A method for constructing a fingerprint spectrum of a Qingfei Decoction compound preparation and a quality detection method

Abstract

This invention relates to the field of quality testing technology for traditional Chinese medicine preparations, specifically providing a method for constructing a fingerprint spectrum and a quality testing method for a Qingfei Decoction compound preparation. In the fingerprint spectrum construction method, the chromatographic conditions include: using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and an aqueous solution containing phosphoric acid as mobile phase B, obtaining 10 common characteristic peaks under a specific elution program. Under the conditions of this invention, the characteristic peaks are well separated, each peak has a good peak shape, and the method can comprehensively, clearly, and effectively detect the quality of the Qingfei Decoction compound preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection of traditional Chinese medicine preparations, and in particular to a method for constructing a fingerprint spectrum of a Qingfei Decoction compound preparation and a quality detection method. Background Art

[0002] Qingfei Decoction was created by Gong Tingxian in the Ming Dynasty. This recipe has the effects of clearing heat and moistening the lungs, descending qi and resolving phlegm, and is mainly used to treat phlegm-heat cough. Symptoms include cough, rough wheezing, phlegm in the throat, thick yellow phlegm and large amount of phlegm, or hoarseness after long-term cough, red tongue with yellow greasy coating, and slippery and rapid pulse. In the recipe, Scutellaria baicalensis and Fritillaria thunbergii are the main drugs, Platycodon grandiflorum, Morus alba, Angelica sinensis, Asparagus cochinchinensis, and Ophiopogon japonicus are the assistant drugs, Tangerine peel, Poria cocos, Gardenia jasminoides, boiled bitter almonds, Schisandra chinensis, ginger, and jujube are the adjuvant drugs, and Licorice is the guiding drug. Flavonoids in Scutellaria baicalensis, alkaloids in Fritillaria thunbergii, saponins and polysaccharides in Platycodon grandiflorum, flavonoids and polysaccharides in Morus alba bark, organic acids and polysaccharides in Angelica sinensis, steroidal saponins and polysaccharides in Asparagus cochinchinensis and Ophiopogon japonicus, polysaccharides and triterpenes in Poria cocos, flavonoids in Citrus aurantium, iridoids and flavonoids in Gardenia jasminoides, lignins in Schisandra chinensis, gingerols in ginger, and polysaccharides and saponins in jujube are related to their effectiveness and are their possible main pharmacodynamic material basis.

[0003] The qualitative identification and quantitative analysis of simple index components are difficult to reflect the overall quality of compound preparations, while fingerprints have the function of overall and characteristic qualitative identification, which can make a comprehensive qualitative evaluation of the taste of compound preparations, reflect the material basis of the efficacy of compound preparations, and reflect the authenticity of compound preparations. Therefore, establishing a new effective, accurate and comprehensive method for detecting the quality of Qingfei Decoction compound preparations has become an urgent problem to be solved. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a fingerprint spectrum of a Qingfei Decoction compound preparation and a method for quality detection. The method establishes a fingerprint spectrum according to the characteristics of the Qingfei Decoction preparation, realizes the effective separation of characteristic peaks, and can comprehensively, clearly and effectively detect the quality of the Qingfei Decoction compound preparation.

[0005] To this end, the present invention discloses a method for constructing a fingerprint spectrum of a Qingfei Decoction compound preparation, comprising the following steps:

[0006] S1. Preparation of test solution;

[0007] S2. The test solution was tested by high performance liquid chromatography. The chromatographic conditions included using octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, and aqueous solution containing phosphoric acid as mobile phase B. Gradient elution was performed according to the following procedure:

[0008]

[0009] Furthermore, the preparation method of the test solution is: take the test sample, add solvent 1 to extract to obtain an extract, separate the solid and liquid, and take the liquid, which is the test sample solution; or, take the test sample, add solvent 1 to extract to obtain an extract, separate the solid and liquid, dry the liquid to obtain a residue, dissolve the residue in solvent 2, separate the solid and liquid again, and take the liquid, which is the test sample solution; or, take the test sample, add solvent 1 to extract to obtain an extract, separate the solid and liquid, dry the liquid to obtain a residue, dissolve the residue in solvent 2, extract with water-saturated n-butanol, dry the n-butanol extract, add solvent 3 to extract, separate the solid and liquid of the extract, and take the liquid, which is the test sample solution.

[0010] Furthermore, the method for preparing the test solution also satisfies any one or more of the following AH:

[0011] A. The ratio of the mass of the sample to the volume of solvent 1 is 0.8-2:25; and / or, the ratio of the mass of the sample to the volume of solvent 2 is 1-5:25-60; and / or, the ratio of the mass of the sample to the volume of solvent 3 is 1-5:20-55; and / or, the relationship between mass and volume is g / mL;

[0012] B. The solvent 1 is selected from at least one of methanol, ethanol, methanol aqueous solution, water, ethanol aqueous solution or water-saturated n-butanol, preferably methanol;

[0013] C. The solvent 2 is selected from at least one of methanol, ethanol and water;

[0014] D. The solvent 3 is selected from at least one of methanol, ethanol and water;

[0015] E. The extraction method is ultrasonic extraction or reflux extraction;

[0016] F. The extraction time is 20-40min;

[0017] G. The drying method is selected from one or more of evaporation, freeze drying, and reduced pressure drying;

[0018] H. The volume ratio of the solution obtained after dissolving the residue in solvent 2 to the water-saturated n-butanol extract is 0.8-1.2:1, and the number of extractions is 2-3 times.

[0019] Furthermore, in step S2, the chromatographic column adopts a chromatographic column with a specification of 4.6×250mm, 5μm; and / or the injection volume is 5-15μL; and / or the volume percentage of the aqueous solution containing phosphoric acid is 0.08-0.12%; and / or the flow rate is 0.8-1.2mL / min; and / or the column temperature is 30-40°C; and / or the detection wavelength is 200-250nm; and / or the high performance liquid chromatography includes HPLC-UV method or HPLC-ELSD method.

[0020] Preferably, the volume percentage of the aqueous solution containing phosphoric acid is 0.10%; and / or, the flow rate is 1.0 mL / min; and / or, the column temperature is 35° C.; and / or, the detection wavelength is 200 nm.

[0021] Furthermore, the construction method also includes the step of using at least one of amygdalin, gardenia glycoside, 3,6,7,2′,6′-pentahydroxydihydroflavonol, hesperidin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronic acid, baicalin, baicalin, and wogonin as a reference substance solubilizing agent to prepare a reference substance solution, and the step of detecting the reference substance solution by high performance liquid chromatography according to the fingerprint spectrum construction method of the Qingfei Decoction compound preparation to obtain a reference substance spectrum.

[0022] Furthermore, the solvent is selected from at least one of methanol, ethanol and water; and / or, 1 mL of the reference substance solution contains 0.05-0.5 mg of each reference substance.

[0023] Furthermore, the fingerprint of the Qingfei Decoction compound preparation has 10 common peaks, peak 1 is amygdalin, peak 2 is gardenia glycoside, peak 4 is hesperidin, peak 5 is baicalin, peak 7 is melaleucain A-7-O-β-D-glucuronic acid, peak 8 is baicalin, peak 9 is baicalin, and peak 10 is wogonin.

[0024] Further, the Qingfei Decoction compound preparation is a compound preparation prepared by conventional technical means with Scutellaria baicalensis, Platycodon grandiflorum, Poria cocos, Dictyophora tangerinae, Fritillaria thunbergii, Morus alba, Angelica sinensis, Asparagus cochinchinensis, Gardenia jasminoides, Schisandra chinensis, Licorice root, Ginger, Boiled Bitter Almond, Ophiopogon japonicus, and Jujube as raw materials. According to weight parts, the Qingfei Decoction compound preparation includes the following raw materials: 4-7 parts of Scutellaria baicalensis, 2-5 parts of Platycodon grandiflorum, 2-5 parts of Poria cocos, 2-5 parts of Dictyophora tangerinae, 2-5 parts of Fritillaria thunbergii, 2-5 parts of Morus alba, 1-4 parts of Angelica sinensis, 1-4 parts of Asparagus cochinchinensis, 1-4 parts of Gardenia jasminoides, 0.5-2 parts of Schisandra chinensis, 0.5-2 parts of Licorice root, 1-3 parts of Ginger, 1-4 parts of Boiled Bitter Almond, 1-4 parts of Ophiopogon japonicus, and 5-8 parts of Jujube. For these compound preparations, the method of the present invention can be used to construct fingerprints.

[0025] Furthermore, the Qingfei Decoction compound preparation includes a solid preparation, a semisolid preparation and a liquid preparation of Qingfei Decoction, preferably at least one of Qingfei Decoction liquid, Qingfei Decoction capsule and Qingfei Decoction dry paste powder. The Qingfei Decoction compound preparation can be prepared by conventional technical means, including but not limited to the methods specified in the Chinese Pharmacopoeia.

[0026] Furthermore, the Qingfei Decoction compound preparation is obtained by crushing, decocting, concentrating and drying 15 traditional Chinese medicines including scutellaria baicalensis, platycodon grandiflorum, tuckahoe, tangerine peel, thunbergia thunbergii, mulberry bark, angelica sinensis, asparagus cochinchinensis, gardenia jasminoides, schisandra chinensis, licorice, ginger, stewed bitter almonds, ophiopogon japonicus and jujube by conventional methods.

[0027] The present invention also provides the application of the method for constructing the fingerprint spectrum of the Qingfei Decoction compound preparation in the quality detection of the Qingfei Decoction compound preparation.

[0028] The invention also provides a quality detection method for the Qingfei Decoction compound preparation product.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1. The method for constructing the fingerprint of the Qingfei Decoction compound preparation of the present invention comprises the following steps: S1, preparation of a test solution; S2, detection of the test solution by high performance liquid chromatography, wherein the chromatographic conditions include gradient elution using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and an aqueous solution containing phosphoric acid as a mobile phase B. Under the elution conditions of the present invention, the characteristic peaks are well separated, and the peak shapes of each peak are good, so that the quality of the Qingfei Decoction compound preparation can be comprehensively, clearly and effectively detected.

[0031] 2. The method for constructing the fingerprint of the Qingfei Decoction compound preparation of the present invention is to prepare different batches of Qingfei Decoction reference samples from different batches of decoction pieces, and then construct the fingerprint of the samples, and the constructed fingerprint has high credibility. The fingerprint method established on the basis of the Qingfei Decoction compound preparation can characterize the information of the medicinal taste of Scutellaria baicalensis, Gardenia jasminoides, Bitter Apricot Kernel and Tangerine Peel, and is suitable for the evaluation of other Qingfei Decoction compound preparations on the market.

[0032] 3. The method for constructing the fingerprint of the Qingfei Decoction compound preparation described in the present invention, by using at least one of amygdalin, gardenia glycoside, 3,6,7,2′,6′-pentahydroxydihydroflavonol, hesperidin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronic acid, baicalin, baicalin, and wogonin to prepare a reference solution and construct a reference fingerprint, can achieve the positioning of 9 common peaks out of 10 common peaks in the fingerprint of the Qingfei Decoction compound preparation, so that the chromatogram obtained by detection is more accurate, stable and reliable, which indirectly shows that the chromatographic conditions of the present invention can be used to achieve effective separation of 10 active ingredients in the Qingfei Decoction compound preparation, and the constructed fingerprint has a high degree of credibility.

[0033] 4. The quality detection method of the Qingfei Decoction compound preparation described in the present invention is to construct a fingerprint of the Qingfei Decoction compound preparation product to be tested according to the method for constructing the fingerprint of the Qingfei Decoction compound preparation of the present invention, and perform quality detection on the characteristic effective ingredients of the Qingfei Decoction compound preparation, so that the quality detection of the Qingfei Decoction compound preparation is more comprehensive, thereby ensuring the effectiveness and controllability of the quality of the Qingfei Decoction compound preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 is the fingerprint spectrum of 30 batches of Qingfei Decoction in Example 1-1; R is the control spectrum; the batches corresponding to S2-S16 are 181105-01, 181105-02, 181105-03, 181105-04, 181105-05, 1811105-06, 181106-07, 181106-08, 181106-09, 181106-10, 181106-11, 181106-12, 181106-13, 181106-14, and 181106-15, respectively.

[0036] Figure 2 It is the fingerprint spectrum -2 of 30 batches of Qingfei Decoction in Example 1; R is the control spectrum; the batches corresponding to S2-S16 are 181106-16, 181106-17, 181106-18, 181107-19, 181107-20, 181107-21, 181107-22, 181107-23, 181107-24, 181107-25, 181107-26, 181107-27, 181107-28, 181107-29, and 181107-30, respectively.

[0037] Figure 3 It is the reference fingerprint of Qingfei Decoction in Example 1; Peak 1-Peak 5 correspond to amygdalin peak, gardenia glycoside peak, 3,6,7,2′,6′-pentahydroxydihydroflavonol, hesperidin, and baicalin, respectively; Peak 7-Peak 10 correspond to melaleuca alternifolia A-7-O-β-D-glucuronic acid, baicalin, baicalein, and wogonin, respectively.

[0038] Figure 4This is the HPLC-UV fingerprint of the Qingfei Decoction benchmark sample of method 1 in Example 2.

[0039] Figure 5 It is the HPLC-UV fingerprint of the single medicinal material and the Qingfei Decoction benchmark sample in method 2 a) in Example 2; S1-S6 correspond to the test sample (batch number: 20180115-2), Scutellaria baicalensis slices, blank solvent, Citrus aurantium slices, Gardenia jasminoides slices, and stewed bitter almond slices, respectively.

[0040] Figure 6 It is the HPLC-UV fingerprint of the single medicinal material and the Qingfei Decoction benchmark sample of method 2 b) in Example 2; S1-S10 correspond to the test sample (batch number: 20180115-2), Platycodon grandiflorum slices, Fritillaria thunbergii slices, Morus alba slices, ginger slices, Schisandra chinensis slices, Scutellaria baicalensis slices, Citrus aurantium slices, Gardenia jasminoides slices, and Licorice slices, respectively.

[0041] Figure 7 It is the HPLC-UV fingerprint spectrum of the single medicinal material and the Qingfei Decoction benchmark sample of method 3 in Example 2-1; S1-S9 correspond to the test sample (batch number: 20180115-2), tangerine peel slices, Poria cocos slices, Gardenia slices, stewed bitter almond slices, Schisandra slices, ginger slices, jujube slices, and licorice slices, respectively.

[0042] Figure 8 It is the HPLC-UV fingerprint spectrum of the single medicinal material and the Qingfei Decoction benchmark sample of method 3 in Example 2-2; S1-S8 correspond to the test sample (batch number: 20180115-2), Scutellaria baicalensis slices, Fritillaria thunbergii slices, Platycodon grandiflorum slices, Morus alba slices, Angelica sinensis slices, Asparagus cochinchinensis slices, and Ophiopogon japonicus slices, respectively.

[0043] Fig. 9 HPLC-UV fingerprints of the single medicinal materials and the Qingfei Decoction reference sample in method a) of Example 2.

[0044] Fig.10 HPLC-UV fingerprints of the single medicinal materials and the Qingfei Decoction reference sample in method b) of Example 2.

[0045] Fig.11 This is the HPLC-ELSD fingerprint of the Qingfei Decoction reference sample of method 5 a) in Example 2.

[0046] Fig.12 This is the HPLC-ELSD fingerprint of the Scutellaria baicalensis slices of method 5 a) in Example 2.

[0047] Fig.13 This is the HPLC-ELSD fingerprint of the Citrus aurantium decoction pieces of method 5 a) in Example 2.

[0048] Fig.14It is the Qingfei Decoction reference sample and single medicinal material HPLC-ELSD fingerprint-1 of method 5 b) in Example 2; S1-S5 correspond to Asparagus cochinchinensis slices, Fritillaria thunbergii slices, Platycodon grandiflorum slices, Scutellaria baicalensis slices, and the test sample, respectively.

[0049] Fig.15 It is the HPLC-ELSD fingerprint of single medicinal materials and Qingfei Decoction benchmark sample b) in method 5 in Example 2; S1-S5 correspond to ginger slices, Schisandra slices, Tangerine peel slices, test sample, and Gardenia slices, respectively.

[0050] Fig.16 HPLC-UV fingerprints of single medicinal materials and Qingfei Decoction benchmark samples in method 6 in Example 2; S1-S5 correspond to the test sample (batch number: 180809-03), stewed bitter almond slices, gardenia slices, scutellaria slices, and tangerine peel slices, respectively.

[0051] Fig.17 HPLC-UV fingerprint of Qingfei Decoction benchmark samples extracted with different solvents according to method 7 in Example 2.

[0052] Fig.18 HPLC-UV fingerprints of Qingfei Decoction reference samples extracted by methanol-water solutions of different concentrations in method 8 in Example 2.

[0053] Fig.19 This is the HPLC spectrum of the Qingfei Decoction reference sample with the detection wavelength selected in Example 2.

[0054] Fig. 20 HPLC comparison chart of the peak attribution and the reference selected in Example 2 -1; S1-S11 correspond to the Fritillaria thunbergii slices, lack of Fritillaria thunbergii negative, Citrus aurantium slices, lack of Citrus aurantium negative, Poria slices, lack of Poria negative, Platycodon grandiflorum slices, lack of Platycodon grandiflorum negative, Scutellaria baicalensis slices, lack of Scutellaria baicalensis negative, and Qingfei Decoction benchmark sample.

[0055] Fig.21 HPLC comparison chart of the peak attribution and the medicinal taste attribution selected for the reference in Example 2-2; S1-S11 correspond to the boiled bitter almond slices, lack of boiled bitter almond negative, gardenia slices, lack of gardenia negative, asparagus slices, lack of asparagus negative, angelica slices, lack of angelica negative, mulberry bark slices, lack of mulberry bark negative, and benchmark samples, respectively.

[0056] Fig. 22 HPLC comparison chart of the peak attribution and the medicinal taste attribution selected for the reference in Example 2-3; S1-S11 correspond to the jujube slices, negative lack of jujube, ginger slices, negative lack of ginger, licorice slices, negative lack of licorice, Schisandra slices, negative lack of Schisandra, Ophiopogon slices, negative lack of Ophiopogon, and benchmark samples, respectively.

[0057] Fig.23 HPLC comparison spectra were used to confirm the relevant components selected for peak attribution and reference in Example 2; S1-S9 corresponded to the benchmark sample, wogonin, baicalin, wogonin, melaleucalycin A-7-O-β-D-glucuronic acid, baicalin, hesperidin, gardenia glycoside, and amygdalin, respectively.

[0058] Fig.24 UPLC-UV chromatogram and UPLC-MS total ion current diagram of the Qingfei Decoction reference sample selected for peak attribution and reference in Example 2; Peaks 1 to 50 correspond to O-β-D-Gentiobiosyl-D-mandelamide, gentiana scabranoside, deacetylcaryophyllene methyl ester, Jasminoside B, phlorothiolane, jujube benzyl glycoside, amygdalin, genipin-1-β-D-gentiobiosyl-D-mandelamide, gardenia glycoside, 3,6,7,2′,6′-pentahydroxydihydroflavonol, Jasminoside K, Epijasminoside A, New Zealand vitexin, Fritillaria spicatin, zeburin glycoside, Fritillaria succsin B, liquiritin, apigenin liquiritin, 6-C-glucose-8-C arabinose chrysin, 8-C-glucose-6-C arabinose chrysin, rutin, zeburin A, 6-C-glucose-8-C arabinose chrysin isomer, verbascoside, zeburinone, naringin, isoverbascoside, hesperidin, 5,7,2′,5′-tetrahydroxy-8,6′-dimethoxyflavonoids, isozeburin A, baicalin-7-O-β-D-pyranoglucoside, baicalin, baicalin-7-O D-glucoside, apigenin-7-O-β-D-glucuronic acid, 5,7,2′-trihydroxy-6-methoxyflavone-7-O-glucuronide, chrysin-7-O-β-D-glucuronide, melaleucatin A-7-O-β-D-glucuronic acid, wogonin, platycodon saponin D, baicalin, glucosylnaringin, wogonin, baicalein I, glycyrrhizic acid, baicalein, nobiletin, schisandrin A, 3,5,6,7,8,3′,4′-heptamethoxyflavone, 5,6,7,3′,4′-pentamethoxyflavone, schisandrin B.

[0059] Fig.25 This is the fingerprint of the specificity test and integrity investigation of Example 3.

[0060] Fig.26 This is the instrument precision test fingerprint of Example 3.

[0061] Fig. 27 This is the stability test fingerprint of Example 3.

[0062] Fig.28 This is the repeatability test fingerprint of Example 3.

[0063] Fig.29 This is a comparison chart of the similarity of different flow rate tests in Example 3.

[0064] Fig.30 This is a comparison chart of the similarity of different column temperature tests in Example 3.

[0065] Fig.31 This is a similarity comparison diagram of different acidity tests in Example 3.

[0066] Fig.32 This is a comparison chart of the similarity of different chromatographic column tests in Example 3.

[0067] Fig.33 This is a comparison chart of the similarity of different instrument tests in Example 3. DETAILED DESCRIPTION

[0068] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0069] The instruments and reagents used in the present invention are as follows:

[0070] High performance liquid chromatograph 1: Shimadzu LC-20AT chromatography system; High performance liquid chromatograph 2: Agilent 1260II chromatography system; 1 / 10,000 balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd., MS204TS / 02); 1 / 1000 balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., SQP); Chromatographic columns: (1) Kromasil 100-5-C18 (4.6×250 mm, 5 μm); (2) Welch Xtimate C18 (4.6×250 mm, 5 μm); (3) Sepax GP-C18 (4.6×250 mm, 5 μm). Methanol (chromatographic grade, Tianjin Saifurui); phosphoric acid (chromatographic grade, MREDA); methanol, n-butanol, chloroform, and sulfuric acid are all analytical grade (Beijing Chemical Plant); ethyl acetate (analytical grade, GENERAL); formic acid (analytical grade, Taicang Hushi Reagent Co., Ltd.); water (purified water, Hangzhou Wahaha Group Co., Ltd.). Amygdalin (batch number: 110820-201607, purity 90.7%), gardenia jasminoides (batch number: 110749-201617, purity 98.4%), hesperidin (batch number: 110721-201617, purity 96.1%), baicalin (batch number: 110715-201720, purity: 93.5%), baicalein (batch number: 111595-2016 07, purity 98.5%), wogonin (batch number: 111514-201605, purity 98.5%), and baicalin (batch number: 112002-201702, purity 98.5%) were all purchased from China Food and Drug Inspection Institutes; melaleucalyptus A-7-O-β-D-glucuronic acid (batch number: 18040202) was purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0071] The preparation method of Qingfei Decoction extract is as follows: except ginger and jujube, the remaining 13 herbs are crushed into coarse particles of 1 to 5 mm, 5.60 g of Scutellaria baicalensis, 3.73 g of Platycodon grandiflorum, 3.73 g of Poria cocos, 3.73 g of Citrus aurantium, 3.73 g of Fritillaria thunbergii, 3.73 g of Morus alba, 2.61 g of Angelica sinensis, 2.61 g of Asparagus cochinchinensis, 2.61 g of Gardenia jasminoides, 2.61 g of Boiled Bitter Almond, 2.61 g of Ophiopogon japonicus, 1.00 g of Schisandra chinensis, and 1.12 g of Licorice are put into a casserole, and 2.00 g of ginger (cut into pieces when used) is added. thick slices), 6.00g of jujube (broken when used), add 12 times the amount of water (based on the total mass of the slices) into a casserole, soak for 30 minutes, heat with an electric ceramic stove, boil over high heat, then simmer for 15 minutes, filter through a 200-mesh nylon filter cloth while hot to obtain a filtrate; add 10 times the amount of water (based on the total mass of the slices) to the filter residue, boil over high heat, then simmer for 10 minutes, filter through a 200-mesh nylon filter cloth while hot to obtain a filtrate, combine the two filtrates to obtain Qingfei Decoction Extract (Batch No.: T-180629-01).

[0072] The preparation method of the Qingfei Decoction reference sample is as follows: after the above-mentioned Qingfei Decoction extract is concentrated under reduced pressure at 65°C to a weight ratio of the amount of the decoction piece to the concentrated liquid of 1:2 (g:g), it is placed in a freeze dryer, pre-frozen at a temperature of -45°C for 20 minutes, and then dried at a cold trap temperature of -60°C and a vacuum degree of 80Pa for 20 hours, and crushed to obtain the Qingfei Decoction reference sample. (Batch number: 20180115-2, 180809-01, 180809-03, 180820-01, 181105-01, 181105-02, 181105-03, 181105-04, 181105-05, 181105-06, 181106-07, 181106-08, 181106-09, 181106-10, 181106-11, 181106-12, 181106-1 3. 181106-14, 181106-15, 181106-16, 181106-17, 181106-18, 181107-19, 181107-20, 181107-21, 181107-22, 181107-23, 181107-24, 181107-25, 181107-26, 181107-27, 181107-28, 181107-29, 181107-30).

[0073] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0074] Example 1

[0075] This embodiment provides a method for constructing a fingerprint of a Qingfei Decoction compound preparation, and the specific steps include:

[0076] Preparation of reference solution: Take baicalin reference substance, weigh accurately, add methanol to make a solution containing 0.4 mg per 1 mL.

[0077] Preparation of test solution: Take about 1.0 g of Qingfei Decoction standard sample, weigh it accurately, put it in a stoppered conical flask, add 25 mL of methanol accurately, weigh it, ultrasonically treat it (power 320 W, frequency 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0078] High performance liquid chromatography detection: using octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, at a detection wavelength of 200 nm, a column temperature of 35°C, a flow rate of 1.0 mL / min and an injection volume of 10 μL, gradient elution was performed according to the procedure in Table 1 below, and the number of theoretical plates calculated based on baicalin should be no less than 2500.

[0079] Table 1 Gradient elution program

[0080]

[0081] The common pattern of the fingerprint of the Qingfei Decoction compound preparation was established according to the above method.

[0082] Thirty batches of Qingfei Decoction reference samples were taken respectively to obtain the fingerprints of the Qingfei Decoction compound preparation according to the above method. The obtained fingerprints were analyzed, and the chromatographic peaks with good stability, appropriate response value and good durability were selected as common peaks. A total of 10 common peaks were calibrated, such as Figure 1 , Figure 2 The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition issued by the Chinese Pharmacopoeia Committee was used to automatically match the fingerprints of 30 batches of Qingfei Decoction benchmark samples to form a common pattern map and establish a reference fingerprint map, as shown in the figure below. Figure 3 As shown in the chromatogram, the retention time of the baicalin peak is moderate, the response value is high, and the baseline separation is achieved, so baicalin is selected as the reference peak, and baicalin is marked as peak S, and the baicalin reference substance is used as the reference substance.

[0083] Depend on Figure 1 , Figure 2 It can be seen that there are 10 common characteristic peaks in the control fingerprint, with the baicalin chromatographic peak as the reference peak, marked as peak S. The fingerprint of the test sample presents a chromatographic peak with the same retention time as the reference chromatographic peak. The fingerprint of the test sample is basically consistent with the control fingerprint, with 10 corresponding common peaks. According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity is calculated based on the common peaks, and the similarity between the fingerprint of the test sample and the control fingerprint shall not be less than 0.90. As shown in Tables 2 and 3, the similarity of the fingerprints of 30 batches of reference samples and the control fingerprint is compared, and the measured range is 0.995-1.000, all greater than 0.90. The difference between the fingerprints of each batch of reference samples is small when evaluated by similarity.

[0084]

[0085]

[0086] Through the construction method of this embodiment, a fingerprint with good separation of each characteristic peak can be obtained, and the S peak baicalin is selected as the internal reference peak in the fingerprint, and the relative retention time of the common characteristic peaks 1-10 in the fingerprint of the Qingfei Decoction compound preparation can be determined. Therefore, the Qingfei Decoction compound preparation can be comprehensively and quickly detected, which is beneficial to the comprehensive quality detection and overall quality control of the Qingfei Decoction compound preparation, thereby helping to improve the safety and stability uniformity of the use of the drug.

[0087] Example 2

[0088] This example provides a process for constructing a fingerprint of the Qingfei Decoction compound preparation, as follows:

[0089] 1. Gradient elution procedure and selection of test sample preparation method

[0090] (1) Method 1:

[0091] Preparation of test solution: Take 4 mL of Qingfei Decoction extract (batch number: T-180629-01), place it in a 10 mL volumetric flask, add water to the scale, shake well, filter, and take the filtrate.

[0092] HPLC conditions: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the provisions in Table 4; detection wavelength 200 nm; column temperature 35 ° C, flow rate 1.0 mL / min. Accurately draw 10 μL of the test solution, inject it into the liquid chromatograph, and record the chromatogram. Results are shown in Figure 4 .

[0093] Table 4 Gradient elution Table 1

[0094]

[0095] Depend on Figure 4 It can be seen that the chromatogram baseline is relatively stable, and the chromatographic peaks are mostly between 35 and 75 minutes, so the gradient is delayed for optimization. However, there is less information on the chromatographic peaks, so it is planned to use organic solvent extraction and organic solvent extraction followed by alkaline washing to enrich and purify the chromatographic peaks.

[0096] (2) Method 2:

[0097] a) Preparation of test solution: Take about 4.0 g of Qingfei Decoction reference sample (batch number: 20180115-2), accurately weigh, add 50 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 50 mL of water to dissolve the residue, shake and extract with water-saturated n-butanol for 3 times, 50 mL each time, combine the n-butanol solution, divide it into two parts, evaporate one part to dryness, add 25 mL of methanol to dissolve the residue, shake well, filter, and obtain the test solution.

[0098] Preparation of single-ingredient medicinal material decoction solution: take about 0.30 g of Scutellaria baicalensis decoction slices, about 0.20 g each of Platycodon grandiflorum decoction slices, Poria cocos decoction slices, Tangerine peel decoction slices, Fritillaria thunbergii decoction slices, and Morus alba decoction slices, about 0.14 g each of Angelica sinensis decoction slices, Asparagus cochinchinensis decoction slices, Gardenia jasminoides decoction slices, boiled bitter almond decoction slices, and Ophiopogon japonicus decoction slices, about 0.05 g of Schisandra chinensis decoction slices, about 0.06 g of Licorice decoction slices, about 0.11 g of ginger decoction slices, and about 0.50 g of jujube decoction slices, accurately weigh, place them in round-bottom flasks respectively, add 30 mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50 mL of water to dissolve the residue, and prepare the rest of the method with the preparation method of the test solution in method 2 a) to obtain single-ingredient medicinal material decoction solution.

[0099] Preparation of blank solution: Measure 50 mL of methanol and ultrasonically treat for 30 minutes. The rest of the preparation method is the same as the preparation method of the test solution in method 2 a).

[0100] HPLC conditions: basically follow the chromatographic conditions in method 1, except that the gradient elution is performed according to the gradient program in Table 5 below. Accurately pipette 10 μL of the above solution and blank solution, inject into the liquid chromatograph, and record the chromatogram. Figure 5 .

[0101] Table 5 Gradient elution Table 2

[0102]

[0103] The above method was used to identify the peaks of 15 herbs, and only four herbs were identified: Scutellaria baicalensis, Dictyophora japonica, Gardenia jasminoides, and Rhizoma Amygdalii. Figure 5 Analysis of the fingerprint spectrum showed that there were 10 chromatographic peaks with good peak shape and separation, among which peak 1 was the chromatographic peak of stewed bitter almonds, peak 2 was the chromatographic peak of gardenia, peaks 3, 5, 6, 7, 8 and 9 were the chromatographic peaks of scutellaria baicalensis, peak 4 was the chromatographic peak of tangerine peel, and peak 10 was the chromatographic peak of scutellaria baicalensis and tangerine peel. A total of four medicines, stewed bitter almonds, gardenia, scutellaria baicalensis and tangerine peel, were identified, and each had its own exclusive chromatographic peak.

[0104] b) Preparation of test solution: Take another portion of n-butanol solution prepared from the Qingfei Decoction reference sample in a) of method 2, wash it twice with ammonia test solution, 40 mL each time, discard the washing solution, wash the n-butanol solution twice with water saturated with n-butanol, 40 mL each time, evaporate the n-butanol solution to dryness, add 5 mL of methanol to the residue to dissolve it, shake well, and filter to obtain the test solution.

[0105] Preparation of single-flavor medicinal material slice solution: Take another portion of n-butanol solution prepared from the single-flavor medicinal material slice in method 2 a), prepare another portion of n-butanol solution according to the preparation method of the single-flavor medicinal material slice solution in method 2 a), and prepare a single-flavor medicinal material slice solution according to the preparation method of the test sample solution in method 2 b).

[0106] HPLC conditions: basically follow the chromatographic conditions in method 1, except that the gradient elution is performed according to the gradient program in Table 6 below. Accurately pipette 10 μL of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. Figure 6 .

[0107] Table 6 Gradient elution Table 3

[0108]

[0109] The above method was used to identify the peaks of 15 herbs, and nine herbs were identified: Platycodon grandiflorum, Fritillaria thunbergii, Morus alba bark, ginger, Schisandra chinensis, Scutellaria baicalensis, Citrus aurantium, Gardenia jasminoides, and Licorice. Figure 6 Analysis on the fingerprint spectrum showed that peaks 1 and 2 were the chromatographic peaks of Gardenia, peaks 3 and 4 were the chromatographic peaks of Licorice, peak 5 was the chromatographic peak of Platycodon, peaks 6, 7 and 10 were the chromatographic peaks of Scutellaria, peaks 13, 14 and 15 were the chromatographic peaks of Schisandra, peak 8 was the common peak of Scutellaria and Tangerine Peel, peak 9 was the common peak of Scutellaria and ginger, peak 11 was the common peak of Schisandra and Tangerine Peel, peak 12 was the common peak of Tangerine Peel and Licorice, peak 16 was the common peak of Fritillaria thunbergii, Morus alba and Gardenia. A total of nine medicines were identified, namely Gardenia, Scutellaria, Tangerine Peel, Schisandra, Morus alba, Licorice, Fritillaria thunbergii, Platycodon and ginger. However, Fritillaria thunbergii, Morus alba, ginger and Tangerine Peel had no exclusive chromatographic peaks and could only be attributed to the common chromatographic peaks, which were of little qualitative and semi-quantitative significance.

[0110] Comparing the above two preparation methods, a) can be attributed to the exclusive chromatographic peaks of four herbs: boiled bitter almond, gardenia, scutellaria, and tangerine peel, and b) can be attributed to the exclusive chromatographic peaks of five herbs: gardenia, scutellaria, schisandra, licorice, and platycodon, but b) has an unstable baseline and poor chromatographic peak separation. Therefore, the method needs to be further optimized.

[0111] (3) Method 3:

[0112] The main components of Platycodon grandiflorum, Poria cocos, Asparagus cochinchinensis, Ophiopogon japonicus, and Jujube are saponins, which have no absorption or mostly terminal absorption under ultraviolet light. The samples are processed according to the properties that saponins react with strong acids to generate aglycones, which may absorb at ultraviolet 200-400nm. The details are as follows:

[0113] Preparation of test solution: Take about 5.0 g of Qingfei Decoction reference sample (batch number: 20180115-2), accurately weigh, add 20 mL of 7% sulfuric acid ethanol-water (volume ratio of 1:3) mixed solution, heat and reflux for 3 hours, cool, extract with chloroform shaking twice, 20 mL each time, combine the chloroform solution, wash with water twice, 30 mL each time, discard the washing solution, evaporate the chloroform solution, add 20 mL of methanol to the residue to dissolve it, shake well, and filter.

[0114] Preparation of single-ingredient decoction solution: take about 0.30 g of Scutellaria baicalensis slices, about 0.20 g each of Platycodon grandiflorum slices, Poria cocos slices, Citrus aurantium slices, Fritillaria thunbergii slices, and Morus alba slices, about 0.14 g each of Angelica sinensis slices, Asparagus cochinchinensis slices, Gardenia jasminoides slices, boiled bitter almond slices, and Ophiopogon japonicus slices, about 0.05 g of Schisandra chinensis slices, about 0.06 g of Licorice slices, about 0.11 g of ginger slices, and about 0.50 g of jujube slices, accurately weigh, place in a round-bottom flask, add 30 mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 20 mL of 7% sulfuric acid ethanol-water (volume ratio of 1:3) mixed solution, and the rest of the method is the same as the preparation method of the test solution in Method 3.

[0115] HPLC conditions: basically follow the chromatographic conditions in method 1, except that the gradient elution is performed according to the gradient program in Table 7 below. Accurately pipette 10 μL of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. Figure 7-8 .

[0116] Table 7 Gradient elution Table 4

[0117]

[0118] The above method was used to attribute the peaks of 15 herbs. Figure 7 , Figure 8 According to the fingerprint analysis, peak 1 is the common peak of the remaining 14 herbs except ginger; peak 2 is the common peak of Platycodon grandiflorum and Angelica sinensis, peak 3 is the common peak of Scutellaria baicalensis and Angelica sinensis; peaks 4, 5, and 10 are the common peaks of Scutellaria baicalensis and Dictyophora japonica; peaks 6, 7, 8, and 9 are the chromatographic peaks of Scutellaria baicalensis; peak 8 is the common peak of Scutellaria baicalensis and Licorice; peak 11 is the spectral peak of Dictyophora japonica; peak 12 is the common peak of the remaining 11 herbs except boiled bitter almond, ginger, Poria cocos, and Dictyophora japonica. The chromatographic peaks obtained by this method only have exclusive peaks for Scutellaria baicalensis and Dictyophora japonica, and the other peaks are all common peaks, which has lost the meaning of quality control, so this method is not feasible.

[0119] (4) Method 4:

[0120] From the above methods, it was found that among the fingerprint conditions that have been explored, the herbs of Fritillaria thunbergii, Platycodon grandiflorum, Morus alba bark, Ophiopogon japonicus, Poria cocos, Asparagus cochinchinensis, Jujube, and Angelica sinensis always have no chromatographic peaks or often appear in the form of multiple herbs. Therefore, the fingerprint of the Qingfei Decoction benchmark sample was explored for the attribution of these herbs.

[0121] a) Preparation of test solution: Take about 9.0 g of Qingfei Decoction reference sample (batch number: 180809-01), accurately weigh, add 30 mL of water, adjust the pH to 10 with ammonia test solution, shake and extract twice with chloroform, 20 mL each time, combine the chloroform solutions, evaporate to dryness, add 5 mL of methanol to the residue to dissolve, shake well, filter, and take the filtrate to obtain the solution.

[0122] Preparation of single-ingredient decoction slice solution: take about 0.30 g of Scutellaria baicalensis decoction slice, about 0.20 g each of Platycodon grandiflorum decoction slice, Poria cocos decoction slice, Tangerine peel decoction slice, Fritillaria thunbergii decoction slice, Morus alba decoction slice, about 0.14 g each of Angelica sinensis decoction slice, Asparagus cochinchinensis decoction slice, Gardenia jasminoides decoction slice, boiled bitter almond decoction slice, Ophiopogon japonicus decoction slice, about 0.05 g of Schisandra chinensis decoction slice, about 0.06 g of Licorice decoction slice, about 0.11 g of ginger decoction slice, about 0.50 g of jujube decoction slice, accurately weigh, put into a round-bottom flask, add 30 mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 30 mL of water, and the rest of the method is the same as the test sample preparation method in method 4 a).

[0123] HPLC conditions: basically follow the chromatographic conditions in method 1, except that the gradient elution is performed according to the gradient program in Table 8 below. Accurately pipette 10 μL of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. Fig. 9 .

[0124] b) Preparation of test solution: Take about 9 g of Qingfei Decoction reference sample (batch number: 180809-01), accurately weigh, add 30 mL of water, adjust the pH to 2 with formic acid, shake and extract twice with ether, 20 mL each time, combine the ether solutions, evaporate to dryness, add 5 mL of methanol to the residue to dissolve, shake well, filter, and take the filtrate to obtain the solution.

[0125] Preparation of single-ingredient decoction slice solutions: take about 0.30 g of Scutellaria baicalensis slices, about 0.20 g each of Platycodon grandiflorum slices, Poria cocos slices, Citrus aurantium slices, Fritillaria thunbergii slices, and Morus alba slices, about 0.14 g each of Angelica sinensis slices, Asparagus cochinchinensis slices, Gardenia jasminoides slices, boiled bitter almond slices, and Ophiopogon japonicus slices, about 0.05 g of Schisandra chinensis slices, about 0.06 g of Licorice slices, about 0.11 g of ginger slices, and about 0.50 g of jujube slices, accurately weigh, place in a round-bottom flask, add 30 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 30 mL of water, and the rest of the method is the same as the test sample preparation method in method 4 b).

[0126] HPLC conditions: basically follow the chromatographic conditions in method 1, except that the gradient elution is performed according to the gradient program in Table 8 below. Accurately pipette 10 μL of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. Fig.10 .

[0127] Table 8 Gradient elution Table 5

[0128]

[0129] The above method was used to identify the peaks of 15 herbs, and only eight herbs were identified, namely, Fritillaria thunbergii, Platycodon grandiflorum, Morus alba, Ophiopogon japonicus, Poria cocos, Asparagus cochinchinensis, Jujube, and Angelica sinensis. Fig. 9 , Fig.10 Fingerprint analysis showed that the chromatograms obtained by the two preparation methods showed that no chromatographic peaks appeared for each herb in method a); in method b), except for Fritillaria thunbergii which had an exclusive chromatographic peak at a retention time of 10 minutes and Angelica sinensis which had an exclusive chromatographic peak at a retention time of 32 minutes, the other herb had no exclusive chromatographic peaks, and the chromatographic information of each herb was too little, so both methods were not feasible.

[0130] (5) Method 5:

[0131] The main active ingredients of the seven herbs in Qingfei Decoction, including Platycodon grandiflorum, Poria cocos, Fritillaria thunbergii, Asparagus cochinchinensis, Radix Ophiopogonis, Jujube, and Morus alba, are saponins and alkaloids, which have no detection information in the HPLC-UV fingerprint. In order to better control the quality of the product, the HPLC-ELSD fingerprint of Qingfei Decoction was explored using a high-performance liquid phase-evaporative light scattering detector, and the HPLC-UV and HPLC-ELSD fingerprints were compared.

[0132] a) Preparation of single-ingredient decoction slice solutions: Take about 0.30 g of Scutellaria baicalensis slices, about 0.20 g each of Poria cocos slices, Citrus aurantium slices, Fritillaria thunbergii slices, and Morus alba slices, about 0.14 g each of Angelica sinensis slices, Asparagus cochinchinensis slices, and Ophiopogon japonicus slices, and about 0.50 g of Jujube slices, accurately weigh, place in a round-bottom flask, add 30 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 50 mL of methanol, and prepare single-ingredient decoction slice solutions according to the method a) in method 2 for the rest of the method.

[0133] HPLC conditions: basically follow the chromatographic conditions in method 1, except that acetonitrile is used as mobile phase A, water is used as mobile phase B, and gradient elution is performed according to the gradient program in Table 9 below. Accurately pipette 10 μL of the above single herbal decoction slice solution and the test sample solution in method 2 a) and inject into the liquid chromatograph to record the chromatogram. Results are shown in Figure 11-13 .

[0134] Table 9 Gradient elution Table 6

[0135]

[0136] The above method was used to attribute the peaks of nine herbs, including scutellaria baicalensis, fritillaria thunbergii, white mulberry bark, angelica sinensis, asparagus cochinchinensis, ophiopogon japonicus, tuckahoe, jujube, and tangerine peel. Only two herbs, scutellaria baicalensis and tangerine peel, were identified. Figure 11-13 Fingerprint analysis shows that in the HPLC-ELSD fingerprint, peaks 2, 4, 6, 7 and 8 are all chromatographic peaks of Scutellaria baicalensis, and peak 3 is the chromatographic peak of Citrus aurantium. Compared with HPLC-UV, this method is not superior, so this method is not feasible.

[0137] b) Preparation of single-ingredient medicinal material decoction slice solutions: Take about 0.30 g of Scutellaria baicalensis decoction slices, about 0.20 g each of Platycodon grandiflorum decoction slices, Poria cocos decoction slices, Tangerine peel decoction slices, Fritillaria thunbergii decoction slices, and Morus alba decoction slices, about 0.14 g each of Angelica sinensis decoction slices, Asparagus cochinchinensis decoction slices, Gardenia jasminoides decoction slices, boiled bitter almond decoction slices, and Ophiopogon japonicus decoction slices, about 0.05 g of Schisandra chinensis decoction slices, about 0.06 g of Licorice decoction slices, about 0.11 g of ginger decoction slices, and about 0.50 g of jujube decoction slices, accurately weigh, place in a round-bottom flask, add 30 mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50 mL of methanol, and prepare n-butanol solution according to the preparation method in a) of method 2 for the rest of the method, and then use the n-butanol solution to prepare single-ingredient medicinal decoction slice solutions according to the method in b) of method 2.

[0138] HPLC conditions: basically follow the chromatographic conditions in method 1, except that acetonitrile is used as mobile phase A, water is used as mobile phase B, and gradient elution is performed according to the gradient program in Table 10 below. Accurately pipette 10 μL of the above single herbal decoction slice solution and the test sample solution in method 2 b) and inject into the liquid chromatograph to record the chromatogram. Results are shown in Fig.14 , Fig.15 .

[0139] Table 10 Gradient elution Table 7

[0140]

[0141] The above method was used to identify the peaks of 15 herbs, and only eight herbs were identified: Scutellaria baicalensis, Platycodon grandiflorum, Fritillaria thunbergii, Asparagus cochinchinensis, Ginger, Schisandra chinensis, Citrus aurantium, and Gardenia jasminoides. Figure 14-15 According to the fingerprint, peak 1 is the chromatographic peak of Gardenia, peak 2 is the chromatographic peak of Asparagus cochinchinensis, peak 3 is the chromatographic peak of Platycodon grandiflorum, peak 4 is the chromatographic peak of Scutellaria baicalensis, peak 5 is the common peak of Scutellaria baicalensis and ginger, peak 6 is the chromatographic peak of Schisandra chinensis, peak 7 is the chromatographic peak of Dictaecarpa, and peak 8 is the chromatographic peak of Gardenia jasminoides and Fritillaria thunbergii. In summary, the HPLC-ELSD fingerprint identified eight herbs, namely, Scutellaria baicalensis, Platycodon grandiflorum, Dictaecarpa, Fritillaria thunbergii, Gardenia jasminoides, Asparagus cochinchinensis, Schisandra chinensis, and ginger, among which ginger and Fritillaria thunbergii did not have exclusive chromatographic peaks, and peak 2 (the only exclusive chromatographic peak of Asparagus cochinchinensis) was too small, the overall chromatographic information of the fingerprint of the test sample was too little, the separation of peak 1 was poor, and the method was not ideal.

[0142] (6) Method 6:

[0143] Analyzing "Method 2" to "Method 5", the different preparation methods identified fewer medicinal flavors. Considering the ease of operation, the HPLC-UV fingerprint of "Method 2" a) is preferred. However, since the preparation method and gradient elution conditions of the test sample are relatively complicated, they need to be simplified in the next step.

[0144] Preparation of test solution: Take about 3.0 g of Qingfei Decoction reference sample (batch number: 180809-03), accurately weigh, add 50 mL of water-saturated n-butanol, ultrasonically treat (power 320 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of methanol to the residue to dissolve, shake well, filter, and take the filtrate to obtain the test solution.

[0145] Preparation of single-ingredient decoction slice solutions: take about 0.30 g of Scutellaria baicalensis slices, about 0.20 g each of Platycodon grandiflorum slices, Poria cocos slices, Citrus aurantium slices, Fritillaria thunbergii slices, and Morus alba slices, about 0.14 g each of Angelica sinensis slices, Asparagus cochinchinensis slices, Gardenia jasminoides slices, boiled bitter almond slices, and Ophiopogon japonicus slices, about 0.05 g of Schisandra chinensis slices, about 0.06 g of Licorice slices, about 0.11 g of ginger slices, and about 0.50 g of jujube slices, accurately weigh, place in a round-bottom flask, add 30 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 50 mL of saturated n-butanol with water, and prepare single-ingredient decoction slice solutions according to the preparation method of the test sample solution in Method 6 for the rest of the preparation method.

[0146] HPLC conditions: The chromatographic conditions in method 1 were basically the same, except that the gradient elution was performed according to the gradient program in Table 11 below. 10 μL of each of the above solutions was accurately pipetted and injected into the liquid chromatograph to record the chromatogram. Results are shown in Fig.16 .

[0147] Table 11 Gradient elution Table 8

[0148]

[0149] The above method was used to identify the peaks of 15 herbs, and only four herbs were identified: Scutellaria baicalensis, Dictyophora japonica, Gardenia jasminoides, and Rhizoma Amygdalii. Fig.16 According to the fingerprint analysis, peak 1 is the chromatographic peak of stewed bitter almonds, peak 2 is the chromatographic peak of gardenia, peaks 3, 5, 6, 7, 8 and 9 are the chromatographic peaks of scutellaria, peak 4 is the chromatographic peak of tangerine peel, and peak 10 is the chromatographic peak of scutellaria and tangerine peel. A total of four herbs are identified: stewed bitter almonds, gardenia, scutellaria and tangerine peel, and each has its own exclusive chromatographic peak. Figure 5 and Fig.16The chromatographic peaks of the two preparation methods were found to have no significant difference, and the peak attribution results were the same. Since the preparation method is simpler to operate, direct ultrasonication with water-saturated n-butanol was used instead of water-saturated n-butanol extraction for further research.

[0150] (7) Method 7:

[0151] Preparation of test solution: Take about 3.0 g (3 groups in total) of Qingfei Decoction benchmark sample (batch number: 180820-01), accurately weigh, add 50 mL of water-saturated n-butanol, ethyl acetate and methanol respectively, ultrasonically treat (power 320 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate, add 25 mL of methanol to the residue to dissolve, shake well, filter, and take the filtrate.

[0152] HPLC conditions: The chromatographic conditions in method 1 were basically the same, except that the gradient elution was performed according to the gradient program in Table 11. 10 μL of each of the above solutions was accurately pipetted and injected into the liquid chromatograph. The chromatogram was recorded. The results are shown in Fig.17 .

[0153] from Fig.17 Fingerprint analysis compared methanol ultrasonic extraction, water-saturated n-butanol ultrasonic extraction and ethyl acetate ultrasonic extraction. It was found that the chromatograms obtained by methanol ultrasonic extraction and water-saturated n-butanol ultrasonic extraction had the same number of chromatographic peaks and similar peak shapes. However, the chromatographic peak response value of the test sample extracted by methanol ultrasonic extraction was higher, so methanol ultrasonic extraction was preferred.

[0154] (8) Method 8:

[0155] Preparation of test solution: Take about 1.0 g (3 groups in total) of Qingfei Decoction reference sample (batch number: 180820-01), accurately weigh, put in a stoppered conical flask, accurately add 25 mL of methanol, 50% methanol aqueous solution, and 10% methanol aqueous solution, respectively, stopper, weigh, ultrasonically treat (power 320 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate to obtain.

[0156] Preparation of reference solution: Take baicalin reference substance, weigh accurately, add methanol to make a solution containing 0.4 mg per 1 mL.

[0157] Preparation of blank solvent: Measure 25 mL of methanol and ultrasonically treat for 30 minutes. The rest of the preparation method is the same as the preparation method of the test solution in Method 8.

[0158] HPLC conditions: The chromatographic conditions in method 1 were basically the same, except that the gradient elution was performed according to the gradient program in Table 11. 10 μL of each of the above solutions was accurately pipetted and injected into the liquid chromatograph. The chromatogram was recorded. The results are shown in Fig.18.

[0159] from Fig.18 Fingerprint analysis showed that the response value of the chromatogram peak at retention time of about 81 minutes in the chromatogram obtained with 50% methanol aqueous solution was low; the peak at retention time of 26 minutes in the chromatogram of 10% methanol aqueous solution disappeared, and the number of chromatographic peaks at retention time of 81 minutes was small; the peak shape of the methanol chromatogram at 81 minutes and 26 minutes was good, and the separation was high, so the preparation method of the sample selected methanol ultrasonic treatment (power 320W, frequency 40kHz) for 30 minutes.

[0160] In summary, the optimal method for preparing the test sample is: take about 1.0 g of Qingfei Decoction reference sample powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of methanol, seal it, weigh it, ultrasonically treat it (power 320 W, frequency 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with methanol solution, shake it well, filter it, and take the filtrate to obtain it.

[0161] 2. Selection of detection wavelength

[0162] Take the sample solution extracted from method 8 in "1. Gradient elution procedure and selection of sample preparation method" with methanol as the solvent, and basically follow the chromatographic conditions in method 1, except that the gradient elution is performed according to the gradient procedure in Table 11. A diode array detector is used, and the sample solution is scanned at a wavelength of 190-400nm. The chromatographic peak is displayed in the wavelength range of 200-250nm, which has a large amount of information. Wavelengths of 200nm, 210nm, 230nm, 240nm, and 250nm are selected for analysis. Fig.19 It was found that at a wavelength of 200 nm, each chromatographic peak had a large amount of information, a moderate response value, good separation, and a relatively stable baseline. Therefore, 200 nm was selected as the detection wavelength.

[0163] 3. Peak attribution and reference selection

[0164] (1) Chromatographic peaks and their medicinal properties

[0165] According to the preparation method determined in Method 8 in "1. Gradient elution procedure and selection of test sample preparation method", the test sample solution, single herbal medicine slice solution and single herbal medicine-deficient negative sample solution are prepared. The chromatographic conditions in Method 1 are basically the same, except that the gradient elution is performed according to the gradient procedure in Table 11. At the same time, the attribution of each peak in the fingerprint spectrum of the reference sample is determined by mass spectrometry. Figure 20 to Figure 22 It can be seen that peak 1 is the chromatographic peak of boiled bitter almond, peak 2 is the chromatographic peak of gardenia, peaks 3, 5, 6, 7, 8, 9 and 10 are chromatographic peaks of scutellaria, and peak 4 is an orange-red peak. The attribution results are shown in Table 12.

[0166] Table 12: Table of medicinal flavors

[0167]

[0168] (2) Chromatographic peak identification

[0169] Preparation of test solution: Prepare the test solution according to the test solution preparation method in method 8 in "1. Gradient elution procedure and selection of test sample preparation method".

[0170] Preparation of reference solution: accurately weigh baicalin reference substance, gardenia jasminoides reference substance, hesperidin reference substance, amygdalin reference substance, baicalin reference substance, wogonin peak reference substance, melaleuca alternifolia reference substance, and wogonin reference substance, add methanol to make a solution containing 0.4 mg per 1 mL.

[0171] HPLC conditions: The chromatographic conditions in method 1 were basically the same, except that the gradient elution was performed according to the gradient program in Table 11. 10 μL of each of the above solutions was accurately pipetted and injected into the liquid chromatograph. The chromatogram was recorded. The results are shown in Fig.23 .

[0172] By comparing with the reference substance, the chromatographic peaks in the fingerprint of the Qingfei Decoction reference sample were identified by mass spectrometry. The total ion flow diagram (positive and negative modes) is shown in Fig.24 Since the liquid phase of the LC-MS instrument is UPLC, its chromatogram is slightly different from the chromatogram of the HPLC fingerprint. The specific peak attribution is shown in Table 13 below. The mass spectrometry results were analyzed and tested, and 8 known components in 10 common peaks were identified and confirmed: baicalin, gardenia glycoside, amygdalin, hesperidin, melaleuca A-7-O-β-D-glucuronic acid, wogonin, baicalin, and wogonin. The correspondence between the mass spectrometry analysis results and the reference identification results is shown in Table 13.

[0173] Table 13 Correspondence between mass spectrometry analysis results and common peak identification results

[0174]

[0175] (3) Selection of reference objects

[0176] From the above chromatogram Fig.23 , Fig.24 The retention time of the chromatographic peak of baicalin was moderate, the response value was high, and baseline separation was achieved. Therefore, baicalin was selected as the reference peak, and baicalin was marked as peak S. At the same time, baicalin reference substance was used as a reference substance.

[0177] Example 3

[0178] This embodiment provides a methodological verification of a method for constructing a fingerprint of a Qingfei Decoction compound preparation, including:

[0179] 1. Specificity test and overall investigation

[0180] Methanol was used as the blank solvent, and the reference solution and the test solution were prepared and measured according to the method in Example 1. Fig.25 It shows that the blank solvent has no interference and the chromatographic peaks have been collected, meeting the principle of maximum information content.

[0181] 2. Instrument precision test

[0182] The test solution was prepared and measured according to the method in Example 1. The test solution was drawn and injected 6 times continuously. The relative retention time and relative peak area of ​​each characteristic peak and S peak were calculated. The measurement results are shown in Tables 14-15 and Fig.26 As can be seen from the above table and figure, the RSD of the relative retention time and relative peak area of ​​the 10 main chromatographic peaks in the fingerprint of the test sample are all less than 2%, indicating that the instrument precision is good.

[0183] Table 14 Relative retention time results of fingerprint precision test

[0184]

[0185]

[0186] Table 15 Relative peak area results of fingerprint precision test

[0187]

[0188] 3. Stability test

[0189] The test solution was prepared and measured according to the method in Example 1. The samples were placed for 0, 2, 4, 8, 12, and 24 hours after preparation and the relative retention time and relative peak area of ​​each characteristic peak and S peak were calculated. The measurement results are shown in Table 16-17. Fig. 27 The results showed that the relative retention time and relative peak area RSD of the 10 main chromatographic peaks in the fingerprint of the test sample were all less than 2%, indicating that the test sample had good stability within 24 hours.

[0190] Table 16 Relative retention time results of fingerprint stability test

[0191]

[0192]

[0193] Table 17 Relative peak area results of fingerprint stability test

[0194]

[0195] 4. Repeatability test

[0196] The test solution was prepared and measured according to the method in Example 1. Six test solutions were prepared and injected for measurement. The relative retention time and relative peak area of ​​each characteristic peak and S peak were calculated. The measurement results are shown in Table 18-19. Fig.28 The results showed that the RSDs of retention time and relative peak area of ​​10 main chromatographic peaks in the fingerprint were less than 2%, indicating that the method had good repeatability.

[0197] Table 18 Relative retention time results of fingerprint repeatability test (n=6)

[0198]

[0199] Table 19 Relative peak area results of fingerprint repeatability test (n=6)

[0200]

[0201] 5. Durability test

[0202] The test solution was prepared and measured basically according to the method under Example 1, except that: (1) in the flow rate investigation test, the flow rates were 1.2 mL / min, 1.0 mL / min, and 0.8 mL / min, respectively; (2) in the column temperature investigation test, the column temperatures were 30° C., 35° C., and 40° C., respectively; (3) in the mobile phase acidity investigation test, the mobile phase B was 0.08% phosphoric acid aqueous solution, 0.10% phosphoric acid aqueous solution, and 0.12% phosphoric acid aqueous solution, respectively; (4) in the chromatographic column investigation test, the chromatographic columns were Welch Xtimate C18 (4.6×250 mm, 5 μm), Kromasil-100-5 C18 (4.6×250 mm, 5 μm), and Sepax GP-C18 (4.6×250 mm, 5 μm), respectively; (5) in the instrument investigation test, the instruments were Agilent 1260II and Shimadzu 20AT, respectively. The remaining measurement conditions in (1)-(5) above are consistent with the measurement method in Example 1. Measurement results are shown in Tables 20-29. Figure 29-33 It shows that the common peaks in the fingerprints obtained under different flow rates, column temperatures, chromatographic columns, acidities, and instrument conditions are sharp, symmetrical, and well separated, with similarities ranging from 0.999 to 1.000. The fingerprints of the test samples all show 10 main chromatographic peaks, and the retention time is consistent with that of the reference chromatographic peaks. This shows that the method has good durability for different flow rates, column temperatures, chromatographic columns, acidities, and instruments.

[0203] Table 20 Similarity of tests at different flow rates

[0204] Fingerprint R S2 S3 S4 R 1.000 0.999 1.000 1.000 S2 0.999 1.000 0.999 0.999 S3 1.000 0.999 1.000 1.000 S4 1.000 0.999 1.000 1.000

[0205] Note: R, reference spectrum; S2, 1.2 mL / min; S3, 1.0 mL / min; S4, 0.8 mL / min.

[0206] Table 21 Relative retention time results of different flow rate tests

[0207]

[0208]

[0209] Table 22 Similarity of experiments at different column temperatures

[0210] Fingerprint R S2 S3 S4 R 1.000 0.999 1.000 1.000 S2 0.999 1.000 0.999 0.999 S3 1.000 0.999 1.000 1.000 S4 1.000 0.999 1.000 1.000

[0211] Note: R, reference spectrum; S2, 40℃ column temperature; S3, 35℃ column temperature; S4, 30℃ column temperature.

[0212] Table 23 Relative retention time results of different column temperature tests

[0213]

[0214] Table 24 Similarity of different acidity tests

[0215] Fingerprint R S2 S3 S4 R 1.000 1.000 1.000 1.000 S2 1.000 1.000 1.000 1.000 S3 1.000 1.000 1.000 1.000 S4 1.000 1.000 1.000 1.000

[0216] Note: R, reference spectrum; S2, 0.08% phosphoric acid aqueous solution; S3, 0.1% phosphoric acid aqueous solution; S4, 0.12% phosphoric acid aqueous solution.

[0217] Table 25 Relative retention time results of different acidity tests

[0218]

[0219] Table 26 Similarity of different chromatographic column tests

[0220] Fingerprint R S2 S3 S4 R 1.000 1.000 1.000 1.000 S2 1.000 1.000 1.000 1.000 S3 1.000 1.000 1.000 1.000 S4 1.000 1.000 1.000 1.000

[0221] Notes: R, reference spectrum; S2, Welch Xtimate C18 (4.6×250 mm, 5 μm); S3, Kromasil-100-5 C18 (4.6×250 mm, 5 μm); S4, Sepax GP-C18 (4.6×250 mm, 5 μm).

[0222] Table 27 Relative retention time results of different chromatographic column tests

[0223]

[0224] Table 28 Similarity of tests with different instruments

[0225] Fingerprint R S2 S3 R 1.000 1.000 1.000 S2 1.000 1.000 1.000 S3 1.000 1.000 1.000

[0226] Note: R, reference spectrum; S2, Agilent 1260II; S3, Shimadzu 20AT.

[0227] Table 29 Relative retention time results of different instrument tests

[0228]

[0229] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a fingerprint of a Qingfei Decoction compound preparation, wherein the Qingfei Decoction compound preparation is prepared from raw materials including scutellaria baicalensis, platycodon grandiflorum, tuckahoe, tangerine peel, thunbergia thunbergii, mulberry bark, angelica sinensis, asparagus cochinchinensis, gardenia jasminoides, schisandra chinensis, liquorice, ginger, stewed bitter almonds, ophiopogon japonicus, and jujube, characterized in that: The following steps are involved: S1. Preparation of test solution; the test sample is prepared from the Qingfei Decoction reference sample, and the preparation method of the test solution is as follows: Method 1, take the test sample, add methanol to ultrasonic extraction to obtain an extract, separate the solid and liquid, and take the liquid, which is the test solution; or Method 2, take the test sample, add methanol and / or water-saturated n-butanol to ultrasonic extraction to obtain an extract, separate the solid and liquid, dry the liquid to obtain a residue, dissolve the residue in methanol, separate the solid and liquid again, and take the liquid, which is the test solution; or Method 3, take the test sample, add methanol to ultrasonic extraction to obtain an extract, separate the solid and liquid, dry the liquid to obtain a residue, dissolve the residue in water, extract with water-saturated n-butanol, dry the n-butanol extract, add methanol to extract, separate the solid and liquid of the extract, and take the liquid, which is the test solution; S2. The test solution was tested by high performance liquid chromatography. The chromatographic conditions included a detection wavelength of 200 nm, octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, and an aqueous solution containing phosphoric acid as mobile phase B. Gradient elution was performed according to the following procedure: The fingerprint of the Qingfei Decoction compound preparation has 10 common peaks, peak 1 is amygdalin, peak 2 is gardenia glycoside, peak 4 is hesperidin, peak 5 is baicalin, peak 7 is melaleucain A-7-O-β-D-glucuronic acid, peak 8 is wogonin, peak 9 is baicalein, and peak 10 is wogonin.

2. The method for constructing the fingerprint of the Qingfei Decoction compound preparation according to claim 1, characterized in that: The method for preparing the test solution also satisfies any one or more of the following A to F: A. In method 1, the ratio of the mass of the test sample to the volume of methanol is 0.8-2:25; the relationship between mass and volume is g / mL; B. In the method 2, in the step of ultrasonically extracting the extract by adding methanol and / or water-saturated n-butanol to obtain the extract, the ratio of the mass of the sample to the volume of methanol and / or water-saturated n-butanol is 0.8-2:25; and / or, in the method 2, in the step of dissolving the residue by adding methanol, the ratio of the mass of the sample to the volume of methanol is 1-5:25-60; the relationship between mass and volume is g / mL; C. In the step of adding methanol to ultrasonically extract to obtain the extract in method 3, the ratio of the mass of the sample to the volume of methanol is 0.8-2:25; and / or, in the step of adding water to dissolve the residue in method 3, the ratio of the mass of the sample to the volume of water is 1-5:25-60; and / or, in the step of adding methanol to extract after drying the n-butanol extract in method 3, the ratio of the mass of the sample to the volume of methanol is 1-5:20-55; The relationship between mass and volume is g / mL; D. The extraction time is 20-40min; E. The drying method is selected from one or more of evaporation, freeze drying, and reduced pressure drying; F. In the method 3, the volume ratio of the solution obtained by dissolving the residue in water to the water-saturated n-butanol extract is 0.8-1.2:1, and the number of extractions is 2-3 times.

3. The method for constructing the fingerprint of the Qingfei Decoction compound preparation according to claim 1, characterized in that: In step S2, the chromatographic column adopts a chromatographic column with a specification of 4.6×250mm, 5μm; and / or the injection volume is 5-15μL; and / or the volume percentage of the aqueous solution containing phosphoric acid is 0.08-0.12%; and / or the flow rate is 0.8-1.2mL / min; and / or the column temperature is 30-40°C; and / or the high performance liquid chromatography includes HPLC-UV method.

4. The method for constructing the fingerprint of the Qingfei Decoction compound preparation according to claim 3, characterized in that: The volume percentage of the aqueous solution containing phosphoric acid is 0.10%; and / or, the flow rate is 1.0 mL / min; and / or, the column temperature is 35° C.

5. The method for constructing the fingerprint of the Qingfei Decoction compound preparation according to any one of claims 1 to 4, characterized in that: The construction method also includes the step of using at least one of amygdalin, gardenia glycoside, 3,6,7,2′,6′-pentahydroxydihydroflavonol, hesperidin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronic acid, baicalin, baicalin, and wogonin as a reference substance solubilizer to prepare a reference substance solution, and the step of detecting the reference substance solution by high performance liquid chromatography according to the construction method described in any one of claims 1 to 4 to obtain a reference substance spectrum.

6. The method for constructing the fingerprint of the Qingfei Decoction compound preparation according to claim 5, characterized in that: The solvent is selected from at least one of methanol, ethanol and water; and / or, 1 mL of the reference substance solution contains 0.05-0.5 mg of each reference substance.

7. Application of the method for constructing the fingerprint of the Qingfei Decoction compound preparation according to any one of claims 1 to 6 in the quality inspection of the Qingfei Decoction compound preparation.

Images