Characteristic chromatogram construction and content determination method of radix asparagi and radix ophiopogonis decoction preparation and thin-layer chromatography identification method of traditional Chinese medicine flavor components in radix asparagi and radix ophiopogonis decoction preparation

Abstract

The invention provides a specific chromatogram construction and content determination method of the radix asparagi and radix ophiopogonis decoction preparation and a thin-layer chromatography identification method of traditional Chinese medicine flavor components in the radix asparagi and radix ophiopogonis decoction preparation. The specific chromatogram construction method of the radix asparagi decoction preparation comprises the following steps: step 1, taking mangiferin, quercetin-3-O-glucuronide, baicalin, wogonoside and ammonium glycyrrhizinate reference substances, and adding a solvent to prepare a reference substance solution; step 2, taking an Eupatorium japonicum-Liupu decoction reference sample, and adding a solvent to prepare a test solution; and 3, respectively detecting the reference substance solution and the test solution by using high performance liquid chromatography, and by taking the characteristic spectrum of the reference substance solution as a reference spectrum, selecting a common peak from the characteristic spectrum of the test solution to construct the characteristic spectrum of the radix asparagi decoction preparation.

Description

Technical Field

[0001] The invention relates to a characteristic spectrum construction and content determination method of an Erdong decoction preparation, a thin layer chromatography identification method of Chinese medicinal components of the Erdong decoction preparation, and an identification method of the Erdong decoction preparation, and belongs to the field of Chinese medicinal component analysis and detection. Background Art

[0002] Erdong Decoction is the name of a Chinese medicine prescription. It comes from Volume 3 of "Medical Enlightenment" written by Cheng Guopeng in the Qing Dynasty. The original book records: "Thirst and drinking a lot is upper consumption, digesting food easily is middle consumption, and thirsty and urinating like paste is lower consumption. The three symptoms of consumption are all caused by the accumulation of dryness and heat. The main method is to treat upper consumption by moistening the lungs and clearing the stomach. Erdong Decoction is the main method." The prescription and preparation method are: "2 qian of Asparagus cochinchinensis (without the core), 3 qian of Ophiopogon japonicus (without the core), 1 qian of Pollen, 1 qian of Scutellaria baicalensis, 1 qian of Anemarrhena asphodeloides, 5 fen of Licorice, 5 fen of Ginseng, 1 qian of Lotus Leaf. Decoction in water."

[0003] Erdong decoction has the effects of nourishing yin, clearing away heat, promoting fluid and quenching thirst, and is often used in the clinic to treat diabetes, whooping cough, tuberculosis, chronic bronchitis, yin deficiency cough, etc. However, the relevant research on Erdong decoction mainly focuses on clinical application, and there is relatively little research on its quality control.

[0004] Patent document CN110658295A discloses a fingerprint method for identifying lotus leaves in Erdong Decoction. Patent document CN110596299A discloses a thin layer chromatography identification method for Asparagus cochinchinensis slices in Erdong Decoction. However, the above patent documents only study the detection of single herbs in Erdong Decoction, and do not involve the detection of multiple other ingredients in Erdong Decoction, that is, they cannot comprehensively evaluate the ingredients of Erdong Decoction, nor can they reflect the overall characteristics of the Chinese medicine compound preparation.

[0005] Patent document CN114689765A discloses a Erdongtang reference extract and its preparation method and quality control method, wherein a characteristic spectrum of Erdongtang extract is established, and 19 characteristic peaks should be presented in the characteristic spectrum of the test sample, wherein peak 1, peak 10, and peak 15 should correspond to the retention time of the reference peaks of L-tryptophan, baicalin, and glycyrrhizic acid reference substances, respectively, and the peak corresponding to the baicalin reference substance peak is taken as the S1 peak, and the relative retention time of peaks 2 to 12 and the S1 peak is calculated, and the relative retention time should be within the range of ±8% of the specified value. Patent document CN114689764A discloses a multi-component quality detection method for Erdongtang extract, wherein the contents of mangiferin, hyperoside, baicalin, and glycyrrhizic acid in Erdongtang extract are determined by high performance liquid chromatography. However, these methods use few index components, and there is a problem that it is difficult to fully reflect the quality status of Erdongtang preparations with multiple medicinal flavors and components, and the accuracy of the detection also has room for improvement. Summary of the invention

[0006] Problem that the invention aims to solve

[0007] At present, it is urgent to develop a method that can comprehensively, objectively and accurately evaluate the quality of Erdong Decoction, as well as a method that can simply and accurately detect the components in Erdong Decoction preparations.

[0008] The subject of the present invention is to provide a characteristic spectrum construction method and a content determination method of Erdong Decoction preparations that can quickly and accurately identify the quality of Erdong Decoction preparations based on high performance liquid chromatography, an identification method of Erdong Decoction preparations that can accurately and reliably evaluate the quality of Erdong Decoction preparations, and a thin layer chromatography identification method that can simply and accurately identify the various components in Erdong Decoction preparations.

[0009] Solutions for solving problems

[0010] In order to solve the above problems, the present invention provides the following technical solutions.

[0011] [1]. A method for constructing a characteristic spectrum of an Erdong decoction preparation, characterized in that it comprises the following steps:

[0012] Step 1: Take mangiferin, quercetin-3-O-glucuronide, baicalin, wogonin and ammonium glycyrrhizinate reference substances, and add a solvent to prepare a reference solution;

[0013] Step 2: Take the Erdongtang reference sample, add solvent, and prepare the test solution;

[0014] Step three: Use high performance liquid chromatography to detect the reference solution and the test solution respectively, use the characteristic spectrum of the reference solution as a reference spectrum, select common peaks from the characteristic spectrum of the test solution, and construct the characteristic spectrum of the Erdong Decoction preparation.

[0015] [2] The method according to item [1], characterized in that in step 1, methanol is added to prepare a reference solution containing 10-30 μg of mangiferin, 25-55 μg of quercetin-3-O-glucuronide, 350-550 μg of baicalin, 40-80 μg of wogonin and 25-65 μg of ammonium glycyrrhizinate per 1 mL.

[0016] [3] The method according to item [1] or [2], characterized in that in step 2, 0.5 to 1.5 g of Erdongtang reference sample is taken, accurately weighed, placed in a centrifuge tube, 40 mL of 30% ethanol is added, shaken to dissolve, centrifuged for 5 minutes, the supernatant is transferred to a 50 mL volumetric flask, 30% ethanol is added to the scale, shaken well, filtered, and the filtrate is taken to prepare the test solution.

[0017] [4] The method according to item [1] or [2], characterized in that in step 3, the HPLC detection conditions are:

[0018] The injection volume is 5 to 20 μl.

[0019] The chromatographic column is filled with octadecylsilane bonded silica gel.

[0020] Use 0.1% phosphoric acid as mobile phase A and methanol as mobile phase B for gradient elution at a flow rate of 1.0 mL / min.

[0021] The column temperature is 20-30°C.

[0022] The detection wavelength is 258 nm.

[0023] [5] The method according to item [4], characterized in that the gradient elution conditions are:

[0024] 0~10min, mobile phase A is 78%→65%, mobile phase B is 22%→35%,

[0025] 10~32min, mobile phase A is 65%→52%, mobile phase B is 35%→48%,

[0026] 32~55min, mobile phase A is 52%→40%, mobile phase B is 48%→60%,

[0027] 55-65min, mobile phase A 40%→28%, mobile phase B 60%→72%,

[0028] 65-70min, mobile phase A is 28%→78%, mobile phase B is 72%→22%,

[0029] 70-80min, mobile phase A is 78%→78%, mobile phase B is 22%→22%.

[0030] [6]. The method according to item [1] or [2], characterized in that in step 3, the peak corresponding to baicalin is taken as the S peak, and the relative retention time of the common peak is calculated to construct the characteristic spectrum of the Erdong Decoction preparation.

[0031] [7]. A characteristic spectrum of an Erdong decoction preparation, characterized in that it is constructed by the method described in any one of items [1] to [6], wherein the characteristic spectrum presents 9 common peaks, with peak 5 corresponding to baicalin as the S peak, and the relative retention times of peaks 1 to 4 and peaks 6 to 9 should be within the range of ±5% of the specified values, and the specified values ​​are: 0.32 (peak 1), 0.44 (peak 2), 0.74 (peak 3), 0.77 (peak 4), 1.13 (peak 6), 1.23 (peak 7), 1.25 (peak 8), and 1.88 (peak 9).

[0032] [8]. A method for determining the content of Erdong decoction preparation, characterized in that it comprises the following steps:

[0033] The contents of mangiferin, baicalin and glycyrrhizic acid in the Erdong decoction preparation were determined by high performance liquid chromatography, wherein:

[0034] The HPLC detection conditions are:

[0035] The injection volume is 5 to 20 μl.

[0036] The chromatographic column is filled with octadecylsilane bonded silica gel.

[0037] Use 0.1% phosphoric acid as mobile phase A and methanol as mobile phase B for gradient elution at a flow rate of 1.0 mL / min.

[0038] The column temperature is 20-30°C.

[0039] The detection wavelength is 258 nm.

[0040] [9] The method according to [8], characterized in that the gradient elution conditions are:

[0041] 0~10min, mobile phase A is 78%→65%, mobile phase B is 22%→35%,

[0042] 10~32min, mobile phase A is 65%→52%, mobile phase B is 35%→48%,

[0043] 32~55min, mobile phase A is 52%→40%, mobile phase B is 48%→60%,

[0044] 55-65min, mobile phase A 40%→28%, mobile phase B 60%→72%,

[0045] 65-70min, mobile phase A is 28%→78%, mobile phase B is 72%→22%,

[0046] 70-80min, mobile phase A is 78%→78%, mobile phase B is 22%→22%.

[0047]

[10] . A method for identifying an Erdong decoction preparation, characterized in that it comprises at least one of the following steps:

[0048] Step A: comparing the characteristic spectrum of the Erdong Decoction preparation to be identified with the characteristic spectrum of the Erdong Decoction preparation constructed by the method described in any one of items [1] to [6], or with the characteristic spectrum of the Erdong Decoction preparation described in item [7], to determine whether the Erdong Decoction preparation is a qualified product;

[0049] Step B: Determine the contents of mangiferin, baicalin and glycyrrhizic acid in each prescription of the Erdong Decoction preparation by the content determination method of the Erdong Decoction preparation described in item [8] or [9] to determine whether the Erdong Decoction preparation is a qualified product.

[0050]

[11] The identification method according to item

[10] is characterized in that the Erdong decoction preparation that meets at least one of the following conditions is judged as a qualified product:

[0051] Condition a: In the characteristic spectrum of the Erdong decoction preparation to be identified, peak 5 corresponding to baicalin is taken as the S peak, and the relative retention times of peaks 1 to 4 and peaks 6 to 9 are all within ±5% of the specified value in the characteristic spectrum described in [7;

[0052] Condition b: The content of mangiferin contained in each prescription of the Erdong decoction preparation is 10-30 mg, the content of baicalin is 250-600 mg, and the content of glycyrrhizic acid is 10-30 mg.

[0053]

[12] . A thin layer chromatography method for identifying the asparagus component in Erdong decoction preparation, characterized in that it comprises the following steps:

[0054] Step 1-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0055] Step 1-2: Take a protodioscin reference substance, add a solvent, and prepare a reference substance solution;

[0056] Step 1-3: According to the thin layer chromatography method, aspirate the test solution and the reference solution, spot them on the same thin layer plate respectively to make strips, use 11:7:1 chloroform-methanol-water as the developing agent, develop, take out, dry, spray with color developer and heat until the spots are clearly colored, and inspect.

[0057]

[13] The method according to item

[12] , characterized in that:

[0058] In the step 1-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of 65% methanol is added, ultrasonic treatment is performed, centrifugation is performed, the supernatant is taken, and transferred to a 25 ml volumetric flask, 65% methanol is added to the mark, evaporated to dryness, 10 ml of water is added to the residue to dissolve it, and oscillation extraction is performed with water-saturated n-butanol for 3 times, 10 ml each time, centrifugation is performed, the n-butanol solution is combined, and the solution is washed with ammonia test solution for 3 times, 10 ml each time, the ammonia test solution is discarded, the n-butanol solution is evaporated to dryness, the residue is dissolved in methanol and transferred to a 5 ml volumetric flask, and methanol is added to the mark as the test solution;

[0059] In step 1-2, methanol is added to prepare a solution containing 0.1-0.8 mg of protodioscin reference substance per 1 ml, which is used as the reference substance solution;

[0060] In the steps 1-3, 1-20 μl of the test solution and 2-10 μl of the reference solution are respectively spotted on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105° C., and the plate is inspected under a 365 nm ultraviolet lamp.

[0061]

[14] . A thin layer chromatography method for identifying the Radix Ophiopogonis in Erdong Decoction preparation, characterized in that it comprises the following steps:

[0062] Step 2-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0063] Step 2-2: Take Ophiopogon japonicus as a reference medicinal material, add a solvent, and prepare a reference medicinal material solution;

[0064] Step 2-3: According to the thin layer chromatography method, aspirate the test sample solution and the control medicinal material solution, respectively spot them on the same thin layer plate to form strips, use 4:1 chloroform-acetone as the developing agent, develop, take out, dry, spray with a color developer, and heat until the spots are clearly colored, and inspect.

[0065]

[15] The method according to item

[14] , characterized in that:

[0066] In the step 2-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of water and 5 ml of hydrochloric acid are added, heated to reflux, cooled, filtered, 20 ml of chloroform is added to the filtrate, and the chloroform solution is extracted by shaking, and the chloroform solution is taken and evaporated to dryness, and 1 ml of chloroform is added to the residue to dissolve it, which is used as the test solution;

[0067] In the step 2-2, 0.5 to 2 g of Ophiopogon japonicus reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as in the step 2-1;

[0068] In step 2-3, 1-30 μl of the test solution and 2-10 μl of the control medicinal material solution are respectively spotted on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105° C., and the plate is inspected under sunlight.

[0069]

[16] . A thin layer chromatography method for identifying the scutellaria baicalensis component in Erdong decoction preparation, characterized in that it comprises the following steps:

[0070] Step 3-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0071] Step 3-2-1: Take a reference medicinal material of Scutellaria baicalensis and add a solvent to prepare a reference medicinal material solution;

[0072] Step 3-2-2: Take baicalin reference substance, add solvent, and prepare reference substance solution;

[0073] Step 3-3: According to the thin layer chromatography method, aspirate the test sample solution, the reference medicinal material solution, and the reference substance solution, and spot them on the same thin layer plate to form strips. Use ethyl acetate-butanone-formic acid-water in a ratio of 5:5:1:1 as the developing solvent, develop, take out, dry, and inspect.

[0074]

[17] The method according to item

[16] , characterized in that

[0075] In the step 3-1, 0.5-4 g of Erdong decoction preparation is taken, 40 ml of ether is added, heated to reflux, filtered, the ether solution is discarded, the residue is evaporated, 30 ml of methanol is added, heated to reflux, filtered, the filtrate is evaporated to dryness, 40 ml of water is added to the residue to dissolve it, 50 ml of n-butanol is added to shake and extract, centrifuged, the n-butanol solution is taken, evaporated to dryness, and 5 ml of methanol is added to the residue to dissolve it as the test solution;

[0076] In the step 3-2-1, 0.2-1 g of Scutellaria baicalensis reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as in the step 3-1;

[0077] In the step 3-2-2, methanol is added to prepare a solution containing 0.5 to 2 mg of baicalin reference substance per 1 ml, which is used as the reference substance solution.

[0078] In step 3-3, 1-20 μl of the test solution, 2-10 μl of the reference medicinal material solution and the reference substance solution are respectively spotted on the same silica gel GF254 thin layer plate and inspected under a 254 nm ultraviolet lamp.

[0079]

[18] . A thin layer chromatography identification method for the Anemarrhena component in Erdong decoction preparation, characterized in that it comprises the following steps:

[0080] Step 4-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0081] Step 4-2-1: Take the Rhizoma Anemarrhenae as a control medicinal material, add a solvent, and prepare a control medicinal material solution;

[0082] Step 4-2-2: Take the reference substance of Anemarrhena saponin BⅡ, add solvent, and prepare the reference substance solution;

[0083] Step 4-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 7:2:3 n-butanol-acetic acid-water as the developing agent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

[0084]

[19] The method according to item

[18] , characterized in that

[0085] In step 4-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of dilute ethanol is added, ultrasonic treatment is performed, and filtration is performed, and the filtrate is used as the test solution;

[0086] In the step 4-2-1, 0.2-1 g of Rhizoma Anemarrhenae reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as in the step 4-1;

[0087] In step 4-2-2, methanol is added to prepare a solution containing 0.5-2 mg of timosaponin BⅡ reference substance per 1 ml, which is used as the reference substance solution;

[0088] In step 4-3, 1-20 μl of the test solution, 2-10 μl of the reference medicinal material solution and the reference substance solution are respectively spotted on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105° C., and the plate is inspected under sunlight.

[0089]

[20] . A thin layer chromatography identification method for lotus leaf components in Erdong decoction preparation, characterized in that it comprises the following steps:

[0090] Step 5-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0091] Step 5-2-1: Take lotus leaf as a reference medicinal material, add a solvent, and prepare a reference medicinal material solution;

[0092] Step 5-2-2: Take the nuciferine reference substance, add a solvent, and prepare a reference substance solution;

[0093] Step 5-3: According to the thin layer chromatography method, aspirate the test sample solution, reference medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 15:5:4 ethyl acetate-methanol-water as the developing solvent, develop, take out, dry, spray with a color developer, and inspect.

[0094]

[21] The method according to item

[20] , characterized in that

[0095] In the step 5-1, 0.5-4 g of Erdong decoction preparation is taken, 40 ml of chloroform is added, heated and refluxed for 1 hour, filtered, the chloroform liquid is discarded, the solvent is evaporated from the residue, 1 ml of water is added and stirred to moisten, 10 ml of water-saturated n-butanol is added, ultrasonic treatment is performed, the supernatant is drawn and 3 times the amount of ammonia test solution is added, shaken, allowed to separate, the upper layer is evaporated to dryness, and 1 ml of methanol is added to the residue to dissolve it as the test solution;

[0096] In step 5-2-1, 0.5 to 2 g of lotus leaf reference medicinal material is taken, and a reference medicinal material solution is prepared by the same method as step 5-1;

[0097] In step 5-2-2, methanol is added to prepare a solution containing 0.5-2 mg of nuciferine reference substance per 1 ml, which is used as the reference substance solution;

[0098] In step 5-3, 5-30 μl of the test solution, 5-20 μl of the reference medicinal material solution, and 1-10 μl of the reference solution are respectively spotted on the same silica gel G thin layer plate, the color developer is a dilute potassium bismuth iodide test solution, and the plate is inspected under sunlight.

[0099]

[22] . A thin layer chromatography method for identifying ginseng components in Erdong decoction preparations, characterized in that it comprises the following steps:

[0100] Step 6-1: Take the Erdong decoction preparation, add a solvent, and prepare a test solution;

[0101] Step 6-2-1: Take ginseng reference medicinal material, add solvent, and prepare reference medicinal material solution;

[0102] Step 6-2-2: Take ginsenoside Rg1 reference substance and ginsenoside Re reference substance, add solvent, and prepare reference substance solution;

[0103] Step 6-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use the upper layer solution of 4:1:5:0.5 n-butanol-ethyl acetate-water-acetic acid as the developing agent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

[0104]

[23] The method according to

[22] , characterized in that:

[0105] In the step 6-1: take 0.5-4g of Erdong decoction preparation, add 40ml of chloroform, heat and reflux for 1 hour, filter, discard the chloroform solution, evaporate the solvent from the residue, add 1ml of water to stir and moisten, add 10ml of water-saturated n-butanol, ultrasonically treat, absorb the supernatant and add 3 times the amount of ammonia test solution, shake well, let it stand and separate, take the upper layer and evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution;

[0106] Step 6-2-1: Take 0.5-2 g of ginseng reference medicinal material and prepare a reference medicinal material solution by the same method as step 6-1;

[0107] Step 6-2-2: Add methanol to prepare a mixed solution containing 0.5-4 mg of ginsenoside Rg1 reference substance and 0.5-4 mg of ginsenoside Re reference substance per 1 ml, as the reference substance solution;

[0108] Step 6-3: Pipette 10-40 μl of the test solution, 1-10 μl of the reference drug solution and the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate. The color developer is 10% sulfuric acid ethanol solution. The heating temperature is 105°C and the plate is inspected under sunlight.

[0109]

[24] . A thin layer chromatography identification method for licorice components in Erdong decoction preparation, characterized in that it comprises the following steps:

[0110] Step 7-1: Take the Erdong decoction preparation, add a solvent, and prepare a test solution;

[0111] Step 7-2-1: Take liquiritin control medicinal material, add solvent, and prepare control medicinal material solution;

[0112] Step 7-2-2: Take the liquiritin reference substance, add a solvent, and prepare a reference substance solution;

[0113] Step 7-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 10:3:2 ethyl acetate-acetic acid-water as the developing solvent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

[0114]

[25] The method according to item

[24] , characterized in that:

[0115] In the step 7-1: take 0.5-4g of Erdong decoction preparation, add 40ml of ether, heat to reflux, filter, discard the ether solution, evaporate the residue, add 30ml of methanol, heat to reflux, filter, evaporate the filtrate to dryness, add 40ml of water to dissolve the residue, add 50ml of n-butanol to shake and extract, centrifuge, take the n-butanol solution, evaporate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test solution;

[0116] Step 7-2-1: Take 0.5-2 g of licorice control medicinal material and prepare a control medicinal material solution by the same method as step 7-1;

[0117] Step 7-2-2: Add methanol to prepare a solution containing 0.2-1.5 mg of liquiritin reference substance per 1 ml as the reference substance solution;

[0118] Step 7-3: Pipette 1-20 μl of the test solution, 2-10 μl of the control medicinal material solution and the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate. The color developer is 10% sulfuric acid ethanol solution. The heating temperature is 105°C and the plate is inspected under a 365nm ultraviolet lamp.

[0119]

[26] . The method according to any one of items

[12] -

[25] , characterized in that the thin layer chromatography method is in accordance with General Chapter 0502 of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0120]

[27] . A method for identifying an Erdong Tang preparation, characterized in that it comprises: identifying the ingredients in the Erdong Tang preparation by the method described in any one of items

[12] -

[26] .

[0121]

[28] The method according to any one of items

[10] to

[27] , characterized in that the Erdong decoction preparation is a freeze-dried powder, an extract powder, or granules, wherein when the Erdong decoction preparation is granules, the granules are ground into powder before identification.

[0122] Effects of the Invention

[0123] According to the characteristic spectrum construction method of the Erdong Decoction preparation of the present invention, multiple index components in the Erdong Decoction preparation are profiled, and the obtained characteristic spectrum has a large amount of information, good specificity, high accuracy, good repeatability, high precision, and good stability. Therefore, the quality of the Erdong Decoction preparation can be comprehensively, objectively, and accurately evaluated.

[0124] According to the content determination method of the Erdong decoction preparation of the present invention, the components of Rhizoma Anemarrhenae, Radix Scutellariae and Radix Glycyrrhizae in the Erdong decoction preparation can be quantified with high accuracy, good repeatability, high precision and good stability. Furthermore, the content determination method of the Erdong decoction preparation of the present invention can simultaneously analyze multiple components in the same sample under the same conditions using high performance liquid chromatography, without the need for additional separation, purification and other operations, and without the need for more complex detection equipment such as mass spectrometry.

[0125] The identification method of the Erdong decoction preparation of the present invention can comprehensively, objectively, accurately and quickly evaluate the quality of the Erdong decoction preparation.

[0126] According to the thin layer identification method of the asparagus components, ophiopogon components, scutellaria components, anemarrhena components, lotus leaf components, ginseng components and licorice components in the Erdong Decoction preparation of the present invention, the asparagus components, ophiopogon components, scutellaria components, anemarrhena components, lotus leaf components, ginseng components and licorice components in the Erdong Decoction preparation can be simply and accurately identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 This is the chromatogram of the reference substances mangiferin, quercetin-3-O-glucuronide, baicalin, wogonin and ammonium glycyrrhizinate in Example 1.

[0128] Figure 2 This is the reference characteristic spectrum and common peak chromatogram in Example 1.

[0129] Figure 3 This is the chromatogram of the reference substances of mangiferin, baicalin and ammonium glycyrrhizinate in Example 1.

[0130] Figure 4 This is the chromatogram of the test sample in Example 1.

[0131] Figure 5 This is the characteristic spectrum measured by different personnel, at different times, and using different instruments for the Erdong Soup benchmark sample in Example 1-4.

[0132] Figure 6 1 is the chromatogram of the Erdong Decoction benchmark sample, each negative lyophilized powder and the reference solution obtained in Example 1-5.

[0133] Figure 7 It is the TLC identification spectrum of the asparagus component in the Erdong Decoction benchmark sample in Example 2-1 and the TLC identification result of the component in Example 2-5 (wherein, 1: asparagus negative, 2: asparagus and ophiopogon negative, 3: asparagus, ophiopogon and Anemarrhena negative, 4: asparagus reference medicinal material, 5: protodioscin, 6: test sample CH310101S, 7: test sample CH310102S, 8: test sample CH310103S).

[0134] Figure 8 This is the thin layer chromatography identification spectrum of the Ophiopogon japonicus component in the Erdong Decoction benchmark sample in Example 2.1 (wherein, 1: Ophiopogon japonicus negative, 2: Ophiopogon japonicus negative, 3: Ophiopogon japonicus reference medicinal material, 4: test sample CH310101S, 5: test sample CH310102S, 6: test sample CH310103S).

[0135] Fig. 9It is the thin layer chromatography identification spectrum of the scutellaria component in the Erdong Decoction benchmark sample in Example 2-1 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: scutellaria negative, 2: scutellaria reference medicinal material, 3: baicalin, 4: test sample CH310101S).

[0136] Fig.10 This is the thin layer chromatography identification spectrum of the Anemarrhena component in the Erdong Decoction benchmark sample in Example 2-1 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: Anemarrhena negative, 2: Anemarrhena reference medicinal material, 3: Anemarrhena saponin BⅡ, 4: test sample CH310101S, 5: test sample CH310102S, 6: test sample CH310103S).

[0137] Fig.11 It is the TLC identification spectrum of the lotus leaf component in the Erdong Decoction benchmark sample in Example 2-1 and the TLC identification result of the component in Example 2-5 (wherein, 1: lotus leaf negative, 2: lotus leaf reference medicinal material, 3: nuciferine, 4: test sample CH310101S, 5: test sample CH310102S, 6: test sample CH310103S).

[0138] Fig.12 This is the TLC identification spectrum of the ginseng component in the Erdong Decoction benchmark sample in Example 2-1 and the TLC identification result of the component in Example 2-5 (wherein, 1: ginseng negative, 2: ginseng reference medicinal material, 3: from top to bottom, ginsenoside Rg1 (the first spot), ginsenoside Re (the third spot), 4: test sample CH310101S, 5: test sample CH310102S, 6: test sample CH310103S).

[0139] Fig.13 It is the thin layer chromatography identification spectrum of the licorice component in the Erdong Decoction benchmark sample in Example 2-1 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: licorice negative, 2: licorice reference medicinal material, 3: glycyrrhizin, 4: test sample CH310101S, 5: test sample CH310102S, 6: test sample CH310103S).

[0140] Fig.14 This is the thin layer chromatography identification spectrum of the asparagus component in the Erdong Decoction extract powder in Example 2.2 and the thin layer chromatography identification result of the component in Example 2.5 (wherein, 1: asparagus negative, 2: asparagus and ophiopogon negative, 3: asparagus, ophiopogon and Anemarrhena negative, 4: asparagus reference medicinal material, 5: protodioscin, 6: test sample 221C213010, 7: test sample 221C213040, 8: test sample 221C213050).

[0141] Fig.15This is the thin layer chromatography identification spectrum of the Ophiopogon japonicus component in the Erdong decoction extract powder in Example 2.2 and the thin layer chromatography identification result of the component in Example 2.5 (1: Ophiopogon japonicus negative, 2: Ophiopogon japonicus negative, 3: Ophiopogon japonicus control medicinal material, 4: test sample 221C213010, 5: test sample 221C213040, 6: test sample 221C213050).

[0142] Fig.16 It is the thin layer chromatography identification spectrum of the scutellaria baicalensis component in the Erdong decoction extract powder in Example 2.2 and the thin layer chromatography identification result of the component in Example 2.5 (wherein, 1: scutellaria baicalensis negative, 2: scutellaria baicalensis control medicinal material, 3: baicalin, 4: test sample 221C213010, 5: test sample 221C213040, 6: test sample 221C213050).

[0143] Fig.17 This is the thin layer chromatography identification spectrum of the Anemarrhena component in the Erdong Decoction extract powder in Example 2.2 and the thin layer chromatography identification result of the component in Example 2.5 (wherein, 1: Anemarrhena negative, 2: Anemarrhena control medicinal material, 3: Anemarrhena saponin BⅡ, 4: test sample 221C213010, 5: test sample 221C213040, 6: test sample 221C213050).

[0144] Fig.18 This is the thin layer chromatography identification spectrum of the lotus leaf component in the Erdong Decoction extract powder in Example 2.2 and the thin layer chromatography identification result of the component in Example 2.5 (wherein, 1: lotus leaf negative, 2: lotus leaf reference medicinal material, 3: nuciferine, 4: test sample 221C213010, 5: test sample 221C213040, 6: test sample 221C213050).

[0145] Fig.19 This is the thin layer chromatography identification spectrum of the ginseng component in the Erdong Decoction extract powder in Example 2.2 and the thin layer chromatography identification result of the component in Example 2.5 (wherein, 1: ginseng negative, 2: ginseng reference medicinal material, 3: from top to bottom, ginsenoside Rg1 (reference substance), ginsenoside Rf (not used as a reference substance), ginsenoside Re (reference substance), ginsenoside Rb1 (not used as a reference substance), 4: test sample 221C213010, 5: test sample 221C213040, 6: test sample 221C213050).

[0146] Fig. 20 It is the thin layer chromatography identification spectrum of the licorice component in the Erdong Decoction extract powder in Example 2.2 and the thin layer chromatography identification result of the component in Example 2.5 (wherein, 1: licorice negative, 2: licorice control medicinal material, 3: glycyrrhizin, 4: test sample 221C213010, 5: test sample 221C213040, 6: test sample 221C213050).

[0147] Fig.21 It is the thin layer chromatography identification spectrum of the asparagus component in the Erdong Decoction granules in Example 2-3 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: asparagus negative, 2: asparagus and ophiopogon negative, 3: asparagus, ophiopogon and Anemarrhena negative, 4: asparagus control medicinal material, 5: protodioscin, 6: test sample 202100901, 7: test sample 2021032B1, 8: test sample 2021032C1).

[0148] Fig. 22 This is the thin layer chromatography identification spectrum of the Ophiopogon japonicus component in the Erdong Decoction granules in Example 2-3 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: Ophiopogon japonicus negative, 2: Ophiopogon japonicus negative, 3: Ophiopogon japonicus control medicinal material, 4: test sample 202100901, 5: test sample 2021032B1, 6: test sample 2021032C1).

[0149] Fig.23 It is the thin layer chromatography identification spectrum of the scutellaria baicalensis component in the Erdong Decoction granules in Example 2-3 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: scutellaria baicalensis negative, 2: scutellaria baicalensis control medicinal material, 3: baicalin, 4: test sample 202100901, 5: test sample 2021032B1, 6: test sample 2021032C1).

[0150] Fig.24 This is the thin layer chromatography identification spectrum of the Anemarrhena component in the Erdong Decoction granules in Example 2-3 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: Anemarrhena negative, 2: Anemarrhena control medicinal material, 3: Anemarrhena saponin BⅡ, 4: test sample 202100901, 5: test sample 2021032B1, 6: test sample 2021032C1).

[0151] Fig.25 This is the thin layer chromatography identification spectrum of the lotus leaf component in the Erdong Decoction granules in Example 2-3 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: lotus leaf negative, 2: lotus leaf control medicinal material, 3: nuciferine, 4: test sample 202100901, 5: test sample 2021032B1, 6: test sample 2021032C1).

[0152] Fig.26This is the thin layer chromatography identification spectrum of the ginseng component in the Erdong Decoction granules in Example 2-3 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: ginseng negative, 2: ginseng control medicinal material, 3: from top to bottom, ginsenoside Rg1 (reference substance), ginsenoside Rf (not used as a reference substance), ginsenoside Re (reference substance), ginsenoside Rb1 (not used as a reference substance), 4: test sample 202100901, 5: test sample 2021032B1, 6: test sample 2021032C1).

[0153] Fig. 27 It is the thin layer chromatography identification spectrum of the licorice component in the Erdong Decoction granules in Example 2-3 and the thin layer chromatography identification result of the component in Example 2-5 (wherein, 1: licorice negative, 2: licorice control medicinal material, 3: glycyrrhizin, 4: test sample 202100901, 5: test sample 2021032B1, 6: test sample 2021032C1). DETAILED DESCRIPTION

[0154] The following is a description of the specific embodiments of the present invention. In order to better illustrate the present invention, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0155] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0156] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0157] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0158] In this specification, the numerical range expressed using "numerical value A to numerical value B" or "numerical value A-numerical value B" means a range including the endpoints numerical values ​​A and B.

[0159] In the present specification, unless otherwise specified, the ratio of each solvent in a mixed solvent refers to a volume ratio.

[0160] One aspect of the present invention provides a method for constructing a characteristic spectrum of an Erdong decoction preparation, comprising the following steps:

[0161] Step 1: Take mangiferin, quercetin-3-O-glucuronide, baicalin, wogonin and ammonium glycyrrhizinate reference substances, and add a solvent to prepare a reference solution;

[0162] Step 2: Take the Erdongtang reference sample, add solvent, and prepare the test solution;

[0163] Step three: Use high performance liquid chromatography to detect the reference solution and the test solution respectively, use the characteristic spectrum of the reference solution as a reference spectrum, select common peaks from the characteristic spectrum of the test solution, and construct the characteristic spectrum of the Erdong Decoction preparation.

[0164] The characteristic spectrum obtained by the method for constructing the characteristic spectrum of the Erdong Decoction preparation of the present invention can be used for quality control of the Erdong Decoction preparation.

[0165] By analyzing the ingredients of the prescription, Erdong Decoction mainly contains saponins, flavonoids and polysaccharides. In the present invention, by selecting components that have ultraviolet absorption, good chromatographic peak separation, large peak area, and can transfer quantity and quality in the HPLC analysis method, namely mangoside, quercetin-3-O-glucuronide, baicalin, baicalin and ammonium glycyrrhizinate, they are used to identify Anemarrhena, Scutellaria, and Licorice, so that qualitative and quantitative analysis can be accurately and reliably performed, thereby achieving the quality control of Erdong Decoction preparations. The 5 reference substances can appropriately use standard substances or high-purity compounds of commercial products, preferably standard substances sold by China Food and Drug Administration.

[0166] The solvent used to prepare the reference solution can be an organic solvent commonly used in the field of Chinese medicine detection, or a mixed solvent of the organic solvent and water. As the organic solvent, for example, petroleum ether, benzene, ether, chloroform, dichloromethane, ethyl acetate, acetone, n-butanol, ethanol, methanol, etc. are listed. Methanol, ethanol or their aqueous solutions are preferably used, and methanol is more preferably used.

[0167] The concentration of each reference substance in the reference solution can be appropriately selected. For example, 10 to 30 μg of mangiferin, 25 to 55 μg of quercetin-3-O-glucuronide, 350 to 550 μg of baicalin, 40 to 80 μg of wogonin, and 25 to 65 μg of ammonium glycyrrhizinate can be contained in each 1 mL of the reference solution. From the perspective that the characteristic peaks of each reference substance can be clearly displayed together in the same chromatogram, it is preferred that 15 to 25 μg of mangiferin, 35 to 50 μg of quercetin-3-O-glucuronide, 400 to 500 μg of baicalin, 50 to 70 μg of wogonin, and 35 to 55 μg of ammonium glycyrrhizinate can be contained in each 1 mL of the reference solution.

[0168] In some preferred embodiments, in step 1, methanol is added to prepare a reference solution containing 10-30 μg of mangiferin, 25-55 μg of quercetin-3-O-glucuronide, 350-550 μg of baicalin, 40-80 μg of wogonin and 25-65 μg of ammonium glycyrrhizinate per 1 mL.

[0169] The preparation method of the test solution can refer to the commonly used method in the field of traditional Chinese medicine detection. The solvent used to prepare the test solution can use an organic solvent commonly used in the field of traditional Chinese medicine detection, or a mixed solvent of the organic solvent and water. As the organic solvent, for example, petroleum ether, benzene, ether, chloroform, dichloromethane, ethyl acetate, acetone, n-butanol, ethanol, methanol, etc. are listed. Methanol and ethanol are preferably used as the organic solvent, and an aqueous solution of ethanol, such as 20% to 50% ethanol, is more preferably used.

[0170] In some preferred embodiments, in the step 2, 0.5-1.5 g of Erdongtang reference sample is taken, accurately weighed, placed in a centrifuge tube, 40 mL of 30% ethanol is added, shaken to dissolve, centrifuged for 5 minutes, the supernatant is transferred to a 50 mL volumetric flask, 30% ethanol is added to the scale, shaken, filtered, and the filtrate is taken to prepare the test solution.

[0171] The form of the Erdongtang reference sample and the Erdongtang preparation to be detected or identified as described later is not particularly limited, and may be, for example, a decoction or a concentrated solution or solid made from the decoction, preferably a lyophilized powder, an extract powder, or granules. From the perspective of being easy to dissolve and make a solution, a lyophilized powder is more preferably used. When using granules, it is preferably ground into a powder before dissolving with a solubilizer.

[0172] In the step three, the HPLC detection conditions can be appropriately set in accordance with the General Rules 0512 of the 2020 edition of the Chinese Pharmacopoeia. From the viewpoint of obtaining a chromatogram in which the characteristic peaks are separated from each other and the peak shape is good, it is preferred that the HPLC detection conditions are: the injection volume is 5 to 20 μl, the chromatographic column is filled with octadecylsilane bonded silica gel, 0.1% phosphoric acid is used as mobile phase A, and methanol is used as mobile phase B, and gradient elution is performed, the flow rate is 1.0 mL / min, the column temperature is 20 to 30 ° C, and the detection wavelength is 258 nm. It is further preferred that the gradient elution conditions are:

[0173] 0~10min, mobile phase A is 78%→65%, mobile phase B is 22%→35%,

[0174] 10~32min, mobile phase A is 65%→52%, mobile phase B is 35%→48%,

[0175] 32~55min, mobile phase A is 52%→40%, mobile phase B is 48%→60%,

[0176] 55-65min, mobile phase A 40%→28%, mobile phase B 60%→72%,

[0177] 65-70min, mobile phase A is 28%→78%, mobile phase B is 72%→22%,

[0178] 70-80min, mobile phase A is 78%→78%, mobile phase B is 22%→22%.

[0179] In some preferred embodiments, in step three, the peak corresponding to baicalin is taken as the S peak, and the relative retention time of the common peak is calculated to construct a characteristic spectrum of the Erdong Decoction preparation.

[0180] Another aspect of the present invention also provides a characteristic spectrum of the Erdong Decoction preparation obtained by the characteristic spectrum construction method of the Erdong Decoction preparation of the present invention, wherein 9 common peaks are presented in the characteristic spectrum, with peak 5 corresponding to baicalin as the S peak, and the relative retention times of peaks 1 to 4 and peaks 6 to 9 should be within ±5% of the specified values, and the specified values ​​are: 0.32 (peak 1), 0.44 (peak 2), 0.74 (peak 3), 0.77 (peak 4), 1.13 (peak 6), 1.23 (peak 7), 1.25 (peak 8), and 1.88 (peak 9).

[0181] Another aspect of the present invention also provides a method for determining the content of Erdong Decoction preparation, which comprises the following steps: determining the content of mangiferin, baicalin and glycyrrhizic acid in the Erdong Decoction preparation by high performance liquid chromatography. The high performance liquid chromatography detection conditions can adopt the high performance liquid chromatography detection conditions in step 3 of the above-mentioned characteristic spectrum construction method.

[0182] Mangiferin, baicalin and glycyrrhizic acid are characteristic components of Rhizoma Anemarrhenae, Radix Scutellariae and Radix Glycyrrhizae, respectively. Therefore, based on the contents of mangiferin, baicalin and glycyrrhizic acid measured by the content determination method of the present invention, the contents of Rhizoma Anemarrhenae, Radix Scutellariae and Radix Glycyrrhizae in Erdong Decoction preparation can be estimated, providing a basis for the quality control of Erdong Decoction preparation.

[0183] In the characteristic spectrum construction method of the present invention, mangiferin, quercetin-3-O-glucuronide, baicalin, wogonin and ammonium glycyrrhizic acid are used as references, while in the content determination method, only mangiferin, baicalin and glycyrrhizic acid are used. This is because quercetin-3-O-glucuronide and wogonin are derived from Scutellaria baicalensis, and baicalin is used to quantify Scutellaria baicalensis in the content determination method, so there is no need to use quercetin-3-O-glucuronide and wogonin additionally.

[0184] Another aspect of the present invention also provides a method for identifying an Erdong decoction preparation, which comprises at least any one of the following steps:

[0185] Step A: comparing the characteristic spectrum of the Erdong Decoction preparation to be identified with the characteristic spectrum of the present invention to determine whether the Erdong Decoction preparation is a qualified product;

[0186] Step B: Determine the contents of mangiferin, baicalin and glycyrrhizic acid in each prescription of the Erdong Decoction preparation by the above-mentioned content determination method of the present invention to determine whether the Erdong Decoction preparation is a qualified product.

[0187] In some preferred embodiments, from the perspective of further accurately evaluating the quality of the Erdong decoction preparation, the Erdong decoction preparation that meets at least any one of the following conditions is determined to be a qualified product:

[0188] Condition a: In the characteristic spectrum of the Erdong Decoction preparation to be identified, peak 5 corresponding to baicalin is taken as the S peak, and the relative retention times of peaks 1 to 4 and peaks 6 to 9 are all within ±5% of the specified value in the characteristic spectrum of the present invention;

[0189] Condition b: The content of mangiferin contained in each prescription of the Erdong Decoction preparation is 10-30 mg, the content of baicalin is 250-600 mg, and the content of glycyrrhizic acid is 10-30 mg. From the viewpoint of further accurately evaluating the quality of the Erdong Decoction preparation, in condition b, it is preferred that the content of mangiferin contained in each prescription of the Erdong Decoction preparation is 12.0-26.6 mg, the content of baicalin is 280-585 mg, and the content of glycyrrhizic acid is 12.5-28.1 mg; it is further preferred that the content of mangiferin contained in each prescription of the Erdong Decoction preparation is 13.4-24.8 mg, the content of baicalin is 302-559 mg, and the content of glycyrrhizic acid is 14.4-26.7 mg.

[0190] Another aspect of the present invention provides a thin layer chromatography identification method for each component in the Erdong Decoction preparation. Specifically, the present invention provides a thin layer chromatography identification method for the components of Asparagus cochinchinensis, Radix Ophiopogonis, Radix Scutellariae, Rhizoma Anemarrhenae, Lotus Leaf, Ginseng and Radix Glycyrrhizae in the Erdong Decoction preparation.

[0191] The Erdong decoction preparation to be detected is preferably freeze-dried powder, extract powder, or granules, wherein when the Erdong decoction preparation is granules, the granules are ground into powder before identification.

[0192] The thin layer chromatography method of the present invention can adopt conventional methods in the field of traditional Chinese medicine detection, and is preferably implemented in accordance with General Chapter 0502 of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0193] The reference substance used in the thin layer chromatography identification method of the present invention can appropriately use the standard substance or high-purity compound of commercial products, preferably the standard substance sold by China Food and Drug Inspection Institute. The solvent used to prepare the reference substance solution can use the organic solvent commonly used in the field of traditional Chinese medicine detection or the mixed solvent of the organic solvent and water. As the organic solvent, for example, petroleum ether, benzene, ether, chloroform, dichloromethane, ethyl acetate, acetone, n-butanol, ethanol, methanol, etc. can be listed. Methanol, ethanol or its aqueous solution is preferably used, and methanol is more preferably used.

[0194] The reference medicinal material used in the thin layer chromatography identification method of the present invention preferably uses the standard product sold by the China Food and Drug Inspection Institute, which is usually in powder form and does not need to be further crushed when used. The preparation method of the reference medicinal material solution is generally processed according to the conventional method in the Chinese Pharmacopoeia, that is, except for the sample weight, it is kept consistent with the preparation method of the test sample solution. This is because the reference medicinal material is essentially a decoction piece, which has the same morphological source as the decoction piece in the preparation. In order to identify the decoction piece in the test solution of the preparation, it is necessary to use the same processing method as the test sample.

[0195] One aspect of the present invention provides a thin layer chromatography identification method for the asparagus component in the Erdong decoction preparation, which comprises the following steps:

[0196] Step 1-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0197] Step 1-2: Take a protodioscin reference substance, add a solvent, and prepare a reference substance solution;

[0198] Step 1-3: According to the thin layer chromatography method, aspirate the test solution and the reference solution, spot them on the same thin layer plate respectively to make strips, use 11:7:1 chloroform-methanol-water as the developing agent, develop, take out, dry, spray with color developer and heat until the spots are clearly colored, and inspect.

[0199] In the thin layer chromatography identification method of the asparagus component of the present invention, 11:7:1 chloroform-methanol-water must be used as the developing solvent, otherwise the separation effect is poor, or it is difficult to obtain clear spots, or the position and / or color of the spots after color development are inconsistent with the reference substance or reference medicinal material.

[0200] In some preferred embodiments, in the step 1-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of 65% methanol is added, ultrasonic treatment is performed, centrifugation is performed, the supernatant is taken, and transferred to a 25 ml volumetric flask, 65% methanol is added to the scale, evaporated to dryness, 10 ml of water is added to the residue to dissolve it, and it is extracted by oscillation with water-saturated n-butanol for 3 times, 10 ml each time, centrifuged, the n-butanol solution is combined, and it is washed with ammonia test solution for 3 times, 10 ml each time, the ammonia test solution is discarded, the n-butanol solution is evaporated to dryness, methanol is added to the residue to dissolve it and transferred to a 5 ml volumetric flask, and methanol is added to the scale as the test solution.

[0201] In some preferred embodiments, in step 1-2, methanol is added to prepare a solution containing 0.1 to 0.8 mg of protodioscin reference substance per 1 ml, which is used as the reference substance solution.

[0202] In some preferred embodiments, in the steps 1-3, 1-20 μl of the test solution and 2-10 μl of the reference solution are respectively spotted on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105°C, and the plate is inspected under a 365nm ultraviolet lamp. Further preferably, in the steps 1-3, 2-10 μl of the test solution is drawn.

[0203] The Erdong decoction preparation to be tested can be a lyophilized powder, an extract powder or granules. When the Erdong decoction preparation is a lyophilized powder, an extract powder or granules, when identifying the asparagus component of the Erdong decoction preparation, the amount of the test solution spotted on the thin layer plate is preferably 2 to 10 μl.

[0204] Another aspect of the present invention provides a thin layer chromatography identification method for the Radix Ophiopogonis component in the Erdong Decoction preparation, which comprises the following steps:

[0205] Step 2-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0206] Step 2-2: Take Ophiopogon japonicus as a reference medicinal material, add a solvent, and prepare a reference medicinal material solution;

[0207] Step 2-3: According to the thin layer chromatography method, aspirate the test sample solution and the control medicinal material solution, respectively spot them on the same thin layer plate to form strips, use 4:1 chloroform-acetone as the developing agent, develop, take out, dry, spray with a color developer, and heat until the spots are clearly colored, and inspect.

[0208] In the thin layer chromatography identification method of the Ophiopogon japonicus components of the present invention, 4:1 chloroform-acetone must be used as the developing solvent, otherwise the separation effect is poor, or it is difficult to obtain clear spots, or the position and / or color of the spots after color development are inconsistent with the reference substance or reference medicinal material.

[0209] In some preferred embodiments, in step 2-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of water and 5 ml of hydrochloric acid are added, heated to reflux, cooled, filtered, 20 ml of chloroform is added to the filtrate, and the filtrate is extracted by oscillation, the chloroform solution is taken, evaporated to dryness, and 1 ml of chloroform is added to the residue to dissolve it, which is used as the test solution.

[0210] In some preferred embodiments, in step 2-2, 0.5 to 2 g of Ophiopogon japonicus reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as step 2-1.

[0211] In some preferred embodiments, in step 2-3, 1-30 μl of the test solution and 2-10 μl of the control medicinal material solution are respectively spotted on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105°C, and the plate is inspected under sunlight. Further preferably, in step 2-3, 5-20 μl of the test solution is drawn.

[0212] The Erdong decoction preparation to be tested can be a lyophilized powder, an extract powder or granules. When the Erdong decoction preparation is a lyophilized powder, when identifying the Radix Ophiopogonis component of the Erdong decoction preparation, the amount of the test solution spotted on the thin layer plate is preferably 5 to 10 μl, and when the Erdong decoction preparation is an extract powder or granules, the amount of the sample spotted on the thin layer plate is preferably 10 to 20 μl.

[0213] Another aspect of the present invention provides a thin layer chromatography identification method for the scutellaria baicalensis component in the Erdong decoction preparation, which comprises the following steps:

[0214] Step 3-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0215] Step 3-2-1: Take a reference medicinal material of Scutellaria baicalensis and add a solvent to prepare a reference medicinal material solution;

[0216] Step 3-2-2: Take baicalin reference substance, add solvent, and prepare reference substance solution;

[0217] Step 3-3: According to the thin layer chromatography method, aspirate the test sample solution, the reference medicinal material solution, and the reference substance solution, and spot them on the same thin layer plate to form strips. Use ethyl acetate-butanone-formic acid-water in a ratio of 5:5:1:1 as the developing solvent, develop, take out, dry, and inspect.

[0218] In the thin layer chromatography identification method of the scutellaria baicalensis components of the present invention, ethyl acetate-butanone-formic acid-water in a ratio of 5:5:1:1 must be used as a developing solvent, otherwise the separation effect is poor, or it is difficult to obtain clear spots, or the position and / or color of the spots after color development are inconsistent with the reference substance or reference medicinal material.

[0219] In some preferred embodiments, in the step 3-1, 0.5-4 g of Erdong decoction preparation is taken, 40 ml of ether is added, the mixture is heated to reflux, filtered, the ether solution is discarded, the residue is evaporated, 30 ml of methanol is added, the mixture is heated to reflux, filtered, the filtrate is evaporated to dryness, 40 ml of water is added to the residue to dissolve it, 50 ml of n-butanol is added to extract by shaking, centrifugation is performed, the n-butanol solution is taken, evaporated to dryness, and 5 ml of methanol is added to the residue to dissolve it as the test solution.

[0220] In some preferred embodiments, in step 3-2-1, 0.2 to 1 g of Scutellaria baicalensis reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as in step 3-1.

[0221] In some preferred embodiments, in step 3-2-2, methanol is added to prepare a solution containing 0.5 to 2 mg of baicalin reference substance per 1 ml, which is used as the reference substance solution.

[0222] In some preferred embodiments, in step 3-3, 1-20 μl of the test solution, 2-10 μl of the control medicinal material solution and the control solution are respectively spotted on the same silica gel GF254 thin layer plate and inspected under a 254 nm ultraviolet lamp. Further preferably, in step 3-3, 5-10 μl of the test solution is drawn.

[0223] The Erdong decoction preparation to be tested can be freeze-dried powder, extract powder or granules. When the Erdong decoction preparation is freeze-dried powder, extract powder or granules, when identifying the scutellaria component of the Erdong decoction preparation, the amount of the test solution spotted on the thin layer plate is preferably 5-10 μl.

[0224] Another aspect of the present invention provides a thin layer chromatography identification method for the Anemarrhena component in the Erdong Decoction preparation, which comprises the following steps:

[0225] Step 4-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0226] Step 4-2-1: Take the Rhizoma Anemarrhenae as a control medicinal material, add a solvent, and prepare a control medicinal material solution;

[0227] Step 4-2-2: Take the reference substance of Anemarrhena saponin BⅡ, add solvent, and prepare the reference substance solution;

[0228] Step 4-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 7:2:3 n-butanol-acetic acid-water as the developing agent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

[0229] In the thin layer chromatography identification method of the Anemarrhena component of the present invention, 7:2:3 n-butanol-acetic acid-water must be used as the developing solvent, otherwise the separation effect is poor, or it is difficult to obtain clear spots, or the position and / or color of the spots after color development are inconsistent with the reference substance or reference medicinal material.

[0230] In some preferred embodiments, in step 4-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of dilute ethanol is added, ultrasonic treatment is performed, and filtration is performed, and the filtrate is used as the test solution. It should be noted that the "dilute ethanol" mentioned in the present invention refers to the dilute ethanol specified in the 2020 edition of the Chinese Pharmacopoeia 8002 test solution. 529 ml of ethanol is taken and diluted to 1000 ml with water, that is, the solution contains 49.5% to 50.5% (ml / ml) of C2H5OH at 20°C.

[0231] In some preferred embodiments, in step 4-2-1, 0.2 to 1 g of Rhizoma Anemarrhenae reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as step 4-1.

[0232] In some preferred embodiments, in step 4-2-2, methanol is added to prepare a solution containing 0.5 to 2 mg of timosaponin BⅡ reference substance per 1 ml, which is used as the reference substance solution.

[0233] In some preferred embodiments, in step 4-3, 1-20 μl of the test solution, 2-10 μl of the control medicinal material solution and the control solution are respectively applied to the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105°C, and the plate is inspected under sunlight. It is further preferred that in step 4-3, 2-20 μl of the test solution is taken; it is further preferred that in step 4-3, 2-10 μl of the test solution is taken.

[0234] The Erdong decoction preparation to be tested can be a lyophilized powder, an extract powder or granules. When the Erdong decoction preparation is a lyophilized powder, an extract powder or granules, when identifying the Anemarrhena component of the Erdong decoction preparation, the amount of the test solution spotted on the thin layer plate is preferably 2 to 10 μl.

[0235] Another aspect of the present invention provides a thin layer chromatography identification method for lotus leaf components in Erdong decoction preparation, characterized in that it comprises the following steps:

[0236] Step 5-1: Take Erdong decoction preparation, add solvent, and prepare test solution;

[0237] Step 5-2-1: Take lotus leaf as a reference medicinal material, add a solvent, and prepare a reference medicinal material solution;

[0238] Step 5-2-2: Take the nuciferine reference substance, add a solvent, and prepare a reference substance solution;

[0239] Step 5-3: According to the thin layer chromatography method, aspirate the test sample solution, reference medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 15:5:4 ethyl acetate-methanol-water as the developing solvent, develop, take out, dry, spray with a color developer, and inspect.

[0240] In the thin layer chromatography identification method of lotus leaf components of the present invention, ethyl acetate-methanol-water with a ratio of 15:5:4 must be used as the developing solvent, otherwise the separation effect is poor, or it is difficult to obtain clear spots, or the position and / or color of the spots after color development are inconsistent with the reference substance or reference medicinal material.

[0241] In some preferred embodiments, in the step 5-1, 0.5-4 g of Erdong decoction preparation is taken, 40 ml of chloroform is added, and the mixture is heated to reflux for 1 hour, filtered, and the chloroform liquid is discarded. The solvent is evaporated from the residue, 1 ml of water is added and stirred to moisten, 10 ml of water-saturated n-butanol is added, and ultrasonic treatment is performed. The supernatant is aspirated and 3 times the amount of ammonia test solution is added, the mixture is shaken, and the mixture is allowed to separate into layers. The upper layer is evaporated to dryness, and 1 ml of methanol is added to the residue to dissolve the mixture, which is used as the test solution.

[0242] In some preferred embodiments, in step 5-2-1, 0.5 to 2 g of lotus leaf reference medicinal material is taken, and a reference medicinal material solution is prepared by the same method as step 5-1.

[0243] In some preferred embodiments, in step 5-2-2, methanol is added to prepare a solution containing 0.5 to 2 mg of nuciferine reference substance per 1 ml, which is used as the reference substance solution.

[0244] In some preferred embodiments, in step 5-3, 5 to 30 μl of the test solution, 5 to 20 μl of the control medicinal material solution, and 1 to 10 μl of the control solution are respectively applied to the same silica gel G thin layer plate, the color developer is a dilute potassium bismuth iodide test solution, and the plate is inspected under sunlight. Further preferably, in step 5-3, 10 to 20 μl of the test solution is applied.

[0245] The Erdongtang preparation to be tested can be a lyophilized powder, an extract powder or a granule. When the Erdongtang preparation is a lyophilized powder, the amount of sample spotted on the thin layer plate is preferably 10 to 15 μl when identifying the lotus leaf component of the Erdongtang preparation, when the Erdongtang preparation is an extract powder, the amount of sample spotted on the thin layer plate is preferably 10 to 20 μl, and when the Erdongtang preparation is a granule, the amount of sample spotted on the thin layer plate is preferably 15 to 20 μl.

[0246] Another aspect of the present invention provides a thin layer chromatography identification method for ginseng components in Erdong decoction preparation, which comprises the following steps:

[0247] Step 6-1: Take the Erdong decoction preparation, add a solvent, and prepare a test solution;

[0248] Step 6-2-1: Take ginseng reference medicinal material, add solvent, and prepare reference medicinal material solution;

[0249] Step 6-2-2: Take ginsenoside Rg1 reference substance and ginsenoside Re reference substance, add solvent, and prepare reference substance solution;

[0250] Step 6-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use the upper layer solution of 4:1:5:0.5 n-butanol-ethyl acetate-water-acetic acid as the developing agent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

[0251] In the thin layer chromatography identification method of ginseng components of the present invention, an upper layer solution of n-butanol-ethyl acetate-water-acetic acid in a ratio of 4:1:5:0.5 must be used as a developing agent, otherwise the separation effect is poor, or it is difficult to obtain clear spots, or the position and / or color of the spots after color development are inconsistent with the reference substance or reference medicinal material.

[0252] In some preferred embodiments, in the step 6-1: take 0.5-4 g of Erdong decoction preparation, add 40 ml of chloroform, heat and reflux for 1 hour, filter, discard the chloroform solution, evaporate the solvent from the residue, add 1 ml of water and stir to moisten, add 10 ml of water-saturated n-butanol, ultrasonically treat, absorb the supernatant and add 3 times the amount of ammonia test solution, shake well, let it stand to separate, take the upper layer and evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0253] In some preferred embodiments, the step 6-2-1 is: taking 0.5 to 2 g of ginseng reference medicinal material, and preparing a reference medicinal material solution by the same method as the step 6-1;

[0254] In some preferred embodiments, the step 6-2-2 is: adding methanol to prepare a mixed solution containing 0.5-4 mg of ginsenoside Rg1 reference substance and 0.5-4 mg of ginsenoside Re reference substance per 1 ml, as a reference substance solution;

[0255] In some preferred embodiments, step 6-3 is: aspirate 10-40 μl of the test solution, 1-10 μl of the control medicinal material solution and the control solution, respectively, and spot them on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105°C, and the plate is inspected under sunlight. Further preferably, in step 6-3, aspirate 10-40 μl of the test solution.

[0256] The Erdongtang preparation to be tested can be freeze-dried powder, extract powder or granules. When the Erdongtang preparation is freeze-dried powder or extract powder, the amount of sample spotted on the thin layer plate is preferably 20 to 30 μl when identifying the ginseng component of the Erdongtang preparation; when the Erdongtang preparation is granules, the amount of sample spotted on the thin layer plate is preferably 25 to 30 μl.

[0257] Another aspect of the present invention provides a thin layer chromatography identification method for liquorice components in Erdong decoction preparation, which comprises the following steps:

[0258] Step 7-1: Take the Erdong decoction preparation, add a solvent, and prepare a test solution;

[0259] Step 7-2-1: Take liquiritin control medicinal material, add solvent, and prepare control medicinal material solution;

[0260] Step 7-2-2: Take the liquiritin reference substance, add a solvent, and prepare a reference substance solution;

[0261] Step 7-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 10:3:2 ethyl acetate-acetic acid-water as the developing solvent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

[0262] In the thin layer chromatography identification method of the licorice components of the present invention, ethyl acetate-acetic acid-water in a ratio of 10:3:2 must be used as the developing solvent, otherwise the separation effect is poor, or it is difficult to obtain clear spots, or the position and / or color of the spots after color development are inconsistent with the reference substance or reference medicinal material.

[0263] In some preferred embodiments, in the step 7-1: take 0.5-4 g of Erdong decoction preparation, add 40 ml of ether, heat to reflux, filter, discard the ether solution, evaporate the residue, add 30 ml of methanol, heat to reflux, filter, evaporate the filtrate to dryness, add 40 ml of water to the residue to dissolve it, add 50 ml of n-butanol to shake and extract, centrifuge, take the n-butanol solution, evaporate to dryness, add 5 ml of methanol to dissolve the residue, and use it as the test solution.

[0264] In some preferred embodiments, the step 7-2-1 is: take 0.5 to 2 g of licorice control medicinal material, and prepare a control medicinal material solution by the same method as the step 7-1.

[0265] In some preferred embodiments, the step 7-2-2 is: adding methanol to prepare a solution containing 0.2 to 1.5 mg of liquiritin reference substance per 1 ml, as the reference substance solution.

[0266] In some preferred embodiments, step 7-3 is: draw 1-20 μl of the test solution, 2-10 μl of the control medicinal material solution and the control solution, respectively, and spot them on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105°C, and the plate is inspected under a 365nm ultraviolet lamp. It is further preferred that in step 7-3, 5-15 μl of the test solution is drawn.

[0267] The Erdong Decoction preparation to be tested may be a lyophilized powder, an extract powder or granules. When the Erdong Decoction preparation is a lyophilized powder, the amount of sample spotted on the thin layer plate is preferably 5 to 10 μl when identifying the licorice component of the Erdong Decoction preparation, when the Erdong Decoction preparation is an extract powder, the amount of sample spotted on the thin layer plate is preferably 10 to 15 μl, and when the Erdong Decoction preparation is granules, the amount of sample spotted on the thin layer plate is preferably 5 to 15 μl.

[0268] The thin layer chromatography identification method of the present invention can identify the various components in the Erdong Decoction preparation, providing a basis for the quality control of the Erdong Decoction. Furthermore, when the thin layer chromatography identification method of the present invention, the characteristic spectrum, and the content determination method based on high performance liquid chromatography are combined, the quality of the Erdong Decoction preparation can be more reliably identified.

[0269] Example

[0270] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, the technical solutions obtained by various obvious changes or substitutions made by those skilled in the art to the present invention all fall within the scope defined by the claims attached to this application.

[0271] The samples used in the examples were prepared as follows.

[0272] The Nidongtang standard sample was produced as follows.

[0273] Preparation method of Erdong Decoction benchmark sample: weigh 11.19g of Ophiopogon japonicus, 7.46g of Asparagus cochinchinensis, 3.73g of Radix Trichosanthis, 3.73g of Scutellaria baicalensis, 3.73g of Anemarrhena asphodeloides, 3.73g of Lotus leaf, 1.87g of Ginseng, and 1.87g of Licorice, soak in water for 40-50 minutes, decoct for 20-30 minutes, decoct 1-2 times, combine the decoctions, and freeze-dry to obtain the Erdong Decoction benchmark sample.

[0274] Six batches of Erdongtang benchmark samples were manufactured using this method, with batch numbers CH20200611, CH20200729, A20211206S, CH310101S, CH310102S, and CH310103S, respectively.

[0275] The Nidongtang extract powder is produced by the following method.

[0276] Preparation method of Erdong decoction extract powder: using modern preparation equipment, put 8 pieces of Erdong decoction granules (9.0kg of Ophiopogon japonicus, 6.0kg of Asparagus cochinchinensis, 3.0kg of Radix Trichosanthis, 3.0g of Radix Scutellariae, 3.0g of Rhizoma Anemarrhenae, 3.0g of Lotus leaf, 1.5kg of Ginseng, 1.5kg of Licorice) into the extraction equipment, add 15-25 times of water, extract 1-2 times, each time for 60 minutes, extract and separate, then perform low temperature concentration and spray drying, so as to obtain extract powder with stable quality. Three batches of Erdong decoction extract powder were manufactured by this method, and the batch numbers were recorded as 221C213010, 221C213040, and 221C213050 respectively.

[0277] The Erdongtang granules are produced by the following method.

[0278] Preparation method of Erdongtang granules: using modern preparation equipment, using the three batches of Erdongtang extract powder with batch numbers 221C213010, 221C213040, and 221C213050, respectively, mixing Erdongtang extract powder 4000g, 800g, and 4000g, lactose 925g, 190g, and 950g, and magnesium stearate 75g, 10g, and 50g, and adopting dry granulation to obtain granules with stable quality. The batch numbers of the three batches of Erdongtang granules manufactured by this method are recorded as 202100901, 2021032B1, and 2021032C1, respectively.

[0279] In addition, a certain medicinal material negative freeze-dried powder is prepared by the same preparation method as the Erdong Decoction reference sample, except that the certain medicinal material is not added. Taking the Anemarrhena asphodeloides negative freeze-dried powder as an example, the Anemarrhena asphodeloides negative freeze-dried powder is prepared by the same preparation method as the Erdong Decoction reference sample, except that Anemarrhena asphodeloides is not added.

[0280] The high performance liquid chromatograph used in the examples was e2695 manufactured by Waters; the chromatographic column was Agilent ZORBAX SB-Aq (Dim: 250×4.6 mm, 5 μm) manufactured by Agilent. Mangiferin, baicalin, wogonin and ammonium glycyrrhizinate as reference substances were purchased from the China Food and Drug Administration; quercetin-3-O-glucuronide was purchased from Chengdu Pufeide Biotechnology Co., Ltd.

[0281] Example 1 Characteristic spectrum and content determination of Erdong decoction preparation (for freeze-dried powder or extract powder)

[0282]

Characteristic spectrum

[0283] The chromatographic conditions and system suitability test are the same as those under [Content].

[0284] Preparation of reference solution: Take appropriate amounts of mangiferin, quercetin-3-O-glucuronide, baicalin, wogonin and ammonium glycyrrhizinate reference substances, weigh them accurately, and add methanol to prepare a mixed solution containing 20 μg of mangiferin, 40 μg of quercetin-3-O-glucuronide, 450 μg of baicalin, 60 μg of wogonin and 45 μg of ammonium glycyrrhizinate per 1 mL.

[0285] The preparation of the test solution is the same as that under [Content].

[0286] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record chromatogram for 80 minutes ( Figure 1 and Figure 2 ) and you’ll get it.

[0287] The characteristic spectrum of the test product should show 9 common peaks ( Figure 2 ), of which 5 peaks have the same retention time as the corresponding reference peaks; the peak corresponding to the baicalin reference peak is taken as the S peak, and the relative retention times of the common peaks Peak 1 to Peak 4, Peak 6 to Peak 9 are calculated, and their relative retention times should be within ±5% of the specified value. The specified values ​​are: 0.32 (Peak 1), 0.44 (Peak 2), 0.74 (Peak 3), 0.77 (Peak 4), 1.13 (Peak 6), 1.23 (Peak 7), 1.25 (Peak 8), 1.88 (Peak 9).

[0288] Figure 2Middle, Peak 2: Mangiferin (C 19 H 18 O 11 ); Peak 3: Quercetin-3-O-glucuronide (Quercetin-3-O-glucuronide, C 21 H 18 O 13 ); Peak 5: Baicalin (C 21 H 18 O 11 ); Peak 8: Wogonoside, C 22 H 20 O 11 ); Peak 9: Glycyrrhizic acid (C 42 H 62 O 16 )

[0289] [Content Determination] Anemarrhena asphodeloides (mangiferin), Scutellaria baicalensis (baicalin), Glycyrrhiza uralensis (glycyrrhizic acid) were determined according to the high performance liquid chromatography method (General Rule 0512).

[0290] The chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); 0.1% phosphoric acid as mobile phase A, methanol as mobile phase B, gradient elution as specified in Table 1; flow rate: 1.0 mL / min; column temperature: 25° C.; detection wavelength: 258 nm.

[0291] System suitability test Theoretical plate number of the chromatographic column: Take the reference solution, inject once as required under [Determination method], and calculate the theoretical plate number of the chromatographic column. The theoretical plate number calculated based on the baicalin peak should not be less than 10,000.

[0292] Repeatability: Take the reference solution and inject it 5 times in succession as required in [Determination Method], and the RSD of the peak area measurement value shall not exceed 2.0%;

[0293] Table 1 Elution conditions

[0294] Time (min) 0.1% phosphoric acid water (A%) Methanol (B%) 0～10 78％→65％ 22％→35％ 10～32 65％→52％ 35％→48％ 32～55 52％→40％ 48％→60％ 55～65 40％→28％ 60％→72％ 65～70 28％→78％ 72％→22％ 70～80 78％→78％ 22％→22％

[0295] Preparation of reference solution: Take appropriate amount of mangiferin, baicalin and ammonium glycyrrhizinate reference substances, weigh accurately, add methanol to prepare a solution containing 20 μg of mangiferin, 450 μg of baicalin and 45 μg of ammonium glycyrrhizinate per 1 ml.

[0296] Preparation of test solution Take an appropriate amount of the product (i.e. Erdong decoction freeze-dried powder or extract powder), mix well, take about 1g, accurately weigh, place in a 100mL centrifuge tube, add 40mL of 30% ethanol, shake (frequency 200) for 20 minutes to dissolve, centrifuge (speed is 3000 revolutions per minute) for 5 minutes, transfer the supernatant to a 50mL volumetric flask, add 30% ethanol to the scale, shake well, filter, and take the filtrate.

[0297] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine ( Figure 3 and Figure 4 ) and you’ll get it.

[0298] Each prescription of this product contains mangiferin (C 19 H 18 O 11 ) in total, the content should be 13.4-24.8 mg; each prescription contains baicalin (C 21 H 18 O 11 ) in terms of content, the content should be 302-559 mg; each prescription contains licorice in the form of glycyrrhizic acid (C 42 H 62 O 16 ) The content should be 14.4-26.7 mg.

[0299] Example 12 Characteristic Spectrum and Content Determination of Erdong Decoction Preparation (for Granules)

[0300] Except for the preparation of the test solution, the same operation as Example 1 was performed.

[0301] In the preparation of the test solution, take the product (i.e., Erdongtang granules) under the item of filling difference, mix well, take an appropriate amount, grind it into powder, take about 1.3 g, accurately weigh it, put it in a 100mL centrifuge tube, add 40mL of 30% ethanol, shake (frequency 200) for 20 minutes to dissolve it, centrifuge (speed is 3000 revolutions per minute) for 5 minutes, transfer the supernatant to a 50mL volumetric flask, add 30% ethanol to the scale, shake well, filter, and take the filtrate to obtain.

[0302] Example 1 3 Specificity verification of characteristic spectrum

[0303] Take 1g each of Erdong Decoction standard sample (batch number: CH20200611), single-flavor Anemarrhena freeze-dried powder (batch number: ZM20200724), single-flavor Lotus leaf freeze-dried powder (batch number: HY20200724), single-flavor Scutellaria baicalensis freeze-dried powder (batch number: HQ20200724) and single-flavor Licorice freeze-dried powder (batch number: GC20200724), accurately weigh, and prepare the test solution, single-flavor Anemarrhena freeze-dried powder solution, single-flavor Lotus leaf freeze-dried powder solution, single-flavor Scutellaria baicalensis freeze-dried powder solution and single-flavor Licorice freeze-dried powder solution according to the preparation method of the test solution under the [Characteristic spectrum] in Example 1-1; prepare the reference solution according to the preparation method of the reference solution under the [Characteristic spectrum]. Take 10μl of each of the above 6 solutions, measure according to the chromatographic conditions under the [Characteristic spectrum], record the retention time, and the results are shown in Table 2.

[0304] Table 2 Summary of sample retention time

[0305]

[0306] The results showed that the retention times of the characteristic peaks of single-flavor freeze-dried powder of Anemarrhena asphodeloides, single-flavor freeze-dried powder of Lotus leaf, single-flavor freeze-dried powder of Scutellaria baicalensis and single-flavor freeze-dried powder of Licorice were basically consistent with those of Erdong Decoction benchmark sample, indicating that this method has good specificity.

[0307] Example 14 Precision verification of characteristic spectrum

[0308] Repeatability Take 6 portions of Erdong Decoction reference sample (batch number: CH20200729) and prepare the test solution according to the test solution preparation method under [Characteristic spectrum]. Take 10 μl of each of the above 6 solutions and measure according to the chromatographic conditions under [Characteristic spectrum] in Example 1-1, record the chromatographic peak area, and calculate the relative retention time and peak area RSD value of each common peak with the retention time and peak area of ​​baicalin chromatographic peak (peak 5) as reference.

[0309] The results showed that: with peak 5 as reference, the relative peak area RSDs of peaks 1 to 4 and 6 to 9 (n=6) were 0.32%, 0.52%, 0.60%, 0.37%, 1.12%, 0.32%, 0.14%, 0.00% respectively; the repeatability relative retention time RSDs (n=6) were 0.26%, 0.14%, 0.10%, 0.07%, 0.04%, 0.07%, 0.04%, 0.23% respectively. The relative retention time of each common peak was within the ±5% range of the specified value, and the relative peak area RSD values ​​of each common peak were no more than 2%, and the relative retention time RSD values ​​were no more than 1%, which met the specified requirements, indicating that the method had good repeatability.

[0310] Intermediate precision Take Erdong Decoction reference sample (batch number: CH20200729) and prepare the test solution according to the test solution preparation method under [Characteristic spectrum]; take 10μl of the test solution and measure it according to the chromatographic conditions under [Characteristic spectrum] in Example 1-1, and compare the influence of different personnel, different time, and different instruments and equipment on the precision. The experimental design is shown in Table 3-1. Each time 10μl is injected, the retention time of the baicalin chromatographic peak (peak 5) is used as a reference to calculate the relative retention time RSD value of each common peak. The results are shown in Table 3-2. Figure 5 .

[0311] Table 3-1 Experimental design table

[0312]

[0313] Table 3-2 Results of the investigation on intermediate precision relative retention time

[0314]

[0315] The results showed that the common peaks had similar characteristic profiles and similar retention times (ie, the RSD value was no greater than 3%), which met the specified requirements and indicated that the intermediate precision of the method was good.

[0316] Example 15 Specificity verification of content determination method

[0317] Take 1g each of Erdong Decoction reference sample (batch number: CH20200611), Anemarrhena negative freeze-dried powder (batch number: Y-ZM20200713), Scutellaria negative freeze-dried powder (batch number: Y-HQ20200709) and Licorice negative freeze-dried powder (batch number: Y-GC20200713), accurately weigh, and prepare the test solution, Anemarrhena negative freeze-dried powder solution, Scutellaria negative freeze-dried powder solution and Licorice negative freeze-dried powder solution according to the test solution preparation method under [Content Determination] in Example 1-1; prepare the reference solution according to the reference solution preparation method under [Content Determination] in Example 1-1. Take 10μl of each of the above 5 solutions and determine them according to the chromatographic conditions under [Content Determination] in Example 1-1. The results are shown in Table 1. Figure 6 .

[0318] In addition, a PDA detector was used to scan the peak spectra of the chromatographic peaks of mangiferin, baicalin and glycyrrhizic acid in the test solution to examine the morphology of the peak apex and inflection point. The results showed that the same peak profile and spectrum as mangiferin (15.585min), baicalin (35.370min) and glycyrrhizic acid reference substance (66.015min) were detected in the test sample but not in the negative sample. The results showed that this method has good specificity.

[0319] Example 16 Accuracy Verification of Content Determination Method

[0320] Take 6 portions of Erdong Decoction reference samples (batch number: CH20200729) with known contents, 0.25g, accurately weigh, add 0.0695mg / ml mangiferin 2.6ml, 1.186mg / ml baicalin 5ml and 0.1248mg / ml glycyrrhizic acid 2.9ml, respectively, prepare the test solution according to the test solution preparation method under [Content Determination] in Example 1-1, take 10μl of each of the above 6 samples, and determine according to the chromatographic conditions under [Content Determination] in Example 1-1, and calculate the recovery rate. The results are shown in Table 4-1, Table 4-2, and Table 4-3.

[0321] Table 4-1 Investigation of recovery rate of mangiferin (n=6)

[0322]

[0323] Table 4-2 Investigation of recovery rate of baicalin (n=6)

[0324]

[0325] Table 4-3 Glycyrrhizic acid recovery rate (n = 6)

[0326]

[0327] The results showed that according to the "Guidelines for Validation of Analytical Methods" of the fourth part of the 2020 edition of the Chinese Pharmacopoeia, when the content of the components to be determined in the sample is 1%, 0.1%, and 0.01%, the recovery limit requirements are 92%-105%, 90%-108%, and 85%-110%, respectively; the average sample recovery rates of mangiferin, baicalin, and glycyrrhizic acid are 98.89%, 101.14%, and 97.69%, respectively, all meeting the requirements. At the same time, their RSD values ​​are 0.35%, 0.80%, and 1.29%, respectively.

[0328] Example 17 Precision Verification of Content Determination Method

[0329] Repeatability Take 6 portions of Erdongtang standard sample (batch number: CH20200729) and prepare the test solution according to the test solution preparation method under [Content Determination] in Example 1. Take 10 μl of each of the above 6 solutions and measure according to the chromatographic conditions under [Content Determination] in Example 1. Record the chromatographic peak area and calculate the content and relative standard deviation RSD value. The results are shown in Table 5.

[0330] Table 5 Repeatability test results

[0331]

[0332] The results show that according to the "Guidelines for Validation of Analytical Methods" of the fourth part of the 2020 edition of the Chinese Pharmacopoeia, when the content of the components to be determined in the sample is 100μg / g, 1mg / g, and 10mg / g, the acceptable range of RSD values ​​is 4%, 3%, and 2%, respectively. The average content of mangiferin is 0.74mg / g, and the repeatability RSD is 0.74%, which is within the acceptable range; the average content of baicalin is 23.69mg / g, and the repeatability RSD is 1.07%, which is within the acceptable range; the average content of glycyrrhizic acid is 1.35mg / g, and the repeatability RSD is 0.30%, which is within the acceptable range. In summary, this method has good repeatability.

[0333] Intermediate precision Take Erdong Decoction reference sample (batch number: CH20200729) and prepare the test solution according to the test solution preparation method under [Content determination] in Example 1. Two different personnel (two people) in the same laboratory carried out the determination at different times (five days apart) using different instruments (two chromatographs of the same model), for a total of 4 determinations. In each determination, 10 μl of each of 6 test solutions was taken, and the contents of mangiferin, baicalin, and glycyrrhizic acid were determined according to the chromatographic conditions under [Content determination] in Example 1.

[0334] The results show that according to the "Guidelines for Validation of Analytical Methods" of the fourth part of the 2020 edition of the Chinese Pharmacopoeia, when the content of the components to be determined in the sample is 100μg / g, 1mg / g, and 10mg / g, the acceptable range of RSD values ​​is 8%, 6%, and 4%, respectively. The average content of mangiferin is 0.42mg / g, and the RSD is 2.21%, which is within the acceptable range; the average content of baicalin is 21.85mg / g, and the RSD is 1.88%, which is within the acceptable range; the average content of glycyrrhizic acid is 1.40mg / g, and the RSD is 2.52%, which is within the acceptable range. The RSD values ​​between the measurement results of different personnel, different times, and different instruments and equipment all meet the requirements, indicating that the intermediate precision of this method is good.

[0335] Example 18 Linear range of content determination method

[0336] Standard curve: 0.52mg, 19.92mg, and 1.01mg of mangiferin, baicalin, and ammonium glycyrrhizinate were accurately weighed and placed in a 10ml volumetric flask. Methanol was added for ultrasonic dissolution. After cooling, the volume was fixed to the mark to serve as the reference substance stock solution. Take an appropriate amount of the reference substance stock solution and dilute it 2, 4, 8, 16, 32, and 64 times in sequence to prepare reference substance solutions of different concentrations. The peak area was determined according to the chromatographic conditions under [Content Determination] in Example 1.

[0337] With the concentration of the reference substance as the horizontal axis (X) and the corresponding peak area integral value as the vertical axis (Y), a standard curve was drawn and the regression equation was calculated. The linear regression equation of mangiferin is Y=45899x-4162.9, R 2 =0.9999, and it showed a good linear relationship in the range of 0.80-51.01 μg / ml; the linear regression equation of baicalin was Y=17987x+89930, R 2 =0.9999, and it has a good linear relationship in the range of 29.69-1900.00 μg / ml; the linear regression equation of glycyrrhizic acid is Y=7947.9x-1080.3, R 2 =0.9999, and it showed a good linear relationship in the range of 1.47 to 93.93 μg / ml.

[0338] Using Excel data analysis, the results showed that the 95% confidence interval passed through the origin.

[0339] Example 19 Quality Identification of Erdong Decoction Preparation

[0340] According to the operation of Example 1, the characteristic spectra of the Erdongtang benchmark samples CH20200611, CH20200729, A20211206S, and the Erdongtang extract powder prepared above were measured respectively. According to the operation of Example 12, the characteristic spectra of the Erdongtang granules prepared above were measured.

[0341] The obtained characteristic spectra showed 9 common peaks, of which 5 peaks had the same retention time as the corresponding reference peaks; the peak corresponding to the baicalin reference peak was taken as the S peak, and the relative retention times of the common peaks 1 to 4 and 6 to 9 were calculated, and the relative retention times were within ±5% of the specified values. The specified values ​​were: 0.32 (peak 1), 0.44 (peak 2), 0.74 (peak 3), 0.77 (peak 4), 1.13 (peak 6), 1.23 (peak 7), 1.25 (peak 8), and 1.88 (peak 9).

[0342] In addition, according to the operation of Example 1, the contents of Anemarrhena (mangiferin), Scutellaria (baicalin), and Licorice (glycyrrhizic acid) were measured for the Erdong Decoction reference samples CH20200611, CH20200729, and A20211206S, respectively. According to the operation of Example 12, the contents of Anemarrhena (mangiferin), Scutellaria (baicalin), and Licorice (glycyrrhizic acid) were measured for the Erdong Decoction granules prepared above. The results of the measurements of each sample were converted to the content in each prescription, and all satisfied the following conditions: Each prescription contained Anemarrhena with mangiferin (C 19 H 18 O 11 ) in total, the content is 13.4-24.8 mg; each prescription contains baicalin (C 21 H 18 O11 ) in terms of content, 302-559 mg; each prescription contains licorice in the form of glycyrrhizic acid (C 42 H 62 O 16 ) is calculated, and the content is 14.4 to 26.7 mg.

[0343] It can be seen from this that Erdong Soup benchmark samples CH20200611, CH20200729, A20211206S, Erdong Soup extract powder and Erdong Soup granules are all qualified products.

[0344] Example 2 Thin layer chromatography identification method of each component in Erdong decoction preparation

[0345] The freeze-dried powder, extract powder and granules of the Erdong Decoction preparation used as the test object in this embodiment are the Erdong Decoction reference sample, Erdong Decoction extract powder and Erdong Decoction granules prepared above, respectively.

[0346] Example 2 1 Thin layer chromatography identification method of Erdongtang freeze-dried powder

[0347]

Identification

[0348] (2) Take 2g of this product, add 20ml of water and 5ml of hydrochloric acid, heat and reflux for 30 minutes, cool, filter, add 20ml of chloroform to the filtrate, shake and extract (frequency is 250 times per minute, 10 minutes), take the chloroform solution, evaporate to dryness, add 1ml of chloroform to the residue to dissolve it, and use it as the test solution. Take 1g of Ophiopogon japonicus control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 5-10μl of the test solution and 5μl of the control medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate to form strips. Use chloroform-acetone (4:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test product, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material.

[0349] (3) Take 0.5 g of Scutellaria baicalensis reference medicinal material and prepare a reference medicinal material solution in the same manner as the preparation method for the test solution under [Identification] (7). Take another reference baicalin and add methanol to prepare a solution containing 1 mg per 1 ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 5-10 μl of the test solution under [Identification] (7), 5 μl of the above-mentioned reference medicinal material solution and 5 μl of the reference solution, and spot them on the same silica gel GF 254 On the thin layer plate, make a strip. Use ethyl acetate-butanone-formic acid-water (5:5:1:1) as the developing agent, develop, take out, dry, and examine under ultraviolet light (254nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference sample.

[0350] (4) Take 2g of this product, add 20ml of dilute ethanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, filter, and use the filtrate as the test solution. Take another 0.5g of Anemarrhena asphodeloides reference medicinal material and prepare the reference medicinal material solution in the same way. Take Anemarrhena asphodeloides saponin BⅡ reference material and add methanol to prepare a solution containing 1mg per 1ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 2-10μl of the test solution, 5μl of the reference medicinal material solution and the reference substance solution, and spot them on the same silica gel G thin layer plate to form strips. Use n-butanol-acetic acid-water (7:2:3) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test product, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

[0351] (5) Take 1g of lotus leaf control medicinal material and prepare the control medicinal material solution in the same way as the preparation method of the test solution under [Identification] (6). Take another reference substance of nuciferine and add methanol to prepare a solution containing 1mg per 1ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 10-15μl of the test solution under [Identification] (6), 10μl of the above-mentioned control medicinal material solution and 1μl of the reference substance solution, and spot them on the same silica gel G thin layer plate to form strips. Use ethyl acetate-methanol-water (15:5:4) as the developing solvent, develop, take out, dry, spray with dilute potassium bismuth iodide test solution, and examine under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the reference substance.

[0352] (6) Take 2g of this product, add 40ml of chloroform, heat and reflux for 1 hour, filter, discard the chloroform liquid, evaporate the solvent from the residue, add 1ml of water to stir and moisten, add 10ml of water-saturated n-butanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, aspirate the supernatant and add 3 times the amount of ammonia test solution, shake well, let it stand for stratification, take the upper layer and evaporate to dryness, add 1ml of methanol to the residue to dissolve it as the test solution. Take 1g of ginseng reference medicinal material and prepare the reference medicinal material solution in the same way. Take ginsenoside Rg1 reference substance and ginsenoside Re reference substance, add methanol to prepare a mixed solution containing 2mg per 1ml, as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 20-30μl of the test solution, 2μl of the reference medicinal material solution and the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate to form strips. Use the upper layer solution of n-butanol-ethyl acetate-water-acetic acid (4:1:5:0.5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference sample.

[0353] (7) Take 2g of this product, add 40ml of ether, heat and reflux for 1 hour, filter, discard the ether solution, evaporate the residue, add 30ml of methanol, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 40ml of water to dissolve the residue, add 50ml of n-butanol for oscillation extraction (frequency is 250 times per minute, 10 minutes), centrifuge (speed is 3000 revolutions per minute, 5 minutes), take the n-butanol solution, evaporate to dryness, and add 5ml of methanol to dissolve the residue as the test solution. Take 1g of licorice control medicinal material and prepare the control medicinal material solution in the same way. Take the liquiritin reference substance and add methanol to prepare a solution containing 0.5mg per 1ml as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 5-10μl of the test solution, 5μl of the control medicinal material solution and the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate to form strips. Use ethyl acetate-acetic acid-water (10:3:2) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference medicinal material and the chromatogram of the reference sample, a fluorescent spot of the same color appears.

[0354] Example 2 Thin layer chromatography identification method of Erdong decoction extract powder

[0355]

Identification

[0356] (2) Take 2g of this product, add 20ml of water and 5ml of hydrochloric acid, heat and reflux for 30 minutes, cool, filter, add 20ml of chloroform to the filtrate, shake and extract (frequency is 250 times per minute, 10 minutes), take the chloroform solution, evaporate to dryness, add 1ml of chloroform to the residue to dissolve it, and use it as the test solution. Take 1g of Ophiopogon japonicus control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 10-20μl of the test solution and 5μl of the control medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate to form strips. Use chloroform-acetone (4:1) as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test product, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material.

[0357] (3) Take 0.5 g of Scutellaria baicalensis reference medicinal material and prepare a reference medicinal material solution in the same manner as the preparation method for the test solution under [Identification] (7). Take another reference baicalin and add methanol to prepare a solution containing 1 mg per 1 ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 5-10 μl of the test solution under [Identification] (7), 5 μl of the above-mentioned reference medicinal material solution and 5 μl of the reference solution, and spot them on the same silica gel GF 254 On the thin layer plate, make a strip. Use ethyl acetate-butanone-formic acid-water (5:5:1:1) as the developing agent, develop, take out, dry, and examine under ultraviolet light (254nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference sample.

[0358] (4) Take 2g of this product, add 20ml of dilute ethanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, filter, and use the filtrate as the test solution. Take another 0.5g of Anemarrhena asphodeloides reference medicinal material and prepare the reference medicinal material solution in the same way. Take Anemarrhena asphodeloides saponin BⅡ reference material and add methanol to prepare a solution containing 1mg per 1ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 2-10μl of the test solution, 5μl of the reference medicinal material solution and the reference substance solution, and spot them on the same silica gel G thin layer plate to form strips. Use n-butanol-acetic acid-water (7:2:3) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test product, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

[0359] (5) Take 1g of lotus leaf control medicinal material and prepare the control medicinal material solution in the same way as the preparation method of the test solution under [Identification] (6). Take another reference substance of nuciferine and add methanol to prepare a solution containing 1mg per 1ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 10-20μl of the test solution under [Identification] (6), 10μl of the above-mentioned control medicinal material solution and 1μl of the reference substance solution, and spot them on the same silica gel G thin layer plate to form strips. Use ethyl acetate-methanol-water (15:5:4) as the developing solvent, develop, take out, dry, spray with dilute potassium bismuth iodide test solution, and examine under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the reference substance.

[0360] (6) Take 2g of this product, add 40ml of chloroform, heat and reflux for 1 hour, filter, discard the chloroform liquid, evaporate the solvent from the residue, add 1ml of water to stir and moisten, add 10ml of water-saturated n-butanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, aspirate the supernatant and add 3 times the amount of ammonia test solution, shake well, let it stand for stratification, take the upper layer and evaporate to dryness, add 1ml of methanol to the residue to dissolve it as the test solution. Take 1g of ginseng reference medicinal material and prepare the reference medicinal material solution in the same way. Take ginsenoside Rg1 reference substance and ginsenoside Re reference substance, add methanol to prepare a mixed solution containing 2mg per 1ml, as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 20-30μl of the test solution, 2μl of the reference medicinal material solution and the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate to form strips. Use the upper layer liquid of n-butanol-ethyl acetate-water-acetic acid (4:1:5:0.5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference sample.

[0361] (7) Take 2g of this product, add 40ml of ether, heat and reflux for 1 hour, filter, discard the ether solution, evaporate the residue, add 30ml of methanol, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 40ml of water to dissolve the residue, add 50ml of n-butanol for oscillation extraction (frequency is 250 times per minute, 10 minutes), centrifuge (speed is 3000 revolutions per minute, 5 minutes), take the n-butanol solution, evaporate to dryness, and add 5ml of methanol to dissolve the residue as the test solution. Take 1g of licorice control medicinal material and prepare the control medicinal material solution in the same way. Take the liquiritin reference substance and add methanol to prepare a solution containing 0.5mg per 1ml as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 10-15μl of the test solution, 5μl of the control medicinal material solution and the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate to form strips. Use ethyl acetate-acetic acid-water (10:3:2) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference medicinal material and the chromatogram of the reference sample, a fluorescent spot of the same color appears.

[0362] Example 23 Thin layer chromatography identification method of Erdongtang granules

[0363]

Identification

[0364] (2) Take 2.5g of this product, grind it into powder, add 20ml of water and 5ml of hydrochloric acid, heat and reflux for 30 minutes, cool it, filter it, add 20ml of chloroform to the filtrate, shake and extract it (frequency is 250 times per minute, 10 minutes), take the chloroform solution, evaporate it to dryness, add 1ml of chloroform to the residue to dissolve it, and use it as the test solution. Take 1g of Ophiopogon japonicus as the control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 10-20μl of the test solution and 5μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate to form strips. Use chloroform-acetone (4:1) as the developing solvent, develop it, take it out, dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly colored, and inspect it under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0365] (3) Take 0.5 g of Scutellaria baicalensis reference medicinal material and prepare a reference medicinal material solution in the same manner as the preparation method for the test solution under [Identification] (7). Take another reference baicalin and add methanol to prepare a solution containing 1 mg per 1 ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 5-10 μl of the test solution under [Identification] (7), 5 μl of the above-mentioned reference medicinal material solution and 5 μl of the reference solution, and spot them on the same silica gel GF 254 On the thin layer plate, make a strip. Use ethyl acetate-butanone-formic acid-water (5:5:1:1) as the developing agent, develop, take out, dry, and examine under ultraviolet light (254nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference sample.

[0366] (4) Take 2.5g of this product, grind it into powder, add 20ml of dilute ethanol, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, filter it, and use the filtrate as the test solution. Take another 0.5g of Anemarrhena asphodeloides reference medicinal material and prepare the reference medicinal material solution in the same way. Take Anemarrhena asphodeloides saponin BⅡ reference material and add methanol to prepare a solution containing 1mg per 1ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 2-10μl of the test solution, 5μl of the reference medicinal material solution and the reference solution, and spot them on the same silica gel G thin layer plate to form strips. Use n-butanol-acetic acid-water (7:2:3) as the developing agent, develop it, take it out, dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly colored, and inspect it under sunlight. In the test sample chromatogram, spots of the same color appear at corresponding positions in the control medicinal material chromatogram and the control sample chromatogram.

[0367] (5) Take 1g of lotus leaf control medicinal material and prepare the control medicinal material solution in the same way as the preparation method of the test solution under [Identification] (6). Take another reference substance of nuciferine and add methanol to prepare a solution containing 1mg per 1ml as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 15-20μl of the test solution under [Identification] (6), 10μl of the above-mentioned control medicinal material solution and 1μl of the reference substance solution, and spot them on the same silica gel G thin layer plate to form strips. Use ethyl acetate-methanol-water (15:5:4) as the developing solvent, develop, take out, dry, spray with dilute potassium bismuth iodide test solution, and examine under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the reference substance.

[0368] (6) Take 2.5g of this product, grind it into powder, add 40ml of chloroform, heat and reflux for 1 hour, filter, discard the chloroform solution, evaporate the solvent from the residue, add 1ml of water to stir and moisten, add 10ml of water-saturated n-butanol, and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, aspirate the supernatant and add 3 times the amount of ammonia test solution, shake well, let it stand for stratification, take the upper layer and evaporate it to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution. Take 1g of ginseng reference medicinal material and prepare the reference medicinal material solution in the same way. Take ginsenoside Rg1 reference substance and ginsenoside Re reference substance, add methanol to prepare a mixed solution containing 2mg of each per 1ml, and use it as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 25-30 μl of the test solution, 2 μl of the control medicinal material solution and the reference substance solution were taken and spotted on the same silica gel G thin layer plate to form strips. The upper layer liquid of n-butanol-ethyl acetate-water-acetic acid (4:1:5:0.5) was used as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the reference substance.

[0369] (7) Take 2.5g of this product, grind it into powder, add 40ml of ether, heat and reflux for 1 hour, filter, discard the ether solution, evaporate the residue, add 30ml of methanol, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 40ml of water to dissolve the residue, add 50ml of n-butanol to shake and extract (frequency is 250 times per minute, 10 minutes), centrifuge (speed is 3000 revolutions per minute, 5 minutes), take the n-butanol solution, evaporate to dryness, and add 5ml of methanol to dissolve the residue to prepare the test solution. Take 1g of licorice control medicinal material and prepare the control medicinal material solution in the same way. Take the liquiritin reference substance and add methanol to prepare a solution containing 0.5mg per 1ml as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 5-15 μl of the test solution, 5 μl of the control medicinal material solution and the reference substance solution were taken and spotted on the same silica gel G thin layer plate to form strips. Ethyl acetate-acetic acid-water (10:3:2) was used as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the reference substance.

[0370] Example 24 Reliability of Thin Layer Chromatography Identification

[0371] According to the operation of Example 2-1, thin layer chromatography was performed on the negative freeze-dried powder of asparagus, negative freeze-dried powder of asparagus and ophiopogon, negative freeze-dried powder of asparagus and ophiopogon, negative freeze-dried powder of asparagus and ophiopogon, negative freeze-dried powder of ophiopogon, negative freeze-dried powder of Scutellaria, negative freeze-dried powder of Anemarrhena, negative freeze-dried powder of lotus leaf, negative freeze-dried powder of ginseng, and negative freeze-dried powder of liquorice. As a result, none of the negative freeze-dried powders showed spots of the same color in the chromatogram of the test sample at the corresponding position of the chromatogram of the reference sample or the reference medicinal material, indicating that there was no negative interference in each component.

[0372] Example 25 Quality Identification of Erdong Decoction Preparation

[0373] According to the operation of Example 2-1, the components in the Erdong Decoction reference samples CH310101S, CH310102S, and CH310103S were identified respectively. As a result, the Erdong Decoction reference samples CH310101S, CH310102S, and CH310103S all showed spots of the same color at the corresponding positions of the chromatograms of the test sample and the reference sample and / or reference medicinal materials in the thin layer chromatography identification of the asparagus component, the ophiopogon component, the scutellaria component, the anemarrhena component, the lotus leaf component, the ginseng component, and the liquorice component.

[0374] According to the operation of Example 2-2, the components in Erdong Decoction extract powders 221C213010, 221C213040, and 221C213050 were identified. As a result, Erdong Decoction extract powders 221C213010, 221C213040, and 221C213050 all showed spots of the same color at the corresponding positions of the chromatograms of the test sample and the reference sample and / or reference medicinal materials in the thin layer chromatography identification of the asparagus component, the ophiopogon component, the scutellaria component, the anemarrhena component, the lotus leaf component, the ginseng component, and the liquorice component.

[0375] According to the operation of Example 2-3, the components in Erdongtang Granules 202100901, 2021032B1, and 2021032C1 were identified respectively. As a result, Erdongtang Granules 202100901, 2021032B1, and 2021032C1 all showed spots of the same color at the corresponding positions of the chromatograms of the test sample and the reference substance and / or reference medicinal materials in the thin layer chromatography identification of the asparagus component, the ophiopogon component, the scutellaria component, the anemarrhena component, the lotus leaf component, the ginseng component, and the liquorice component.

[0376] It can be seen that the Erdong Decoction benchmark samples CH310101S, CH310102S, CH310103S, Erdong Decoction extract powder 221C213010, 221C213040, 221C213050, Erdong Decoction granules 202100901, 2021032B1, and 2021032C1 all contain asparagus cochinchinensis, ophiopogon japonicus, Scutellaria baicalensis, Anemarrhena asphodeloides, lotus leaf, ginseng, and licorice components, and are qualified products.

[0377] Comparative Example 1-1

[0378] The same operation as in Example 2-1 was performed to identify the components of Asparagus cochinchinensis except that the developing solvent was changed to chloroform-methanol-water (13:7:2) for the lower layer. As a result, the chromatographic spots were poorly separated and located too low.

[0379] Comparative Example 1-2

[0380] The same operation as in Example 2-1 was performed to identify the components of Asparagus cochinchinensis except that the developing solvent was changed to a lower layer solution of chloroform-methanol-water (13:7:2) placed below 10°C. As a result, the sample chromatogram had too many spots and serious interference.

[0381] Comparative Example 2-1

[0382] The identification of the components of Ophiopogon japonicus was carried out in the same manner as in Example 2-1, except that the developing solvent was changed to chloroform-ethyl acetate-acetone (8:1:1). As a result, there were too few chromatographic spots and the negative interference was serious.

[0383] Comparative Example 2-2

[0384] The identification of the components of Ophiopogon japonicus was carried out in the same manner as in Example 2-1, except that the developing solvent was changed to n-hexane-ethyl acetate (1:1). As a result, there were too few chromatographic spots and the negative interference was serious.

[0385] Comparative Example 3-1

[0386] The identification of the components of Scutellaria baicalensis was carried out in the same manner as in Example 2-1, except that the developing solvent was changed to toluene-ethyl acetate-methanol-formic acid (10:3:3:3). As a result, the chromatographic stratification phenomenon was severe and could not be observed.

[0387] Comparative Example 3-2

[0388] The identification of the Scutellaria baicalensis components was carried out in the same manner as in Example 2-1 except that the developing solvent was changed to toluene-ethyl acetate-methanol-acetic acid (5:3:3:3). As a result, the spot tailing phenomenon was severe.

[0389] Comparative Example 3-3

[0390] The identification of the Scutellaria baicalensis components was carried out in the same manner as in Example 2-1 except that the developing solvent was changed to toluene-ethyl acetate-methanol-formic acid (5:3:1:2). As a result, the spot separation effect was not good.

[0391] Comparative Examples 3-4

[0392] The identification of the components of Scutellaria baicalensis was carried out in the same manner as in Example 2-1, except that the developing solvent was changed to toluene-ethyl acetate-methanol-formic acid (5:1:3:2). As a result, the chromatographic spots showed a discontinuity phenomenon and could not be observed.

[0393] Comparative Examples 3-5

[0394] The identification of the components of Scutellaria baicalensis was carried out in the same manner as in Example 2-1, except that the developing solvent was changed to ethyl acetate-butanone-acetic acid-water (6:3:1:1). As a result, the chromatographic spot development effect was poor.

[0395] Comparative Example 4-1

[0396] The identification of the components of Anemarrhena Rhizoma was carried out in the same manner as in Example 2-1, except that the developing solvent was changed to n-butanol-acetic acid-water (4:2:3) and mangiferin was used as a reference substance. As a result, the sample did not show a spot corresponding to mangiferin, and the negative test had interference.

[0397] Comparative Example 4-2

[0398] The identification of the components of Anemarrhena asphodeloides was carried out in the same manner as in Example 2-1, except that the developing solvent was changed to n-butanol-acetic acid-water (2:1:1), mangiferin was used as a reference substance, and the color developer was changed to vanillin sulfuric acid test solution. As a result, the chromatographic spots were blurred, the separation effect was poor, and it was impossible to observe.

[0399] Comparative Example 5-1

[0400] The identification of ginseng components was carried out in the same manner as in Example 2-1 except that the developing solvent was changed to ethyl acetate-methanol-water (15:5:4). As a result, the spot separation effect was not good.

[0401] Comparative Example 5-2

[0402] The identification of ginseng components was carried out in the same manner as in Example 2-1 except that the developing solvent was changed to an upper layer solution of ethyl acetate-methanol-water-acetic acid (15:5:4:0.1). As a result, the spot separation effect was poor.

[0403] Comparative Example 5-3

[0404] The identification of ginseng components was carried out in the same manner as in Example 2-1 except that the developing solvent was changed to an upper layer solution of n-butanol-ethyl acetate-water (4:1:5). As a result, the spot separation effect was poor.

[0405] Comparative Example 6

[0406] The identification of ginseng components was carried out in the same manner as in Example 2-1, except that the developing solvent was changed to ethyl acetate-acetic acid-water (10:3:2) and ammonium glycyrrhizinate was used as a reference substance. As a result, no spots corresponding to ammonium glycyrrhizinate appeared in the sample.

[0407] Industrial Applicability

[0408] Based on the above, the characteristic spectrum construction method of the Erdong Decoction preparation of the present invention and the characteristic spectrum obtained by the method, the content determination method based on high performance liquid chromatography, and the thin layer chromatography identification method of each component in the Erdong Decoction preparation, and the identification method of the Erdong Decoction preparation can be used for the quality control and management of Erdong Decoction, which is of great significance in the production and manufacturing of traditional Chinese medicine.

Claims

1. A method for constructing a characteristic spectrum of an Erdong decoction preparation, characterized in that: The following steps are involved: Step 1: Take mangiferin, quercetin-3-O-glucuronide, baicalin, wogonin and ammonium glycyrrhizinate reference substances, and add a solvent to prepare a reference solution; Step 2: Take the Erdongtang reference sample, add solvent, and prepare the test solution; Step three: Use high performance liquid chromatography to detect the reference solution and the test solution respectively, use the characteristic spectrum of the reference solution as a reference spectrum, select common peaks from the characteristic spectrum of the test solution, and construct the characteristic spectrum of the Erdong Decoction preparation.

2. The method according to claim 1, characterized in that In the step 1, methanol is added to prepare a reference solution containing 10-30 μg of mangiferin, 25-55 μg of quercetin-3-O-glucuronide, 350-550 μg of baicalin, 40-80 μg of wogonin and 25-65 μg of ammonium glycyrrhizinate per 1 mL.

3. The method according to claim 1 or 2, characterized in that: In the step 2, 0.5-1.5 g of Erdongtang reference sample is taken, accurately weighed, placed in a centrifuge tube, 40 mL of 30% ethanol is added, shaken to dissolve, centrifuged for 5 minutes, the supernatant is transferred to a 50 mL volumetric flask, 30% ethanol is added to the scale, shaken well, filtered, and the filtrate is taken to prepare the test solution.

4. The method according to claim 1 or 2, characterized in that: In step 3, the HPLC detection conditions are: The injection volume is 5 to 20 μl. The chromatographic column is filled with octadecylsilane bonded silica gel. Use 0.1% phosphoric acid as mobile phase A and methanol as mobile phase B for gradient elution at a flow rate of 1.0 mL / min. The column temperature is 20-30°C. The detection wavelength is 258 nm.

5. The method according to claim 4, characterized in that The gradient elution conditions are: 0~10min, mobile phase A is 78%→65%, mobile phase B is 22%→35%, 10~32min, mobile phase A is 65%→52%, mobile phase B is 35%→48%, 32~55min, mobile phase A is 52%→40%, mobile phase B is 48%→60%, 55-65min, mobile phase A 40%→28%, mobile phase B 60%→72%, 65-70min, mobile phase A is 28%→78%, mobile phase B is 72%→22%, 70-80min, mobile phase A is 78%→78%, mobile phase B is 22%→22%.

6. The method according to claim 1 or 2, characterized in that: In the step three, the peak corresponding to baicalin is taken as the S peak, and the relative retention time of the common peak is calculated to construct the characteristic spectrum of the Erdong Decoction preparation.

7. A characteristic spectrum of an Erdong decoction preparation, characterized in that: It is constructed by the method according to any one of claims 1 to 6, and 9 common peaks are presented in the characteristic spectrum, with peak 5 corresponding to baicalin as the S peak, and the relative retention times of peaks 1 to 4 and peaks 6 to 9 should be within the range of ±5% of the specified value, and the specified value is: 0.32 (peak 1), 0.44 (peak 2), 0.74 (peak 3), 0.77 (peak 4), 1.13 (peak 6), 1.23 (peak 7), 1.25 (peak 8), and 1.88 (peak 9).

8. A method for determining the content of Erdong decoction preparation, characterized in that: The following steps are involved: The contents of mangiferin, baicalin and glycyrrhizic acid in the Erdong decoction preparation were determined by high performance liquid chromatography, wherein: The HPLC detection conditions are: The injection volume is 5 to 20 μl. The chromatographic column is filled with octadecylsilane bonded silica gel. Use 0.1% phosphoric acid as mobile phase A and methanol as mobile phase B for gradient elution at a flow rate of 1.0 mL / min. The column temperature is 20-30°C. The detection wavelength is 258 nm.

9. The method according to claim 8, characterized in that The gradient elution conditions are: 0~10min, mobile phase A is 78%→65%, mobile phase B is 22%→35%, 10~32min, mobile phase A is 65%→52%, mobile phase B is 35%→48%, 32~55min, mobile phase A is 52%→40%, mobile phase B is 48%→60%, 55-65min, mobile phase A 40%→28%, mobile phase B 60%→72%, 65-70min, mobile phase A is 28%→78%, mobile phase B is 72%→22%, 70-80min, mobile phase A is 78%→78%, mobile phase B is 22%→22%.

10. A method for identifying an Erdong decoction preparation, characterized in that: Includes at least any of the following steps: Step A: comparing the characteristic spectrum of the Erdong Decoction preparation to be identified with the characteristic spectrum of the Erdong Decoction preparation constructed by the method described in any one of claims 1 to 6, or with the characteristic spectrum of the Erdong Decoction preparation described in claim 7, to determine whether the Erdong Decoction preparation is a qualified product; Step B: Determine the contents of mangiferin, baicalin and glycyrrhizic acid in each prescription of the Erdong Decoction preparation by the content determination method of the Erdong Decoction preparation according to claim 8 or 9, and determine whether the Erdong Decoction preparation is a qualified product.

11. The identification method according to claim 10, characterized in that: Erdong decoction preparations that meet at least one of the following conditions are considered qualified products: Condition a: In the characteristic spectrum of the Erdong Decoction preparation to be identified, peak 5 corresponding to baicalin is taken as the S peak, and the relative retention times of peaks 1 to 4 and peaks 6 to 9 are all within ±5% of the specified value in the characteristic spectrum of claim 7; Condition b: The content of mangiferin contained in each prescription of the Erdong decoction preparation is 10-30 mg, the content of baicalin is 250-600 mg, and the content of glycyrrhizic acid is 10-30 mg.

12. A thin layer chromatography identification method for the asparagus component in Erdong decoction preparation, characterized in that: The following steps are involved: Step 1-1: Take Erdong decoction preparation, add solvent, and prepare test solution; Step 1-2: Take a protodioscin reference substance, add a solvent, and prepare a reference substance solution; Step 1-3: According to the thin layer chromatography method, aspirate the test solution and the reference solution, spot them on the same thin layer plate respectively to make strips, use 11:7:1 chloroform-methanol-water as the developing agent, develop, take out, dry, spray with color developer and heat until the spots are clearly colored, and inspect.

13. The method according to claim 12, characterized in that In the step 1-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of 65% methanol is added, ultrasonic treatment is performed, centrifugation is performed, the supernatant is taken, and transferred to a 25 ml volumetric flask, 65% methanol is added to the mark, evaporated to dryness, 10 ml of water is added to the residue to dissolve it, and oscillation extraction is performed with water-saturated n-butanol for 3 times, 10 ml each time, centrifugation is performed, the n-butanol solution is combined, and the solution is washed with ammonia test solution for 3 times, 10 ml each time, the ammonia test solution is discarded, the n-butanol solution is evaporated to dryness, the residue is dissolved in methanol and transferred to a 5 ml volumetric flask, and methanol is added to the mark as the test solution; In step 1-2, methanol is added to prepare a solution containing 0.1-0.8 mg of protodioscin reference substance per 1 ml, which is used as the reference substance solution; In the steps 1-3, 1-20 μl of the test solution and 2-10 μl of the reference solution are respectively spotted on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105° C., and the plate is inspected under a 365 nm ultraviolet lamp.

14. A thin layer chromatography identification method for Radix Ophiopogonis in Erdong Decoction preparation, characterized in that: The following steps are involved: Step 2-1: Take Erdong decoction preparation, add solvent, and prepare test solution; Step 2-2: Take Ophiopogon japonicus as a reference medicinal material, add a solvent, and prepare a reference medicinal material solution; Step 2-3: According to the thin layer chromatography method, aspirate the test sample solution and the control medicinal material solution, respectively spot them on the same thin layer plate to form strips, use 4:1 chloroform-acetone as the developing agent, develop, take out, dry, spray with a color developer, and heat until the spots are clearly colored, and inspect.

15. The method according to claim 14, characterized in that In the step 2-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of water and 5 ml of hydrochloric acid are added, heated to reflux, cooled, filtered, 20 ml of chloroform is added to the filtrate, and the chloroform solution is extracted by shaking, and the chloroform solution is taken and evaporated to dryness, and 1 ml of chloroform is added to the residue to dissolve it, which is used as the test solution; In the step 2-2, 0.5 to 2 g of Ophiopogon japonicus reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as in the step 2-1; In step 2-3, 1-30 μl of the test solution and 2-10 μl of the control medicinal material solution are respectively spotted on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105° C., and the plate is inspected under sunlight.

16. A thin layer chromatography identification method for the scutellaria baicalensis component in Erdong decoction preparation, characterized in that: The following steps are involved: Step 3-1: Take Erdong decoction preparation, add solvent, and prepare test solution; Step 3-2-1: Take a reference medicinal material of Scutellaria baicalensis and add a solvent to prepare a reference medicinal material solution; Step 3-2-2: Take baicalin reference substance, add solvent, and prepare reference substance solution; Step 3-3: According to the thin layer chromatography method, aspirate the test sample solution, the reference medicinal material solution, and the reference substance solution, and spot them on the same thin layer plate to form strips. Use ethyl acetate-butanone-formic acid-water in a ratio of 5:5:1:1 as the developing solvent, develop, take out, dry, and inspect.

17. The method according to claim 16, characterized in that In the step 3-1, 0.5-4 g of Erdong decoction preparation is taken, 40 ml of ether is added, heated to reflux, filtered, the ether solution is discarded, the residue is evaporated, 30 ml of methanol is added, heated to reflux, filtered, the filtrate is evaporated to dryness, 40 ml of water is added to the residue to dissolve it, 50 ml of n-butanol is added to shake and extract, centrifuged, the n-butanol solution is taken, evaporated to dryness, and 5 ml of methanol is added to the residue to dissolve it as the test solution; In the step 3-2-1, 0.2-1 g of Scutellaria baicalensis reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as in the step 3-1; In step 3-2-2, methanol is added to prepare a solution containing 0.5 to 2 mg of baicalin reference substance per 1 ml, which is used as the reference substance solution; In step 3-3, 1-20 μl of the test solution, 2-10 μl of the reference medicinal material solution and the reference substance solution are respectively applied to the same silica gel GF 254 The thin layer plate was placed under a 254 nm ultraviolet lamp for inspection.

18. A thin layer chromatography identification method for the Anemarrhena component in Erdong decoction preparation, characterized in that: The following steps are involved: Step 4-1: Take Erdong decoction preparation, add solvent, and prepare test solution; Step 4-2-1: Take the Rhizoma Anemarrhenae as a control medicinal material, add a solvent, and prepare a control medicinal material solution; Step 4-2-2: Take the reference substance of Anemarrhena saponin BⅡ, add solvent, and prepare the reference substance solution; Step 4-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 7:2:3 n-butanol-acetic acid-water as the developing agent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

19. The method according to claim 18, characterized in that In step 4-1, 0.5-4 g of Erdong decoction preparation is taken, 20 ml of dilute ethanol is added, ultrasonic treatment is performed, and filtration is performed, and the filtrate is used as the test solution; In the step 4-2-1, 0.2-1 g of Rhizoma Anemarrhenae reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as in the step 4-1; In step 4-2-2, methanol is added to prepare a solution containing 0.5-2 mg of timosaponin BⅡ reference substance per 1 ml, which is used as the reference substance solution; In step 4-3, 1-20 μl of the test solution, 2-10 μl of the reference medicinal material solution and the reference substance solution are respectively spotted on the same silica gel G thin layer plate, the color developer is 10% sulfuric acid ethanol solution, the heating temperature is 105° C., and the plate is inspected under sunlight.

20. A thin layer chromatography identification method for lotus leaf components in Erdong decoction preparation, characterized in that: The following steps are involved: Step 5-1: Take Erdong decoction preparation, add solvent, and prepare test solution; Step 5-2-1: Take lotus leaf as a reference medicinal material, add a solvent, and prepare a reference medicinal material solution; Step 5-2-2: Take the nuciferine reference substance, add a solvent, and prepare a reference substance solution; Step 5-3: According to the thin layer chromatography method, aspirate the test sample solution, reference medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 15:5:4 ethyl acetate-methanol-water as the developing solvent, develop, take out, dry, spray with a color developer, and inspect.

21. The method according to claim 20, characterized in that In the step 5-1, 0.5-4 g of Erdong decoction preparation is taken, 40 ml of chloroform is added, heated and refluxed for 1 hour, filtered, the chloroform liquid is discarded, the solvent is evaporated from the residue, 1 ml of water is added and stirred to moisten, 10 ml of water-saturated n-butanol is added, ultrasonic treatment is performed, the supernatant is drawn and 3 times the amount of ammonia test solution is added, shaken, allowed to separate, the upper layer is evaporated to dryness, and 1 ml of methanol is added to the residue to dissolve it as the test solution; In step 5-2-1, 0.5 to 2 g of lotus leaf reference medicinal material is taken and a reference medicinal material solution is prepared by the same method as step 5-1; In step 5-2-2, methanol is added to prepare a solution containing 0.5-2 mg of nuciferine reference substance per 1 ml, which is used as the reference substance solution; In step 5-3, 5-30 μl of the test solution, 5-20 μl of the reference medicinal material solution, and 1-10 μl of the reference solution are respectively spotted on the same silica gel G thin layer plate, the color developer is a dilute potassium bismuth iodide test solution, and the plate is inspected under sunlight.

22. A thin layer chromatography identification method for ginseng components in Erdong decoction preparations, characterized in that: The following steps are involved: Step 6-1: Take the Erdong decoction preparation, add a solvent, and prepare a test solution; Step 6-2-1: Take ginseng reference medicinal material, add solvent, and prepare reference medicinal material solution; Step 6-2-2: Take ginsenoside Rg1 reference substance and ginsenoside Re reference substance, add solvent, and prepare reference substance solution; Step 6-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use the upper layer solution of 4:1:5:0.5 n-butanol-ethyl acetate-water-acetic acid as the developing agent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

23. The method according to claim 22, characterized in that In the step 6-1: take 0.5-4g of Erdong decoction preparation, add 40ml of chloroform, heat and reflux for 1 hour, filter, discard the chloroform solution, evaporate the solvent from the residue, add 1ml of water to stir and moisten, add 10ml of water-saturated n-butanol, ultrasonically treat, absorb the supernatant and add 3 times the amount of ammonia test solution, shake well, let it stand and separate, take the upper layer and evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution; Step 6-2-1: Take 0.5-2 g of ginseng reference medicinal material and prepare a reference medicinal material solution by the same method as step 6-1; Step 6-2-2: Add methanol to prepare a mixed solution containing 0.5-4 mg of ginsenoside Rg1 reference substance and 0.5-4 mg of ginsenoside Re reference substance per 1 ml, as the reference substance solution; Step 6-3: Pipette 10-40 μl of the test solution, 1-10 μl of the reference drug solution and the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate. The color developer is 10% sulfuric acid ethanol solution. The heating temperature is 105°C and the plate is inspected under sunlight.

24. A thin layer chromatography identification method for liquorice components in Erdong decoction preparations, characterized in that: The following steps are involved: Step 7-1: Take Erdong decoction preparation, add solvent, and prepare test solution; Step 7-2-1: Take liquiritin control medicinal material, add solvent, and prepare control medicinal material solution; Step 7-2-2: Take the liquiritin reference substance, add a solvent, and prepare a reference substance solution; Step 7-3: According to the thin layer chromatography method, aspirate the test sample solution, control medicinal material solution, and reference substance solution, and spot them on the same thin layer plate to form strips. Use 10:3:2 ethyl acetate-acetic acid-water as the developing solvent, develop, take out, dry, spray with color developer, and heat until the spots are clearly colored, and inspect.

25. The method according to claim 24, characterized in that In the step 7-1: take 0.5-4g of Erdong decoction preparation, add 40ml of ether, heat to reflux, filter, discard the ether solution, evaporate the residue, add 30ml of methanol, heat to reflux, filter, evaporate the filtrate to dryness, add 40ml of water to dissolve the residue, add 50ml of n-butanol to shake and extract, centrifuge, take the n-butanol solution, evaporate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test solution; Step 7-2-1: Take 0.5-2 g of licorice control medicinal material and prepare a control medicinal material solution by the same method as step 7-1; Step 7-2-2: Add methanol to prepare a solution containing 0.2-1.5 mg of liquiritin reference substance per 1 ml as the reference substance solution; Step 7-3: Pipette 1-20 μl of the test solution, 2-10 μl of the control medicinal material solution and the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate. The color developer is 10% sulfuric acid ethanol solution. The heating temperature is 105°C and the plate is inspected under a 365nm ultraviolet lamp.

26. The method according to any one of claims 12 to 25, characterized in that The thin layer chromatography method is in accordance with General Chapter 0502 of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

27. A method for identifying an Erdong decoction preparation, characterized in that: It includes: The ingredients in the Erdongtang preparation are identified by the method described in any one of claims 12-26.

28. The method according to any one of claims 10 to 27, characterized in that The Erdong decoction preparation is freeze-dried powder, extract powder, or granules, wherein when the Erdong decoction preparation is granules, the granules are ground into powder before identification.

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