A characteristic map construction method for quality control of non-volatile components of high galangal samples

Abstract

This invention belongs to the field of traditional Chinese medicine analysis technology, and relates to a method for constructing characteristic chromatograms for the quality control of non-volatile components in Alpinia galanga samples. The construction of the basic characteristic chromatogram includes the following steps: 1) preparation of a basic test solution; 2) preparation of a basic reference solution; 3) performing UPLC analysis on the basic test solution and the basic reference solution respectively to construct the basic characteristic chromatogram. Furthermore, the method also includes the construction of supplementary characteristic chromatograms. The constructed characteristic chromatograms can comprehensively reflect the characteristics of Alpinia galanga samples, avoiding the shortcomings of measuring only a single or a few peaks, and providing a new analytical means for the intrinsic quality control of Alpinia galanga samples.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, and relates to a method for constructing characteristic spectra for the quality control of non-volatile components in Alpinia galanga samples (especially Alpinia galanga medicinal materials). Background Technology

[0002] Galangal is the dried rhizome of *Alpinia officinarum* Hance, a plant in the ginger family. It is also known as small galangal, galangal, and wild ginger. First recorded in *Mingyi Bielu* (Records of Famous Physicians), it is included in all editions of the *Chinese Pharmacopoeia*. It is warm in nature, pungent in taste, and enters the spleen and stomach meridians. It has the effects of warming the stomach and relieving vomiting, dispelling cold and relieving pain. Clinically, it is often used to treat symptoms such as cold pain in the epigastrium and abdomen, belching and acid reflux, and vomiting due to stomach cold. [1] Galangal is rich in nutrients, containing protein, polysaccharides, trace elements, and other components. [2] Alpinia galanga is one of the traditional Chinese medicines listed by the Ministry of Health as both food and medicine. As a seasoning, it is widely used in the preparation of folk delicacies and cooked foods, and has a large market both domestically and internationally. Modern research shows that Alpinia galanga has strong pharmacological activity, possessing 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 galangal are mainly divided into two categories: volatile oils (or volatile components) and non-volatile components. For volatile oils, the composition varies depending on the extraction method and origin, making it difficult to incorporate into quality control. Non-volatile components, based on polarity, can be broadly divided into water-soluble and alcohol-soluble components. Among these, alcohol-soluble components (mainly flavonoids and diphenylheptane compounds)... [4] (This has always been one of the main research directions for the quality control of Alpinia galanga.)

[0004] Studies on the characteristic fingerprint spectra of non-volatile components of Alpinia galanga mainly include: Ma Liping [5] High-performance liquid chromatography (HPLC) was used to establish the HPLC fingerprint of galangal slices. Simultaneously, the content of galangin in the samples was determined, nine common peaks were identified, and a common chromatogram of ethanol extracts from 14 batches of galangal was established. (Han Liang) [6] The fingerprint spectra of 10 batches of Alpinia galanga methanol extract were analyzed by HPLC, and 10 common peaks were identified. (Long Qin) [7] Eleven batches of characteristic chromatograms from different cultivated varieties of Alpinia galanga (Zanthoxylum bungeanum and Zingiber officinale) were compared using HPLC, and twelve common peaks of the methanol extract of Alpinia galanga were identified. Three chemical components, diphenylheptane A, galangin, and kaempferol, were identified using reference standards. (Deng Yifeng) [8]Fingerprints of 12 batches of Alpinia galanga were constructed using HPLC, and 10 characteristic peaks of the ethanol extract of Alpinia galanga were identified. Galangin was then identified using reference standards. (Wang Lifeng) [9] A method for HPLC fingerprint analysis of Alpinia galanga was established, and 13 common peaks in the ethanol fraction of Alpinia galanga were identified. (Cui Weili)

[10] Using HPLC, 15 batches of Alpinia galanga medicinal materials were analyzed, and 6 common peaks of the petroleum ether extract of Alpinia galanga medicinal materials were identified as fingerprint characteristic peaks. Diphenylheptane A was identified by reference standard.

[0005] The Chinese Pharmacopoeia (2020 edition) lists galangin as one of the quality evaluation indicators for galangal. However, a single indicator component cannot comprehensively evaluate quality. Most existing literature reports characteristic chromatograms only show the chemical components (diphenylheptane A, galangin, kaempferol) in the alcohol extract of galangal, and only identify one or a few common chromatographic peaks, failing to show water-soluble components. Since galangal also contains a large number of water-soluble components, characterizing galangal solely based on fat-soluble or alcohol-soluble components is not comprehensive.

[0006] Therefore, it is necessary to construct a characteristic spectral method for the quality control of non-volatile components in Alpinia galanga samples (especially Alpinia galanga medicinal materials) in order to comprehensively reflect the water-soluble and alcohol-soluble components in Alpinia galanga samples and provide a comprehensive quality control means to standardize the uniformity and stability of the intrinsic quality of Alpinia galanga samples.

[0007] References

[0008] [1] National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China, 2020 Edition, Part I [S]. Beijing: China Medical Science and Technology Press.

[0009] Society, 2020:300.

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

[0011] [3] Huang Huizhen, Yang Dan. Research progress on chemical constituents and pharmacological activities of Alpinia galanga [J]. Guangdong Chemical Industry, 2009, 36(1):77-80.

[0012] [4] Lü Wei, Jiang Linghuo. Chemical constituents and pharmacological effects of Alpinia galanga [J]. China Pharmaceutical Industry, 2006, 15(3):19-21.

[0013] [5] Ma Liping, Qiu Xiaohui, Min Jiang. Study on HPLC fingerprint of galangal slices and determination of galangin content [J]. Chinese Traditional and Herbal Drugs, 2008, 30(11):1565-1569.

[0014] [6] Han Liang, Shi Zhongfeng, Rui Wen, et al. Characteristic spectral analysis and component identification of galangal flavonoid extract [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2011, 17(21):82-85.

[0015] [7] Long Qin, Lin Dingguang, Hu Jiali, et al. Study on HPLC fingerprint of different cultivated varieties of Alpinia galanga and determination of index components [J]. Journal of Guangzhou University of Chinese Medicine, 2019, 36(1):109-114.

[0016] [8] Deng Yifeng, Feng Lina, Luo Hui. Study on high phase liquid chromatography fingerprint of authentic Alpinia galanga medicinal material [J]. Chinese Medicinal Herbs, 2011, 34(9):1351-1355.

[0017] [9] Wang Lifeng, Peng Jingdong, Wang Ning. Study on high performance liquid fingerprint of Alpinia galanga [J]. Journal of Southwest Normal University (Natural Science Edition), 2008, 33(6):33-38.

[0018]

[10] Cui Weili, Feng Yifan, Guo Xiaoling, et al. Determination of diphenylheptane A content and fingerprint spectroscopy in Alpinia galanga [J]. Journal of Guangdong Pharmaceutical University, 2007, 23(6):635-637. Summary of the Invention

[0019] The problem the invention aims to solve

[0020] To address the problems existing in the prior art, the purpose of this invention is to establish a characteristic spectrum for the quality control of non-volatile components in Alpinia galanga samples (especially Alpinia galanga medicinal materials), simultaneously reflecting both water-soluble and alcohol-soluble components. This elevates the quality control of Alpinia galanga samples from the determination of a few common peaks to a comprehensive grasp of the intrinsic quality of the samples. It can more comprehensively reflect the characteristics of Alpinia galanga samples, avoiding the shortcomings of determining a single or a few peaks, and providing a new analytical method for the intrinsic quality control of Alpinia galanga samples.

[0021] Solution for solving the problem

[0022] This invention provides a method for constructing characteristic spectra of non-volatile components in galangal samples, comprising the following steps:

[0023] 1) Preparation of the basic test solution: Take a sample of Alpinia galanga, crush it accurately, add water accurately and extract it, make up the weight loss with water, take the filtrate to obtain the basic test solution.

[0024] 2) Preparation of basic reference solutions: Take appropriate amounts of each basic reference standard, dissolve them separately in solvent to obtain the basic reference solution;

[0025] 3) Establishment of basic characteristic chromatograms: The basic test solution prepared in step 1) and the basic reference solution prepared in step 2) are subjected to ultra-high performance liquid chromatography analysis to obtain the basic characteristic chromatograms.

[0026] In step 1), the sample is selected from medicinal materials, processed medicinal slices, standard decoctions and formula granules, with medicinal materials being preferred.

[0027] Preferably, the extraction method in step 1) is selected from ultrasonic extraction, heating reflux extraction and shaking extraction, with heating reflux extraction being preferred.

[0028] Preferably, the extraction time in step 1) is 30-120 min, more preferably 60-90 min, and even more preferably 60 min.

[0029] Preferably, the ratio of sample to water in step 1) is 1g:25-50mL, more preferably 1g:25mL.

[0030] Preferably, the basic reference standard in step 2) is selected from at least one of proanthocyanidin B1, proanthocyanidin B2, catechin, epicatechin and protocatechuic acid, and more preferably proanthocyanidin B1, proanthocyanidin B2, catechin, epicatechin and protocatechuic acid.

[0031] Preferably, the solvent in step 2) is an alcohol, and more preferably methanol.

[0032] Preferably, the concentrations of proanthocyanidins B1, proanthocyanidins B2, catechins, epicatechins, and protocatechuic acid in the basic reference solution in step 2) are 20-60 μg / mL.

[0033] More preferably, the concentrations of proanthocyanidin B1, proanthocyanidin 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.

[0034] Preferably, the ultra-high performance liquid chromatography in step 3) includes the following chromatographic conditions: using a surface porous packing material based on octadecylsilane bonded silica as the stationary phase; using acetonitrile-phosphoric acid aqueous solution as the mobile phase; and employing gradient elution.

[0035] More preferably, the ultra-high performance liquid chromatography in step 3) includes the following chromatographic conditions: using an Agilent InfinityLab Poroshell 120AQ-C18 column as the stationary phase; using acetonitrile-0.1% v / v phosphoric acid aqueous solution as the mobile phase; the gradient elution program is as follows: 0-5 min, acetonitrile volume percentage is 3%; 5-6 min, acetonitrile volume percentage is 3% → 6%; 6-18 min, acetonitrile volume percentage is 6%; 18-30 min, acetonitrile volume percentage is 6% → 9%; 30-33 min, acetonitrile volume percentage is 9%.

[0036] More preferably, the ultra-high performance liquid chromatography in step 3) further includes the following chromatographic conditions:

[0037] The flow rate is 0.25-0.35 mL / min, preferably 0.3 mL / min;

[0038] The column temperature is 30-40℃, preferably 35℃;

[0039] The detection wavelength is 215nm.

[0040] Preferably, the basic feature map in step 3) contains at least 8 feature peaks.

[0041] More preferably, the basic characteristic chromatogram described in step 3) contains 8 characteristic peaks. Taking the chromatographic peak corresponding to catechin as the reference peak, the relative retention times of the 8 characteristic peaks within ±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), wherein: peak 2 is protocatechuic acid, peak 4 is proanthocyanidin B1, peak 5 is catechin, peak 7 is proanthocyanidin B2, and peak 8 is epicatechin.

[0042] Preferably, the feature map construction method further includes the following steps:

[0043] 1') Preparation of supplementary test solution: Take the same Alpinia galanga sample as in the basic feature map construction method, select one, crush it accurately, add extraction solvent accurately and extract, use extraction solvent to make up the weight loss, take the filtrate to obtain the supplementary test solution;

[0044] 2') Preparation of supplementary reference solutions: Take appropriate amounts of each supplementary reference standard, dissolve them separately in solvent to obtain the supplementary reference solution;

[0045] 3') Establishment of supplementary characteristic chromatograms: The supplementary test solution prepared in step 1') and the supplementary reference solution prepared in step 2') are subjected to ultra-high performance liquid chromatography analysis to obtain the supplementary characteristic chromatograms.

[0046] Preferably, the extraction solvent in step 1') is selected from alcohols and aqueous solutions of alcohols.

[0047] More preferably, the alcohol is methanol.

[0048] More preferably, the volume percentage of alcohol in the aqueous solution of the alcohol is 30%-70%, preferably 70%.

[0049] Preferably, the extraction method described in step 1') is selected from ultrasonic extraction, heating reflux extraction and shaking extraction, with ultrasonic extraction being preferred.

[0050] Preferably, the extraction time in step 1') is 30-120 min, more preferably 60-90 min, and even more preferably 60 min.

[0051] Preferably, the ratio of sample to extraction solvent in step 1') is 1g:25-50mL, more preferably 1g:25mL.

[0052] Preferably, the supplementary reference standard in step 2') is selected from at least one of galangin, galangin-3-methyl ether and galangin, with galangin, galangin-3-methyl ether and galangin being the most preferred.

[0053] Preferably, the solvent in step 2') is an alcohol, and more preferably methanol.

[0054] Preferably, the concentrations of galangin, galangin-3-methyl ether, and galangin in the supplementary reference solution in step 2') are 10-30 μg / mL, more preferably 20 μg / mL.

[0055] Preferably, the ultra-high performance liquid chromatography in step 3') includes the following chromatographic conditions: using a fully porous packing material based on octadecylsilane-bonded silica gel as the stationary phase; using acetonitrile-phosphoric acid aqueous solution as the mobile phase; and employing gradient elution.

[0056] More preferably, the ultra-high performance liquid chromatography in step 3') includes the following chromatographic conditions: using an Agilent ZORBAX Eclipse Plus C18 RRHD column as the stationary phase; using acetonitrile-0.1% v / v phosphoric acid aqueous solution as the mobile phase; and the gradient elution program is as follows: 0-13 min, acetonitrile volume percentage 28% → 50%; 13-18 min, acetonitrile volume percentage 50% → 100%; 18-22 min, acetonitrile volume percentage 100%.

[0057] More preferably, the ultra-high performance liquid chromatography in step 3') further includes the following chromatographic conditions:

[0058] The flow rate is 0.35-0.45 mL / min, preferably 0.4 mL / min;

[0059] The column temperature is 25-35℃, preferably 30℃;

[0060] The detection wavelength is 215nm.

[0061] Preferably, the supplementary feature map described in step 3') contains at least 11 feature peaks.

[0062] More preferably, the supplementary characteristic spectrum described in step 3') contains 11 characteristic peaks. Taking the chromatographic peak corresponding to galangin as the reference peak, the relative retention times of the 11 characteristic peaks within ±5% 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 galangin, peak 5 is galangin, and peak 7 is galangin-3-methyl ether.

[0063] The effects of the invention

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] (1) This invention establishes a characteristic spectrum for the quality control of non-volatile components in Alpinia galanga samples (especially Alpinia galanga medicinal materials) by using ultra-high performance liquid chromatography. The basic characteristic spectrum reflects water-soluble components and identifies 8 characteristic peaks. The supplementary characteristic spectrum reflects alcohol-soluble components and identifies 11 characteristic peaks. This improves the quality control of Alpinia galanga samples from the original determination of the content of a single component or a few common peaks to the control of the intrinsic quality of the entire Alpinia galanga sample. It can more comprehensively reflect the characteristics of Alpinia galanga samples and avoid the defects of single or a few peak determinations. It can provide a new analytical means for the intrinsic quality control of Alpinia galanga samples.

[0066] (2) The method of the present invention is simple, reproducible, accurate and reliable, easy to operate, saves time, consumes less solvent and causes less environmental pollution compared with HPLC. Attached Figure Description

[0067] Figure 1 The UPLC basic characteristic spectrum of Alpinia galanga medicinal material is shown, where: 2 is protocatechuic acid, 4 is proanthocyanidin B1, 5 is catechin, 7 is proanthocyanidin B2, and 8 is epicatechin.

[0068] Figure 2 The UPLC basic characteristic maps (S1~S16) of 16 batches of Alpinia galanga medicinal materials constructed according to Example 1 are shown.

[0069] Figure 3 UPLC basic characteristic maps of Alpinia galanga under different extraction methods.

[0070] Figure 4 The results of the investigation on the extraction method factors in the construction of the basic characteristic map of Alpinia galanga medicinal material.

[0071] Figure 5 UPLC basic characteristic maps of Alpinia galanga under different extraction time conditions.

[0072] Figure 6 The results of the investigation on the extraction time factor in the construction method of the basic characteristic map of Alpinia galanga medicinal material.

[0073] Figure 7 This is a 3D UPLC basic feature map of Alpinia galanga under full wavelength scanning.

[0074] Figure 8 The basic UPLC characteristic spectrum of Alpinia galanga under 215 nm wavelength conditions.

[0075] Figure 9 The basic UPLC characteristic spectra of Alpinia galanga under different column conditions.

[0076] Figure 10 UPLC basic characteristic spectra of Alpinia galanga under different elution conditions.

[0077] Figure 11 Supplementary UPLC characterization of Alpinia galanga medicinal material, wherein: 4 represents galpin, 5 represents galangin, and 7 represents galangin-3-methyl ether.

[0078] Figure 12 Supplementary UPLC characterization maps (S1-S16) of 16 batches of Alpinia officinarum medicinal materials constructed according to Example 2.

[0079] Figure 13 Supplementary UPLC characteristic spectra of Alpinia galanga under different extraction solvent conditions.

[0080] Figure 14 Results of the investigation on the extraction solvent factor in the method for constructing supplementary characteristic maps of Alpinia galanga.

[0081] Figure 15 Supplementary UPLC feature maps of Alpinia galanga under different extraction methods.

[0082] Figure 16 Results of an investigation into the extraction method factors in the construction of supplementary feature maps for Alpinia galanga medicinal materials.

[0083] Figure 17Supplementary UPLC feature maps for Alpinia galanga under different extraction time conditions.

[0084] Figure 18 Results of the investigation on the extraction time factor in the method for constructing supplementary feature maps of Alpinia galanga.

[0085] Figure 19 This provides supplementary feature maps for 3D UPLC analysis of Alpinia galanga under full-wavelength scanning.

[0086] Figure 20 Supplementary UPLC characteristic spectra of Alpinia galanga under 215 nm wavelength conditions.

[0087] Figure 21 Supplementary UPLC characteristic maps of Alpinia galanga under different elution conditions. Detailed Implementation

[0088] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific experimental conditions and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0089] Example 1: Construction of basic characteristic map of Alpinia galanga medicinal material

[0090] 1. Instruments, reagents and samples

[0091] (1) Instruments: Waters Acquity UPLC (Waters Corporation); Waters Empower3 workstation (Waters Corporation); Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent Corporation); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent Corporation); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); HY-4 variable speed multi-purpose shaker (Jintan Kexing Instrument Factory); temperature-controlled water bath (Nantong Huatai Experimental Instruments Co., Ltd.); pure water system (Sartorius Corporation); AS165W centrifuge (Asia Pacific (Shanghai) Trading Co., Ltd.).

[0092] (2) Reagents: Acetonitrile (chromatographic grade, Thermo Fisher Scientific); phosphoric acid (chromatographic grade, Aladdin Scientific); water was ultrapure water; other reagents were all analytical grade.

[0093] (3) Samples: Reference standards: 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 the China National Institutes for Food and Drug Control; proanthocyanidin B1 (number: 10439, CAS: 20315-25-7), proanthocyanidin B2 (number: 10269, CAS: 29106-49-8), purchased from Shanghai Shidande Standard Technical Service Co., Ltd. Test sample: Alpinia officinarum medicinal materials were collected from Guangdong, Hainan and other regions, and identified by Jiangyin Tianjiang Pharmaceutical Co., Ltd. as the dried rhizomes of Alpinia officinarum Hance, a plant of the ginger family, totaling 16 batches.

[0094] 2. Preparation of reference solution

[0095] Take appropriate amounts of protocatechuic acid, proanthocyanidin B1, catechin, proanthocyanidin B2, and epicatechin reference standards, accurately weigh them, and add methanol to prepare solutions containing 20 μg of protocatechuic acid, 60 μg of proanthocyanidin B1, 50 μg of catechin, 40 μg of proanthocyanidin B2, and 30 μg of epicatechin per 1 mL, as reference solutions.

[0096] 3. Preparation of the test solution

[0097] Take about 1g of Alpinia officinarum powder (passed through a No. 4 sieve) (batch number: YC1805098), weigh it accurately, place it in a stoppered conical flask, add 25mL of water accurately, seal tightly, heat under reflux for 60min, cool, weigh it again, make up the lost weight with water, shake well, filter, and collect the filtrate to obtain the product.

[0098] 4. Ultra-high performance liquid chromatography analysis

[0099] Chromatographic column: Agilent InfinityLab Poroshell 120AQ-C18 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℃; detection wavelength: 215 nm; gradient elution program is shown in Table 1. The theoretical plate number, calculated based on the catechin peak, should be no less than 5000.

[0100] Table 1. Gradient Elution Table

[0101]

[0102] Under these conditions, accurately pipette 1 μL of the reference solution and 2 μL of the test solution, and inject them separately into the ultra-high performance liquid chromatograph to obtain the chromatograms of the reference solution and each test sample (Alpinia galanga medicinal material), as shown in the figure below. Figure 1 and Figure 2 As shown.

[0103] 5. Analyze the spectrum

[0104] Following the method described in this embodiment, 16 batches of Alpinia galanga medicinal materials were analyzed by ultra-high performance liquid chromatography (UPLC). The UPLC chromatograms were obtained and imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (Version 2012.0)" issued by the National Pharmacopoeia Commission. A time window width of 0.1 min was selected, and a control chromatogram was generated using the median. After multi-point correction and Mark peak matching, a common pattern of the basic characteristic UPLC chromatogram of Alpinia galanga medicinal materials was generated (see details). Figure 1 The UPLC chromatogram of Alpinia galanga was obtained, containing eight characteristic peaks. High-resolution mass spectrometry (HPLC) data were acquired using HPLC conditions based on the basic characteristic chromatogram of Alpinia galanga. The molecular formulas of the compounds in Alpinia galanga were deduced by isotope analysis, and the characteristic peaks were assigned based on references and relevant standards. Peaks 2, 4, 5, 7, and 8 were identified as protocatechuic acid, proanthocyanidin B1, catechin, proanthocyanidin B2, and epicatechin, respectively. Using the chromatographic peak corresponding to catechin as a reference peak, the relative retention times of the eight characteristic peaks within ±10% 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 constituted the basic UPLC characteristic chromatogram of Alpinia galanga.

[0105] The UPLC basic characteristic spectra of 16 batches of Alpinia galanga medicinal materials were overlaid using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (Version 2012.0)" promulgated by the National Pharmacopoeia Commission (e.g., ...). Figure 2 As shown in the figure, the consistency of chromatographic peak similarity was examined, and 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 chromatogram and the control chromatogram was high. The eight characteristic peaks appeared stably in each sample and had good agreement. Therefore, they can be used as characteristic chromatograms of Alpinia officinarum.

[0106] 6. Methodological Examination

[0107] (1) Examination of extraction methods:

[0108] This study investigated the effects of different extraction methods on the UPLC characteristic spectra of Alpinia galanga during the preparation of the test solution. Ultrasonic extraction (250 W, 40 kHz), reflux extraction, and shaking extraction were used, with an extraction time of 30 min. The effects of different extraction methods on the UPLC characteristic spectra of Alpinia galanga were evaluated by analyzing the peak area / sample weight of eight characteristic peaks and comparing chromatograms. The results are shown in Table 2. Figure 3 and Figure 4 As shown.

[0109] Table 2. Comparison of extraction efficiency of different extraction methods (peak area / sample volume)

[0110]

[0111] The results showed that the number of chromatographic peaks obtained by the three extraction methods was consistent. Considering the extraction efficiency of each chromatographic peak, heating reflux was finally selected as the preferred extraction method.

[0112] (2) Examination of extraction time:

[0113] This study investigated the effect of different extraction times on the UPLC characteristic spectra of Alpinia galanga during the preparation of the test solution. Four different extraction times (30 min, 60 min, 90 min, and 120 min) were selected, and the extraction method was reflux extraction. The effect of different extraction times on the UPLC characteristic spectra of Alpinia galanga was evaluated by analyzing the peak area / sample weight of eight characteristic peaks and comparing chromatograms. The results are shown in Table 3. Figure 5 and Figure 6 As shown.

[0114] Table 3. Comparison of extraction efficiency at different extraction times (peak area / sample volume)

[0115]

[0116] The results showed that the number of chromatographic peaks obtained at each extraction time was consistent. Considering the extraction efficiency of each chromatographic peak, 60 min was finally selected as the optimal extraction time.

[0117] (3) Determination of detection wavelength:

[0118] This study investigated the effect of different detection wavelengths on the UPLC characteristic spectra of Alpinia galanga during ultra-high performance liquid chromatography (UHPLC). Spectra within the wavelength range of 210–400 nm were recorded to evaluate the influence of different detection wavelengths on the UPLC characteristic spectra of Alpinia galanga. The results are as follows: Figure 7 and Figure 8 As shown.

[0119] The results showed that the chromatographic peaks of the Alpinia galanga sample solution responded well at a wavelength of 215 nm, therefore 215 nm was selected as the preferred detection wavelength.

[0120] (4) Determination of the chromatographic column:

[0121] This study investigated the effects of different chromatographic columns on the UPLC characteristic chromatograms of Alpinia galanga during ultra-high performance liquid chromatography (UHPLC) analysis. The chromatograms of Waters ACQUITY UPLC HSS T3 column (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size) under gradient elution conditions (0–4 min 0% (A), 4–18 min 0%→10% (A), 18–30 min 10%→20% (A)) and Agilent InfinityLab Poroshell 120AQ-C18 column (150 mm length, 2.1 mm inner diameter, 2.7 μm particle size) 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 influence of different chromatographic columns on the UPLC characteristic chromatograms of Alpinia galanga. The results are as follows: Figure 9 As shown.

[0122] The results showed that the chromatographic peaks analyzed under the AQ-C18 column conditions had better baseline, peak shape, resolution, and symmetry. Therefore, AQ-C18 was selected as the preferred chromatographic column.

[0123] (5) Determination of elution conditions:

[0124] This study investigated the effects of different elution conditions on the UPLC characteristic chromatograms of Alpinia galanga in the ultra-high performance liquid chromatography (UHPLC) analysis procedure. Using acetonitrile (A)-0.1% v / v phosphoric acid aqueous solution (B) as the mobile phase, the chromatograms 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 0% (A), 5-20 min 0%→10% (A), 20-30 min 10%→18% (A); Condition 5: 0-5 min 3% (A), 5-6 min 0%→10% (A), 5-20 min 0%→10% (A), 20-30 min 10%→18% (A); Condition 6: 0-5 min 0% (A), 5-20 min 0%→10% (A), 20-30 min 10%→12% (A); Condition 7: 0-5 min 0% (A), 5-20 min 0%→10% (A), 20-30 min 10%→12% (A); Condition 8: 0-5 min 0% (A), 5-20 min 0%→10% (A), 20-30 min 10%→12% The effects of different elution conditions on the UPLC characteristic spectra of Alpinia galanga were evaluated using the following methods: 3%→6% (A), 6–18 min 6% (A), 18–30 min 6%→9% (A), 30–33 min 9% (A). The results are as follows: Figure 10 As shown.

[0125] The results show that the target peak of condition 5 is clearer, has a better peak shape, better separation effect, no tailing phenomenon, and a stable baseline. Therefore, condition 5 is selected as the preferred elution condition.

[0126] Example 2: Method for constructing supplementary characteristic maps of Alpinia galanga medicinal material

[0127] 1. Instruments, reagents and samples

[0128] (1) Instruments: Waters Acquity UPLC (Waters Corporation); Waters Empower3 workstation (Waters Corporation); Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent Corporation); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent Corporation); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); HY-4 variable speed multi-purpose shaker (Jintan Kexing Instrument Factory); temperature-controlled water bath (Nantong Huatai Experimental Instruments Co., Ltd.); pure water system (Sartorius Corporation); AS165W centrifuge (Asia Pacific (Shanghai) Trading Co., Ltd.).

[0129] (2) Reagents: Acetonitrile (chromatographic grade, Thermo Fisher Scientific); phosphoric acid (chromatographic grade, Aladdin Scientific); water was ultrapure water; other reagents were all analytical grade.

[0130] (3) Samples: Reference standards: Galangin (batch number: 111699-201703, CAS: 548-83-4) was purchased from the China National Institutes for Food and Drug Control; galangin (number: 5238, CAS: 480-39-7) and galangin-3-methyl ether (number: 9507, CAS: 6665-74-3) were purchased from Shanghai Shidande Standard Technical Service Co., Ltd. Test samples: Galangal medicinal materials were collected from Guangdong, Hainan and other regions, and identified by Jiangyin Tianjiang Pharmaceutical Co., Ltd. as the dried rhizomes of Alpinia officinarum Hance, a plant of the ginger family, totaling 16 batches.

[0131] 2. Preparation of reference solution

[0132] Take appropriate amounts of galangin, galangin-3-methyl ether reference standards, accurately weigh them, and add methanol to prepare solutions containing 20 μg of each per 1 mL as reference solutions.

[0133] 3. Preparation of the test solution

[0134] Take approximately 1g of Alpinia galanga powder (passed through a No. 4 sieve) (batch number: YC1805098), accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 70% methanol, seal tightly, weigh, sonicate (power 250W, frequency 40kHz) for 60min, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0135] 4. Ultra-high performance liquid chromatography analysis

[0136] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 RRHD 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℃; detection wavelength: 215 nm; gradient elution program is shown in Table 4. The theoretical plate number, calculated based on the galangin peak, should be no less than 5000.

[0137] Table 4. Gradient Elution Table

[0138]

[0139] Under these conditions, accurately pipette 1 μL of the reference solution and 2 μL of the test solution, and inject them separately into the ultra-high performance liquid chromatograph to obtain the chromatograms of the reference solution and each test sample (Alpinia galanga medicinal material), as shown in the figure below. Figure 11 and Figure 12 As shown.

[0140] 5. Analyze the spectrum

[0141] Following the method described in this embodiment, 16 batches of Alpinia galanga medicinal materials were analyzed by ultra-high performance liquid chromatography (UPLC). The UPLC chromatograms were obtained and imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (Version 2012.0)" issued by the National Pharmacopoeia Commission. A time window width of 0.1 min was selected, and a control chromatogram was generated using the median. After multi-point correction and Mark peak matching, a common pattern of the basic characteristic UPLC chromatogram of Alpinia galanga medicinal materials was generated (see details). Figure 11 The UPLC spectrum of Alpinia galanga (from *Alpinia galanga*) contains 11 characteristic peaks. High-resolution mass spectrometry (HPLC) data were acquired using HPLC conditions based on the basic characteristic chromatograms of Alpinia galanga. Molecular formulas of compounds in Alpinia galanga were deduced by isotope analysis, and the characteristic peaks were assigned based on comparisons with references and relevant standards. Peaks 4, 5, and 7 were identified as galangin, galangin, and galangin-3-methyl ether, respectively. Using the chromatographic peak corresponding to galangin as a reference peak, the relative retention times of the 11 characteristic peaks within ±5% 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 a supplementary UPLC characteristic spectrum of galangal medicinal material.

[0142] The UPLC supplementary characteristic spectra of 16 batches of Alpinia galanga medicinal materials were overlaid using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (Version 2012.0)" issued by the National Pharmacopoeia Commission. Figure 12 As shown in the figure, the consistency of chromatographic peak similarity was examined, and 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 chromatogram and the control chromatogram was high. The 11 characteristic peaks appeared stably in each sample and had good agreement. Therefore, they can be used as the characteristic chromatograms of Alpinia officinarum.

[0143] 6. Methodological Examination

[0144] (1) Investigation of extraction solvent:

[0145] This study investigated the effect of different extraction solvents on the UPLC characteristic spectra of Alpinia galanga during the preparation of the test solution. 30% methanol-water solution, 50% methanol-water solution, 70% methanol-water solution, methanol, and water were selected as extraction solvents (by volume percentage). The effect of different extraction solvents on the UPLC characteristic spectra of Alpinia galanga was evaluated by analyzing the peak area / sample weight of 11 characteristic peaks and comparing chromatograms. The results are as follows: Figure 13 and Figure 14 As shown.

[0146] The results showed that the extraction efficiency was high when methanol and 70% methanol were used as extraction solvents. Considering the need to reduce the amount of organic reagents used, 70% methanol was selected as the preferred extraction solvent.

[0147] (2) Examination of extraction methods:

[0148] This study investigated the effects of different extraction methods on the UPLC characteristic spectra of Alpinia galanga during the preparation of the test solution. Ultrasonic extraction (250 W, 40 kHz), reflux extraction, and shaking extraction were used, with an extraction time of 60 min. The effects of different extraction methods on the UPLC characteristic spectra of Alpinia galanga were evaluated by analyzing the peak area / sample weight of 11 characteristic peaks and comparing chromatograms. The results are as follows: Figure 15 and Figure 16 As shown.

[0149] The results showed that the number of chromatographic peaks obtained by the three extraction methods was consistent and the extraction efficiency was similar. Considering that ultrasonic treatment is more convenient, ultrasonic extraction was selected as the preferred extraction method.

[0150] (3) Examination of extraction time:

[0151] This study investigated the effect of different extraction times on the UPLC characteristic spectra of Alpinia galanga during the preparation of the test solution. Four different extraction times (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 effect of different extraction times on the UPLC characteristic spectra of Alpinia galanga was evaluated by analyzing the peak area / sample weight of 11 characteristic peaks and comparing chromatograms. The results are as follows: Figure 17 and Figure 18 As shown.

[0152] The results showed that the number of chromatographic peaks obtained at each extraction time was consistent and the extraction efficiency was relatively similar, indicating that complete extraction was achieved at 30 min. To ensure sufficient extraction, the ultrasonic extraction time was ultimately selected as 60 min.

[0153] (4) Examination of detection wavelength:

[0154] This study investigated the effect of different detection wavelengths on the UPLC characteristic spectra of Alpinia galanga during ultra-high performance liquid chromatography (UHPLC). Spectra within the wavelength range of 190–400 nm were recorded to evaluate the influence of different detection wavelengths on the UPLC characteristic spectra of Alpinia galanga. The results are as follows: Figure 19 and Figure 20 As shown.

[0155] The results showed that, under the wavelength of 215 nm, the response of each chromatographic peak of the Alpinia galanga sample solution was good, and the separation of each chromatographic peak was also good. Therefore, 215 nm was selected as the preferred detection wavelength.

[0156] (5) Examination of elution conditions:

[0157] This study investigated the effects of different elution conditions on the UPLC characteristic chromatograms of Alpinia galanga in the ultra-high performance liquid chromatography (UHPLC) analysis procedure. Using acetonitrile (A)-0.1% v / v phosphoric acid aqueous solution (B) as the mobile phase, the chromatograms 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)). The effects of different elution conditions on the UPLC characteristic chromatograms of Alpinia galanga were evaluated. The results are as follows: Figure 21 As shown.

[0158] The results show that the target peak in condition 3 is clearer, has a better peak shape, better separation effect, no tailing phenomenon, and a stable baseline. Therefore, condition 3 is selected as the preferred elution condition.

[0159] The above-described embodiments merely illustrate several implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the claims.

Claims

1. A method for constructing characteristic spectra of non-volatile components in a galangal sample, comprising the following steps: 1) Preparation of the basic test solution: Take a sample of Alpinia galanga, weigh it accurately, add water accurately and extract it, make up the weight loss with water, take the filtrate to obtain the basic test solution; the sample is selected from medicinal materials, decoction pieces, standard decoctions and formula granules; 2) Preparation of basic reference solutions: Take appropriate amounts of each basic reference standard, dissolve them separately in solvent to obtain the basic reference solution; The basic reference standards are proanthocyanidins B1, proanthocyanidins B2, catechins, epicatechins, and protocatechuic acid; 3) Establishment of basic characteristic chromatograms: The basic test solution prepared in step 1) and the basic reference solution prepared in step 2) were subjected to ultra-high performance liquid chromatography (UHPLC) analysis to obtain the basic characteristic chromatograms. The UHPLC included the following chromatographic conditions: an Agilent InfinityLab Poroshell 120AQ-C18 column as the stationary phase; acetonitrile-0.1% v / v phosphoric acid aqueous solution as the mobile phase; gradient elution was used, and the gradient elution program was as follows: 0-5 min, acetonitrile volume percentage 3%; 5-6 min, acetonitrile volume percentage 3% → 6%; 6-18 min, acetonitrile volume percentage 6%; 18-30 min, acetonitrile volume percentage 6% → 9%; 30-33 min, acetonitrile volume percentage 9%; detection wavelength was 215 nm.

2. The feature map construction method according to claim 1, characterized in that, The extraction method described in step 1) is selected from ultrasonic extraction, heating and reflux extraction, and shaking extraction; and / or, the extraction time is 30-120 min; And / or, The ratio of sample to water used in step 1) is 1g:25-50mL.

3. The feature map construction method according to claim 2, characterized in that, The extraction method is heating and reflux extraction.

4. The feature map construction method according to claim 2, characterized in that, The extraction time is 60-90 minutes.

5. The feature map construction method according to claim 2, characterized in that, The extraction time was 60 minutes.

6. The feature map construction method according to claim 2, characterized in that, The ratio of the sample to water was 1g:25mL.

7. The feature map construction method according to claim 1 or 2, characterized in that, The solvent mentioned in step 2) is an alcohol; And / or, The concentrations of proanthocyanidins B1, proanthocyanidins B2, catechins, epicatechins, and protocatechuic acid in the basic reference solution described in step 2) are 20-60 μg / mL.

8. The feature map construction method according to claim 7, characterized in that, The solvent is methanol.

9. The feature map construction method according to claim 7, characterized in that, The concentrations of proanthocyanidins B1, proanthocyanidins B2, catechins, epicatechins, and protocatechuic acid in the basic reference solution were 60 μg / mL, 40 μg / mL, 50 μg / mL, 30 μg / mL, and 20 μg / mL, respectively.

10. The feature map construction method according to claim 1 or 2, characterized in that, The ultra-high performance liquid chromatography described in step 3) also includes the following chromatographic conditions: flow rate of 0.25-0.35 mL / min; column temperature of 30-40℃.

11. The feature map construction method according to claim 10, characterized in that, The flow rate is 0.3 mL / min.

12. The feature map construction method according to claim 10, characterized in that, The column temperature is 35°C.

13. The feature map construction method according to claim 1 or 2, characterized in that, The basic characteristic chromatogram described in step 3) contains 8 characteristic peaks; using the chromatographic peak corresponding to catechin as the reference peak, the relative retention times of the 8 characteristic peaks within ±10% are as follows: peak 1 is 0.14, peak 2 is 0.38, peak 3 is 0.88, peak 4 is 0.92, peak 5 is 1.00, peak 6 is 1.61, peak 7 is 1.71 and peak 8 is 1.85, wherein: peak 2 is protocatechuic acid, peak 4 is proanthocyanidin B1, peak 5 is catechin, peak 7 is proanthocyanidin B2 and peak 8 is epicatechin.

14. The feature map construction method according to claim 1 or 2, characterized in that, It also includes the following steps: 1') Preparation of supplementary test solution: Take the same Alpinia galanga sample as in claim 1, weigh it accurately, add the extraction solvent accurately and extract, replenish the weight loss with the extraction solvent, take the filtrate to obtain the supplementary test solution; 2') Preparation of supplementary reference solution: Take appropriate amounts of each supplementary reference standard, dissolve them separately in solvent to obtain the supplementary reference solution; the supplementary reference standard is at least one of galangin, galangin-3-methyl ether and galangin; 3') Establishment of supplementary characteristic chromatograms: The supplementary test solution prepared in step 1') and the supplementary reference solution prepared in step 2') were subjected to ultra-high performance liquid chromatography (UHPLC) analysis to obtain the supplementary characteristic chromatograms. The UHPLC included 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; gradient elution was used, and the gradient elution program was as follows: 0-13 min, acetonitrile volume percentage 28% → 50%; 13-18 min, acetonitrile volume percentage 50% → 100%; 18-22 min, acetonitrile volume percentage 100%; detection wavelength 215 nm.

15. The feature map construction method according to claim 14, characterized in that, The supplementary reference standards are galangin, galangin-3-methyl ether, and galangin.

16. The feature map construction method according to claim 14, characterized in that, The extraction solvent mentioned in step 1') is selected from alcohols and aqueous solutions of alcohols; And / or, The extraction method described in step 1') is selected from ultrasonic extraction, reflux extraction and shaking extraction; and / or, the extraction time is 30-120 min; And / or, The ratio of sample to extraction solvent in step 1') is 1g:25-50mL.

17. The feature map construction method according to claim 16, characterized in that, The alcohol is methanol.

18. The feature map construction method according to claim 16, characterized in that, The volume percentage of alcohol in the aqueous solution of the alcohol is 30%-70%.

19. The feature map construction method according to claim 16, characterized in that, The volume percentage of alcohol in the aqueous solution of the alcohol is 70%.

20. The feature map construction method according to claim 16, characterized in that, The extraction method is ultrasonic extraction.

21. The feature map construction method according to claim 16, characterized in that, The extraction time is 60-90 minutes.

22. The feature map construction method according to claim 16, characterized in that, The extraction time was 60 minutes.

23. The feature map construction method according to claim 16, characterized in that, The ratio of sample to extraction solvent is 1g:25mL.

24. The feature map construction method according to claim 14, characterized in that, The solvent mentioned in step 2') is an alcohol; And / or, The concentrations of galangin, galangin-3-methyl ether, and galangin in the supplementary reference solution described in step 2') are 10-30 μg / mL.

25. The feature map construction method according to claim 24, characterized in that, The solvent is methanol.

26. The feature map construction method according to claim 24, characterized in that, The concentrations of galangin, galangin-3-methyl ether, and galangin in the supplementary reference solution were 20 μg / mL.

27. The feature map construction method according to claim 14, characterized in that, The ultra-high performance liquid chromatography described in step 3') also includes the following chromatographic conditions: flow rate of 0.35-0.45 mL / min; column temperature of 25-35℃.

28. The feature map construction method according to claim 27, characterized in that, The flow rate is 0.4 mL / min.

29. The feature map construction method according to claim 27, characterized in that, The column temperature is 30°C.

30. The feature map construction method according to claim 14, characterized in that, The supplementary characteristic chromatogram described in step 3') contains 11 characteristic peaks; using the chromatographic peak corresponding to galangin as the reference peak, the relative retention times of the 11 characteristic peaks within ±5% are as follows: peak 1 is 0.48, peak 2 is 0.82, peak 3 is 0.86, peak 4 is 0.97, peak 5 is 1.00, peak 6 is 1.03, peak 7 is 1.11, peak 8 is 1.41, peak 9 is 1.46, peak 10 is 1.50 and peak 11 is 1.88, wherein: peak 4 is galangin, peak 5 is galangin, and peak 7 is galangin-3-methyl ether.

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