Identification method and content determination method for anti-colon cancer active ingredients of Geqinlian pills

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to a method for identifying anti-colon cancer active ingredients of a Gegen Qinlian pill and a method for measuring the content of the anti-colon cancer active ingredients of the Gegen Qinlian pill. Taking berberine hydrochloride and wogonin to prepare a reference solution; according to a thin-layer chromatography test, a berberine hydrochloride reference substance solution, a wogonin reference substance solution and a test solution are taken and dripped on the same silica gel G thin-layer plate and in a test chromatogram respectively, and fluorescent spots with the same color are shown at the positions corresponding to the berberine hydrochloride reference chromatogram, namely the berberine hydrochloride is contained in the test solution; when dark spots with the same color appear at the position corresponding to the wogonin contrast chromatogram, the test sample contains wogonin. According to the method, wogonin and berberine in the Gegen Qinlian pill can be measured at the same time, the color development effect and the separation degree are good, and the content of 16 active ingredients such as wogonin and berberine can be measured at the same time.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to an identification method and a content determination method of an anti-colon cancer active ingredient of Gegenqinlian pills. Background Art

[0002] Like chemical drugs, the pharmacodynamics of traditional Chinese medicine compound prescriptions with unique and significant therapeutic effects also depends on their inherent chemical substance basis. Although traditional Chinese medicine compound prescriptions contain many ingredients, not all ingredients are effective for specific diseases. There is a group of equivalent ingredients with clear structure, measurable content, and specific efficacy that is basically equivalent to the original prescription.

[0003] Gegenqinlian Wan is a traditional Chinese medicine, mainly used to treat symptoms such as damp-heat dysentery, diarrhea and gastrointestinal dysfunction. In recent years, some studies have used Gegenqinlian Wan as an anti-colon cancer drug, but more studies have focused on pharmacological efficacy. At present, there is no report on the efficient identification and content determination of potential anti-colon cancer active ingredients such as baicalin, berberine, wogonin, apigenin, chrysin, coptisine, daidzein, formononetin, melaleuca A, scutellariae, wogonin, teratrine, glycyrrhizic coumarin, genistin, liquiritin, and magnolin when Gegenqinlian Wan is used as an anti-colon cancer drug.

[0004] In the field of content determination of traditional Chinese medicine, Gegenqinlian pills are an object of great concern. For this traditional Chinese medicine preparation, researchers have used a variety of high-performance liquid chromatography (HPLC) methods, thin-layer scanning methods, and spectroscopy methods to determine the content. Through these methods, the content of key ingredients in Gegenqinlian micropills can be accurately evaluated to ensure the quality and effectiveness of the drug.

[0005] However, the existing methods are relatively simple in function, especially the thin-layer scanning method, which can only measure one of the components. In order to fully measure multiple components, it is often necessary to conduct multiple tests to achieve this. In addition, the existing thin-layer identification method of Gegenqinlian Pills wogonin and berberine hydrochloride uses a polyamide film and a developing agent as the thin-layer plate, which has poor spot separation and the test effect is not ideal. Summary of the invention

[0006] In view of the defects existing in the above-mentioned prior art, "baicalin is an acidic compound, berberine is an alkaline compound, and it is difficult to detect them simultaneously on the same thin layer plate", the present invention provides a thin layer identification method for simultaneously determining baicalin and berberine in Gegenqinlian Pills, which can simultaneously determine baicalin and berberine in Gegenqinlian Pills, and has good color development effect and separation degree.

[0007] To achieve the above object, the present invention proposes the following technical solutions:

[0008] A thin layer chromatography identification method for simultaneously determining wogonin and berberine in Gegenqinlian pills comprises the following steps:

[0009] Step 1: Take Gegenqinlian Pills, grind them into powder, take 1g, add 30ml of ethyl acetate:methanol=3:1 (V / V), heat and reflux for 30min, filter, recover the solvent from the filtrate until dry, add 5ml of methanol to the residue to dissolve it, and use it as the test solution;

[0010] Step 2: adding methanol to berberine hydrochloride to prepare a reference solution containing 1 mg of berberine hydrochloride per 1 ml, and adding methanol to wogonin to prepare a reference solution containing 1 mg of wogonin per 1 ml;

[0011] Step 3, according to the thin layer chromatography test, take berberine hydrochloride reference solution, baicalin reference solution, and test solution, and spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-methanol-formic acid as the developing solvent, pre-saturate for 20 minutes, develop, and develop with a development distance of 12 to 13 cm. Take it out, dry it, spray it with a 3% ferric chloride ethanol solution, and examine it under ultraviolet light. In the test sample chromatogram, at the corresponding position of the berberine hydrochloride control chromatogram, a fluorescent spot of the same color appears, indicating that the test sample contains berberine hydrochloride; at the corresponding position of the baicalin control chromatogram, a dark spot of the same color appears, indicating that the test sample contains baicalin.

[0012] Furthermore, in the developing solvent, the volume ratio of toluene-ethyl acetate-methanol-formic acid is 5:8:2:2. Furthermore, the spotting volume of the test solution is 5-8 μl.

[0013] Furthermore, the spotting volume of the reference solution is 5 to 8 μl.

[0014] Furthermore, the ultraviolet light lamp is ultraviolet light with a wavelength of 365nm.

[0015] In view of the fact that there is no method for simultaneously determining the contents of 16 potential anti-colon cancer active ingredients in Gegen Qinlian Pills in the prior art, the present invention also provides a HPLC-MS / MS method for simultaneously determining the contents of 16 potential anti-colon cancer active ingredients in Gegen Qinlian Pills, and the specific steps are as follows:

[0016] Step 1, preparation of test solution, weigh 50 mg of Gegenqinlian pill powder, place in 10 mL volumetric flask, add appropriate amount of methanol-water (7:3, v / v), then add 2 mL DMSO, vortex for 3 min to dissolve, ultrasonicate at room temperature for 40 min, cool to make up for weight loss, take 1 mL of suspension and centrifuge at 4 ° C, 14000 rpm for 10 min, take 150 μL of supernatant in a new EP tube, dilute the other part 10 times with 70% methanol, take 150 μL of supernatant, add 5 μL of naringenin standard solution (IS, 5 mM) to each, mix well, centrifuge again, and set aside;

[0017] Step 2: LC-MS / MS liquid phase mass spectrometry is used to analyze the sample solution obtained in step 1. 测 The content of 16 active ingredients in the herb was determined, including baicalin, berberine, wogonin, apigenin, chrysin, coptisine, daidzein, formononetin, melaleuca A, flavonoids, wogonin, fangfang pine, glycyrrhizic coumarin, genistein, licorice glycoside, and magnolia glycoside; among them:

[0018] Chromatographic conditions were: chromatographic columns: Waters ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm) and ACQUITY UPLC BEH C18 VanGuard Pre-Column (2.1×5 mm, 1.7 μm); column temperature: 45°C; flow rate: 0.2 mL / min; injection volume: 5 μL; mobile phase: 5 mM ammonium formate + 0.1% formic acid aqueous solution (phase A)-methanol (phase B) binary gradient elution system, gradient elution program: 0-20 min, 35%-60% B; 20-23 min, 60%-100% B; 23-27 min, 100% B; 27-28 min, 100%-35% B; 28-33 min, 35% B;

[0019] The mass spectrometry conditions were as follows: spray voltage 4.5 kV, nebulizer gas 3 L / min, drying gas 10 L / min, heater 10 L / min, heating block temperature 400 °C, capillary temperature 250 °C; in ESI positive and negative ion modes, multiple reaction monitoring (MRM) mode, the mass spectrometry MRM parameters of the analyte and internal standard are shown in Table 1.

[0020] Table 1 MRM parameters of analytes and internal standards

[0021]

[0022]

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses silica gel G thin layer plates and adjusts the ratio of developing agents through continuous development experiments to determine the optimal method, so that the determination test has the advantages of good color development effect and separation. The present invention also investigates different spot amounts of the test solution in the method. The color development effect and separation can be further enhanced when the spot amount is 5 to 8 μl, so the spot amount of the test solution is determined to be 5 to 8 μl. The specificity and durability of the thin layer identification method described in the present invention are in compliance with the regulations, and wogonin and berberine in Gegenqinlian Pills can also be determined simultaneously, with higher efficiency. The HPLC-MS / MS method of the present invention for simultaneously determining the contents of 16 potential anti-colon cancer active ingredients in Gegenqinlian Pills is simple and fast, consumes little resources, has low detection cost, and has good precision and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Thin layer chromatography of berberine hydrochloride and wogonin in Gegenqinlian Pills when confirming the sample quantity in the implementation method.

[0026] Figure 2 : Another thin layer chromatography spectrum of berberine hydrochloride and wogonin in Gegenqinlian Pills when the sample quantity is confirmed in the implementation mode.

[0027] Figure 3 : TLC spectrum of berberine hydrochloride and wogonin in Gegenqinlian Pills during specificity investigation in the implementation method.

[0028] Figure 4 : TLC spectra of berberine hydrochloride and wogonin in Gegenqinlian pills (different manufacturers - Jiyida) in the implementation mode.

[0029] Figure 5 : TLC spectra of berberine hydrochloride and wogonin in Gegenqinlian pills (different manufacturers - Jiaoao).

[0030] Figure 6 : TLC of berberine hydrochloride and wogonin in Gegenqinlian pills (high temperature and high humidity).

[0031] Figure 7 : TLC of berberine hydrochloride and wogonin in Gegenqinlian pills (low temperature and low humidity).

[0032] Figure 8 : HPLC-MS / MS content determination of Gegenqinlian Pills (A) Representative spectrum of mixed reference substance; (B) Representative spectrum of undiluted Gegenqinlian Pills sample; (C) Representative spectrum of Gegenqinlian Pills sample diluted 10 times. Figure 8Chinese: 1. Magnoliatin; 2. Licorice glycoside; 3. Genistin; 4. Coptisine; 5. Tetrandrine; 6. Berberine; 7. Fructus Aconitifolii; 8. Baicalin; 9. Daidzein; 10. Baicalin; 11. Apigenin; 12. Formononetin; 13. Baicalin; 14. Chrysin; 15. Melaleuca A; 16. Licorice Coumarin. DETAILED DESCRIPTION

[0033] The following is further described with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0034] This embodiment implements a thin layer identification method for simultaneously determining wogonin and berberine in Gegenqinlian Pills, which is implemented according to the following steps:

[0035] Step 1: Take Gegenqinlian Pills, grind them into powder, take 1g, add 30ml of ethyl acetate:methanol=3:1 (V / V), heat and reflux for 30min, filter, recover the solvent from the filtrate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test solution;

[0036] Step 2: add methanol to berberine hydrochloride to prepare a reference solution containing 1 mg per 1 ml, and add methanol to wogonin to prepare a reference solution containing 1 mg per 1 ml;

[0037] Step 3, according to the thin layer chromatography test, 3 μl of berberine hydrochloride reference solution, 5 μl of wogonin reference solution, and 5-8 μl of test solution are respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-methanol-formic acid (volume ratio of 5:8:2:2) is used as the developing agent. Pre-saturation for 20 minutes, development, development, development, development distance 12-13 cm, take out, dry, spray with 3% ferric chloride ethanol solution, and examine under ultraviolet light (365nm). In the test sample chromatogram, at the corresponding position of the berberine hydrochloride reference chromatogram, a fluorescent spot of the same color appears, indicating that the test sample contains berberine hydrochloride; at the corresponding position of the wogonin reference chromatogram, a dark spot of the same color appears, indicating that the test sample contains wogonin.

[0038] In this embodiment, the test sample, standard substance and reagent, reference substance solution and instruments and equipment are as follows:

[0039] Test product

[0040] Take an appropriate amount of Gegenqinlian Pills, grind them into powder, take 1g, add 30ml of ethyl acetate:methanol=3:1 (V / V), heat and reflux for 30min, filter, recover the solvent from the filtrate to dryness, add 5ml of methanol to the residue to dissolve it, and use it as the test solution.

[0041] Standards:

[0042] Berberine hydrochloride (CAS No. 633-65-8) and wogonin (CAS No. 632-85-9) were purchased from China Food and Drug Administration.

[0043] Reagents: ethyl acetate, methanol, toluene, formic acid, 3% ferric chloride ethanol solution.

[0044] Preparation of reference solution:

[0045] Berberine hydrochloride reference solution: Accurately weigh 5.14 mg of berberine hydrochloride reference, place in a 5 ml volumetric flask, add methanol to dissolve and dilute to the scale (concentration: 0.94 mg / ml).

[0046] Wogonin reference solution: Accurately weigh 5.00 mg of wogonin reference, place in a 5 ml volumetric flask, add methanol to dissolve and dilute to the scale (concentration: 1.00 mg / ml).

[0047] All reference solutions were stored in a -20°C refrigerator for future use.

[0048] Instruments and equipment:

[0049] ZF-2 three-purpose UV instrument (Shanghai Anting Electronic Instrument Factory);

[0050] HH digital display constant temperature water bath (Changzhou Ronghua Instrument Manufacturing Co., Ltd.).

[0051] 1. Sample quantity inspection

[0052] Different sample amounts of the test solution were investigated. Figure 1-2 ,in, Figure 1 The order of sampling is as follows: from left to right, berberine hydrochloride control 3μl, wogonin control 5μl, Gegenqinlian Pills 20230802 batch sample 1μl, Gegenqinlian Pills 20230802 batch sample 2μl, Gegenqinlian Pills 20230802 batch sample 3μl, Gegenqinlian Pills 20230802 batch sample 4μl, Gegenqinlian Pills 20230802 batch sample 5μl. Figure 2 The order of spotting is: from left to right, berberine hydrochloride control 3μl, wogonin control 5μl, Gegenqinlian pill 20230802 batch sample 6μl, Gegenqinlian pill 20230802 batch sample 8μl, Gegenqinlian pill 20230802 batch sample 10μl, Gegenqinlian pill 20230802 batch sample 12μl. As can be seen from the figure, the color development effect and separation degree are best when the spotting amount is 5-8μl, so the spotting amount of the test solution is set to 5-8μl.

[0053] 2. Exclusive inspection

[0054] Negative control solution lacking Scutellaria baicalensis and Coptis chinensis, negative control solution lacking Scutellaria baicalensis, negative control solution lacking Coptis chinensis, berberine hydrochloride reference solution (concentration: 0.94 mg / ml), and wogonin reference solution (concentration: 1.00 mg / ml) were prepared. At the same time, 3 batches (batch numbers: 20230802, 20230902, and 20230903) of test solution were prepared and tested according to the test method in 2.1 above. The results showed that: there was no interference in the negative control lacking Scutellaria baicalensis and Coptis chinensis; there was no interference in the negative control lacking Scutellaria baicalensis; there was no interference in the negative control lacking Coptis chinensis; this indicated that the specificity of this method met the requirements, and the tests on the 3 batches of large-scale production samples all met the requirements. The results are shown in Table 2. Figure 3 , Figure 3 The order of spotting in the figure is, from left to right, 5 μl of negative sample lacking Coptis chinensis, 5 μl of negative sample lacking Scutellaria baicalensis, 5 μl of negative sample lacking Coptis chinensis and Scutellaria baicalensis, 3 μl of berberine hydrochloride reference, 5 μl of baicalin reference, 5 μl of Gegenqinlian Pills batch 20230802 sample, 5 μl of Gegenqinlian Pills batch 20230902 sample, and 5 μl of Gegenqinlian Pills batch 20230903 sample.

[0055] 3. Durability inspection

[0056] Three batches (batch numbers: 20230802, 20230902, and 20230903) of test solution were prepared and durability tests were carried out to investigate the influencing factors such as thin-layer silicone G plates from different manufacturers and different development temperatures and humidities.

[0057] 3.1 Investigation of Thin Layer Silicone G Sheets from Different Manufacturers

[0058] According to the above operation method, the thin layer silicone plate of Qingdao Jiyida factory and the thin layer silicone plate of Qingdao Jiaoao factory were investigated. The results are shown in Figure 4-5 ,in Figure 4 The order of spotting is from left to right: 5 μl of negative sample lacking Coptis chinensis, 5 μl of negative sample lacking Scutellaria baicalensis, 5 μl of negative sample lacking Coptis chinensis and Scutellaria baicalensis, 3 μl of berberine hydrochloride reference substance, 5 μl of wogonin reference substance, 5 μl of Gegenqinlian Pills 20230802nd batch sample, 5 μl of Gegenqinlian Pills 20230902nd batch sample, 5 μl of Gegenqinlian Pills 20230903rd batch sample; Figure 5 The order of spotting is from left to right: 5μl of negative samples lacking coptis chinensis, 5μl of negative samples lacking scutellaria baicalensis, 5μl of negative samples lacking coptis chinensis, 3μl of berberine hydrochloride reference, 5μl of wogonin reference, 5μl of Gegenqinlian Pills 20230802nd batch sample, 5μl of Gegenqinlian Pills 20230902nd batch sample, 5μl of Gegenqinlian Pills 20230903rd batch sample. The results showed that silica gel plates from different manufacturers had no significant effect on the experimental results.

[0059] 3.2. Investigation of different temperature and humidity conditions

[0060] According to the above operation method, the different development conditions of high temperature and high humidity (40℃±2℃; RH75%±5%) and low temperature and low humidity (2~8℃; RH45%±5%) were investigated. The results are shown in Figure 6-7 ,in Figure 6 The order of spotting is: from left to right, 5 μl of negative sample lacking coptis chinensis and scutellaria baicalensis, 3 μl of berberine hydrochloride reference substance, 5 μl of wogonin reference substance, 5 μl of Gegenqinlian Pills 20230802nd batch sample, 5 μl of Gegenqinlian Pills 20230902nd batch sample, 5 μl of Gegenqinlian Pills 20230903rd batch sample; Figure 7 The order of spotting is: from left to right, 5μl of negative samples lacking coptis and scutellaria, 3μl of berberine hydrochloride reference, 5μl of wogonin reference, 5μl of Gegenqinlian Pills 20230802 batch sample, 5μl of Gegenqinlian Pills 20230902 batch sample, and 5μl of Gegenqinlian Pills 20230903 batch sample. The results showed that different development temperature and humidity had no significant effect on the experimental results.

[0061] From the above experimental investigations, it can be seen that the specificity and durability of the thin-layer chromatography identification method for simultaneously determining wogonin and berberine in Gegen Qinlian Pills described in the present invention are in compliance with the regulations, and it is an effective thin-layer chromatography identification method for wogonin and berberine in Gegen Qinlian Pills.

[0062] This embodiment also carries out a HPLC-MS / MS method for simultaneously and accurately determining the contents of 16 potential anti-colon cancer active ingredients in Gegen Qinlian Pills. The 16 potential anti-colon cancer active ingredients include: baicalin, berberine, wogonin, apigenin, chrysin, coptisine, daidzein, formononetin, melaleuca A, calendula officinalis, wogonin, fangfang pine, glycyrrhizic coumarin, genistin, liquiritin, and magnolia tin. The specific steps are as follows:

[0063] Step 1, preparation of test solution, weigh 50 mg of Gegenqinlian pill powder, place in 10 mL volumetric flask, add appropriate amount of methanol-water (7:3, v / v), then add 2 mL DMSO, vortex for 3 min to dissolve, ultrasonicate at room temperature for 40 min, cool to make up for weight loss, take 1 mL of suspension and centrifuge at 4 ° C, 14000 rpm for 10 min, take 150 μL of supernatant in a new EP tube, dilute the other part 10 times with 70% methanol, take 150 μL of supernatant, add 5 μL of naringenin standard solution (IS, 5 mM) to each, mix well, centrifuge again, and set aside;

[0064] Step 2: LC-MS / MS liquid phase mass spectrometry is used to analyze the sample solution obtained in step 1. 测The content of 16 active ingredients in the herb was determined, including baicalin, berberine, wogonin, apigenin, chrysin, coptisine, daidzein, formononetin, melaleuca A, flavonoids, wogonin, fangfang pine, glycyrrhizic coumarin, genistein, licorice glycoside, and magnolia glycoside; among them:

[0065] Chromatographic conditions were: chromatographic columns: Waters ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm) and ACQUITY UPLC BEH C18 VanGuard Pre-Column (2.1×5 mm, 1.7 μm); column temperature: 45°C; flow rate: 0.2 mL / min; injection volume: 5 μL; mobile phase: 5 mM ammonium formate + 0.1% formic acid aqueous solution (phase A)-methanol (phase B) binary gradient elution system, gradient elution program: 0-20 min, 35%-60% B; 20-23 min, 60%-100% B; 23-27 min, 100% B; 27-28 min, 100%-35% B; 28-33 min, 35% B;

[0066] The mass spectrometry conditions were as follows: spray voltage 4.5 kV, nebulizer gas 3 L / min, drying gas 10 L / min, heater 10 L / min, heating block temperature 400 °C, capillary temperature 250 °C; in ESI positive and negative ion modes, multiple reaction monitoring (MRM) mode, the mass spectrometry MRM parameters of the analyte and internal standard are shown in Table 1.

[0067] Table 1 MRM parameters of analytes and internal standards

[0068]

[0069]

[0070] In this embodiment, the medicinal materials include: Pueraria root, Scutellaria baicalensis, Coptis chinensis, and Glycyrrhiza uralensis.

[0071] Standards: Berberine (CAS No.2086-83-1), Wogonin (CAS No.632-85-9), Wogonoside (CAS No.51059-44-0), Baicalin (CAS No.21967-41-9), Liquiritin (CAS No.551-15-5), Chrysin (CAS No.480-40-0), Palmatine (CAS No.3486-67-7), Apigenin (CAS No.520-36-5), Oroxylin A (CAS No.480-11-5), Formononetin (CAS No. No.485-72-3) was purchased from Chengdu Ruifensi Biotechnology Co., Ltd.; Coptisine (CAS No.3486-66-6), Columbamine (CAS No.3621-36-1), Daidzein (CAS No.486-66-8), Genistin (CAS No.529-59-9), Glycycoumarin (CAS No.94805-82-0), and Magnoflorine (CAS No.2141-09-5) were purchased from Chengdu Desit Biotechnology Co., Ltd.

[0072] Internal standard: Naringenin (IS, CAS No. 10236-47-2), purchased from China Food and Drug Inspection Institutes.

[0073] Reagents: Methanol (chromatographic grade) was purchased from Merck (Germany); formic acid (chromatographic grade) was purchased from ROE SCIENTIFIC INC.; ammonium formate (analytical grade) was purchased from Nanjing Chemical Reagent Co., Ltd.; dimethyl sulfoxide (DMSO, chromatographic grade) was purchased from Shanghai Eon Chemical Technology Co., Ltd.; fresh ultrapure water was prepared by the laboratory Milli-Q system.

[0074] In this embodiment, the standard solution is prepared as follows:

[0075] Standard stock solutions of the test substances: accurately weigh appropriate amounts of baicalin, berberine, wogonin, apigenin, chrysin, coptisine, daidzein, formononetin, melaleuca A, calendula, wogonin, tertiapine, glycyrrhizic coumarin, genistin, liquiritin, and magnoliavine standard substances, and dissolve them in methanol or DMSO to prepare standard stock solutions of 3, 3, 5, 5, 4, 5, 4, 4, 5, 5, 3, 5, 5, 5, 5, 5 mM according to their solubility properties. Then accurately dilute with methanol to standard working solutions of various concentrations.

[0076] Internal standard stock solution: Accurately weigh an appropriate amount of naringenin standard and dissolve it in methanol to quantitatively prepare a 5 mM standard stock solution.

[0077] All stock solutions, standard working solutions and internal standard solutions were stored in a -20°C refrigerator for future use.

[0078] In this embodiment, the equipment includes:

[0079] LC-MS / MS 8040 triple quadrupole liquid chromatography-mass spectrometer (Shimadzu, Japan); low-temperature high-speed centrifuge (Eppendorf, Germany); ultra-low temperature refrigerator (Zhongke Meiling, China); pure water preparation system (Millipore Milli-Q, USA); vortex mixer (Shanghai Huyueming Scientific Instrument Co., Ltd., China); analytical balance (Sartorius, China).

[0080] The methodological investigation process of HPLC-MS / MS for simultaneously and accurately determining the contents of 16 potential anti-colon cancer active ingredients in Gegenqinlian Pills described in this embodiment is as follows:

[0081] 1Standard curve and linear range

[0082] Accurately pipette appropriate amount of each reference stock solution, mix well, and prepare a mixed reference solution (containing coptisine 200μM, berberine 300μM, flavonoids 100μM, tertralidine 200μM, magnolia 40μM, glycyrrhizic coumarin 4μM, apigenin 32μM, chrysin 32μM, melaleuca A80μM, wogonin 320μM, baicalin 200μM, genistin 100μM, baicalin 800μM, liquiritin 200μM, daidzein 40μM, formononetin 80μM). Use 35% methanol solution to gradient dilute to a series of solutions, take 150μL of each concentration of the mixed reference solution, add 5μL of naringenin standard solution (IS, 5mM), mix well, centrifuge and analyze, and record the sample peak area (As) and the internal standard chromatographic peak area (Ar). The peak area ratio R (R = As / Ar) of each component was used for linear regression against the concentration (C, μM) to obtain the regression equation of each component. Table 2 shows that the linear relationship of each component was good within the corresponding detection range.

[0083] Table 2 Linear range and detection limit of components determined by LC-MS / MS

[0084]

[0085]

[0086] 2. Precision inspection

[0087] According to the concentration range of the working solution determined in the "Standard Curve and Linear Range" above, prepare the mixed standard working solution at three concentration levels: low, medium and high. Determine 6 times within one analytical batch, calculate the concentration of each component, and examine the intra-batch precision; at the same time, prepare and measure three analytical batches continuously on different days to examine the inter-batch precision. The results are shown in Table 3, indicating that this test method has good precision.

[0088] Table 3 Precision of components determined by LC-MS / MS

[0089]

[0090]

[0091] 3 Repeatability study

[0092] Six test solutions were prepared in parallel according to the test solution preparation method, and the samples were injected and analyzed to calculate the concentration of each component. The results are shown in Table 4, and the reproducibility of each component was good.

[0093] Table 6 Repeatability of components determined by LC-MS / MS

[0094]

[0095] 4. Stability investigation

[0096] The same test solution was sampled at 0h and 24h, the concentration of each component was determined, and the concentration ratio of 0h and 24h (C24 / C0)×100% was calculated to investigate the stability. The results showed that each component had good stability within 24h (Table 5).

[0097] Table 5 Stability of components determined by LC-MS / MS

[0098]

[0099]

[0100] 5. Dilution reliability investigation

[0101] A mixed standard solution with a concentration higher than the upper limit of quantitation was added to the blank solution, and the blank solution was diluted 10 times for analysis, and the concentration of each component was recorded to investigate the dilution reliability. The results are shown in Table 6. After the mixed standard solution was diluted 10 times, the precision and accuracy were good, indicating that the determination results were reliable.

[0102] Table 6 Dilution reliability of components determined by LC-MS / MS

[0103]

[0104] 6. Sample recovery rate investigation

[0105] Six portions of GQD extracts with known contents of each component were accurately measured, and 100% of the standard solution of each component was accurately added for sampling and analysis. The recovery rate of each component was calculated according to the standard of sample addition recovery rate % = (content of each component after sample addition - content of each component in traditional Chinese medicine) / spiked amount * 100%. The results are shown in Table 7, indicating that the recovery rate of each component was between 92% and 105%.

[0106] Table 7 LC-MS / MS determination of the sample recovery rate of the components

[0107]

[0108]

[0109] 7 Determination of potential active ingredients

[0110] Considering that the concentrations of potential active ingredients in GQD and Gegenqinlian Pills are quite different, the mass spectrometry response signals are also inconsistent, and some ingredients will produce mass spectrometry response saturation in sample detection. Therefore, these ingredients were monitored after the samples were diluted 10 times. The representative spectra of the mixed reference and the representative spectra of the Gegenqinlian Pills samples are shown in Figure 2. Figure 8 shown.

[0111] The extract of Gegenqinlian Pills was prepared as described above, and the contents of 16 potential active ingredients were determined according to the chromatographic and mass spectrometric conditions described above. The results are shown in Table 8. Although apigenin has been reported to have good anticancer effects, its content is lower than the minimum detection limit of the instrument and does not meet the measurability requirements, indicating that it cannot be used as a quality marker for this prescription. Based on the measurability analysis, the contents of 15 potential synergistic active ingredients in Gegenqinlian Pills were finally determined. The minimum content requirements of the 15 potential synergistic active ingredients are as follows: Berberine per gram of this product shall not be less than 15.376 mg; Baicalin per gram of this product shall not be less than 11.245 mg; Chrysin per gram of this product shall not be less than 0.144 mg; Coptisine per gram of this product shall not be less than 0.565 mg; Daidzein per gram of this product shall not be less than 1.619 mg; Formononetin per gram of this product shall not be less than 0.071 mg; Genistin per gram of this product shall not be less than 1. 341mg; each gram of this product contains oroxylin A of not less than 0.907mg; each gram of this product contains palmatine of not less than 3.568mg; each gram of this product contains wogonin of not less than 1.704mg; each gram of this product contains wogonoside of not less than 1.86mg; each gram of this product contains columbamine of not less than 1.96mg; each gram of this product contains glycycoumarin of not less than 0.009mg; each gram of this product contains liquiritin of not less than 0.094mg; each gram of this product contains magnoflorine of not less than 0.835mg.

[0112] Table 8 Determination results of potential active ingredients in Gegenqinlian pills

[0113]

[0114] Note: The content is expressed as the amount contained in each gram of Gegenqinlian Pills (mg); nd, not detected.

Claims

1. A method for identifying the anti-colon cancer active ingredients of Gegenqinlian Pills, characterized in that: The following steps are involved: Step 1: Take Gegenqinlian Pills, grind them into powder, take 1g, add 30ml of ethyl acetate:methanol=3:1 (V / V), heat and reflux for 30min, filter, recover the solvent from the filtrate until dry, add 5ml of methanol to the residue to dissolve it, and use it as the test solution; Step 2: adding methanol to berberine hydrochloride to prepare a reference solution containing 1 mg of berberine hydrochloride per 1 ml, and adding methanol to wogonin to prepare a reference solution containing 1 mg of wogonin per 1 ml; Step 3, according to the thin layer chromatography test, take berberine hydrochloride reference solution, baicalin reference solution, and test solution, and spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-methanol-formic acid as the developing solvent, pre-saturate for 20 minutes, develop, and develop with a development distance of 12 to 13 cm. Take it out, dry it, spray it with a 3% ferric chloride ethanol solution, and examine it under ultraviolet light. In the test sample chromatogram, at the corresponding position of the berberine hydrochloride control chromatogram, a fluorescent spot of the same color appears, indicating that the test sample contains berberine hydrochloride; at the corresponding position of the baicalin control chromatogram, a dark spot of the same color appears, indicating that the test sample contains baicalin.

2. The method for identifying the anti-colon cancer active ingredient of Gegenqinlian Pills according to claim 1, characterized in that: The volume ratio of toluene-ethyl acetate-methanol-formic acid is 5:8:2:

2.

3. The method for identifying the anti-colon cancer active ingredient of Gegenqinlian Pills according to claim 1, characterized in that: The spotting volume of the test solution is 5 to 8 μl.

4. The method for identifying the anti-colon cancer active ingredient of Gegenqinlian Pills according to claim 1, characterized in that: The spotting volume of the reference solution is 5 to 8 μl.

5. The method for identifying the anti-colon cancer active ingredients of Gegenqinlian Pills according to claim 1, characterized in that: The ultraviolet light lamp is ultraviolet light with a wavelength of 365nm.

6. A method for determining the content of the anti-colon cancer active ingredient of Gegenqinlian Pills, characterized in that: The following steps are included: Step 1, preparation of test solution, weigh 50 mg of Gegenqinlian pill powder, place in 10 mL volumetric flask, add methanol-water with a volume ratio of 7:3, then add 2 mL DMSO, vortex for 3 min to dissolve, ultrasonicate at room temperature for 40 min, cool to make up for weight loss, take 1 mL of suspension, centrifuge at 4 ° C, 14000 rpm for 10 min, take 150 μL of supernatant in EP tube, dilute 10 times with 70% methanol in another part, take 150 μL of supernatant, add 5 μL of naringenin standard solution (IS, 5 mM) to each, mix well, centrifuge again, and set aside; Step 2: Using HPLC-MS / MS liquid phase mass spectrometry, the test solution obtained in step 1 is used to determine the contents of 16 active ingredients, including baicalin, berberine, wogonin, apigenin, chrysin, coptisine, daidzein, formononetin, melaleuca A, flavonoids, wogonin, fangfang pine, glycyrrhizic coumarin, genistin, liquiritin, and magnolia tin; wherein: Chromatographic conditions were: chromatographic columns: Waters ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm) and ACQUITY UPLC BEH C18 VanGuard Pre-Column (2.1×5 mm, 1.7 μm); column temperature: 45° C.; flow rate: 0.2 mL / min; injection volume: 5 μL; mobile phase: 5 mM ammonium formate + 0.1% formic acid aqueous solution (phase A)-methanol (phase B) binary gradient elution system, gradient elution program: 0-20 min, 35%-60% B; 20-23 min, 60%-100% B; 23-27 min, 100% B; 27-28 min, 100%-35% B; 28-33 min, 35% B; The mass spectrometry conditions were as follows: spray voltage 4.5 kV, nebulizer gas 3 L / min, drying gas 10 L / min, heater 10 L / min, heating block temperature 400 °C, capillary temperature 250 °C; in ESI positive and negative ion modes, multiple reaction monitoring (MRM) mode, the mass spectrometry MRM parameters of the analyte and internal standard are shown in Table 1. Table 1 MRM parameters of analytes and internal standards

Images