The chromatographic conditions are as follows: using a CORTECS UPLC T3 column as a chromatographic column with specifications of 100*2.1 mm, 1.6 μm; using acetonitrile as mobile phase A, and using 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution, the flow rate is set to 0.3 mL / min, and the detection wavelength is set to 0.0-10.0 min, 286 nm; 10.1-25.1 min, 365 m; and the column temperature is 35°C.

[0022] The column flushing conditions are: 22.1-25.0min, the acetonitrile ratio is maintained at 100%, and pure acetonitrile is used to flush the column for 2.9min, which can flush out the impurities remaining in the column within an analysis time period for each sample injection, thereby extending the service life of the chromatographic column and ensuring the repeatability and stability of the characteristic spectrum of cockscomb and its preparations.

[0023] The beneficial effects produced by the present invention are:

[0024] 1. The present invention uses ultra-high performance liquid chromatography and variable wavelength technology to measure the samples of Celosia and its preparations to obtain the characteristic spectrum of Celosia. The ethanol heating reflux and concentrated hydrochloric acid hydrolysis extraction technology are adopted to greatly improve the dissolution efficiency of the effective ingredients of Celosia and its preparations, and improve the preparation effect of the test solution.

[0025] 2. The present invention overcomes the problem that the existing technology needs to locate the flavonoids, organic acids and amino acids in cockscomb separately under different chromatographic conditions, resulting in a complicated detection method. The present invention provides a detection method for ethanol and concentrated hydrochloric acid hydrolysis extracts of cockscomb, which can simultaneously achieve qualitative identification of flavonoids, organic acids and amino acids, ensuring the quality control of cockscomb and its preparations.

[0026] 3. The separation degree and peak shape between the characteristic peaks obtained by the present invention are good, the method has strong repeatability, and has the advantages of high separation efficiency and good precision. Moreover, the method can detect a wide range of samples, for example, cockscomb medicinal materials, cockscomb decoction pieces, cockscomb standard decoction and cockscomb formula granules can all be effectively detected.

[0027] 4. The characteristic spectrum constructed by the present invention uses Peak 6 (parahydroxycinnamic acid reference substance) and Peak 11 (kaempferol) as reference peaks, both of which are representative peaks of the effective ingredients in Celosia scomb, and the separation is good, the peak area is moderate, and the quality of Celosia scomb and its preparation can be accurately controlled qualitatively. At the same time, Peak 4 refers to tryptophan, Peak 8 is ferulic acid, Peak 10 is quercetin, Peak 12 is isorhamnetin, and Peak 15 is kaempferol-4'-O-methyl ether. The 15 characteristic peaks determined in the characteristic spectrum of the present invention are stable common peaks of 15 batches of Celosia scomb from different origins and different batches, which can accurately represent the characteristic components of Celosia scomb and its preparation, thereby ensuring the effectiveness and accuracy of Celosia scomb quality control, and the 15 characteristic peaks have good peak shapes and high separation, and can accurately control the retention time and relative retention time of the characteristic peaks of Celosia scomb, thereby improving the reproducibility and scope of application of the present invention.

[0028] 5. The present invention constructs the optimal elution conditions of the variable wavelength UPLC characteristic spectrum of Celosia and its preparations, which greatly shortens the detection time of Celosia and its preparations. The characteristic spectrum has a stable baseline, little external noise interference, good peak shape and separation degree, and strong separation efficiency.

[0029] 6. The present invention uses pure acetonitrile to flush the chromatographic column for 3 minutes. The chromatographic column is flushed while being tested, which can accelerate the rapid flushing of the residues of each injection of the sample out of the chromatographic column, avoid the residues from clogging the chromatographic column, thereby extending its service life and ensuring the high repeatability and stability of the characteristic spectrum of cockscomb and its preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the variable wavelength UPLC characteristic spectrum of 15 batches of Celosia cristata standard decoction dry paste powder in Example 1 of the present invention;

[0031] Figure 2 is the characteristic spectrum of the mixed reference substance in Example 1 of the present invention;

[0032] Figure 3 It is a comparative characteristic spectrum generated from 15 batches of Celosia cristata standard decoction dry paste powder in Example 1 of the present invention;

[0033] Figure 4 is the ultraviolet absorption chromatogram of 7 components scanned by ultra-high performance liquid chromatography at full wavelength in Experimental Example 1 of the present invention;

[0034] Figure 5 is a chromatogram of the standard decoction of Celosia cristata at different wavelengths in Experimental Example 1 of the present invention;

[0035] Figure 6 This is the chromatogram of the standard decoction of Celosia cristata in Experimental Example 2 of the present invention under different gradient elution programs

[0036] Figure 7 is a chromatogram of the standard decoction of Celosia cristata in Experimental Example 3 of the present invention under different mobile phases;

[0037] Figure 8 is a chromatogram of the standard decoction of Celosia cristata in Experimental Example 4 of the present invention under different extraction reagents;

[0038] Fig. 9 It is the identification of the chromatogram of the standard decoction of Celosia cristata in Experimental Example 5 of the present invention;

[0039] Fig.10 is the chromatogram of the standard decoction of Celosia cristata at different column temperatures in Experimental Example 6 of the present invention;

[0040] Fig.11 is a chromatogram of the standard decoction of Celosia cristata at different flow rates in Experimental Example 6 of the present invention;

[0041] Fig.12: is the chromatogram of the standard decoction of Celosia cristata in Experimental Example 6 of the present invention under different chromatographic columns;

[0042] Fig.13 This is the variable wavelength UPLC characteristic spectrum of the cockscomb medicinal material and its preparation in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0043] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0044] Reagent and instrument of the present invention:

[0045] Test drug: Celosia cristata control drug (batch number: 121110-202004, China Food and Drug Inspection Institutes), marked with R;

[0046] Tryptophan reference substance (batch number: 140686-202205, purity 100%, China Food and Drug Inspection Institute), p-hydroxycinnamic acid (batch number: 110770-201702, purity 99.4%, China Food and Drug Inspection Institute), ferulic acid reference substance (batch number: 110773-201915, purity 99.4%, China Food and Drug Inspection Institute), quercetin (batch number: 10081-2016 10, purity 99.1%, China Food and Drug Inspection Institutes), kaempferol (batch number: 110861-202013, purity 93.2%, China Food and Drug Inspection Institutes), isorhamnetin (batch number: 110860-202012, purity 99.1%, China Food and Drug Inspection Institutes), kaempferol-4'-O-methyl ether (batch number: 21-12-0317, purity 100%, SINCO Company);

[0047] The preparation method of Cockscomb slices is to remove impurities and residual stems from the Cockscomb medicinal materials and cut them into irregular segments.

[0048] The preparation method of the standard decoction dry paste powder of cockscomb is as follows: weigh 200g of cockscomb slices, place them in a decoction pot, add 3000mL of pure water, soak for 30min, boil over high heat, and then simmer for 60min, filter the extract through a 300-mesh filter, add 3000mL of pure water to the residue, boil over high heat, and then simmer for 60min, add 2000ml of water for the third time and simmer for 30 minutes. Combine the three filtrates, concentrate them by rotary evaporator to a solid-liquid ratio of about 1:1, set the concentration temperature to 60°C, place the concentrate in a tray, dry it in a vacuum at low temperature, and crush it to obtain the concentrate.

[0049] The batch numbers and origins of 15 batches of Celosia cristata standard decoction dry paste powder used in the present invention are shown in Table 1.

[0050] Table 1 Batch numbers, origins and standard decoction, dry paste and powder numbers of 15 batches of Celosia cristata

[0051]

[0052] The preparation method of cockscomb formula granules is as follows: take an appropriate amount of cockscomb slices, extract 3 times, add 15 times the amount of water for the first time, soak for 30 minutes, add 15 times the amount of water for the second time, decoct for 1 hour each time, add 10 times the amount of water for the third time, decoct for half an hour. After the decoction is filtered through a 300-mesh double filter, the filtrate is concentrated to an extract with a solid content of 5% to 15% by a double-effect concentrator under reduced pressure, and the extract is transferred to a spray raw material tank. After spray drying, add an appropriate amount of maltodextrin, mix well, and dry granulate to obtain the product.

[0053] The cockscomb decoction pieces and cockscomb medicinal materials used in the present invention are produced in Yingshan County, Huanggang City, Hubei Province.

[0054] Reagents: ethanol (batch number: 10009164, analytical grade); hydrochloric acid (batch number: 10011018, analytical grade); acetonitrile (batch number: F2403M2011, chromatographic grade); methanol (batch number: F2403D203, chromatographic grade); chromatographic grade from Sinopharm Chemical Reagent Co., Ltd.; pure water from ELGA option-Q ultrapure water system.

[0055] Instruments: Waters series ultra-high performance liquid chromatograph (ACQUITY UPLC H-Class Plus), American PDA detector; Agilent 1290 series ultra-high performance liquid chromatograph (1290 quaternary gradient pump, 1290 autosampler, 1290 column oven), American 1290 VWD detector; 1 / 100,000 electronic balance (Sartorius); XMTD-7000 constant temperature water bath (Beijing Guangming Medical Instrument Co., Ltd.).

[0056] Example 1

[0057] This embodiment provides a method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparations, and the specific steps are as follows:

[0058] (1) Preparation of reference substance solution: Take appropriate amounts of tryptophan, p-hydroxycinnamic acid, ferulic acid, quercetin, kaempferol, isorhamnetin, and kaempferol-4'-O-methyl ether, accurately weigh them, and add methanol to prepare a mixed solution containing 10 μg of tryptophan, 10 μg of ferulic acid, 10 μg of p-hydroxycinnamic acid, 20 μg of quercetin, 20 μg of kaempferol, 5 μg of isorhamnetin, and 5 μg of kaempferol-4'-O-methyl ether per 1 ml.

[0059] (2) Preparation of reference medicinal material solution: 1.0 g of Celosia cristata reference medicinal material was taken, accurately weighed, placed in a stoppered conical flask, accurately added with 50 mL of 70% ethanol and 10 mL of concentrated hydrochloric acid solution, weighed, heated under reflux at 95° C. for 1.5 h, cooled, reweighted, filtered, and the filtrate was taken as the reference medicinal material solution;

[0060] (3) Preparation of test solution: Take 0.5 g of the Celosia cristata preparation sample to be tested, grind it into powder, accurately add 50 mL of 70% ethanol and 10 mL of concentrated hydrochloric acid solution, weigh it, heat it under reflux at 95°C for 1.0 h, weigh it, filter it, cool it, add weight, shake it well, filter it, and take the filtrate to obtain the solution;

[0061] Take 1.0g of the sample of Celosia cristata to be tested, grind it into powder, accurately add 50mL of 70% ethanol and 10mL of concentrated hydrochloric acid solution, weigh it, heat and reflux it at 95℃ for 1.5h, weigh it, filter it, cool it, add weight, shake it well, filter it, and take the filtrate to obtain the final product;

[0062] (4) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.6 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 2; the flow rate was 0.3 mL per minute; the column temperature was 35°C; 0-10.0 min, the detection wavelength was 286 nm; 10.1-25.1 min, the detection wavelength was 365 nm;. The theoretical plate number calculated based on the kaempferol peak should be no less than 5000;

[0063] (5) Determination: Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

[0064] Table 2 Gradient elution program

[0065]

[0066] (6) Characteristic peak determination: Take 15 batches of Celosia cristata standard decoction dry powder, prepare 15 batches of Celosia cristata standard decoction dry powder test sample solutions according to the method in step (3), and measure according to steps (4) and (5) to obtain variable wavelength UPLC characteristic spectra of 15 batches of Celosia cristata standard decoction dry powder, as shown in detail. Figure 1Among them, R marks the reference map of Celosia scomb. The "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation Software System (2012 Edition)" promulgated by the National Pharmacopoeia Committee was used. The chromatographic peaks of 15 batches of Celosia scomb standard decoction UPLC maps were matched with the number S1 map as the reference map to form a common peak pattern map. As a result, 15 batches of Celosia scomb standard decoction dry paste powder test samples all showed 15 common peaks, and the reference characteristic map was established according to the median calculation. Among them, peak 6 was positioned as the reference substance of p-hydroxycinnamic acid, and peak 11 was kaempferol. The two components had strong signals, moderate peak areas, and good separation, so they were marked as S1 and S2 peaks. Peak 4 was tryptophan, peak 8 was ferulic acid, peak 10 was quercetin, peak 12 was isorhamnetin, and peak 15 was kaempferol-4'-O-methyl ether. Figure 2 There are 15 peaks corresponding to the reference medicinal materials, which are marked separately to establish the standard reference characteristic spectrum, see Figure 3 , calculated the relative retention time and RSD value, and the results are shown in Table 3.

[0067]

[0068]

[0069] From Table 3 and Figure 1 It can be seen that there is no significant difference in the relative retention times of the 15 characteristic peaks of Celosia cristata. Taking the average value of each relative retention time as the measured value, the relative retention time limit values ​​of Peak 1-Peak 9 and S1 peak are 0.252 (Peak 1), 0.336 (Peak 2), 0.432 (Peak 3), 0.546 (Peak 4), 0.698 (Peak 5), 1.054 (Peak 7), 1.093 (Peak 8), and 1.113 (Peak 9), and the relative retention time limit values ​​of Peak 10-Peak 15 and S2 peak are 0.825 (Peak 10), 1.041 (Peak 12), 1.098 (Peak 13), 1.217 (Peak 14), and 1.413 (Peak 15), respectively. Their relative retention times are all within the range of ±10% of the specified value. Then, the characteristic spectra of 15 batches of Celosia scomb standard decoction dry paste powder and Celosia scomb reference medicinal material reference were imported into the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation Software System (2012 Edition)" promulgated by the State Pharmacopoeia Committee, and the similarities between the characteristic spectra of 15 batches of Celosia scomb standard decoction dry paste powder and the generated common pattern (R) as the reference characteristic spectrum were 0.998, 0.969, 0.990, 0.989, 0.978, 0.997, 1.000, 0.995, 0.999, 0.988, 0.982, 0.991, 0.998, 0.993, and 0.997, respectively, indicating that the chromatograms of Celosia scomb quasi-decoction dry paste powder from different origins have a high degree of consistency, the 15 characteristic peaks have clear peak shapes, and the separation is good. The use of this construction method can better achieve precise control of Celosia scomb quality.

[0070] Experimental Example 1: Investigation of wavelength

[0071] A diode array detector was used to perform a 3D full wavelength scan of the Celosia cristata standard decoction dry powder test solution (S1) at a wavelength of 190-400 nm, see Figure 4 . Through the analysis of the isobsorptivity diagram, the maximum absorption wavelengths of the seven components are tryptophan 217.8nm, p-hydroxycinnamic acid 309.1nm, ferulic acid 322.3nm, quercetin 369.6nm, kaempferol 366.0nm, isorhamnetin 369.6nm, kaempferol-4'-O-methyl ether 364.8nm. Therefore, 5 wavelengths of 220nm, 260nm, 286nm, 315nm, and 365nm were selected for investigation. Figure 5 At 220nm, the solvent peak signal at 1min is extremely strong, resulting in the weakest overall peak signal; at 260nm, the overall peak signal is weak; at 286nm, the peak area of ​​the first half is the largest, but the peak area of ​​peak 15 is extremely small, and peak 14 is missing; at 365nm, the first half is lost, and the flavonoid peak area of ​​the second half is the largest; at 315nm, tryptophan is lost, and the quercetin peak shape is abnormal; therefore, considering the comprehensive selection of the first half wavelength of 286nm and the second half wavelength of flavonoids of 365nm, the response values ​​of each chromatographic peak are moderate, the separation is good, and the peak area can be guaranteed to be maximum.

[0072] Experimental Example 2 Investigation of Gradient Elution Procedure

[0073] Three samples S1 of the same batch of standard Celosia cristata decoction dry paste powder were taken, and the sample solutions were obtained according to the preparation method in step (3) of Example 1. Experimental group 1 was measured according to the chromatographic conditions in steps (4) and (5) of Example 1.

[0074] The difference between control group 1 and experimental group 1 is that the gradient elution program is different. The gradient elution program of control group 1 is shown in Table 4.

[0075] Table 4 Gradient elution program of control group 1

[0076]

[0077] The difference between control group 2 and experimental group 1 is that the gradient elution procedure is different. See Table 5 for control group 2.

[0078] Table 5 Gradient elution program of control group 2

[0079]

[0080] The gradient elution program of the mobile phase in the prior art CN110441445A is used as the control group 3, which is different from the experimental group 1 in that the gradient elution program is different. The control group 3 is shown in Table 6.

[0081] Table 6 Gradient elution program of control group 3

[0082]

[0083] The characteristic spectra of experimental group 1, control group 1, control group 2 and control group 3 are shown in Figure 6 The peak order and number of the three groups were consistent in experimental group 1, control group 1 and control group 2. However, as shown in Table 7, the peak areas of peak 4 (tryptophan), peak 6 (p-hydroxycinnamic acid), peak 8 (ferulic acid), peak 10 (quercetin), peak 11 (kaempferol), peak 12 (isorhamnetin) and peak 15 (kaempferol-4'-O-methyl ether) of the characteristic spectrum of the standard decoction dry powder of Celosia cristata obtained in experimental group 1 were all higher than those in control group 1 and control group 2.

[0084] After investigation, the mobile phase gradient elution program in the prior art CN110441445A showed that, in addition to the detection time being doubled, peaks 1, 2, and 3 appeared earlier, were closer together, and had poor separation; peak 4 (tryptophan) and peak 13 were not detected; peak 6 (p-hydroxycinnamic acid) and peak 8 (ferulic acid) appeared at 4.0 min and 4.9 min, respectively, and peaks 5, 7, 9, and 10 could not be located; the peak areas of peak 6 (p-hydroxycinnamic acid), peak 8 (ferulic acid), peak 10 (quercetin), peak 11 (kaempferol), peak 12 (isorhamnetin), and peak 15 (kaempferol-4'-O-methyl ether) were all smaller than those of the experimental group.

[0085] Experimental Example 3 Investigation of mobile phase types

[0086] The test solution (S1) of the standard decoction of cockscomb was measured under four mobile phases according to the chromatographic conditions in step (4) and step (5) of Example 1, respectively. Figure 7 .

[0087] Compared with acetonitrile-0.1% phosphoric acid (experimental group) as the standard mobile phase, the peak signal is weaker under methanol-0.1% phosphoric acid (comparative group 1), and peaks 2, 6, 7, 11, 13, 14, and 15 are missing. Under acetonitrile-0.1% acetic acid (comparative group 2) and acetonitrile-0.1% formic acid (comparative group 3), the peak shape of peak 15 (kaempferol-4'-O-methyl ether) is obviously abnormal, and peak 4 (tryptophan) in acetonitrile-0.1% acetic acid has a peak. The peak areas of each group are shown in Table 8.

[0088]

[0089] Experimental Example 4 Investigation of reagents for preparing test solution

[0090] The test sample solution (S1) of the standard decoction of cockscomb was extracted with 8 reagents according to step (3) of Example 1, and then measured according to the chromatographic conditions in steps (4) and (5). Figure 8 .

[0091] 70% ethanol solution (comparison group 1), pure water + concentrated hydrochloric acid solution (comparison group 7), 70% methanol solution + concentrated hydrochloric acid solution (comparison group 3), methanol + concentrated hydrochloric acid solution (comparison group 5) extraction, there are 9 peaks, 6 peaks, 2 peaks, 2 peaks missing respectively. Although the other four groups have no missing peaks, compared with the standard group 70% ethanol solution + concentrated hydrochloric acid solution, 50% ethanol solution + concentrated hydrochloric acid solution (comparison group 4), 95% ethanol solution + concentrated hydrochloric acid solution (comparison group 6), and 70% ethanol solution refluxed for 1 hour, and then the filtered solution was refluxed with concentrated hydrochloric acid solution for 1.0 hour (comparison group 2), the peak areas of characteristic spectrum peaks 4 (tryptophan), peak 10 (quercetin), peak 12 (isorhamnetin), and peak 15 (kaempferol-4'-O-methyl ether) in the standard group are the largest, see Table 9, and 70% ethanol solution + concentrated hydrochloric acid solution (experimental group 1) is only heated and refluxed once, which is convenient to operate. Therefore, 70% ethanol solution + concentrated hydrochloric acid solution was selected as the preparation solvent.

[0092]

[0093] Experimental Example 5 Methodological Investigation

[0094] 5.1. Identification of chromatographic peaks

[0095] Preparation of the test solution: Take the dry powder of the standard decoction of Celosia cristata to be tested sample S1, and obtain the test sample solution according to the preparation method in step (3) of Example 1.

[0096] Preparation of reference solution: Preparation of reference solution: Take appropriate amount of tryptophan, p-hydroxycinnamic acid, ferulic acid, quercetin, kaempferol, isorhamnetin, and kaempferol-4'-O-methyl ether, weigh accurately, and add methanol to make a mixed solution containing 10 μg of tryptophan, 10 μg of ferulic acid, 10 μg of p-hydroxycinnamic acid, 20 μg of quercetin, 20 μg of kaempferol, 5 μg of isorhamnetin, and 5 μg of kaempferol-4'-O-methyl ether per 1 ml.

[0097] Preparation of control medicinal material solution: Take 1.0 g of cockscomb control medicinal material, accurately weigh it, put it in a stoppered conical flask, add 50 mL of 70% ethanol and 10 mL of hydrochloric acid, weigh it, heat it under reflux at 95°C for 1.5 h, cool it, make up the weight, filter it, and take the filtrate as the control medicinal material reference solution.

[0098] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking the standard decoction of Celosia cristata was prepared.

[0099] The determination was carried out according to the chromatographic conditions in step (4) and step (5) of Example 1.

[0100] The characteristic peaks of the standard decoction of Celosia cristata were located. Fig. 9 shown.

[0101] 5.2 Precision test

[0102] Take the test solution (S1) of the dry powder of the standard decoction of Celosia cristata, and perform the measurement according to the method in step (4) and step (5) of Example 1. Inject 6 times continuously, 1 μl each time, and calculate the retention time and peak area of ​​each characteristic peak. As shown in Table 10 and Table 11. The results show that the instrument has good precision.

[0103] 5.3 Repeatability test

[0104] Six samples of the dry powder of standard decoction of Celosia cristata were prepared (S1) and tested according to the proposed experimental method, as shown in Table 12. The results showed that the method had good repeatability.

[0105] 5.4 Stability test

[0106] Based on the above proposed experimental conditions, the Celosia cristata standard decoction dry paste powder test sample (S1) solution was taken and measured at 0h, 3h, 6h, 9h, 12h, and 24h, respectively, as shown in Table 13. The results show that the method has good stability.

[0107]

[0108]

[0109] Experimental Example 6 Durability Study

[0110] 6.1. Column temperature investigation

[0111] Based on the experimental conditions proposed above, the column temperatures of 30℃, 35℃, and 40℃ were investigated. Fig.10 As shown in Table 14.

[0112] The results showed that when the column temperature was 30℃, peaks 5 and 9 were overlapping; at 40℃, the separation between peaks 7 and 8 was poor; at 35℃, the chromatogram peaks were symmetrical, the separation was good, and the RSD of the relative retention time of the peaks was less than 2.2%, indicating that the method had good durability.

[0113] 6.2 Flow rate investigation

[0114] Based on the experimental conditions proposed above, the flow rates of 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min were investigated. Fig.11 As shown in Table 15, at three flow rates, the RSD of the relative retention time of each peak was less than 2.8%, indicating that the invention has good durability to the flow rate.

[0115] 6.3. Chromatographic column durability investigation

[0116] Based on the above experimental conditions, the following chromatographic columns were investigated: chromatographic column 1: CORTECS UPLC T3, 2.1mm*100mm, 1.6μm; chromatographic column 2: ZORBAX SB-C18, 2.1mm*100mm, 1.8μm; chromatographic column 3: ACQUITY HSST3, 2.1mm*100mm, 1.8μm. Fig.12 , as shown in Table 16.

[0117] The results show that the RSD of the characteristic peak relative retention time is between 0.40% and 10.77% when the samples are detected by the above three chromatographic columns, but both chromatographic columns 2 and 3 lack a peak, and the baseline of ACQUITY UPLC HSS T3 chromatographic column is not flat at 10min of wavelength change. Therefore, the durability of the chromatographic column of this method is poor, and CORTECS UPLC T3, 2.1mm*100mm, 1.6μm chromatographic column is better.

[0118]

[0119] Experimental Example 7 Investigation of Celosia scomb samples to be tested

[0120] Group S1: Accurately weigh 0.5 g of dry powder of standard decoction of Celosia cristata, place it in a stoppered conical flask, accurately add 50 mL of 70% ethanol solution and 10 mL of concentrated hydrochloric acid solution, weigh, heat and reflux at 95°C for 1.0 h, weigh, filter, cool, add weight, shake well, filter, and obtain the test solution.

[0121] Group S2: Accurately weigh 0.5 g of Celosia cristata formula granules, place in a stoppered conical flask, add 50 mL of 70% ethanol and 10 mL of concentrated hydrochloric acid solution, weigh, heat under reflux at 95°C for 1.0 h, weigh, filter, cool, add weight, shake well, filter, and obtain the test solution.

[0122] S3 group: accurately weigh 1.0 g of Celosia cristata medicinal material, place it in a stoppered conical flask, add 50 mL of 70% ethanol solution and 10 mL of concentrated hydrochloric acid solution, weigh it, heat and reflux at 95°C for 1.5 h, weigh it, filter it, cool it, add weight, shake it well, filter it, and get the test solution.

[0123] Group S4: Accurately weigh 1.0 g of Celosia cristata slices, place in a stoppered conical flask, add 50 mL of 70% ethanol solution and 10 mL of concentrated hydrochloric acid solution, weigh, heat under reflux at 95°C for 1.5 h, weigh, filter, cool, add weight, shake well, filter, and obtain the test solution.

[0124] Then the test solutions obtained from Group S1, Group S2, Group S3 and Group S4 were measured according to the chromatographic method of step (4) and step (5) of Example 1, and the characteristic spectra obtained were shown in Fig.13 .

[0125] Fig.13 It can be seen that the variable-wavelength UPLC characteristic spectrum of Celosia scomb constructed by the present invention can be applied to Celosia scomb standard decoction, Celosia scomb formula granules, Celosia scomb medicinal materials and Celosia scomb decoction pieces at the same time, and has a wide range of applications.

[0126] In summary, the method for constructing the variable-wavelength UPLC characteristic spectrum of Celosia scomb disclosed in the present invention has the advantages of high sample solution extraction efficiency, clear peak shape, high separation and good repeatability; at the same time, the method for constructing the characteristic spectrum of Celosia scomb disclosed in the present invention can also expand the detection range of the effective ingredients of Celosia scomb, greatly improve the detection efficiency, save detection time, and can significantly increase the cleaning function of the chromatographic column and extend the service life of the chromatographic column.

[0127] Obviously, the above embodiments are only examples of the preferred embodiments of the present invention and do not include all the implementation scopes of the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be based on the scope defined in the claims.

Claims

1. A method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparations, characterized in that: The following steps are involved: Preparation of test solution: weigh 0.5g-1.0g of the sample of Celosia cristata and its preparation to be tested, accurately add the extraction solvent, weigh, heat and reflux for 1.0h-1.5h, cool, weigh again, and make up the weight with the extraction reagent to prepare the test solution; Using tryptophan, p-hydroxycinnamic acid, ferulic acid, quercetin, kaempferol, isorhamnetin and kaempferol-4'-O-methyl ether as reference substances, the characteristic spectra of Celosia cristata and its preparations were obtained by ultra performance liquid chromatography and variable wavelength technology.

2. According to the method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparations as described in claim 1, it is characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: octadecylsilane bonded silica gel as filler, column length 100 mm, inner diameter 2.1 mm, silica gel particle size 1.6 μm as chromatographic column; acetonitrile as mobile phase A, 0.1-0.3% phosphoric acid aqueous solution as mobile phase B for gradient elution, flow rate set to 0.28-0.32 mL / min, detection wavelengths set to 286 nm and 365 nm, column temperature 35°C; The gradient elution conditions are as follows: 0-2.0 min, the volume ratio of mobile phase A to mobile phase B is 5-8%:95-92%; 2.0-4.0 min, the volume ratio of mobile phase A to mobile phase B is 8-10%:92-90%; 4.0-7.0 min, the volume ratio of mobile phase A to mobile phase B is 10-21%:90-79%; 7.0-9.0min, the volume ratio of mobile phase A to mobile phase B is 21-24%:79-76%; 9.0-10.0min, the volume ratio of mobile phase A to mobile phase B is 24-26%:76-74%; 10.0-15.0min, the volume ratio of mobile phase A to mobile phase B is 26-30%:74-70%; 15.0-16.0min, the volume ratio of mobile phase A to mobile phase B is 30%:70%; 16.0-17.0min, the volume ratio of mobile phase A to mobile phase B is 30-38%:70-62%; 17.0-22.0min, the volume ratio of mobile phase A to mobile phase B is 38%:62%; 22.0-22.1min, the volume ratio of mobile phase A to mobile phase B is 38-100%:74-70%; 22.1-25.0min, the volume ratio of mobile phase A to mobile phase B is 100%:0%; 25.0-25.1min, the volume ratio of mobile phase A to mobile phase B is 100-5%:0-95%.

3. The method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparation according to claim 1 or 2, characterized in that: The preparation method of the reference substance is as follows: taking appropriate amounts of tryptophan, p-hydroxycinnamic acid, ferulic acid, quercetin, kaempferol, isorhamnetin, and kaempferol-4'-O-methyl ether reference substances, adding methanol to dissolve and preparing a mixed reference substance solution; The preparation method of the reference medicinal material reference solution is as follows: taking an appropriate amount of cockscomb reference medicinal material into a stoppered conical bottle, adding a certain volume of 70% ethanol solution or 50% ethanol solution or 95% ethanol solution and concentrated hydrochloric acid solution, heating and refluxing for extraction for 1-1.5 hours, cooling, adding weight, filtering, and taking the filtrate as the reference medicinal material solution; The preparation method of the test solution is as follows: take an appropriate amount of cockscomb and its preparation samples into a stoppered conical flask, add a certain volume of 70% ethanol solution or 50% ethanol solution or 95% ethanol solution and concentrated hydrochloric acid solution, weigh, heat and reflux for 1-1.5h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution.

4. The method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparation according to claim 3, characterized in that: The characteristic spectrum of the cockscomb and its preparation includes 15 characteristic peaks, with peak No. 6 (p-hydroxycinnamic acid) as reference peak S1 and peak No. 11 (kaempferol) as reference peak S2. The relative retention time limit values ​​of peaks 1 to 9 and peak S1 are respectively: 0.252 (peak 1), 0.336 (peak 2), 0.432 (peak 3), 0.546 (peak 4), 0.698 (peak 5), 1.054 (peak 7), 1.093 (peak 8), 1.113 (peak 9); the relative retention time limit values ​​of peaks 10 to 15 and peak S2 are respectively: 0.825 (peak 10), 1.041 (peak 12), 1.098 (peak 13), 1.217 (peak 14), 1.413 (peak 15). The relative retention time of each peak is within the range of ±10% of the specified value. The remaining peaks were located as follows: peak 4 for tryptophan, peak 8 for ferulic acid, peak 10 for quercetin, peak 12 for isorhamnetin, and peak 15 for kaempferol-4'-O-methyl ether.

5. A method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparation according to any one of claims 1 to 4, characterized in that: The reflux temperature is 90-98° C., and the concentration of the test solution is 8.0-16.0 mg / mL.

6. A method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparation according to any one of claims 1-5, characterized in that: The preparation method of the test solution specifically includes taking an appropriate amount of the sample of Celosia cristata and its preparation to be tested, grinding it, accurately adding 50 mL of 70% ethanol and 10 mL of concentrated hydrochloric acid solution, weighing it, heating it under reflux at 95° C. for 1-1.5 h, cooling it, adding weight, shaking it well, filtering it, and taking the filtrate to obtain the solution; The preparation method of the reference substance solution specifically comprises taking appropriate amounts of seven reference substances, namely, tryptophan, p-hydroxycinnamic acid, ferulic acid, quercetin, kaempferol, isorhamnetin, and kaempferol-4'-O-methyl ether, accurately weighing, adding methanol to prepare a mixed solution, shaking well, filtering, and taking a filtrate to obtain; The preparation method of the control medicinal material reference solution and medicinal material specifically includes taking 1.0 g of cockscomb control medicinal material, accurately weighing, placing in a stoppered conical flask, accurately adding 50 mL of 70% ethanol and 10 mL of concentrated hydrochloric acid solution, weighing, heating and reflux at 95° C. for 1.5 hours, cooling, re-weighting, shaking, filtering, and taking the filtrate as the control medicinal material reference solution.

7. A method for constructing a wavelength UPLC characteristic spectrum of Celosia cristata and its preparation according to any one of claims 1-6, characterized in that: The cockscomb and its preparations include cockscomb medicinal materials, cockscomb decoction pieces, cockscomb standard decoction dry products, and cockscomb Chinese medicine formula granules.

8. A method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparation according to any one of claims 1-7, characterized in that: The concentrations of the reference substance solution are tryptophan 10 μg / mL, ferulic acid 10 μg / mL, p-hydroxycinnamic acid 10 μg / mL, quercetin 20 μg / mL, kaempferol 20 μg / mL, isorhamnetin 5 μg / mL, and kaempferol-4'-O-methyl ether 5 μg / mL; The concentration of the control medicinal material reference solution is 16.0 mg / mL.

9. A method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparation according to any one of claims 1-8, characterized in that: The chromatographic conditions are as follows: using a CORTECS UPLC T3 column as a chromatographic column with specifications of 100*2.1 mm, 1.6 μm; using acetonitrile as mobile phase A, and 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution, the flow rate is set to 0.3 mL / min, the detection wavelength is set to 286 nm and 365 m, and the switching time point is 10 min; the column temperature is 35°C.

10. A method for constructing a variable wavelength UPLC characteristic spectrum of Celosia cristata and its preparation according to any one of claims 1 to 9, characterized in that: From 22.1 to 25.0 min, the proportion of acetonitrile was maintained at 100%, and the column was flushed with pure acetonitrile for 2.9 min to flush out the impurities remaining in the column within one analysis period for each injection of sample.

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