CN114624349B - Method for analyzing traditional Chinese medicinal materials in lung-heat clearing and toxin expelling decoction formula - Google Patents
Method for analyzing traditional Chinese medicinal materials in lung-heat clearing and toxin expelling decoction formula Download PDFInfo
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Abstract
The invention relates to a rapid identification method of prescription decoction pieces of a traditional Chinese medicine compound preparation, in particular to a rapid identification method of prescription decoction pieces of a lung-clearing and toxin-expelling decoction established based on ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). The lung-clearing and toxin-expelling decoction consists of 21 traditional Chinese medicines, specific index components of 20 traditional Chinese medicine decoction pieces except mineral medicine gypsum are identified by using accurate molecular weight and characteristic fragment information of a high-resolution mass spectrum and are used as index components of corresponding decoction pieces in the lung-clearing and toxin-expelling decoction, and whether the decoction pieces are contained in a compound is judged by using the existence of the index components. The method is simple and efficient, can realize rapid identification of the decoction pieces of the lung-clearing and toxin-expelling decoction within 13min, and lays a foundation for quality control of the lung-clearing and toxin-expelling decoction.
Description
Technical Field
The invention belongs to the field of quality control of traditional Chinese medicine compound preparations, and relates to a quality control method of a compound preparation lung-clearing and toxin-expelling decoction, in particular to a rapid identification method of lung-clearing and toxin-expelling decoction formula decoction pieces based on ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS).
Background
The lung-clearing and toxin-expelling soup is an epidemic-resisting traditional Chinese medicine recommended by the office of the national health commission and the national traditional Chinese medicine administration, and the clinical effectiveness of the lung-clearing and toxin-expelling soup is proved when 1262 patients who receive and treat the lung-clearing and toxin-expelling soup are treated without mild symptoms and severe symptoms and ordinary symptoms. In the sixth and seventh diagnosis and treatment schemes of the new coronary pneumonia, the lung-clearing and toxin-expelling soup is the only universal prescription for treating light, common, heavy and critical patients, however, at present, no national standard and relevant research reports about the quality control of the lung-clearing and toxin-expelling soup exist, and the safe use and clinical efficacy of the lung-clearing and toxin-expelling soup cannot be guaranteed.
The qualitative and quantitative analysis of main components by High Performance Liquid Chromatography (HPLC) is a commonly used quality control method for Chinese patent medicines at present, but the HPLC depends on the ultraviolet absorption of analytes, generally needs to use analyte standards and has certain limitations. Furthermore, there is also some inaccuracy in the mere use of the retention time parameter to identify the compound. The combination of liquid chromatography and mass spectrometry (LC-MS) can ameliorate this problem very well, and accurate molecular weight and characteristic fragment information of high resolution mass spectrometry can be used to identify compounds in the absence of standards. Meanwhile, the sensitivity of the mass spectrum is far higher than that of liquid chromatogram, and compounds with lower content can be detected. [ Su C, li C, sun K, et al. Food Chemistry,2020 ]
The Chinese patent medicine generally comprises a plurality of Chinese traditional medicines, and the first step of the quality control of the product is to judge the correctness of the prepared slices and check whether the products lack necessary prepared slices. The qualitative inspection methods reported in the prior art for the constituent drugs of Chinese patent medicine include vibrational spectroscopy, [ Chen J B, sun S Q, zhou Q. Analytical and Bioanalytical Chemistry,2014,406 (18): 4513-4525 ], metagenomic methods [ Bai H, li X, li H, et al. Scientific reports,2019,9 (1): 5853 ]; cheng X, su X, chen X, et al, scientific Reports,2014,4 (1): 5147.] and molecular diagnostic scans [ Cao M, wang JK, yao L, et al, molecular Biology Reports,2014,41 (4): 2485-2491 ]. The vibration spectral imaging method utilizes the nonuniformity of the spatial distribution of medicinal powder particles to identify the spectral signals of certain specific components in the medicinal materials, and cannot distinguish mixed extracts; the metagenomics method and the molecular diagnosis scanning technology rely on DNA extraction and analysis, have high operation difficulty and high cost, and are not suitable for the rapid identification of large-scale traditional Chinese medicine extracts. Therefore, the method for developing a universal compound composition decoction piece identification method by utilizing the advantages of UPLC-Q-TOF-MS/MS in the aspects of separation and compound identification has important significance for the overall quality control of lung clearing and toxin expelling decoction and other Chinese patent medicines.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a quality control method of lung-clearing and toxin-expelling soup, which identifies the varieties of decoction pieces added in a compound through index components and lays a foundation for the subsequent quality control of the lung-clearing and toxin-expelling soup.
The invention is realized by the following technical scheme: the method for resolving the traditional Chinese medicinal materials in the lung clearing and toxin expelling decoction formula is characterized by comprising the following steps of:
1. acquisition of index component
(1) Preparing lung-heat clearing and toxin expelling soup: taking 9 parts by mass of decoction pieces of ephedra, 6 parts by mass of honey-fried licorice root, 9 parts by mass of almond, 15 parts by mass of gypsum (needing to be decocted first), 9 parts by mass of cassia twig, 9 parts by mass of oriental waterplantain rhizome, 9 parts by mass of polyporus umbellatus, 9 parts by mass of largehead atractylodes rhizome, 15 parts by mass of poria cocos, 16 parts by mass of radix bupleuri, 6 parts by mass of scutellaria baicalensis, 9 parts by mass of ginger-processed pinellia tuber, 9 parts by mass of dried ginger, 9 parts by mass of aster, 9 parts by mass of flos farfarae, 9 parts by mass of blackberry lily, 6 parts by mass of asarum, 12 parts by mass of Chinese yam, 6 parts by mass of immature bitter orange, 6 parts by mass of dried orange peel and 9 parts by mass of wrinkled gianthyssop herb, mixing to obtain decoction pieces of a prescription dosage of 196 parts by mass, adding 2000 parts by mass of pure water, soaking for 30-40min, heating to boil, decocting for 20-30min, and collecting liquid medicine; adding 1600 parts by mass of pure water, heating to boil, and decocting for 15-20min; mixing decoctions, filtering with 200 mesh filter cloth, cooling to room temperature, diluting the decoction to 20mg/mL (by weight of decoction pieces), to obtain decoction extract for clearing lung-heat and removing toxic substance;
(2) Extracting single decoction pieces: respectively weighing 9 parts by mass of ephedra herb, 6 parts by mass of honey-fried licorice root, 9 parts by mass of almond, 9 parts by mass of cassia twig, 9 parts by mass of rhizoma alismatis, 9 parts by mass of polyporus umbellatus, 9 parts by mass of bighead atractylodes rhizome, 15 parts by mass of poria cocos, 16 parts by mass of radix bupleuri, 6 parts by mass of radix scutellariae, 9 parts by mass of ginger pinellia tuber, 9 parts by mass of ginger, 9 parts by mass of radix asteris, 9 parts by mass of flos farfarae, 9 parts by mass of blackberrylily rhizome, 6 parts by mass of asarum, 12 parts by mass of Chinese yam, 6 parts by mass of immature bitter orange, 6 parts by mass of dried orange peel and 9 parts by mass of wrinkled gianthyssop herb, respectively adding 2000 parts by mass of pure water, soaking for 30-40min, heating to boil, decocting for 20-30min, and collecting liquid medicine; adding 1600 parts by mass of pure water, heating to boil, and decocting for 15-20min; mixing decoctions, filtering with 200 mesh filter cloth, and cooling to room temperature; respectively concentrating to 20mg/mL (by the mass of the decoction pieces), or concentrating under reduced pressure to dryness, and re-dissolving with pure water to 20mg/mL (by the mass of the decoction pieces), respectively to obtain 20 kinds of decoction piece extractive solutions except for mineral medicine gypsum;
(3) Analysis of the extract: respectively analyzing the extracting solutions in the steps (1) and (2) by adopting ultra-high performance liquid chromatography-series quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS/MS); the chromatographic column is a reversed phase C18 chromatographic column, the column temperature is 20-45 ℃, the flow rate is 0.2-0.6 mL/min, the mobile phase A is methanol or acetonitrile, the mobile phase B is formic acid/water solution with the volume concentration of 0.05-0.5%, and the elution mode is gradient elution; the mass spectrometry ion source is an electrospray ion source (ESI), and the ion source parameters are as follows: the temperature of the drying gas is 300-350 ℃, the flow rate of the drying gas is 5-15L/min, the pressure of the atomizing gas is 30-50psi, the temperature of the sheath gas is 300-400 ℃, the flow rate of the sheath gas is: 5-15L/min, capillary voltage under a positive ion mode is 3000-4000V, capillary voltage under a negative ion mode is 2500-3500V, fragmentation voltage is 50-150V, collision voltage is 20-80V, the primary mass scanning range m/z is 100-1200, and the secondary mass scanning range m/z is 50-1100;
(4) Identification of index components of each component in the prescription decoction pieces: and (3) acquiring index components of the 20 decoction pieces except the mineral medicine gypsum in the formula by using the accurate molecular weight and characteristic fragment information of the high-resolution mass spectrum of the extracting solution in the steps (1) and (2). Wherein the index components are defined as follows: the two types of decoction pieces are divided into two types, wherein one type comprises the components contained in the single decoction piece and the lung-clearing and toxin-expelling decoction, and the components not contained in the other decoction pieces except the single decoction piece, and the existence of the components can be used for judging whether the decoction pieces are contained in the compound; the other is a characteristic component of the single-flavor decoction pieces, even if the characteristic component is present in the decoction pieces other than the single-flavor decoction pieces, the characteristic component can be used as an auxiliary component for identification of the single-flavor decoction pieces in the formula, and when the auxiliary component is not present, even if the first component is present, the fact that the single-flavor decoction pieces are contained in the formula cannot be proved. The two components are jointly used as index components for identifying decoction pieces in the prescription, and whether the decoction pieces are contained in the lung-clearing and toxin-expelling decoction can be identified by using the accurate molecular weight and the characteristic fragment information of the index components.
Index components of 20 decoction pieces except for the mineral medicine gypsum are as follows:
6 components of ephedrine, pseudoephedrine, catechin, epicatechin, demethyl ephedrine and demethyl pseudoephedrine are index components of herba Ephedrae in the decoction for clearing lung-heat and removing toxic substance;
5 components of liquiritin, isoliquiritin, glycyrrhizic acid, apioside liquiritin and apioside isoliquiritin are index components of liquorice in the lung-heat clearing and toxin expelling soup;
the 2 components of amygdalin and prunasin are index components of almond in the lung-heat clearing and toxin expelling soup;
4 components of baicalein, wogonoside, baicalein I and baicalein II are index components of Scutellariae radix in the decoction for clearing lung-heat and removing toxic substance;
trans-o-hydroxycinnamic acid, a compound with a mass-to-charge ratio of 415.1254 in an anion mode (structure unknown), and a compound with a mass-to-charge ratio of 429.1406 in an anion mode (structure unknown) are index components of cassia twig in the lung-clearing and toxin-expelling decoction;
2 components of saikosaponin A and saikosaponin B2 are index components of radix bupleuri in the lung-heat clearing and toxin expelling decoction;
the 4 ingredients of astin A, astin C, astin G and asterin A are index ingredients of aster in the lung-heat clearing and toxin expelling soup;
2 components of hyperin and crimson groundsel herb are index components of winter flowers in the lung-heat clearing and toxin expelling soup;
the 3 components of mangiferin, isomonoside and belamcandin are index components of belamcanda chinensis in the lung clearing and toxin expelling soup;
the 3 components of verbascoside, linarin and bignonia neo-sweet II are index components of wrinkled gianthyssop in the lung-heat clearing and toxin expelling decoction;
the 2 components of the citrin and the compound (with unknown structure) with the mass-to-charge ratio of 371.0628 in the negative ion mode are index components of the immature bitter orange in the lung-clearing and toxin-expelling decoction;
natsudaidaidan-3-O- (5-alpha-glucosyl-HMG) -beta-glucoside, monohydroxypentamethoxy flavonol 3-O- (5-alpha-glucosyl-HMG) -beta-glucoside, 7,4-dihydroxy-5,6,8,3-tetramethylhydroxyflavanol-3-O- (5-alpha-glucosyl-HMG) -beta-glucoside, which are 3 components of the tangerine peel in the lung-clearing and toxin-expelling soup;
atractyloside A is an index component of rhizoma atractylodis macrocephalae in the lung-heat clearing and toxin expelling decoction;
the compound (with unknown structure) with the mass-to-charge ratio of 506.2313 in the positive ion mode is an index component of asarum in the lung-clearing and toxin-expelling decoction;
the compound (with unknown structure) with the mass-to-charge ratio of 712.8736 in the positive ion mode is an index component of rhizoma pinelliae in the lung-heat clearing and toxin expelling decoction;
the 2 components of 6-shogaol and 6-gingerol are index components of ginger in the lung-clearing and toxin-expelling soup;
allantoin and a compound (with unknown structure) with the mass-to-charge ratio of 460.2402 in the positive ion mode, wherein the 2 components are index components of Chinese yam in the lung-clearing and toxin-expelling decoction;
23-acetyl alisol C, and a compound (with unknown structure) with the mass-to-charge ratio of 557.3096 in the negative ion mode, wherein the 2 components are index components of alisma in the lung-clearing and toxin-expelling decoction;
polyporus umbellatus ketone A is index component of Polyporus umbellatus in decoction for clearing lung-heat and removing toxic substance;
3 components of a compound with a mass-to-charge ratio of 558.2777 in the positive ion mode (structure unknown), a compound with a mass-to-charge ratio of 517.2987 in the positive ion mode (structure unknown), and a compound with a mass-to-charge ratio of 497.3264 in the negative ion mode (structure unknown) are index components of poria in the lung-clearing and toxin-expelling soup.
2. Identification of traditional Chinese medicinal materials in prescription
Index components of Shang Zhongshan decoction pieces for clearing lung and expelling toxin are adopted to identify 20 Chinese medicinal materials of formula except mineral medicine gypsum:
taking lung-clearing and toxin-expelling soup (or particles) to be analyzed, diluting or concentrating the lung-clearing and toxin-expelling soup (or particles) to a solution with the concentration of 20mg/mL (by the mass of the decoction pieces), analyzing the solution by adopting ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS), and extracting index component information of each medicinal material in the prescription from the mass spectrum data of the obtained lung-clearing and toxin-expelling soup (or particles);
if all index components of a certain medicinal material in the formula are extracted, the lung-clearing and toxin-expelling decoction (or the granules) to be analyzed is considered to be added with the medicinal material specified in the formula during preparation; otherwise, the medicinal material is not added during the preparation;
when the index components of the decoction pieces are more than two, the index components of each decoction piece need to exist at the same time to judge that the decoction pieces really contain in the lung-clearing and toxin-expelling decoction;
the lung-heat clearing and toxin expelling soup to be analyzed is decoction or granules containing the lung-heat clearing and toxin expelling soup.
The invention has the following advantages:
(1) The identification method of the lung-heat clearing and toxin expelling decoction formula decoction pieces based on UPLC-Q-TOF-MS/MS is established for the first time, and the technical basis is laid for the quality control of the lung-heat clearing and toxin expelling decoction
(2) The accurate molecular weight and fragment information of the high-resolution mass spectrum are used for identifying the composition of the decoction pieces in the compound for the first time, and the method is suitable for the accurate quality control of complex traditional Chinese medicine samples
(3) The method is rapid and simple, can be completed in 13 minutes, and is suitable for quality monitoring of large-batch lung-clearing and toxin-expelling soup
Drawings
FIG. 1 EIC chart of herba Ephedrae index components in herba Ephedrae extractive solution and decoction for clearing lung-heat and removing toxic substance
FIG. 2 is a primary mass spectrum of herba Ephedrae index component
FIG. 3 is a secondary mass spectrum of herba Ephedrae index component
FIG. 4 EIC chart of index components of herba asari in herba asari extractive solution and decoction for clearing lung-heat and removing toxic substance
FIG. 5 is a first-order mass spectrum of index components of herba asari
FIG. 6 second-order mass spectrogram of index component of herba asari
FIG. 7 EIC chart of rhizoma Pinelliae Preparada index components in rhizoma Pinelliae Preparada extractive solution and decoction for clearing Lung and removing toxic substance
FIG. 8 is a primary mass spectrum of rhizoma Pinelliae Preparatum index component
FIG. 9 secondary mass spectrogram of index components of ginger processed pinellia Tuber
FIG. 10 EIC chart of index components of ramulus Cinnamomi in ramulus Cinnamomi extractive solution and decoction for clearing lung-heat and removing toxic substances
FIG. 11 first-order mass spectrogram of index components of ramulus Cinnamomi
FIG. 12 Secondary mass spectrum of index components of ramulus Cinnamomi
FIG. 13 index components of ion mode Shang Zheng for clearing lung and expelling toxin
FIG. 14 index components of lung-heat clearing and toxin expelling decoction in negative ion mode
FIG. 15 shows index components of some plants in ion mode of Shang Zheng for clearing lung and expelling toxin
FIG. 16 index components of lung-heat clearing and toxin expelling decoction in negative ion mode
FIG. 17 shows index components of lung heat clearing and toxin expelling Shang Zheng made by some hospitals in ion mode
FIG. 18 shows index components of lung-heat clearing and toxin expelling decoction prepared in certain hospital under anion mode
Detailed Description
The present invention will now be further described with reference to the following examples, which are intended to be illustrative of the invention and not limiting.
The first embodiment is as follows: index component search
(1) Preparing lung-heat clearing and toxin expelling soup: taking 4.5g of decoction pieces ephedra, 3g of honey-fried licorice root, 4.5g of almond, 7.5g of gypsum (decocted first), 4.5g of cassia twig, 4.5g of rhizoma alismatis, 4.5g of polyporus umbellatus, 4.5g of bighead atractylodes rhizome, 7.5g of poria cocos, 8g of radix bupleuri, 3g of scutellaria baicalensis, 4.5g of ginger processed pinellia tuber, 4.5g of dried ginger, 4.5g of aster, 4.5g of winter flower, 4.5g of blackberry lily, 3g of asarum, 6g of Chinese yam, 3g of immature bitter orange, 3g of dried orange peel and 4.5g of wrinkled gianthyssop herb, and mixing to obtain the decoction pieces with the total dosage of 98 g; decocting: adding 1L pure water into the mixed decoction pieces, soaking for 30min, heating with strong fire to boil, decocting with slow fire for 20min, and collecting medicinal liquid; adding 0.8L pure water, heating with strong fire to boil, and decocting with slow fire for 15min. Mixing decoctions, filtering with 200 mesh filter cloth, cooling, diluting to 2.5L, and diluting to 20mg/mL (based on the mass of the decoction pieces).
(2) Extracting single decoction pieces: weighing decoction pieces of 4.5g of ephedra, 3g of honey-fried licorice root, 4.5g of almond, 7.5g of gypsum, 4.5g of cassia twig, 4.5g of rhizoma alismatis, 4.5g of polyporus umbellatus, 4.5g of bighead atractylodes rhizome, 7.5g of poria cocos, 8g of radix bupleuri, 3g of scutellaria baicalensis, 4.5g of ginger processed pinellia tuber, 4.5g of dried ginger, 4.5g of aster, 4.5g of flos farfarae, 4.5g of blackberry lily, 3g of asarum, 6g of Chinese yam, 3g of immature bitter orange, 3g of dried orange peel and 4.5g of wrinkled gianthyssop herb respectively; and (2) respectively decocting the single decoction pieces separately according to the decoction method of the lung clearing and toxin expelling decoction in the step (1), concentrating under reduced pressure to dryness, and re-dissolving with pure water to 20mg/mL (by mass of the decoction pieces).
(3) Analysis of the extract: respectively analyzing the extracting solution in the steps (1) and (2) by adopting ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS); the UPLC-Q-TOF-MS/MS method comprises the following steps: the chromatographic column is a reversed phase C18 chromatographic column, the column temperature is 25 ℃, the flow rate is 0.2mL/min, the mobile phase A is methanol, the mobile phase B is formic acid/water solution with the volume concentration of 0.2 percent, the elution gradient is 0-13min, and the content of the mobile phase A is 5-90 percent; the mass spectrometry ion source is an electrospray ion source (ESI), and the ion source parameters are as follows: the temperature of the drying gas is 300 ℃, the flow rate of the drying gas is 10L/min, the pressure of the atomizing gas is 30psi, the temperature of the sheath gas is 350 ℃, the flow rate of the sheath gas is: 8L/min, the capillary voltage under the positive ion mode is 4000V, the capillary voltage under the negative ion mode is 3500V, the fragmentation voltage is 100V, the collision voltage is 40V, the primary mass scanning range m/z is 100-1200, and the secondary mass scanning range m/z is 50-1100;
(4) Extraction of index components: firstly, analyzing and identifying mass spectrum data of 20 single decoction pieces except for mineral medicine gypsum, extracting the identified components in the lung-clearing and toxin-expelling decoction and other decoction pieces by using an EIC mode, wherein the identified components can be extracted from the lung-clearing and toxin-expelling decoction, and the identified components can be judged as index components of the decoction pieces when the identified components cannot be extracted from the other decoction pieces or can be extracted from the other decoction pieces but have small peak area ratio, and whether the decoction pieces are contained in the lung-clearing and toxin-expelling decoction can be identified by using accurate molecular weight and characteristic fragment information of the index components.
For example, the searching process of the index components of the ephedra decoction pieces is as follows: firstly, analyzing mass spectrum data of the ephedra extract, identifying 47 compounds from the ephedra extract, extracting the accurate molecular weight of the 47 compounds in the ephedra from the lung-heat clearing and toxin expelling decoction and the other 19 decoction pieces, and finding that demethylephedrine and demethylpseudoephedrine can be extracted from the lung-heat clearing and toxin expelling decoction (figure 1) and can not be extracted from the other 19 decoction pieces; catechin and epicatechin can be extracted from the lung-heat clearing and toxin expelling soup (figure 1) and can also be extracted from Chinese yam decoction pieces, but the peak area ratio is less than 5%, so that the catechin and epicatechin are not extracted from the rest 18 decoction pieces; ephedrine and pseudoephedrine can be extracted from decoction for clearing lung-heat and removing toxic substance (figure 1), and can also be extracted from rhizoma Pinelliae decoction pieces, but the peak area ratio is less than 1%, so that ephedrine and pseudoephedrine are not extracted from the rest 18 decoction pieces. Through the extraction process, 6 components of demethylephedrine, demethylpseudoephedrine, catechin, epicatechin, ephedrine and pseudoephedrine can be used as index components of herba Ephedrae in the decoction for clearing lung-heat and removing toxic substance, and the rest 41 components identified from herba Ephedrae extractive solution have part of content which can not be extracted in the decoction for clearing lung-heat and removing toxic substance, and part of content in other medicinal materials is too high to be used as index components of herba Ephedrae. The primary and secondary mass spectrograms of 6 index components in herba Ephedrae are shown in figures 2 and 3.
For example, the searching process of index components in asarum and pinellia ternata comprises the following steps: firstly, analyzing mass spectrum data of the asarum and ginger pinellia extracting solution, identifying 30 compounds from the asarum extracting solution, identifying 53 compounds from the ginger pinellia extracting solution, and respectively extracting the identified components in the asarum and the ginger pinellia in the lung-heat clearing and toxin expelling decoction and the rest decoction pieces. As a result, the contents of the components in the lung-clearing and toxin-expelling soup are low, or the interference of other medicinal materials is too large, so that the components are not suitable to be used as index components of asarum and pinellia ternate. Extracting the rest components of herba asari and rhizoma Pinelliae Preparata, which can not be identified temporarily, to find that the compound with mass-to-charge ratio of 506.2313 in positive ion mode in herba asari contains both herba asari and lung-heat clearing and toxin expelling decoction (figure 4), and has no interference of other medicinal materials; the compound with the mass-to-charge ratio of 712.8736 in the positive ion mode in the ginger pinellia is contained in both the ginger pinellia and the lung-heat clearing and toxin expelling soup (shown in figure 7), and has no interference of other medicinal materials, so the two components can be used as index components of the asarum and the ginger pinellia in the lung-heat clearing and toxin expelling soup although the structures are temporarily unknown. The primary and secondary mass spectra are shown in figure 5, figure 6, figure 8 and figure 9.
For example, in the process of searching index components in the cassia twig, mass spectrum data of the cassia twig extract is analyzed, 28 compounds are identified from the asarum extract, and the identified components in the cassia twig are respectively extracted from the lung-clearing and toxin-expelling decoction and other decoction pieces. As a result, the content of the components in the lung-clearing and toxin-expelling decoction is low, or the interference of other medicinal materials is too large, wherein a characteristic component trans-o-hydroxycinnamic acid is also contained in ephedra, and the ratio of the peak area in the ephedra to the peak area in cassia twig is 11%, so that the composition is not suitable for being used as a judgment standard for judging whether cassia twig exists in a formula, but can assist the analysis of the cassia twig, and is reserved. The extraction of the rest components which can not be identified temporarily in the cassia twig is continued, and the two compounds with the mass-to-charge ratios of 415.1254 and 429.1406 in the negative ion mode in the cassia twig are found to be contained in the cassia twig and the lung-clearing and toxin-expelling decoction (shown in figure 10) and have no interference of other medicinal materials. Therefore, although the structures of the two components are temporarily unknown, the two components can also be used as index components of the cassia twig in the lung clearing and toxin expelling decoction. The primary and secondary mass spectrograms of the three index components in ramulus Cinnamomi are shown in figures 11 and 12.
The extraction process of the index components in the other 16 decoction pieces is similar to that of ephedra, asarum, ginger processed pinellia and cassia twig. The index components of 20 decoction pieces are shown in Table 1, figure 13 and figure 14.
TABLE 1 index ingredients of 20 decoction pieces in Lung-heat-clearing and toxin-expelling decoction
Example two: identification of prescription decoction pieces in lung-clearing and toxin-expelling decoction (granules) of certain manufacturer
The decoction (granules) for clearing lung and expelling toxin of a certain manufacturer is packaged with 30g (equivalent to 105.5g of decoction pieces) per bag. Precisely weighing 1.00g of the decoction (granule) for clearing lung-heat and removing toxin, adding 175mL of pure water to obtain a sample with a concentration of about 20mg/mL (based on the mass of the decoction pieces), dissolving the sample by ultrasonic treatment, and analyzing the sample by a 0.22-micron water film under the following liquid quality analysis conditions: the instrument is ultra-high performance liquid chromatography-series quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS), the chromatographic column is a BEH C18 chromatographic column, the column temperature is 25 ℃, the flow rate is 0.2mL/min, the mobile phase A is methanol, the mobile phase B is formic acid/water solution with the volume concentration of 0.2%, the elution gradient is 0-13min, and the concentration is 5% -90% A; the mass spectrometry ion source is an electrospray ion source (ESI), and the ion source parameters are as follows: the temperature of the drying gas is 300 ℃, the flow rate of the drying gas is 10L/min, the pressure of the atomizing gas is 30psi, the temperature of the sheath gas is 350 ℃, the flow rate of the sheath gas is: 8L/min, the capillary voltage under the positive ion mode is 4000V, the capillary voltage under the negative ion mode is 3500V, the fragmentation voltage is 100V, the collision voltage is 40V, the primary mass scanning range m/z is 100-1200, and the secondary mass scanning range m/z is 50-1100;
extracting index components of each medicinal material obtained in the first embodiment from the mass spectrum data of the lung-clearing and toxin-expelling decoction (granule), wherein the index components are shown in different medicinal material indexes marked in the attached fig. 15 and the attached fig. 16;
and (4) conclusion: all index components of all medicinal materials can be extracted, and 20 decoction pieces specified by the formula are added in the lung-clearing and toxin-expelling decoction (granules) of the pharmaceutical factory during preparation.
Example three: identification of prescription decoction pieces in self-made lung-clearing and toxin-expelling decoction in certain hospital
Taking 5mL of lung-clearing and toxin-expelling decoction prepared by a hospital, diluting the decoction pieces to 20mg/mL (by mass of the decoction pieces) according to the feeding amount of the decoction pieces, and analyzing the decoction pieces after passing through a 0.22-micron water film, wherein the liquid quality analysis conditions are as follows: the instrument is ultra-high performance liquid chromatography-series quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS), the chromatographic column is a BEH C18 chromatographic column, the column temperature is 25 ℃, the flow rate is 0.2mL/min, the mobile phase A is methanol, the mobile phase B is formic acid/water solution with the volume concentration of 0.2%, the elution gradient is 0-13min, and the concentration is 5% -90% A; the mass spectrometry ion source is an electrospray ion source (ESI), and the ion source parameters are as follows: the temperature of the drying gas is 300 ℃, the flow rate of the drying gas is 10L/min, the pressure of the atomizing gas is 30psi, the temperature of the sheath gas is 350 ℃, the flow rate of the sheath gas is: 8L/min, the capillary voltage under the positive ion mode is 4000V, the capillary voltage under the negative ion mode is 3500V, the fragmentation voltage is 100V, the collision voltage is 40V, the primary mass scanning range m/z is 100-1200, and the secondary mass scanning range m/z is 50-1100;
extracting index components of each medicinal material obtained in the first embodiment from the obtained lung-clearing and toxin-expelling decoction mass spectrum data, wherein the index components are shown in different medicinal material index components marked in the attached figures 17 and 18;
and (4) conclusion: all index components of all medicinal materials can be extracted, and 20 decoction pieces specified by the formula are considered to be added into the lung-clearing and toxin-expelling decoction prepared by the hospital.
Claims (2)
1. An analysis method of a traditional Chinese medicine with a lung-heat clearing and toxin expelling decoction formula is characterized in that,
index components of Shang Zhongshan decoction pieces for clearing lung and expelling toxin are adopted to identify 20 Chinese medicinal materials of formula except mineral medicine gypsum:
taking lung-clearing and toxin-expelling soup or particles to be analyzed, diluting or concentrating the soup or particles to a solution with the concentration of 20mg/mL by the mass of decoction pieces, analyzing the solution by adopting ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS), and extracting index component information of each medicinal material in the prescription from mass spectrum data of the obtained lung-clearing and toxin-expelling soup or particles;
if all index components of a certain medicinal material in the formula are extracted, the lung-clearing and toxin-expelling decoction or the granule to be analyzed is added with the medicinal material specified in the formula during preparation; otherwise, the medicinal material is not added during the preparation;
all index components extracted from a certain medicinal material in the formula refer to:
when the index components of the decoction pieces are two or more, the index components of each decoction piece need to exist at the same time to judge that the decoction pieces really contain in the decoction for clearing away the lung-heat and expelling the toxin;
index components of 20 decoction pieces except for the mineral medicine gypsum are as follows:
6 components of ephedrine, pseudoephedrine, catechin, epicatechin, demethylephedrine and demethylpseudoephedrine are index components of herba Ephedrae in the decoction for clearing lung-heat and removing toxic substance;
5 components of liquiritin, isoliquiritin, glycyrrhizic acid, apioside liquiritin and apioside isoliquiritin are index components of liquorice in the lung-heat clearing and toxin expelling soup;
the 2 components of amygdalin and prunasin are index components of almond in the lung-heat clearing and toxin expelling soup;
4 components of baicalein, wogonoside, baicalein I and baicalein II are index components of Scutellariae radix in the decoction for clearing lung-heat and removing toxic substance;
trans-o-hydroxycinnamic acid, a compound with a mass-to-charge ratio of 415.1254 in an anion mode, and 3 components with a mass-to-charge ratio of 429.1406 in an anion mode are index components of cassia twig in the lung-clearing and toxin-expelling decoction;
2 components of saikosaponin A and saikosaponin B2 are index components of radix bupleuri in the lung-heat clearing and toxin expelling decoction;
the 4 ingredients of astin A, astin C, astin G and asterin A are index ingredients of aster in the lung-heat clearing and toxin expelling soup;
2 components of hyperin and crimson groundsel herb are index components of winter flowers in the lung-heat clearing and toxin expelling soup;
3 components of mangiferin, isomangiferin and belamcanda chinensis glycoside are index components of belamcanda chinensis in the lung-heat clearing and toxin expelling soup;
the 3 components of verbascoside, linarin and bignonia neo-sweet II are index components of wrinkled gianthyssop in the lung-heat clearing and toxin expelling decoction;
the 2 components of the citrin and the compound with the mass-to-charge ratio of 371.0628 in the negative ion mode are index components of immature bitter orange in the lung-heat clearing and toxin expelling decoction;
natsudaidaidan-3-O- (5-alpha-glucosyl-HMG) -beta-glucoside, monohydroxypentamethoxy flavonol 3-O- (5-alpha-glucosyl-HMG) -beta-glucoside, 7,4-dihydroxy-5,6,8,3-tetramethoxyflavanol-3-O- (5-alpha-glucosyl-HMG) -beta-glucoside, which are 3 components used as index components of dried orange peel in the lung-clearing and toxin-expelling soup;
atractyloside A is an index component of rhizoma atractylodis macrocephalae in the lung-heat clearing and toxin expelling decoction;
the compound with the mass-to-charge ratio of 506.2313 in the positive ion mode is an index component of asarum in the lung clearing and toxin expelling soup;
the compound with the mass-to-charge ratio of 712.8736 in the positive ion mode is an index component of rhizoma pinelliae in the lung-heat clearing and toxin expelling decoction;
the 2 components of 6-shogaol and 6-gingerol are index components of ginger in the lung-clearing and toxin-expelling soup;
2 components of allantoin and a compound with the mass-to-charge ratio of 460.2402 in a positive ion mode are index components of Chinese yam in the lung-clearing and toxin-expelling decoction;
23-acetyl alisol C, and 2 components of a compound with the mass-to-charge ratio of 557.3096 in a negative ion mode are index components of alisma in the lung clearing and toxin expelling decoction;
polyporus umbellatus ketone A is index component of Polyporus umbellatus in decoction for clearing lung-heat and removing toxic substance;
3 components of a compound with the mass-to-charge ratio of 558.2777 in the positive ion mode, a compound with the mass-to-charge ratio of 517.2987 in the positive ion mode and a compound with the mass-to-charge ratio of 497.3264 in the negative ion mode are index components of poria in the lung-clearing and toxin-expelling soup;
the acquisition process of the index components comprises the following steps:
(1) Preparing lung-heat clearing and toxin expelling soup: taking 9 parts by mass of decoction pieces of ephedra, 6 parts by mass of honey-fried licorice root, 9 parts by mass of almond, 15 parts by mass of gypsum, 9 parts by mass of cassia twig, 9 parts by mass of oriental waterplantain rhizome, 9 parts by mass of polyporus umbellatus, 9 parts by mass of largehead atractylodes rhizome, 15 parts by mass of poria cocos, 16 parts by mass of radix bupleuri, 6 parts by mass of scutellaria baicalensis, 9 parts by mass of ginger-processed pinellia tuber, 9 parts by mass of dried ginger, 9 parts by mass of aster, 9 parts by mass of common coltsfoot flower, 9 parts by mass of blackberry lily, 6 parts by mass of asarum, 12 parts by mass of Chinese yam, 6 parts by mass of immature bitter orange, 6 parts by mass of dried orange peel and 9 parts by mass of wrinkled gianthyssop herb, mixing to obtain decoction pieces of a total prescription dosage of 196 parts by mass, adding 2000 parts by mass of pure water, soaking for 30-40min, heating to boil, decocting for 20-30min, and collecting liquid medicine; adding 1600 parts by mass of pure water, heating to boil, and decocting for 15-20min; mixing decoctions, sieving with 200 mesh filter cloth, cooling to room temperature, diluting the decoctions to 20mg/mL by weight of decoction pieces to obtain decoction extract for clearing lung-heat and removing toxic substance;
(2) Extracting single decoction pieces: respectively weighing 9 parts by mass of ephedra herb, 6 parts by mass of honey-fried licorice root, 9 parts by mass of almond, 9 parts by mass of cassia twig, 9 parts by mass of rhizoma alismatis, 9 parts by mass of polyporus umbellatus, 9 parts by mass of bighead atractylodes rhizome, 15 parts by mass of poria cocos, 16 parts by mass of radix bupleuri, 6 parts by mass of radix scutellariae, 9 parts by mass of ginger pinellia tuber, 9 parts by mass of ginger, 9 parts by mass of radix asteris, 9 parts by mass of flos farfarae, 9 parts by mass of blackberrylily rhizome, 6 parts by mass of asarum, 12 parts by mass of Chinese yam, 6 parts by mass of immature bitter orange, 6 parts by mass of dried orange peel and 9 parts by mass of wrinkled gianthyssop herb, respectively adding 2000 parts by mass of pure water, soaking for 30-40min, heating to boil, decocting for 20-30min, and collecting liquid medicine; adding 1600 parts by mass of pure water, heating to boil, and decocting for 15-20min; mixing decoctions, filtering with 200 mesh filter cloth, and cooling to room temperature; respectively concentrating to 20mg/mL by the mass of the decoction pieces, or concentrating under reduced pressure to dryness, re-dissolving to 20mg/mL by the mass of the decoction pieces with pure water to respectively obtain 20 kinds of decoction piece extract except mineral medicine gypsum;
(3) Analysis of the extract: respectively analyzing the extracting solution in the steps (1) and (2) by adopting ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS);
(4) Identification of index components of each component in the prescription decoction pieces: acquiring index components of the 20 decoction pieces in the formula except the mineral medicine gypsum by using the accurate molecular weight and characteristic fragment information of the high-resolution mass spectrum of the extracting solution in the steps (1) and (2); the UPLC-Q-TOF-MS/MS method comprises the following steps: the chromatographic column is a reversed phase C18 chromatographic column, the column temperature is 20-45 ℃, the flow rate is 0.2mL/min-0.6mL/min, the mobile phase A is methanol, the mobile phase B is formic acid/water solution with the volume concentration of 0.2 percent,
elution gradient 0-13min,5% -90% A; the mass spectrometry ion source is an electrospray ion source (ESI), and the ion source parameters are as follows: the temperature of the drying gas is 300-350 ℃, the flow rate of the drying gas is 5-15L/min, the pressure of the atomizing gas is 30-50psi, the temperature of the sheath gas is 300-400 ℃, the flow rate of the sheath gas is: 5-15L/min, 3000-4000V in a positive ion mode, 2500-3500V in a negative ion mode, 50-150V in a fragmentation voltage, 20-80V in an impact voltage, 100-1200 parts of primary mass scanning range m/z and 50-1100 parts of secondary mass scanning range m/z.
2. The method of claim 1, wherein the designation element in step (4) is defined as: the two types of decoction pieces are divided into two types, wherein one type comprises the components contained in the single decoction piece and the lung-clearing and toxin-expelling decoction, and the components not contained in the other decoction pieces except the single decoction piece, and the existence of the components can be used for judging whether the decoction pieces are contained in the compound; the other is the characteristic component of the single-ingredient decoction piece, even if the characteristic component exists in other decoction pieces except the single-ingredient decoction piece, the characteristic component can be used as an auxiliary component for identifying the single-ingredient decoction piece in the prescription, and when the auxiliary component does not exist, even if the first component exists, the prescription cannot be proved to contain the single-ingredient decoction piece; the two components are jointly used as index components for identifying decoction pieces in the prescription, and whether the decoction pieces are contained in the lung-clearing and toxin-expelling decoction can be identified by using the accurate molecular weight and the characteristic fragment information of the index components.
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