CN112903845B - Method for establishing fingerprint of lung ventilating and toxin removing prescription - Google Patents
Method for establishing fingerprint of lung ventilating and toxin removing prescription Download PDFInfo
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Abstract
The invention discloses a method for establishing a fingerprint of a lung ventilating and toxin removing prescription. The invention establishes the fingerprint of the lung-ventilating and toxin-vanquishing prescription by a UHPLC method, and performs content determination on 9 effective components in the fingerprint so as to provide reference for research on drug effect substance basis and quality control of the lung-ventilating and toxin-vanquishing prescription.
Description
Technical Field
The invention belongs to the technical field of formula component detection, and particularly relates to a lung ventilating and toxin removing formula fingerprint spectrum establishment method.
Background
New coronary pneumonia belongs to the plague category taking damp toxin as disease property, and can be called as damp toxin plague. The traditional Chinese medicine prescription used in the lung-ventilating and toxin-vanquishing decoction is drawn up according to clinical manifestation differentiation and is used for treating common type new coronary pneumonia with syndrome of damp toxin stagnating in lung in the seventh edition of diagnosis and treatment scheme. The prescription is prepared by adding and reducing a plurality of classical famous prescriptions, including Maxingshigan decoction, maxingyigan decoction, semen Lepidii and jujube lung-purging decoction, qianjin Phragmites Stem decoction and the like, and is prepared by combining 13 traditional Chinese medicines. Under the background of new coronary pneumonia pandemics, the prescription for dispersing lung qi and relieving toxicity has a relatively large research value as a clinically effective prescription.
The traditional Chinese medicine compound is prepared by compatibility of various traditional Chinese medicines, has the principles of overall appearance and treatment based on syndrome differentiation, and cannot comprehensively control the quality because the traditional Chinese medicine compound has complex components and the detection of a single active ingredient is difficult to show the overall efficacy of the traditional Chinese medicine compound. The traditional Chinese medicine fingerprint can comprehensively reflect the information of chemical components contained in the traditional Chinese medicine compound, and is an important method for controlling the quality of traditional Chinese medicines recognized at home and abroad at present, so that a method for establishing a fingerprint of a lung-ventilating and toxin-vanquishing prescription is needed.
Disclosure of Invention
The invention provides a method for establishing a fingerprint of a lung ventilating and detoxifying prescription.
The invention comprises the following steps:
the method comprises the steps of calculating the RSD value of the relative retention time and the relative peak area of each common peak by taking a mixed component as a standard substance, wherein the mixed component comprises ephedrine, amygdalin, verbascoside, verbena glycoside, polydatin, liquiritin, acteoside, naringin and glycyrrhizic acid.
Further, the steps are as follows:
a, preparing a test solution: adding methanol into the formula for ventilating the lung and removing toxicity, performing ultrasonic extraction, and filtering to obtain a solution to be detected;
b preparation of control solution: adding methanol into the mixed component reference substance dried under reduced pressure to constant weight to obtain mixed reference substance solution;
c, measurement: respectively and precisely measuring a test solution and a reference solution, performing HPLC analysis, and performing gradient elution to obtain a mobile phase A: 0.1% formic acid, mobile phase B: acetonitrile (B), analyzing and processing the atlas obtained by the high performance liquid chromatography determination, and obtaining the fingerprint atlas of the lung ventilating and toxin removing prescription;
further, the preparation method of the mixed reference solution in the step b is as follows: accurately weighing the mixed component standard substance, placing the mixed component standard substance in a volumetric flask, adding methanol to dissolve and diluting the mixed component standard substance to prepare a mixed standard substance solution.
Further, the column in step C is Agilent ZORBAX RRHD Eclipse XDB-C18 (2.1X 100mm,1.8 μm).
Further, the gradient elution procedure in step c is 0-8 min, and 5% -10% of B;8 to 13min,10 to 15 percent of B;13 to 18min,15 to 17% by weight B; 18-30min, 17% -45% B; 30-35min, 45-95 percent B; the detection wavelength is 254nm; the volume flow is 0.3mL/min; the sample injection volume is 2 mu L; the column temperature was 40 ℃.
Further, the application of the fingerprint in the quality control of the lung ventilating and toxin removing prescription is provided.
A quality control method of a lung-ventilating and toxin-vanquishing prescription, which comprises the following steps: a plurality of batches of lung ventilating and toxin relieving formulas are taken to obtain fingerprint spectrums of the lung ventilating and toxin relieving formulas according to the method, and a plurality of batches of the fingerprint spectrums of the lung ventilating and toxin relieving formulas are generated according to an averaging method to obtain a comparison fingerprint spectrum, wherein the method can be used for preparing other different dosage forms such as decoction, granules, tablets and capsules for ventilating and releasing lung and toxin relieving.
A quality control method of a lung-ventilating and toxin-vanquishing prescription, which comprises the following steps: taking multiple batches of lung-ventilating and toxin-vanquishing formulas to carry out multi-component content measurement, respectively measuring the content of ephedrine, amygdalin, penoniflorin, verbenin, polydatin, liquiritin, acteoside, naringin and glycyrrhizic acid, and comparing with the content data of a standard substance.
One or more embodiments of the present invention may have the following advantages over the prior art:
1. the invention establishes the fingerprint of the lung-ventilating and toxin-vanquishing prescription by a UHPLC method, and performs content determination on 9 effective components in the fingerprint so as to provide reference for research on drug effect substance basis and quality control of the lung-ventilating and toxin-vanquishing prescription.
2. The invention avoids the one-sidedness of judging the whole quality of the preparation by only measuring a single chemical component, reduces the possibility of manual treatment for reaching the quality standard, and simultaneously can more comprehensively and scientifically evaluate the quality of the decoction of the lung-diffusing and toxin-vanquishing prescription by systematically analyzing a plurality of batches of samples, thereby ensuring the quality and the curative effect of the product, providing a good solution for the quality control of the lung-diffusing and toxin-vanquishing prescription, and having great value in practical application.
Drawings
FIG. 1 shows the comparison fingerprint (A) of UHPLC of the lung-ventilating and toxin-vanquishing prescription and the fingerprint (B) of UHPLC of 12 batches of the lung-ventilating and toxin-vanquishing prescription;
wherein: 6-pennyroyal, 7-pennyroyal, 10-polydatin, 11-liquiritin, 14-acteoside, 15-naringin and 21-glycyrrhizic acid.
FIG. 2 is a chromatogram of 210nm (A), 254nm (B) mixed reference substance and 210nm (C), 254nm (D) test substance of 9 components of lung ventilating and toxin removing formula;
wherein the raw materials comprise 1-ephedrine, 2-amygdalin, 3-pennyroyal, 4-pennyroyal, 5-polydatin, 6-liquiritin, 7-acteoside, 8-naringin and 9-glycyrrhizic acid.
Detailed Description
The technical solutions in the drawings and the embodiments of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
The instrument used in this example was a Waters Acquity UHPLC ultra high performance liquid chromatograph, waters corporation, usa; a one hundred thousand scale of the type MSA225P-0CE-DU, sartorius, germany; BP121S type ten thousandth balance, sartorius, germany; 5430R model high speed refrigerated centrifuge, eppendorf, germany; milli-Q academic ultrapure water system, millipore USA.
The materials selected in this example are ephedrine hydrochloride (batch number: 171241-201809) and polydatin (batch number: 111575-200502) as reference substances purchased from Chinese institute for drug and food testing; the reference amygdalin (batch: DST 190829-004), verbascoside (batch: DST 191011-072), spurge verbascoside (batch: DST 191119-063), acteoside (batch: DST 200709-061), naringin (batch: DST 191011-099) were all purchased from Goodpastel Limited; glycyrrhizin (batch number: MUST-14020911) and glycyrrhizic acid (batch number: MUST-14081812) were purchased from Kyormant Biotechnology Ltd. All reference samples had a purity of 98% or more. Methanol, acetonitrile, chromatographically pure, fisher corporation, usa; formic acid, mass spectrometric alcohol, anaqua Chemicals Supply, USA; the water is ultrapure water.
Raw ephedra herb, bitter apricot seed, gypsum, raw coix seed, atractylis lancea rhizome, cablin potchouli herb, sweet wormwood herb, giant knotweed rhizome, verbena, dry reed rhizome, semen lepidii, pummelo peel and raw liquorice root 13 medicinal materials are purchased from the genuine producing area or the main producing area, and a plurality of batches of the medicinal materials are identified by the Marin professor of the Chinese academy of medicine of Tianjin Chinese medicine university and all accord with the following regulation under the next term of the national pharmacopoeia 2015 edition: the herba Ephedrae is dried grass stem of Ephedra sinica Sinica Stapf, ephedra intermedia Schrenk et C.A.Mey, or Ephedra equisetifolia equisetum Bge; the semen Armeniacae amarum is dried mature seed of Prunus armeniaca L.var.ansu Maxim, prunus sibirica L.in Siberian, prunus mandshurica (Maxim.) Koehne or Prunus Primus armeniaca L.in Rosaceae; the gypsum is sulfate mineral gypsum group gypsum; coicis semen is dried mature kernel of Coicis semen Coix lacryma-jobi L.var.ma-yuen (Roman.) Stapf of Gramineae; atractylodes lancea is dried rhizome of Atractylodes lancea DC of Compositae; herba Agastaches is dry aerial part of herba Agastaches Pogostemon cablin (Blanco) Benth of Labiatae; the Artemisia annua L is dried aerial part of Artemisia annua L of Compositae; rhizoma Polygoni Cuspidati is dried rhizome and root of Polygonum cuspidatum Sieb. Et Zucc. Of Polygonaceae; the herba Verbenae is dry aerial parts of Verbena officinalis L. of Verbenaceae; the dry reed root is the dry rhizome of Phragmitis communis Trin of Gramineae plant; the semen Lepidii is dry mature seed of Descurainia sophia (L.) Sophia in Brassicaceae or Lepidium apetalum Willd; exocarpium Citri Grandis is immature or near mature dry outer pericarp of Osbeck of Citrus grandis (Citrus grandis L.) of Rutaceae; the raw Glycyrrhrizae radix is dried root and rhizome of Glycyrrhiza uralensis Fisch, glycyrrhiza inflata Bat or Glycyrrhiza glabra L of Leguminosae.
Method and results
2.1 preparation of Standard decoction
The lung-ventilating and toxin-vanquishing formula consists of 6g of raw ephedra, 15g of bitter apricot seed, 30g of gypsum, 30g of raw coix seed, 10g of atractylis lancea, 15g of pogostemon cablin, 12g of artemisia apiacea, 20g of polygonum cuspidatum, 30g of verbena, 30g of dry reed rhizome, 15g of semen lepidii, 15g of exocarpium citri grandis and 10g of raw liquorice, and 12 batches of lung-ventilating and toxin-vanquishing formulas are randomly combined by utilizing SPSS 26.0 in different batches. Setting the random seeds of the 13 Chinese medicaments as fixed values 20209201-20209213 in sequence through a menu 'conversion → random number generator', and generating random numbers; the different batches of each traditional Chinese medicine are arranged according to the ascending order of random numbers and are divided into 1 to 12 batches of lung-ventilating and toxin-vanquishing formulas in sequence.
Crushing each batch of decoction pieces, sieving with a 60-mesh sieve, precisely weighing 5.00g of each batch of medicinal herbs according to the prescription proportion, uniformly mixing, adding 50mL of water, weighing, soaking for 30min, heating, refluxing and extracting for 60min, complementing weight loss, and sieving with 3 layers of gauze to obtain 12 batches of standard decoction of the formula for freeing lung, detoxifying, freezing and storing at minus 80 ℃ and numbering S1-S12.
2.2 chromatographic conditions
The column was Agilent ZORBAX RRHD Eclipse XDB-C18 (2.1X 100mm,1.8 μm). 0.1% formic acid (A) -acetonitrile (B) as a mobile phase, 0 to 8min as a gradient elution procedure, 5 to 10% by weight of B; 8-13min, 10% -15% of C; 13 to 18min,15 to 17% by weight B; 18-30min, 17-45% of B; 30-35min, 45-95 percent B; the detection wavelength is 254nm; the volume flow is 0.3mL/min; the sample injection volume is 2 mu L; the column temperature was 40 ℃.
2.3 solution preparation
2.3.1 Standard solution
Preparing a standard substance mixed solution: accurately weighing appropriate amount of standard substances, respectively, dissolving with methanol, and preparing ephedrine, polydatin, amygdalin, verbascoside, acteoside, glycyrrhizic acid mother liquor with concentration of 1.0mg/mL, and naringin mother liquor with concentration of 4.0mg/mL; the above mother solutions were each precisely aspirated, mixed with methanol to give mixed standard solutions having concentrations of 50. Mu.g/mL, 150. Mu.g/mL, 100. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, and 1mg/mL, respectively, and placed in a refrigerator at 4 ℃ for further use.
Preparing a quality control stock solution: precisely transferring appropriate amount of each standard substance stock solution respectively, adding 50% methanol, continuously diluting, and making into quality control stock solution containing ephedrine (2, 10, 25 μ g/mL), amygdalin (4, 20, 50 μ g/mL), verbascoside (4, 20, 50 μ g/mL), verbenaside (4, 20, 50 μ g/mL), polydatin (6, 30, 75 μ g/mL), acteoside (2, 10, 25 μ g/mL), liquiritin (2, 10, 25 μ g/mL), naringin (40, 200, 500 μ g/mL), glycyrrhizic acid (4, 20, 50 μ g/mL) with high, medium, and low concentrations (3 different concentrations). All solutions were stored in a 4 ℃ freezer for use.
2.3.2 test article solutions
Precisely measuring 2mL of standard decoction of the lung ventilating and toxin removing formula under the item '2.1', placing in a 10mL volumetric flask, adding 50% methanol to dilute to a scale mark, shaking up, performing ultrasonic treatment for 30min, diluting to a constant volume, and filtering through a 0.22 mu m microporous membrane to obtain a test solution.
2.4 establishment of fingerprint of Lung-ventilating and toxin-vanquishing prescription
2.4.1 precision test
Taking the same lung-ventilating and toxin-vanquishing prescription sample solution (S6), continuously sampling for 6 times according to the chromatographic condition under the item of 2.2, recording an UHPLC (ultra high performance liquid chromatography) diagram, taking naringin as a reference peak, and respectively calculating the relative retention time of each common peak and the RSD value of the relative peak area to be 0.205-0.580% and 0.603-1.211%, which indicates that the instrument has good precision.
2.4.2 stability test
Taking the same lung-ventilating and toxin-vanquishing prescription test solution (S6), respectively injecting samples at 0, 2, 4, 8, 12 and 24 hours according to the chromatographic condition under the item of 2.2, taking naringin as a reference peak, and indicating that the test solution is stable within 24 hours, wherein the RSD value of each common peak is 0.214-0.390%, and the RSD value of each relative peak area is 0.150-0.579%.
2.4.3 repeatability tests
Taking S6 batches of lung ventilating and toxin removing formulas, preparing 6 parts of test solution in parallel according to the method under the item 2.3.2, injecting sample according to the chromatographic condition under the item 2.2, recording a UHPLC (ultra high performance liquid chromatography) diagram, taking naringin as a reference peak, and calculating to obtain the relative retention time of each common peak and the RSD (differential pressure) value of the relative peak area to be 0.038-0.715% and 0.238-2.343%, which indicates that the method has good repeatability.
2.4.4 fingerprint establishment and similarity evaluation
Taking 12 batches of lung ventilating and toxin counteracting prescription test sample solutions, carrying out sample injection detection under the chromatographic condition of the item 2.2, and recording a chromatogram map. Data analysis is carried out by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), S1 is taken as a reference spectrum, the time window width is set to be 0.1min, multipoint correction and full spectrum peak matching (peak area accounts for more than 0.5 percent of the total peak area to participate in matching) are carried out by adopting an average method, and a channel warming decoction fingerprint and a comparison fingerprint are generated, and are shown in figure 1. The total 21 common peaks are marked, wherein 7 chromatographic peaks are identified, and the chromatographic peaks are 6-peak verbascoside, 7-peak verbascoside, 10-peak polydatin, 11-peak liquiritin, 14-peak acteoside, 15-peak naringin and 21-peak glycyrrhizic acid respectively. By taking the comparison fingerprint as reference, the similarity of 12 samples of the lung ventilating and toxin removing formula is calculated to be respectively 0.983, 0.959, 0.937, 0.986, 0.979, 0.983, 0.946, 0.986, 0.990, 0.982, 0.941 and 0.960, and is more than 0.90, the similarity is good, which indicates that the established fingerprint material standard of the lung ventilating and toxin removing formula has good stability and can reflect the fingerprint characteristics thereof, and the detail is shown in table 1.
TABLE 1 similarity of UHPLC fingerprint spectra of the materials of the Lung-ventilating and toxin-vanquishing formula
2.5 multicomponent assay
2.5.1 chromatographic conditions
The chromatographic column, the mobile phase, the gradient elution program, the column temperature, the volume flow and the sample injection volume are all the same as the term of 2.2; the detection wavelengths were 210nm and 254nm.
2.5.2 specificity test
Taking a proper amount of the mixed standard solution under the item '2.3' and the test solution (S6), carrying out sample injection measurement under the chromatographic condition under the item '2.5.1', recording a chromatogram, and as shown in figure 2, according to results, chromatographic peaks with the same retention time appear in the test solution at corresponding positions of ephedrine, amygdalin, verbenaside, polydatin, liquiritin, acteoside, naringin and glycyrrhizic acid reference substances, wherein the known components have good separation degree, and the results show that the specificity of the method is good.
2.5.3 Linear relationship investigation
Taking the mixed standard solution under the item 2.3.1, preparing a series of mixed reference substance solutions by a multiple dilution method, respectively injecting samples according to the chromatographic condition under the item 2.5.1, measuring, recording peak areas, drawing a standard curve by taking the reference mass concentration as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y), and respectively calculating a detection Limit (LOD) and a quantification Limit (LOQ) by using signal to noise ratios (S/N) 3 and 10, wherein the results are shown in a table 2.
TABLE 2 examination of the Linear relationship
2.5.4 precision test
The precision examination is to take a proper amount of three quality control samples (n = 6) with different concentrations under the item of '2.3.1', sample injection measurement is carried out according to the chromatographic condition under the item of '2.5.1', and samples within one day and continuously three days are analyzed as the precision within the day and the precision between the days and are expressed by the accuracy (the ratio of the measured concentration to the theoretical concentration) and the RSD (the variation value of the measured concentration). The results are shown in Table 3, the RSD of the precision in the day and the day is 0.09% -5.14%, and the accuracy is 94.3% -105%, which shows that the method has good precision.
TABLE 3 precision and stability of 9 ingredients of the Lung-ventilating and toxin-vanquishing formula
2.5.5 stability test
Taking appropriate amount of quality control samples with three different concentrations under the item of 2.3.1, respectively measuring at 0, 2, 4, 8, 12 and 24h under the chromatographic condition under the item of 2.5.1, calculating and comparing the ratio of the actually measured concentration to the theoretical concentration, and expressing the ratio by accuracy and RSD. The results are shown in Table 3, with RSD of 0.44% -4.86% for stability and 95.6% -105% for accuracy, indicating good stability of the sample at 24h of injector placement.
2.5.6 repeatability tests
Taking S6 batches of lung-ventilating and toxin-vanquishing formulas, preparing 6 parts of test sample solution in parallel according to the method under the item 2.3.2, and then measuring the RSD of the average content of each component of ephedrine, amygdalin, verbascoside, polydatin, liquiritin, acteoside, naringin and glycyrrhizic acid by sample injection under the item 2.5.1, wherein the RSD of each component of ephedrine, amygdalin, verbenaside, polydatin, liquiritin, acteoside, naringin and glycyrrhizic acid is 1.03%, 0.63%, 2.33%, 2.13%, 2.30%, 3.94%, 0.65%, 1.65% and 0.82% in sequence, which shows that the method has good repeatability.
2.5.7 sample recovery test
Precisely absorbing 6 parts of a lung-ventilating and toxin-vanquishing formula (S6) with a known content, precisely adding a reference substance solution with a level of 50%, preparing a test sample solution in parallel according to the method under the item 2.3.2, then carrying out sample injection according to the chromatographic condition under the item 2.5.1, and calculating the average sample injection recovery rate of ephedrine, amygdalin, verbascoside, polydatin, liquiritin, acteoside, naringin and glycyrrhizic acid, wherein the results are 96.8%, 94.5%, 97.5%, 97.6%, 101%, 93.7%, 94.2%, 105% and 103% in sequence; RSD is 0.90%, 0.80%, 4.66%, 1.18%, 3.71%, 0.32%, 0.62%, 0.82%, 0.90%, respectively.
2.5.8 sample assay
Taking each batch of lung ventilating and toxin removing formula, preparing a test solution according to the method under the item 2.3.2, injecting samples under the chromatographic condition of the item 2.5.1, and measuring the content of 12 batches of test solutions. The results are shown in Table 4, the contents of ephedrine, amygdalin, verbena glycoside, polygonin, liquiritin, acteoside, naringin and glycyrrhizic acid are 0.21-0.32 mg/g, 0.25-1.13 mg/g, 0.74-1.17 mg/g, 0.60-0.92 mg/g, 0.48-1.08 mg/g, 0.15-0.43 mg/g, 0.48-1.14 mg/g, 1.83-6.44 mg/g and 0.25-0.49 mg/g in sequence, wherein the naringin content is the highest, and the content difference among different batches is larger.
TABLE 4 determination of contents of 9 components in the formula for ventilating the lung and relieving toxicity
3 conclusion
The fingerprint is an effective quality control method for comprehensively and integrally reflecting the information of the traditional Chinese medicine compound, the UHPLC fingerprint of the lung-ventilating and toxin-vanquishing formula is established for the first time in the experiment, and the multi-index component content determination of ephedrine, amygdalin, verbascoside, verbena glycoside, polygonin, liquiritin, acteoside, naringin and glycyrrhizic acid is carried out. The method is efficient, simple and convenient, has good repeatability, and provides a better reference basis for the material basis and the comprehensive quality control of the lung ventilating and toxin removing prescription.
It should be noted that the above examples are only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
Claims (4)
1. A method for establishing a fingerprint of a lung-ventilating and toxin-vanquishing formula is characterized in that a mixed component is used as a reference substance, and the RSD value of the relative retention time and the relative peak area of each common peak is calculated, wherein the mixed component is ephedrine, amygdalin, verbena glycoside, polydatin, liquiritin, acteoside, naringin and glycyrrhizic acid; the method comprises the following steps:
a. preparing a test solution: adding methanol into the prescription for ventilating the lung and removing toxicity, performing ultrasonic extraction, and filtering to obtain a solution to be detected;
b. preparation of a reference solution: adding methanol into the mixed component reference substance dried under reduced pressure to constant weight to obtain mixed reference substance solution;
c. and (3) determination: respectively and precisely measuring a test solution and a reference solution, carrying out UPLC analysis, and carrying out gradient elution to obtain a mobile phase A: 0.1% formic acid, mobile phase B: acetonitrile, analyzing and processing the atlas obtained by the ultra-high performance liquid chromatography determination to obtain the lung ventilating and toxin counteracting prescription fingerprint atlas, wherein the chromatographic column is Agilent ZORBAX RRHD Eclipse XDB-C18, the specification is 2.1X 100mm,1.8 mu m, the gradient elution procedure is 0-8min, and the proportion is 5-10 percent B; 8-13min, 10% -15% of C; 13 to 18min,15 to 17% by weight B; 18-30min, 17-45% of B; 30-35min, 45-95 percent B; the detection wavelength is 254nm; the volume flow is 0.3mL/min; the sample injection volume is 2 mu L; the column temperature was 40 ℃.
2. The method for establishing the fingerprint of the lung ventilating and toxin vanquishing prescription according to claim 1, wherein the preparation method of the mixed reference solution in the step b is as follows: accurately weighing a plurality of mixed component reference substances, placing the mixed component reference substances into a volumetric flask, adding methanol to dissolve and diluting the mixed component reference substances to prepare a mixed reference substance solution.
3. Use of the method according to any one of claims 1-2 for quality control of lung ventilating and toxicity removing prescription.
4. A quality control method for a lung ventilating and toxin vanquishing formula is characterized by comprising the following steps: taking a plurality of batches of lung-ventilating and toxin-vanquishing formulas to obtain fingerprint spectra of the lung-ventilating and toxin-vanquishing formulas according to the method of any one of claims 1-2, and generating the fingerprint spectra of the lung-ventilating and toxin-vanquishing formulas according to an averaging method or a median method to obtain a comparison fingerprint spectrum.
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