Characteristic chromatogram construction method for quality control of non-volatile components of galangal sample

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and relates to a specific chromatogram construction method for quality control of non-volatile components of a galangal sample. In the construction method, the construction of the basic characteristic spectrum comprises the following steps: 1) preparing a basic test sample solution; 2) preparing a basic reference substance solution; and (3) respectively carrying out UPLC analysis on the basic test solution and the basic reference substance solution, and constructing a basic characteristic spectrum. In addition, the construction method further comprises construction of a supplementary characteristic spectrum. The constructed characteristic chromatogram can comprehensively reflect the characteristics of the galangal sample, the defect of single or few peak determination is avoided, and a new analysis means is provided for internal quality control of the galangal sample.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine analysis, and relates to a characteristic spectrum construction method for quality control of non-volatile components of galangal samples (especially galangal medicinal materials). Background Art

[0002] Alpinia officinarum Hance is the dried rhizome of Alpinia officinarum Hance, a plant of the ginger family. It is also known as small galangal, galangal, and wild ginger. It was first recorded in the "Famous Doctors' Records" and has been included in all editions of the "Chinese Pharmacopoeia". It is hot in nature, pungent in taste, and enters the spleen and stomach meridians. It has the effects of warming the stomach and stopping vomiting, dispersing cold and relieving pain. It is often used clinically to treat cold pain in the abdomen, belching and acid regurgitation, and stomach cold vomiting. [1] Galangal is rich in nutrients, containing protein, polysaccharides, trace elements and other ingredients. [2] It is one of the traditional Chinese medicines that are both medicinal and edible, and has been widely used as a seasoning in the production of folk flavor foods and cooked foods. It has a large market not only in China but also abroad. Modern research shows that galangal has strong pharmacological activity and has antibacterial, antiviral, antioxidant, hypoglycemic, and hypolipidemic effects. [3] In recent years, it has attracted widespread attention due to its unique medicinal and edible value.

[0003] The chemical components of Alpinia officinalis can be divided into two categories, namely volatile oil components (or volatile components) and non-volatile components. For volatile oil components, due to different extraction methods and different origins, the composition of the obtained volatile components is also different, which is difficult to include in quality control. For non-volatile components, according to the size of polarity, they can be roughly divided into two categories: water-soluble components and alcohol-soluble components. Among them, alcohol-soluble components (mainly flavonoids, diphenylheptane compounds) [4] ) has always been one of the main research directions of quality control of Alpinia officinalis.

[0004] The research on the characteristic fingerprint of non-volatile components of Alpinia officinalis mainly includes: Ma Liping [5] et al. used high performance liquid chromatography to establish the HPLC fingerprint of Alpinia officinalis decoction pieces, and simultaneously determined the content of galangin in the samples, identified 9 common peaks, and established the common spectrum of 14 batches of Alpinia officinalis medicinal material ethanol extracts. [6] et al. used HPLC to analyze the fingerprints of 10 batches of galangal methanol extracts and identified 10 common peaks. [7] et al. used HPLC to compare the characteristic spectra of 11 batches of different cultivars of Alpinia galanga (bamboo ginger and honeycomb ginger), determined the 12 common peaks of Alpinia galanga methanol extract, and identified three chemical components, diphenylheptane A, galangin, and kaempferol, through reference substances. [8]et al. constructed fingerprints of 12 batches of Alpinia officinalis using HPLC, identified 10 characteristic peaks of the ethanol extract of Alpinia officinalis, and identified galangin through reference substances. [9] et al. established an HPLC fingerprint analysis method for Alpinia officinalis and identified 13 common peaks in the ethanol fraction of Alpinia officinalis.

[10] et al. used HPLC to determine 15 batches of Alpinia officinalis and identified 6 common peaks in the petroleum ether extraction part of Alpinia officinalis as fingerprint characteristic peaks, and identified diphenylheptane A through reference substances.

[0005] The Chinese Pharmacopoeia (2020 edition) lists galangin as one of the quality evaluation indicators of galangal, but a single indicator component cannot comprehensively evaluate the quality. Most of the characteristic spectra reported in existing literature only show the chemical components (diphenylheptane A, galangin, kaempferol) in the alcohol extract of galangal medicinal materials, and only one or a few chromatographic peaks among the common peaks are identified, and the water-soluble components are not displayed. Because there are also many water-soluble components in galangal, the characteristic spectrum of galangal that only uses fat-soluble components or alcohol-soluble components to characterize galangal is not comprehensive.

[0006] Therefore, it is necessary to construct a characteristic spectrum method for quality control of non-volatile components of galangal samples (especially galangal medicinal materials) so as to comprehensively reflect the water-soluble and alcohol-soluble components in galangal samples and provide a comprehensive quality control method for regulating the uniformity and stability of the intrinsic quality of galangal samples.

[0007] References

[0008] [1] Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China 2020 Edition Volume 1[S]. Beijing: China Medical Science and Technology Press, 2020: 300.

[0010] [2] Liao Kunmei, Bai Tianhe, Chen Chuhua, et al. Study on the cholesterol-lowering effect of galangal crude polysaccharide in vitro [J]. Agricultural Products Processing, 2017(10):4~6+17.

[0012] [3] Huang Huizhen, Yang Dan. Research progress on chemical components and pharmacological activities of Alpinia officinalis[J]. Guangdong Chemical Industry, 2009, 36(1): 77-80.

[0014] [4] Lv Wei, Jiang Linghuo. Chemical components and pharmacological effects of Alpinia officinalis[J]. China Pharmaceutical Industry, 2006, 15(3): 19-21.

[0015] [5] Ma Liping, Qiu Xiaohui, Min Jiang. Study on HPLC fingerprint of Alpinia officinalis slices and determination of galangin content[J]. Chinese Traditional Patent Medicine, 2008, 30(11): 1565-1569.

[0017] [6] Han Liang, Shi Zhongfeng, Rui Wen, et al. Characteristic spectrum analysis and component confirmation of flavonoid extract of Alpinia galanga[J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2011, 17(21): 82-85.

[0019] [7] Long Qin, Lin Dingguang, Hu Jiali, et al. Study on HPLC fingerprint of different cultivars of Alpinia officinalis and determination of index component content[J]. Journal of Guangzhou University of Chinese Medicine, 2019, 36(1): 109-114.

[0021] [8] Deng Yifeng, Feng Lina, Luo Hui. Study on the fingerprint of authentic medicinal materials of Alpinia officinalis by high performance liquid chromatography [J]. Chinese Medicinal Materials, 2011, 34(9): 1351-1355.

[0023] [9] Wang Lifeng, Peng Jingdong, Wang Ning. Study on HPLC fingerprint of Alpinia officinalis[J]. Journal of Southwest Normal University (Natural Science Edition), 2008, 33(6): 33-38.

[0025]

[10] Cui Weili, Feng Yifan, Guo Xiaoling, et al. Determination of diphenylheptane A in Alpinia officinalis and its fingerprint study [J]. Journal of Guangdong Pharmaceutical College, 2007, 23(6): 635-637. Summary of the invention

[0027] Problem that the invention aims to solve

[0028] To solve the problems existing in the above-mentioned prior art. The purpose of the present invention is to establish a characteristic spectrum for the quality control of non-volatile components of galangal samples (especially galangal medicinal materials), which reflects water-soluble components and alcohol-soluble components at the same time, so that the quality control of galangal samples is upgraded from the original determination of a few common peaks to the overall control of the intrinsic quality of galangal samples, which can more comprehensively reflect the characteristics of galangal samples, avoid the defects of single or a few peak determinations, and provide a new analytical means for the intrinsic quality control of galangal samples.

[0029] Solutions for solving problems

[0030] The present invention provides a method for constructing a characteristic spectrum of non-volatile components of an Alpinia officinalis sample, which comprises the following steps:

[0031] 1) Preparation of basic test solution: Take a sample of galangal, optionally crush it and weigh it accurately, accurately add water and extract it, make up the weight loss with water, take the filtrate, and obtain the basic test solution;

[0032] 2) Preparation of basic reference substance solution: taking appropriate amount of basic reference substances, respectively dissolving them in solvents to obtain the basic reference substance solution;

[0033] 3) Establishment of basic characteristic spectrum: The basic test solution prepared in step 1) and the basic reference solution prepared in step 2) are respectively subjected to ultra-high performance liquid chromatography analysis to obtain the basic characteristic spectrum.

[0034] Wherein, the sample in step 1) is selected from medicinal materials, decoction pieces, standard decoctions and formula granules, preferably medicinal materials.

[0035] Preferably, the extraction method in step 1) is selected from ultrasonic extraction, heating reflux extraction and shaking extraction, preferably heating reflux extraction.

[0036] Preferably, the extraction time in step 1) is 30-120 min, preferably 60-90 min, more preferably 60 min.

[0037] Preferably, in step 1), the ratio of the sample to water is 1 g:25-50 mL, preferably 1 g:25 mL.

[0038] Preferably, the basic reference substance in step 2) is selected from at least one of procyanidin B1, procyanidin B2, catechin, epicatechin and protocatechuic acid, preferably procyanidin B1, procyanidin B2, catechin, epicatechin and protocatechuic acid.

[0039] Preferably, the solvent in step 2) is alcohol, preferably methanol.

[0040] Preferably, the concentration of procyanidin B1, procyanidin B2, catechin, epicatechin and protocatechuic acid in the basic reference solution in step 2) is 20-60 μg / mL.

[0041] More preferably, the concentrations of procyanidin B1, procyanidin B2, catechin, epicatechin and protocatechuic acid in the basic reference solution in step 2) are 60 μg / mL, 40 μg / mL, 50 μg / mL, 30 μg / mL and 20 μg / mL, respectively.

[0042] Preferably, the ultra-high performance liquid chromatography in step 3) includes the following chromatographic conditions: using a surface porous filler based on octadecylsilane bonded silica gel as the stationary phase; using acetonitrile-phosphoric acid aqueous solution as the mobile phase, and using gradient elution as the elution method.

[0043] More preferably, the ultra-high performance liquid chromatography in step 3) includes the following chromatographic conditions: an Agilent InfinityLab Poroshell 120AQ-C18 chromatographic column as the stationary phase; acetonitrile-0.1% v / v phosphoric acid aqueous solution as the mobile phase; the gradient elution program is as follows: 0-5 min, the volume percentage of acetonitrile is 3%; 5-6 min, the volume percentage of acetonitrile is 3%→6%; 6-18 min, the volume percentage of acetonitrile is 6%; 18-30 min, the volume percentage of acetonitrile is 6%→9%; 30-33 min, the volume percentage of acetonitrile is 9%.

[0044] Further preferably, the ultra-high performance liquid chromatography in step 3) further comprises the following chromatographic conditions:

[0045] The flow rate is 0.25-0.35 mL / min, preferably 0.3 mL / min;

[0046] The column temperature is 30-40°C, preferably 35°C;

[0047] The detection wavelength is 215 nm.

[0048] Preferably, the basic characteristic spectrum in step 3) contains at least 8 characteristic peaks.

[0049] More preferably, the basic characteristic spectrum in step 3) comprises 8 characteristic peaks, and the chromatographic peak corresponding to catechin is used as the reference peak. Within the range of ±10%, the relative retention times of the 8 characteristic peaks are as follows: 0.14 (peak 1), 0.38 (peak 2), 0.88 (peak 3), 0.92 (peak 4), 1.00 (peak 5), 1.61 (peak 6), 1.71 (peak 7) and 1.85 (peak 8), wherein: peak 2 is protocatechuic acid, peak 4 is procyanidin B1, peak 5 is catechin, peak 7 is procyanidin B2, and peak 8 is epicatechin.

[0050] Preferably, the feature map construction method further comprises the following steps:

[0051] 1') Preparation of supplementary test solution: Take the same galangal sample as in the basic characteristic spectrum construction method, optionally crush it and accurately weigh it, accurately add an extraction solvent and then extract it, make up the weight loss with the extraction solvent, take the filtrate, and obtain the supplementary test solution;

[0052] 2') Preparation of supplementary reference substance solution: taking appropriate amount of each supplementary reference substance, respectively dissolving in solvent to obtain the supplementary reference substance solution;

[0053] 3') Establishment of the supplementary characteristic spectrum: The supplementary test solution prepared in step 1') and the supplementary reference solution prepared in step 2') are respectively subjected to ultra-high performance liquid chromatography analysis to obtain the supplementary characteristic spectrum.

[0054] Preferably, the extraction solvent in step 1') is selected from alcohol and an aqueous solution of alcohol.

[0055] More preferably, the alcohol is methanol.

[0056] More preferably, the volume percentage of alcohol in the alcohol aqueous solution is 30%-70%, preferably 70%.

[0057] Preferably, the extraction method in step 1') is selected from ultrasonic extraction, heating reflux extraction and shaking extraction, preferably ultrasonic extraction.

[0058] Preferably, the extraction time in step 1') is 30-120 min, preferably 60-90 min, more preferably 60 min.

[0059] Preferably, in step 1'), the ratio of the sample to the extraction solvent is 1 g:25-50 mL, preferably 1 g:25 mL.

[0060] Preferably, the supplementary reference substance in step 2') is selected from at least one of galangin, galangin-3-methyl ether and pinocytol, preferably galangin, galangin-3-methyl ether and pinocytol.

[0061] Preferably, the solvent in step 2') is an alcohol, preferably methanol.

[0062] Preferably, the concentration of galangin, galangin-3-methyl ether and galangin in the supplementary reference solution in step 2') is 10-30 μg / mL, preferably 20 μg / mL.

[0063] Preferably, the ultra-high performance liquid chromatography in step 3') comprises the following chromatographic conditions: using a fully porous filler based on octadecylsilane bonded silica gel as the stationary phase; using acetonitrile-phosphoric acid aqueous solution as the mobile phase; and using gradient elution as the elution method.

[0064] More preferably, the ultra-high performance liquid chromatography in step 3') comprises the following chromatographic conditions: an Agilent ZORBAXEclipse Plus C18 RRHD column as the stationary phase; acetonitrile-0.1% v / v phosphoric acid aqueous solution as the mobile phase; the gradient elution program is as follows: 0-13 min, the volume percentage of acetonitrile is 28%→50%; 13-18 min, the volume percentage of acetonitrile is 50%→100%; 18-22 min, the volume percentage of acetonitrile is 100%.

[0065] Further preferably, the ultra-high performance liquid chromatography in step 3') further comprises the following chromatographic conditions:

[0066] The flow rate is 0.35-0.45 mL / min, preferably 0.4 mL / min;

[0067] The column temperature is 25-35°C, preferably 30°C;

[0068] The detection wavelength is 215 nm.

[0069] Preferably, the supplementary characteristic spectrum in step 3') contains at least 11 characteristic peaks.

[0070] More preferably, the supplementary characteristic spectrum in step 3') contains 11 characteristic peaks, and the chromatographic peak corresponding to galangin is used as the reference peak. Within the range of ±5%, the relative retention times of the 11 characteristic peaks are as follows: 0.48 (peak 1), 0.82 (peak 2), 0.86 (peak 3), 0.97 (peak 4), 1.00 (peak 5), 1.03 (peak 6), 1.11 (peak 7), 1.41 (peak 8), 1.46 (peak 9), 1.50 (peak 10) and 1.88 (peak 11), wherein: peak 4 is pinocytol, peak 5 is galangin, and peak 7 is galangin-3-methyl ether.

[0071] Effects of the Invention

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] (1) The present invention establishes a characteristic spectrum for quality control of non-volatile components of galangal samples (especially galangal medicinal materials) by adopting ultra-high performance liquid chromatography, wherein the basic characteristic spectrum reflects water-soluble components and determines 8 characteristic peaks, and the supplementary characteristic spectrum reflects alcohol-soluble components and determines 11 characteristic peaks, so that the quality control of galangal samples is upgraded from the original content determination of a single component or the determination of a few common peaks to the control of the intrinsic quality of the entire galangal sample, which can more comprehensively reflect the characteristics of the galangal sample, avoid the defects of single or a few peak determination, and provide a new analytical means for the intrinsic quality control of the galangal sample.

[0074] (2) The method of the present invention is simple, reproducible, accurate, reliable, and easy to operate. Compared with HPLC, it saves time, consumes less solvent, and has little pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is the UPLC basic characteristic spectrum of the medicinal material Alpinia officinalis, where: 2 is protocatechuic acid, 4 is proanthocyanidin B1, 5 is catechin, 7 is proanthocyanidin B2, and 8 is epicatechin.

[0076] Figure 2 UPLC basic characteristic spectra (S1-S16) of 16 batches of Alpinia officinalis medicinal materials constructed according to Example 1.

[0077] Figure 3 This is the UPLC basic characteristic spectrum of Alpinia officinalis under different extraction conditions.

[0078] Figure 4 This is the result of investigating the extraction method factors in the construction of the basic characteristic map of Alpinia officinalis.

[0079] Figure 5 This is the UPLC basic characteristic spectrum of Alpinia officinalis under different extraction time conditions.

[0080] Figure 6 This is the investigation result of the extraction time factor in the construction method of basic characteristic map of Alpinia officinalis.

[0081] Figure 7 This is the 3D UPLC basic characteristic spectrum of Alpinia officinalis under full wavelength scanning.

[0082] Figure 8 This is the UPLC basic characteristic spectrum of Alpinia officinalis at a wavelength of 215 nm.

[0083] Fig. 9 This is the UPLC basic characteristic spectrum of Alpinia officinalis under different chromatographic column conditions.

[0084] Fig.10 This is the UPLC basic characteristic spectrum of Alpinia officinalis under different elution conditions.

[0085] Fig.11 This is the UPLC supplementary characteristic spectrum of the medicinal material Alpinia officinalis, where: 4 is galangin, 5 is galangin, and 7 is galangin-3-methyl ether.

[0086] Fig.12 UPLC supplementary characteristic spectra (S1-S16) of 16 batches of Alpinia officinalis medicinal materials constructed according to Example 2.

[0087] Fig.13 Supplementary characteristic spectrum of UPLC of Alpinia officinalis under different extraction solvent conditions.

[0088] Fig.14 To supplement the results of the investigation on the extraction solvent factors in the characteristic spectrum construction method of Alpinia officinalis.

[0089] Fig.15 It is the UPLC supplementary characteristic spectrum of Alpinia officinalis under different extraction conditions.

[0090] Fig.16 To supplement the results of the investigation on the extraction method factors in the characteristic spectrum construction method of Alpinia officinalis.

[0091] Fig.17Supplementary characteristic spectra of UPLC of Alpinia officinalis under different extraction time conditions.

[0092] Fig.18 To supplement the results of the investigation on the extraction time factor in the characteristic spectrum construction method of Alpinia officinalis.

[0093] Fig.19 This is the 3D UPLC supplementary characteristic spectrum of Alpinia officinalis under full wavelength scanning.

[0094] Fig. 20 This is the UPLC supplementary characteristic spectrum of Alpinia officinalis under 215nm wavelength conditions.

[0095] Fig.21 Supplementary characteristic spectra of UPLC of Alpinia officinalis under different elution conditions. DETAILED DESCRIPTION

[0096] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific experimental conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0097] Example 1: Construction of the basic characteristic map of Alpinia officinalis

[0098] 1. Instruments, reagents and samples

[0099] (1) Instruments: Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters); Waters Empower 3 workstation (Waters); Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent); electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); HY-4 speed-adjustable multi-purpose oscillator (Jintan Kexing Instrument Factory); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); pure water system (Sartorius); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.).

[0100] (2) Reagents: acetonitrile (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Aladdin); ultrapure water; other reagents were of analytical grade.

[0101] (3) Samples: Reference substances: protocatechuic acid (batch number: 110809-201906, CAS: 99-50-3), catechin (batch number: 110877-202005, CAS: 154-23-4), epicatechin (batch number: 110878-201703, CAS: 490-46-0), purchased from China Food and Drug Administration; proanthocyanidin B1 (batch number: 10439, CAS: 20315-25-7), proanthocyanidin B2 (batch number: 10269, CAS: 29106-49-8), purchased from Shanghai Standard Standard Technical Service Co., Ltd. Test substances: Alpinia officinarum Hance, a plant of the ginger family, was collected from Guangdong and Hainan and identified by Jiangyin Tianjiang Pharmaceutical Co., Ltd. as dried rhizomes, totaling 16 batches.

[0102] 2. Preparation of reference solution

[0103] Take appropriate amount of protocatechuic acid, procyanidin B1, catechin, procyanidin B2, and epicatechin reference substances, weigh accurately, and add methanol to prepare solutions containing 20 μg of protocatechuic acid, 60 μg of procyanidin B1, 50 μg of catechin, 40 μg of procyanidin B2, and 30 μg of epicatechin per 1 mL as reference substance solutions.

[0104] 3. Preparation of test solution

[0105] Take about 1g of Alpinia officinalis powder (passed through No. 4 sieve) (batch number: YC1805098), accurately weigh it, put it in a stoppered conical flask, accurately add 25mL of water, seal it, heat and reflux for 60min, cool it, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it.

[0106] 4. Ultra-high performance liquid chromatography analysis

[0107] Chromatographic column: Agilent InfinityLab Poroshell 120AQ-C18 chromatographic column (150 mm×2.1 mm, 2.7 μm); mobile phase: acetonitrile (mobile phase A)-0.1% v / v phosphoric acid aqueous solution (mobile phase B); flow rate: 0.3 mL / min; column temperature: 35°C; detection wavelength: 215 nm; gradient elution program as shown in Table 1. The theoretical plate number calculated based on the catechin peak should not be less than 5000.

[0108] Table 1. Gradient elution table

[0109]

[0110] Under this condition, 1 μL of the reference solution and 2 μL of the test solution were accurately aspirated and injected into the ultra-high performance liquid chromatograph to obtain the chromatograms of the reference solution and each test solution (galangal medicinal material). Figure 1 and Figure 2 shown.

[0111] 5. Analyze the spectrum

[0112] According to the method in this embodiment, 16 batches of galangal medicinal materials were subjected to ultra-high performance liquid chromatography analysis to obtain ultra-high performance liquid chromatograms, which were introduced into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)" promulgated by the State Pharmacopoeia Committee, and the time window width was selected as 0.1 min. The median was used to generate a reference spectrum, and after multi-point correction, the Mark peaks were matched to generate the common mode of the UPLC basic characteristic spectrum of galangal medicinal materials (see Figure 1 UPLC spectrum of Alpinia officinalis), which contains 8 characteristic peaks. Using LC-MS technology, according to the liquid chromatography conditions of the basic characteristic spectrum of Alpinia officinalis medicinal materials, high-resolution mass spectrometry data was collected, and the molecular formula of the compounds in Alpinia officinalis medicinal materials was inferred by combining isotope analysis. The characteristic peaks were attributed by combining references and related reference substances. Among them, peaks 2, 4, 5, 7, and 8 were identified as protocatechuic acid, proanthocyanidin B1, catechin, proanthocyanidin B2, and epicatechin, respectively. The chromatographic peak corresponding to catechin was used as the reference peak. Within the range of ±10%, the relative retention times of the 8 characteristic peaks were as follows: 0.14 (peak 1), 0.38 (peak 2), 0.88 (peak 3), 0.92 (peak 4), 1.00 (peak 5), 1.61 (peak 6), 1.71 (peak 7), and 1.85 (peak 8). These characteristic peaks constructed the UPLC basic characteristic spectrum of Alpinia officinalis medicinal materials.

[0113] The UPLC basic characteristic spectra of 16 batches of Alpinia galanga were superimposed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)" issued by the Chinese Pharmacopoeia Committee (such as Figure 2 As shown), the consistency of the chromatographic peak similarity was examined and the similarity evaluation was performed. The results showed that S1 was 0.98, S2 was 0.99, S3 was 0.99, S4 was 0.99, S5 was 0.98, S6 was 0.99, S7 was 0.99, S8 was 0.99, S9 was 0.98, S10 was 0.99, S11 was 0.99, S12 was 0.98, S13 was 0.98, S14 was 0.99, S15 was 0.99, and S16 was 0.99 (all greater than 0.95), indicating that the similarity between the sample characteristic spectrum and the control spectrum was high, and the 8 characteristic peaks could stably appear in each sample with good consistency, so it can be used as the characteristic spectrum of Alpinia officinalis.

[0114] 6. Methodological investigation

[0115] (1) Investigation of extraction methods:

[0116] This study investigated the effects of different extraction methods on the UPLC characteristic spectrum of Alpinia officinalis in the preparation of the test solution. Ultrasonic treatment (power 250W, frequency 40kHz), heating reflux and shaking extraction methods were selected, and the extraction time was 30min. The peak area / sample weight of 8 characteristic peaks and the chromatograms were analyzed to evaluate the effects of different extraction methods on the UPLC characteristic spectrum of Alpinia officinalis. The results are shown in Table 2. Figure 3 and Figure 4 shown.

[0117] Table 2. Comparison of extraction efficiency of different extraction methods (peak area / sample weight)

[0118]

[0119] The results showed that the number of chromatographic peaks obtained by the three extraction methods was consistent. Taking into account the extraction efficiency of each chromatographic peak, heating reflux was finally selected as the preferred extraction method.

[0120] (2) Investigation of extraction time:

[0121] This study investigated the effect of different extraction times on the UPLC characteristic spectrum of Alpinia officinalis in the preparation of the test solution. Four different extraction times of 30 min, 60 min, 90 min and 120 min were selected. The extraction method was heating reflux extraction. The effect of different extraction times on the UPLC characteristic spectrum of Alpinia officinalis was evaluated by analyzing the peak area / sample weight of 8 characteristic peaks and comparing the chromatograms. The results are shown in Table 3. Figure 5 and Figure 6 shown.

[0122] Table 3. Comparison of extraction efficiency at different extraction times (peak area / sample weight)

[0123]

[0124] The results showed that the number of chromatographic peaks obtained at each extraction time was consistent. Taking into account the extraction efficiency of each chromatographic peak, 60 min was finally selected as the optimal extraction time.

[0125] (3) Determination of detection wavelength:

[0126] This study investigates the effect of different detection wavelengths on the UPLC characteristic spectrum of Alpinia officinalis in the ultra-high performance liquid chromatography analysis step. The spectrum with a wavelength range of 210-400nm was recorded to evaluate the effect of different detection wavelengths on the UPLC characteristic spectrum of Alpinia officinalis. The results are shown in Figure 2. Figure 7 and Figure 8 shown.

[0127] The results showed that under the wavelength of 215 nm, the responses of the various chromatographic peaks of the galangal medicinal material test solution were better, so 215 nm was selected as the preferred detection wavelength.

[0128] (4) Determination of chromatographic column:

[0129] This study investigated the effects of different chromatographic columns on the UPLC characteristic spectrum of Alpinia officinalis during the ultra-high performance liquid chromatography analysis step. The spectrum analyzed by Waters ACQUITY UPLC HSS T3 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) under gradient elution conditions (0-4 min 0% (A), 4-18 min 0% → 10% (A), 18-30 min 10% → 20% (A)) and the spectrum analyzed by Agilent InfinityLab Poroshell 120AQ-C18 (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm) under gradient elution conditions (0-5 min 0% (A), 5-20 min 0% → 10% (A), 20-30 min 10% → 18% (A)) were recorded to evaluate the effects of different chromatographic columns on the UPLC characteristic spectrum of Alpinia officinalis. The results are shown in Figure 2. Fig. 9 shown.

[0130] The results show that the baseline, peak shape, separation, symmetry and other aspects of the chromatographic peak analyzed under the conditions of AQ-C18 chromatographic column are all good, so AQ-C18 is selected as the preferred chromatographic column.

[0131] (5) Determination of elution conditions:

[0132] This study investigated the effects of different elution conditions on the UPLC characteristic spectrum of Alpinia officinalis in the ultra-high performance liquid chromatography analysis step. Acetonitrile (A)-0.1% v / v phosphoric acid aqueous solution (B) was used as the mobile phase, and the spectra under elution conditions 1-5 were recorded (Condition 1: 0-5 min 0% (A), 5-20 min 0%→10% (A), 20-30 min 10%→18% (A); Condition 2: 0-5 min 5% (A), 5-28 min 5%→9% (A), 28-35 min 9%→13% (A); Condition 3: 0-6 min 0%→3% (A), 6-30 min 3%→7% (A), 30-43 min 7%→12% (A); Condition 4: 0-5 min 0% (A), 5-10 min 0%→5% (A), 10-33 min 5%→8% (A); Condition 5: 0-5 min 3% (A), 5-6 min 3%→6%(A), 6~18min6%(A), 18~30min 6%→9%(A), 30~33min 9%(A)) were used to evaluate the effect of different elution conditions on the UPLC characteristic spectrum of Alpinia officinalis. The results are shown in Fig.10 shown.

[0133] The results showed that the target peak of condition 5 was clearer, with a better peak shape, better separation effect, no tailing phenomenon, and a stable baseline. Therefore, condition 5 was selected as the preferred elution condition.

[0134] Example 2: Method for constructing a supplementary characteristic profile of Alpinia officinalis

[0135] 1. Instruments, reagents and samples

[0136] (1) Instruments: Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters); Waters Empower 3 workstation (Waters); Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent); electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); HY-4 speed-adjustable multi-purpose oscillator (Jintan Kexing Instrument Factory); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); pure water system (Sartorius); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.).

[0137] (2) Reagents: acetonitrile (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Aladdin); ultrapure water; other reagents were of analytical grade.

[0138] (3) Samples: Reference substance: galangin (batch number: 111699-201703, CAS: 548-83-4) was purchased from the China Food and Drug Administration; galangin (batch number: 5238, CAS: 480-39-7) and galangin-3-methyl ether (batch number: 9507, CAS: 6665-74-3) were purchased from Shanghai Standard Standard Technical Services Co., Ltd. Test sample: Alpinia officinarum Hance was collected from Guangdong, Hainan and other regions and identified by Jiangyin Tianjiang Pharmaceutical Co., Ltd. as the dried rhizome of Alpinia officinarum Hance, a plant of the ginger family, with a total of 16 batches.

[0139] 2. Preparation of reference solution

[0140] Take appropriate amount of galangin, galangin-3-methyl ether reference substances, weigh accurately, and add methanol to prepare solutions containing 20 μg of each per 1 mL as reference substance solutions.

[0141] 3. Preparation of test solution

[0142] Take about 1g of Alpinia officinalis powder (passed through No. 4 sieve) (batch number: YC1805098), accurately weigh it, put it in a stoppered conical flask, accurately add 25mL of 70% methanol, seal it, weigh it, treat it with ultrasound (power 250W, frequency 40kHz) for 60min, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0143] 4. Ultra-high performance liquid chromatography analysis

[0144] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 RRHD chromatographic column (100 mm×2.1 mm, 1.8 μm); mobile phase: acetonitrile (mobile phase A)-0.1% v / v phosphoric acid aqueous solution (mobile phase B); flow rate: 0.4 mL / min; column temperature: 30°C; detection wavelength: 215 nm; gradient elution program as shown in Table 4. The theoretical plate number calculated based on the galangin peak should not be less than 5000.

[0145] Table 4. Gradient elution table

[0146]

[0147] Under this condition, 1 μL of the reference solution and 2 μL of the test solution were accurately aspirated and injected into the ultra-high performance liquid chromatograph to obtain the chromatograms of the reference solution and each test solution (galangal medicinal material). Fig.11 and Fig.12 shown.

[0148] 5. Analyze the spectrum

[0149] According to the method in this embodiment, 16 batches of galangal medicinal materials were subjected to ultra-high performance liquid chromatography analysis to obtain ultra-high performance liquid chromatograms, which were introduced into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)" promulgated by the State Pharmacopoeia Committee, and the time window width was selected as 0.1 min. The median was used to generate a reference spectrum, and after multi-point correction, the Mark peaks were matched to generate the common mode of the UPLC basic characteristic spectrum of galangal medicinal materials (see Fig.11 UPLC spectrum of Alpinia officinalis), which contains 11 characteristic peaks. Using LC-MS technology, according to the liquid chromatography conditions of the basic characteristic spectrum of Alpinia officinalis medicinal materials, high-resolution mass spectrometry data was collected, and the molecular formula of the compounds in Alpinia officinalis medicinal materials was inferred by combining isotope analysis. Combined with reference literature and related reference substances, the characteristic peaks were attributed. Among them, peak 4, peak 5, and peak 7 were identified as pinocytol, galangin, and galangin-3-methyl ether, respectively. Taking the chromatographic peak corresponding to galangin as the reference peak, within the range of ±5%, the relative retention times of the 11 characteristic peaks were as follows: 0.48 (peak 1), 0.82 (peak 2), 0.86 (peak 3), 0.97 (peak 4), 1.00 (peak 5), 1.03 (peak 6), 1.11 (peak 7), 1.41 (peak 8), 1.46 (peak 9), 1.50 (peak 10), and 1.88 (peak 11). These characteristic peaks constructed the UPLC supplementary characteristic map of galangal medicinal materials.

[0150] The UPLC supplementary characteristic spectra of 16 batches of Alpinia galanga were superimposed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)" issued by the Chinese Pharmacopoeia Committee (e.g. Fig.12 As shown), the consistency of the chromatographic peak similarity was investigated and the similarity evaluation was performed. The results showed that S1 was 0.94, S2 was 0.95, S3 was 0.96, S4 was 0.99, S5 was 0.98, S6 was 0.96, S7 was 0.99, S8 was 0.96, S9 was 0.98, S10 was 0.99, S11 was 0.97, S12 was 0.98, S13 was 0.98, S14 was 0.99, S15 was 0.95, and S16 was 0.99 (all greater than 0.90), indicating that the similarity between the sample characteristic spectrum and the control spectrum was high, and the 11 characteristic peaks could stably appear in each sample with good consistency, so it can be used as the characteristic spectrum of Alpinia officinalis.

[0151] 6. Methodological investigation

[0152] (1) Investigation of extraction solvent:

[0153] This study investigated the effects of different extraction solvents on the UPLC characteristic spectrum of Alpinia officinalis during the preparation of the test solution. In terms of volume percentage, 30% methanol-water solution, 50% methanol-water solution, 70% methanol-water solution, methanol and water were selected as extraction solvents. The effects of different extraction solvents on the UPLC characteristic spectrum of Alpinia officinalis were evaluated by analyzing the peak area / sample weight of 11 characteristic peaks and comparing the chromatograms. The results are shown in the figure below. Fig.13 and Fig.14 shown.

[0154] The results show that when methanol and 70% methanol are used as extraction solvents, the extraction efficiency is higher. Considering the reduction of the amount of organic reagents, 70% methanol is selected as the preferred extraction solvent.

[0155] (2) Investigation of extraction methods:

[0156] This study investigated the effects of different extraction methods on the UPLC characteristic spectrum of Alpinia officinalis in the preparation of the test solution. Ultrasonic treatment (power 250W, frequency 40kHz), heating reflux and shaking extraction methods were selected, and the extraction time was 60min. The peak area / sample weight of 11 characteristic peaks and the chromatograms were analyzed to evaluate the effects of different extraction methods on the UPLC characteristic spectrum of Alpinia officinalis. The results are as follows: Fig.15 and Fig.16 shown.

[0157] The results showed that the number of chromatographic peaks obtained by the three extraction methods was consistent and the extraction efficiency was relatively close. Considering that ultrasonic treatment was more convenient, ultrasonic extraction was selected as the preferred extraction method.

[0158] (3) Investigation of extraction time:

[0159] This study investigated the effect of different extraction times on the UPLC characteristic spectrum of Alpinia officinalis in the preparation of the test solution. Four different extraction times of 30 min, 60 min, 90 min and 120 min were selected, and the extraction method was ultrasonic treatment (power 250 W, frequency 40 kHz). The peak area / sample weight of 11 characteristic peaks were analyzed and the chromatograms were compared to evaluate the effect of different extraction times on the UPLC characteristic spectrum of Alpinia officinalis. The results are as follows: Fig.17 and Fig.18 shown.

[0160] The results showed that the number of chromatographic peaks obtained at each extraction time was consistent and the extraction efficiency was relatively close, indicating that complete extraction was achieved at 30 minutes. In order to ensure sufficient extraction, the ultrasonic extraction time was finally selected as 60 minutes.

[0161] (4) Investigation of detection wavelength:

[0162] This study investigates the effect of different detection wavelengths on the UPLC characteristic spectrum of Alpinia officinalis in the ultra-high performance liquid chromatography analysis step. The spectrum with a wavelength range of 190 to 400 nm was recorded to evaluate the effect of different detection wavelengths on the UPLC characteristic spectrum of Alpinia officinalis. The results are shown in Figure 2. Fig.19 and Fig. 20 shown.

[0163] The results showed that under the wavelength of 215 nm, the response of each chromatographic peak of the galangal medicinal material test solution was better, and the separation of each chromatographic peak was better, so 215 nm was selected as the preferred detection wavelength.

[0164] (5) Investigation of elution conditions:

[0165] This study investigated the effects of different elution conditions on the UPLC characteristic spectrum of Alpinia officinalis in the ultra-high performance liquid chromatography analysis step. Acetonitrile (A)-0.1% v / v phosphoric acid aqueous solution (B) was used as the mobile phase, and the spectra under elution conditions 1-3 were recorded (Condition 1: 0-4 min 40%→45% (A), 4-7 min 45%→50% (A), 7-9 min 50%→70% (A), 9-12 min 70%→100% (A); Condition 2: 0-8 min 35%→45% (A), 8-10 min 45%→50% (A), 10-12 min 50%→70% (A), 12-14 min 70%→100% (A); Condition 3: 0-13 min 28%→50% (A), 13-18 min 50%→100% (A), 18-22 min 100% (A)) to evaluate the effects of different elution conditions on the UPLC characteristic spectra of Alpinia officinalis. The results are shown in Figure 2. Fig.21 shown.

[0166] The results showed that the target peak of condition 3 was clearer, with a better peak shape, better separation effect, no tailing phenomenon, and a stable baseline. Therefore, condition 3 was selected as the preferred elution condition.

[0167] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention's patent shall be based on the claims.

Claims

1. A method for constructing a characteristic spectrum of non-volatile components of an Alpinia officinalis sample, comprising the following steps: 1) Preparation of basic test solution: Take a sample of Alpinia officinalis, crush it and weigh it accurately, add water accurately and extract it, make up the weight loss with water, take the filtrate, and obtain the basic test solution; the sample is selected from medicinal materials, decoction pieces, standard decoctions and formula granules, preferably medicinal materials; 2) Preparation of basic reference substance solution: taking appropriate amounts of basic reference substances, respectively dissolving them in solvents to obtain the basic reference substance solution; preferably, the basic reference substance is selected from at least one of procyanidin B1, procyanidin B2, catechin, epicatechin and protocatechuic acid, preferably procyanidin B1, procyanidin B2, catechin, epicatechin and protocatechuic acid; 3) Establishment of basic characteristic spectrum: The basic test solution prepared in step 1) and the basic reference solution prepared in step 2) are respectively subjected to ultra-high performance liquid chromatography analysis to obtain the basic characteristic spectrum; preferably, the ultra-high performance liquid chromatography includes the following chromatographic conditions: using a surface porous filler based on octadecylsilane bonded silica gel as the stationary phase; using acetonitrile-phosphoric acid aqueous solution as the mobile phase, and using gradient elution as the elution method.

2. The method for constructing a characteristic map according to claim 1, characterized in that: The extraction method in step 1) is selected from ultrasonic extraction, heating reflux extraction and shaking extraction, preferably heating reflux extraction; and / or, the extraction time is 30-120 min, preferably 60-90 min, more preferably 60 min; and / or, In step 1), the dosage ratio of the sample to water is 1 g:25-50 mL, preferably 1 g:25 mL.

3. The method for constructing a characteristic spectrum according to claim 1 or 2, characterized in that: In step 2), the solvent is an alcohol, preferably methanol; and / or, The concentrations of procyanidin B1, procyanidin B2, catechin, epicatechin and protocatechuic acid in the basic reference solution in step 2) are 20-60 μg / mL, preferably 60 μg / mL, 40 μg / mL, 50 μg / mL, 30 μg / mL and 20 μg / mL, respectively.

4. The method for constructing a characteristic spectrum according to any one of claims 1 to 3, characterized in that: The ultra-high performance liquid chromatography in step 3) includes the following chromatographic conditions: an Agilent InfinityLabPoroshell 120AQ-C18 chromatographic column as the stationary phase; acetonitrile-0.1% v / v phosphoric acid aqueous solution as the mobile phase; the gradient elution program is as follows: 0-5min, the volume percentage of acetonitrile is 3%; 5-6min, the volume percentage of acetonitrile is 3%→6%; 6-18min, the volume percentage of acetonitrile is 6%; 18-30min, the volume percentage of acetonitrile is 6%→9%; 30-33min, the volume percentage of acetonitrile is 9%; Preferably, the ultra-high performance liquid chromatography also includes the following chromatographic conditions: flow rate of 0.25-0.35 mL / min, preferably 0.3 mL / min; column temperature of 30-40° C., preferably 35° C.; detection wavelength of 215 nm.

5. The method for constructing a characteristic spectrum according to any one of claims 1 to 4, characterized in that: The basic characteristic spectrum in step 3) contains at least 8, preferably 8 characteristic peaks; preferably, taking the chromatographic peak corresponding to catechin as the reference peak, the relative retention times of the 8 characteristic peaks within the range of ±10% are as follows: 0.14 (peak 1), 0.38 (peak 2), 0.88 (peak 3), 0.92 (peak 4), 1.00 (peak 5), 1.61 (peak 6), 1.71 (peak 7) and 1.85 (peak 8), among which: peak 2 is protocatechuic acid, peak 4 is procyanidin B1, peak 5 is catechin, peak 7 is procyanidin B2, and peak 8 is epicatechin.

6. The method for constructing a characteristic spectrum according to any one of claims 1 to 5, characterized in that: It also includes the following steps: 1') Preparation of a supplementary test solution: taking the same galangal sample as in claim 1, optionally crushing it and accurately weighing it, accurately adding an extraction solvent and then extracting it, using the extraction solvent to make up for the loss in weight, and taking the filtrate to obtain the supplementary test solution; 2') Preparation of supplementary reference substance solution: taking appropriate amounts of supplementary reference substances, respectively dissolving them in solvents to obtain the supplementary reference substance solution; preferably, the supplementary reference substance is selected from at least one of galangin, galangin-3-methyl ether and pinocytol, preferably galangin, galangin-3-methyl ether and pinocytol; 3') Establishment of a supplementary characteristic spectrum: The supplementary test solution prepared in step 1') and the supplementary reference solution prepared in step 2') are respectively subjected to ultra-high performance liquid chromatography analysis to obtain the supplementary characteristic spectrum; preferably, the ultra-high performance liquid chromatography comprises the following chromatographic conditions: a fully porous filler based on octadecylsilane bonded silica gel is used as the stationary phase; an acetonitrile-phosphoric acid aqueous solution is used as the mobile phase; The elution method adopted was gradient elution.

7. The method for constructing a characteristic map according to claim 6, characterized in that: In step 1'), the extraction solvent is selected from alcohol and an aqueous solution of alcohol; Preferably, the alcohol is methanol; and / or, Preferably, the volume percentage of alcohol in the aqueous solution of alcohol is 30%-70%, preferably 70%; and / or, The extraction method in step 1') is selected from ultrasonic extraction, reflux extraction and shaking extraction, preferably ultrasonic extraction; and / or, the extraction time is 30-120 min, preferably 60-90 min, more preferably 60 min; and / or, In step 1'), the ratio of the sample to the extraction solvent is 1 g:25-50 mL, preferably 1 g:25 mL.

8. The method for constructing a characteristic spectrum according to claim 6 or 7, characterized in that: In step 2'), the solvent is an alcohol, preferably methanol; and / or, The concentration of galangin, galangin-3-methyl ether and galangin in the supplementary reference solution in step 2') is 10-30 μg / mL, preferably 20 μg / mL.

9. The method for constructing a characteristic spectrum according to any one of claims 6 to 8, characterized in that: The ultra-high performance liquid chromatography in step 3') includes the following chromatographic conditions: an Agilent ZORBAX Eclipse Plus C18 RRHD column as the stationary phase; acetonitrile-0.1% v / v phosphoric acid aqueous solution as the mobile phase; the gradient elution program is as follows: 0-13min, the volume percentage of acetonitrile is 28%→50%; 13-18min, the volume percentage of acetonitrile is 50%→100%; 18-22min, the volume percentage of acetonitrile is 100%; Preferably, the ultra-high performance liquid chromatography also includes the following chromatographic conditions: flow rate of 0.35-0.45 mL / min, preferably 0.4 mL / min; column temperature of 25-35° C., preferably 30° C.; detection wavelength of 215 nm.

10. The method for constructing a characteristic spectrum according to any one of claims 6 to 9, characterized in that: The supplementary characteristic spectrum in step 3') comprises at least 11, preferably 11 characteristic peaks; preferably, taking the chromatographic peak corresponding to galangin as the reference peak, within the range of ±5%, the relative retention times of the 11 characteristic peaks are as follows: 0.48 (peak 1), 0.82 (peak 2), 0.86 (peak 3), 0.97 (peak 4), 1.00 (peak 5), 1.03 (peak 6), 1.11 (peak 7), 1.41 (peak 8), 1.46 (peak 9), 1.50 (peak 10) and 1.88 (peak 11), where: Peak 4 is pinocytol, Peak 5 is galangin, and Peak 7 is galangin-3-methyl ether.

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