A method for identifying ophiopogon japonicus, forsythia suspensa and baicalin in Qinghouyan granules

Abstract

The present invention discloses a method for identifying Ophiopogon japonicus, Forsythia suspensa and Baicalin in Qinghouyan Granules, and relates to the technical field of quality control of traditional Chinese medicine. The present invention mainly improves the method for preparing the test solution, specifically: the Ophiopogon japonicus test solution is first boiled with methanol and sodium hydroxide, and then extracted with chloroform and n-butanol; the Forsythia suspensa test solution is first boiled with calcium carbonate and water, and then evaporated with magnesium oxide, and then extracted with ethanol reflux; the Baicalin test solution is first ultrasonically treated in alkaline 50% ethanol, and then supplemented with 50% ethanol to make up for the weight loss. The present invention improves the purity and concentration of the sample to be tested by improving the preparation method of the test solution, and solves the problem that the existing technology for identifying Ophiopogon japonicus, Forsythia suspensa and Baicalin in Qinghouyan Granules has negative interference, negative tailing, and is difficult to judge.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicines, and in particular to a method for identifying ophiopogon japonicus, forsythia suspensa and baicalin in Qinghouyan granules. Background Art

[0002] Qinghouyan Granules are a traditional Chinese medicine whose main ingredients are Rehmannia root, Ophiopogon japonicus, Scrophularia ningpoensis, Forsythia suspensa, and Scutellaria baicalensis. They have the effects of nourishing yin, clearing the throat, and detoxifying. They are primarily used to treat acute and chronic pharyngitis, laryngitis, acute and chronic tonsillitis, localized diphtheria, and mild to moderate diphtheria, relieving throat pain and inflammation caused by these conditions.

[0003] The existing method for identifying Ophiopogon japonicus in Qinghouyan granules is as follows: Prepare the test solution: Take 5g of the product, add 20ml of water, add 1ml of hydrochloric acid (6mol / L), boil for 5 minutes, cool, and extract twice with 10ml of chloroform. Combine the extracts, evaporate to dryness, and dissolve the residue in 1ml of chloroform to prepare the test solution. Prepare the control solution: Take 1g of Ophiopogon japonicus control herb, add 20ml of water, boil for 10 minutes, filter, and prepare the control solution using the same method. Thin-layer chromatography (TLC) is performed using the same method. 5μl of each solution is spotted on the same silica gel G thin-layer plate, developed with chloroform-acetone (3:2) as the developing solvent, removed, cooled, sprayed with 10% ethanolic sulfuric acid solution, and dried at 105°C for approximately 5 minutes (Guo Qun et al., "Research on the Quality Standard of Qinghouyan Sugar-Free Granules," Tianjin Pharmacy, Issue 2, 2000). This identification result is negative and indicates interference.

[0004] The existing technology for identifying Forsythia in Qinghouyan Granules is as follows: weigh 10g of sample powder, add 50mL of methanol, ultrasonically treat for 30min, filter, evaporate the filtrate in a water bath, and dissolve the residue in 1mL of methanol to obtain the test solution. Take 1g of Forsythia reference medicinal material and prepare a control medicinal material solution in the same way. Take the Forsythia suspense reference substance, add methanol to prepare a solution containing 1mg per 1mL, which is the reference solution. Take the control sample prepared according to the production process without Forsythia suspense, and prepare a negative control solution in the same way. The above three solutions are spotted on the same silica gel G thin layer plate with a microcapillary, 20μL of the test solution and 10μL of the reference solution, the developing agent system is chloroform-methanol (5:1), and after development, spray with 5% vanillin sulfuric acid solution for color development, and heat at 105°C until the spots are clear (

[0005] Yao Shixia, Niu Yuting, et al. “Study on the quality standard of Qinghouyan sugar-free granules” Journal of Traditional Chinese Medicine, 2021, 32(07). The identification result is negative with a tail, which is difficult to judge.

[0006] The existing high-performance liquid chromatography (HPLC) method for detecting baicalin in Qinghouyan granules involves taking approximately 0.5 g of sample, accurately weighing it, placing it in a 100 ml volumetric flask, adding an appropriate amount of 50% ethanol, ultrasonically treating it, cooling it, diluting it to the mark with 50% ethanol, shaking it well, filtering it, and collecting the filtrate. The results of this method require further optimization.

[0007] In summary, the present invention provides a method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granules. Summary of the Invention

[0008] To address the above shortcomings, the present invention provides a method for identifying Radix Ophiopogonis, Fructus Forsythiae, and Baicalin in Qinghouyan Granules, which solves the problem of negative interference, negative tailing, and difficulty in judging in the existing technology for identifying Radix Ophiopogonis and Fructus Forsythiae in Qinghouyan Granules. The specific technical solution is as follows:

[0009] A method for identifying Radix Ophiopogonis, Fructus Forsythiae, and Baicalin in Qinghouyan Granules comprises the following steps:

[0010] (1) Thin layer identification of Radix Ophiopogonis in Qinghouyan Granules:

[0011] a. Preparation of test solution: Take the sample to be tested, add methanol, add sodium hydroxide solution, heat to a boil, keep boiling, let cool, filter, extract the filtrate with chloroform by shaking, adjust the pH of the aqueous layer to 3-5, extract 2-4 times with n-butanol by shaking, combine the n-butanol layers, evaporate to dryness, dissolve the residue in methanol to obtain the test solution;

[0012] b. Preparation of control herbal solution: Take 1-3g of Radix Ophiopogonis control herbal solution according to the preparation method of the test solution;

[0013] c. 5-10 μl of the test solution and 5-10 μl of the control herbal solution were spotted on the same silica gel G thin layer plate, developed with n-butanol-acetic acid-water, dried, sprayed with 10% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored; the ratio of n-butanol-acetic acid-water was (4-6):(0.5-1):1;

[0014] (2) Identification of Forsythia suspensa in Qinghouyan Granules:

[0015] a. Preparation of test solution: take the sample to be tested, add calcium carbonate, add water, heat to a boil, let cool, filter, concentrate the filtrate, add magnesium oxide, stir evenly, evaporate to dryness, reflux and extract the residue with ethanol 2-3 times, combine the ethanol solution, evaporate the residue and add methanol to dissolve it to obtain the test solution;

[0016] b. Preparation of reference solution: Take the forsythiaside reference substance and add methanol to make a solution containing 1 mg per 1 ml as the reference solution;

[0017] c. Pipette 5-10 μl of the test solution and 5-10 μl of the reference solution onto the same high-performance silica gel G thin layer plate, develop with chloroform-ethyl acetate-methanol-formic acid as the developing solvent, remove, dry, spray with vanillin sulfuric acid test solution, and heat until the spots are clearly colored; the ratio of chloroform-ethyl acetate-methanol-formic acid is (30-45):(2-7):(8-15):(0.1-0.3);

[0018] (3) Identification of baicalin in Qinghouyan granules by HPLC:

[0019] a. Chromatographic conditions: An Elite column was used with methanol-water-glacial acetic acid as the mobile phase and the detection wavelength was 276 nm.

[0020] b. Preparation of test solution: Grind the sample to be tested, accurately weigh it, place it in a flask, accurately add ethanol solution adjusted with sodium hydroxide solution to the pH, weigh it, sonicate, let it cool, add ethanol solution to make up the loss, shake well, filter, and collect the filtrate.

[0021] c. Preparation of reference solution: Prepare baicalin reference solution in the same manner as the test solution;

[0022] d. High performance liquid chromatography determination: Accurately aspirate the test solution and reference solution and inject them into the high performance liquid chromatography instrument for determination.

[0023] Preferably, in the step (1), a. preparation of the test solution: take 10-15g of the sample to be tested, add 35-45ml of 20% methanol and 4-8ml of 20% sodium hydroxide solution in proportion, heat to boil, keep boiling for 5-10min, cool, filter, and extract the filtrate by shaking with 10-30ml of chloroform. After adjusting the pH of the aqueous layer to 3-5, shake and extract with n-butanol 2-4 times, 10-30ml each time, combine the n-butanol layers, evaporate to dryness, and dissolve the residue in 0.5-1.5ml of methanol to obtain the test solution.

[0024] Preferably, in step (1), the ratio of the developing agent n-butanol-acetic acid-water is 4:1:1.

[0025] Preferably, in the step (2), a. preparation of the test solution: take 15-20g of the sample to be tested, add 1-3g of calcium carbonate in proportion, add 40-55ml of water, heat to boil, cool, filter, concentrate the filtrate, add 2-5g of magnesium oxide, stir evenly, evaporate to dryness, add 5-15ml of ethanol to the residue and reflux extract 2-3 times, combine the ethanol solution, evaporate the residue and add 0.5-1.5ml of methanol to dissolve it to obtain the test solution.

[0026] Preferably, the filtrate is concentrated to 1 / 2 of the original volume.

[0027] Preferably, in step (2), the ratio of the developing agent chloroform-ethyl acetate-methanol-formic acid is 40:5:10:0.2.

[0028] Preferably, in the step (3), b. Preparation of the test solution: take the sample to be tested, grind it, accurately weigh 0.2-0.4g, place it in a 100ml flask, accurately add 40-60ml of 50% ethanol solution adjusted to pH 9-11 with sodium hydroxide solution, weigh the weight, ultrasonically treat, let cool, add 50% ethanol solution to make up the lost weight, shake well, filter, and take the filtrate to obtain.

[0029] Preferably, in step (3), the specification of the chromatographic column is 250*4.6 mm.

[0030] Preferably, in step (3), the ratio of methanol-water-glacial acetic acid is 50:50:1.

[0031] Preferably, in step (3), the ultrasonic power is 250-300w, the frequency is 25-33kHz, and the time is 30-50min.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. When thin-layer identification of Ophiopogon japonicus of the present invention is carried out, the test solution is first boiled with methanol and sodium hydroxide, and then extracted with chloroform and n-butanol to prepare the solution, and n-butanol-acetic acid-water (4:1:1) is used as the developing agent, which can reduce impurities in the test sample, eliminate negative interference, and improve the accuracy of the identification result.

[0034] 2. When thin-layer identification of the forsythia of the present invention is performed, the test solution is first boiled with calcium carbonate and water, then magnesium oxide is added and evaporated, and then prepared by reflux extraction with ethanol, and chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) is used as the developing agent, which can prevent the negative control from having a tailing phenomenon and improve the accuracy of the identification result.

[0035] 3. When identifying baicalin by high-performance liquid chromatography of the present invention, the test solution is prepared by first adding alkaline 50% ethanol for ultrasonic treatment and then adding 50% ethanol to make up for the weight loss. This can improve the transfer rate of the component to be tested, improve the purity and concentration of the sample, and make the results more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0037] Figure 1is the thin layer chromatogram of Radix Ophiopogonis in Example 1;

[0038] Figure 2 The thin layer chromatogram of Radix Ophiopogonis in Comparative Example 1 is shown;

[0039] Figure 3 is the thin layer chromatogram of Forsythia suspensa in Example 1;

[0040] Figure 4 The thin layer chromatogram of Forsythia suspensa in Comparative Example 2;

[0041] Figure 5 is the liquid phase spectrum of baicalin in Example 1;

[0042] Figure 6 This is the liquid phase spectrum of baicalin in Comparative Example 3. DETAILED DESCRIPTION

[0043] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0044] The medicinal formula of the Qinghouyan granules involved in the examples and comparative examples is: 180g of Rehmannia root, 160g of Ophiopogon japonicus, 260g of Scrophularia ningpoensis, 315g of Forsythia suspensa, and 315g of Scutellaria baicalensis.

[0045] Example 1

[0046] A method for identifying Radix Ophiopogonis, Fructus Forsythiae, and Baicalin in Qinghouyan Granules comprises the following steps:

[0047] (1) Thin layer identification of Radix Ophiopogonis in Qinghouyan Granules:

[0048] a. The test solution was prepared by taking 12 g of the test sample Qinghouyan Granules, adding 40 ml of 20% methanol and 5 ml of 20% sodium hydroxide solution, heating to a boil, maintaining boiling for 5 minutes, cooling, filtering, and extracting the filtrate by shaking with 20 ml of chloroform. After adjusting the pH of the aqueous layer to 4, extract the filtrate by shaking with n-butanol twice, each time with 20 ml. The n-butanol layers were combined and evaporated to dryness. The residue was dissolved in 1 ml of methanol to obtain the test solution.

[0049] b. Preparation of control medicinal solution: Take 1 g of Ophiopogon japonicus control medicinal material and prepare a control medicinal material solution according to the preparation method of the test solution; Ophiopogon japonicus medicinal material solution and Ophiopogon japonicus negative sample solution are also prepared in the same way;

[0050] c. Pipette 5 μl of the test solution and 5 μl of the control medicinal material solution onto the same silica gel G thin layer plate, develop with n-butanol-acetic acid-water (4:1:1), dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. The identification results are shown in the table. Figure 1In the chromatogram of the test sample, a main spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material, and there is no negative interference.

[0051] (2) Identification of Forsythia suspensa in Qinghouyan Granules:

[0052] a. The test solution was prepared by taking 18 g of the test sample Qinghouyan granules, adding 2 g of calcium carbonate, adding 50 ml of water, heating to a boil, cooling, filtering, concentrating the filtrate to 1 / 2 of the original volume, adding 3 g of magnesium oxide, stirring, evaporating to dryness, and reflux extracting the residue with 10 ml of ethanol twice. The ethanol solution was combined, and the residue was evaporated to dryness and dissolved in 1 ml of methanol to obtain the test solution;

[0053] b. Preparation of reference solution: Take the forsythiaside reference substance and add methanol to prepare a solution containing 1 mg per 1 ml as the reference solution; the negative sample solution lacking forsythia is also prepared in the same way;

[0054] c. Pipette 5 μl of the test solution and 5 μl of the reference solution onto the same high-performance silica gel G thin layer plate, develop with chloroform-ethyl acetate-methanol-formic acid, remove, dry, spray with vanillin sulfuric acid test solution, and heat until the spots are clearly colored; the ratio of chloroform-ethyl acetate-methanol-formic acid is (40:5:10:0.2). Results are shown in Figure 3 (Test solution 1, test solution 2, and test solution 3 in the figure are made from different batches of Qinghouyan Granules respectively). In the test sample chromatogram, spots of the same color appear at the corresponding positions in the control sample chromatogram. The spots are clear and have no tailing phenomenon.

[0055] (3) Identification of baicalin in Qinghouyan granules by HPLC:

[0056] a. Chromatographic conditions: A 250*4.6 mm Elite column was used with a mobile phase of methanol-water-glacial acetic acid (50:50:1) and a detection wavelength of 276 nm.

[0057] b. Prepare the test solution by finely grinding the Qinghouyan granules (Qinghouyan granules) to be tested. Accurately weigh 0.3 g and place in a 100 ml flask. Accurately add 50 ml of 50% ethanol solution adjusted to pH 9 with sodium hydroxide solution, weigh the solution, sonicate, cool, and make up the lost weight with 50% ethanol solution. Shake well, filter, and collect the filtrate.

[0058] c. Preparation of reference solution: Prepare baicalin reference solution in the same manner as the test solution; prepare negative sample solution lacking baicalin in the same manner;

[0059] d. High performance liquid chromatography determination: Accurately aspirate the test solution, reference solution and negative sample solution lacking Scutellaria baicalensis into the high performance liquid chromatography instrument for determination. The results are shown in Figure 5 , the figure shows that the sample has few impurities and the negative result has no interference.

[0060] Example 2

[0061] A method for identifying Radix Ophiopogonis, Fructus Forsythiae, and Baicalin in Qinghouyan Granules comprises the following steps:

[0062] (1) Thin layer identification of Radix Ophiopogonis in Qinghouyan Granules:

[0063] a. Preparation of test solution: Take 10 g of the sample to be tested, add 35 ml of 20% methanol, add 4 ml of 20% sodium hydroxide solution, heat to a boil, keep boiling for 5 minutes, let cool, filter, and extract the filtrate with 10 ml of chloroform by shaking. After adjusting the pH of the aqueous layer to 3, extract it twice with n-butanol by shaking, each time with 10 ml. Combine the n-butanol layers, evaporate to dryness, and dissolve the residue in 0.5 ml of methanol to obtain the test solution;

[0064] b. Preparation of control herbal solution: Take 3g of Ophiopogon japonicus control herbal solution according to the method for preparing the test solution; Ophiopogon japonicus herbal solution and Ophiopogon japonicus negative sample were also prepared in the same way;

[0065] c. Pipette 10 μl of the test solution and 10 μl of the control medicinal material solution onto the same silica gel G thin layer plate, develop with n-butanol-acetic acid-water (4:0.5:1) as the developing solvent, dry, spray with 10% ethanolic sulfuric acid solution, and heat at 105°C until the spots are clearly colored.

[0066] (2) Identification of Forsythia suspensa in Qinghouyan Granules:

[0067] a. Preparation of test solution: Take 15g of the sample to be tested, add 1g of calcium carbonate, add 40ml of water, heat to a boil, let cool, filter, concentrate the filtrate to 1 / 2 of the original volume, add 2g of magnesium oxide, stir evenly, evaporate to dryness, add 5ml of ethanol to the residue and reflux and extract twice, combine the ethanol solution, evaporate the residue to dryness and add 0.5ml of methanol to dissolve it to obtain the test solution;

[0068] b. Preparation of reference solution: Take the forsythiaside reference substance and add methanol to prepare a solution containing 1 mg per 1 ml as the reference solution; the negative sample solution lacking forsythia is also prepared in the same way;

[0069] c. Pipette 10 μl of the test solution and 10 μl of the reference solution onto the same high-performance silica gel G thin-layer plate. Use chloroform-ethyl acetate-methanol-formic acid (30:2:8:0.1) as the developing solvent. Develop the plate, remove the plate, dry it, spray it with vanillin sulfuric acid test solution, and heat it until the spots are clearly colored.

[0070] (3) Identification of baicalin in Qinghouyan granules by HPLC:

[0071] a. Chromatographic conditions: A 250*4.6 mm Elite column was used with a mobile phase of methanol-water-glacial acetic acid (50:50:1) and a detection wavelength of 276 nm.

[0072] b. Preparation of test solution: Grind the sample to be tested, accurately weigh 0.2 g, and place in a 100 ml flask. Accurately add 40 ml of 50% ethanol solution adjusted to pH 9 with sodium hydroxide solution, weigh the solution, and sonicate at 250 W, 25 kHz for 30 min. Allow to cool, and add 50% ethanol solution to make up the loss. Shake well, filter, and collect the filtrate.

[0073] c. Preparation of reference solution: Prepare baicalin reference solution in the same manner as the test solution; prepare negative samples lacking baicalin in the same manner;

[0074] d. High performance liquid chromatography determination: Accurately aspirate the test solution, reference solution and negative sample solution lacking Scutellaria baicalensis into the high performance liquid chromatography instrument for determination.

[0075] Example 3

[0076] A method for identifying Radix Ophiopogonis, Fructus Forsythiae, and Baicalin in Qinghouyan Granules comprises the following steps:

[0077] (1) Thin layer identification of Radix Ophiopogonis in Qinghouyan Granules:

[0078] a. Preparation of test solution: Take 15 g of the sample to be tested, add 45 ml of 20% methanol, add 8 ml of 20% sodium hydroxide solution, heat to a boil, keep boiling for 10 min, let cool, filter, and extract the filtrate with 30 ml of chloroform by shaking. Adjust the pH of the aqueous layer to 5, and extract it with n-butanol by shaking 4 times, 30 ml each time. Combine the n-butanol layers, evaporate to dryness, and dissolve the residue in 1.5 ml of methanol to obtain the test solution;

[0079] b. Preparation of control medicinal solution: Take 2 g of Ophiopogon japonicus control medicinal material and prepare a control medicinal solution according to the test solution preparation method; Ophiopogon japonicus medicinal solution and Ophiopogon japonicus negative sample were also prepared in the same way;

[0080] c. Pipette 8 μl of the test solution and 8 μl of the control medicinal material solution, spot them on the same silica gel G thin layer plate, develop with n-butanol-acetic acid-water (6:1:1), dry, spray with 10% ethanolic sulfuric acid solution, and heat at 105°C until the spots are clearly colored; the ratio of n-butanol-acetic acid-water is.

[0081] (2) Identification of Forsythia suspensa in Qinghouyan Granules:

[0082] a. Preparation of test solution: Take 20 g of the sample to be tested, add 3 g of calcium carbonate, add 55 ml of water, heat to a boil, let cool, filter, concentrate the filtrate to 1 / 2 of the original volume, add 5 g of magnesium oxide, stir evenly, evaporate to dryness, add 15 ml of ethanol to the residue and reflux and extract three times, combine the ethanol solution, evaporate the residue and add 1.5 ml of methanol to dissolve it to obtain the test solution;

[0083] b. Preparation of reference solution: Take the forsythiaside reference substance and add methanol to prepare a solution containing 1 mg per 1 ml as the reference solution; the negative sample solution lacking forsythia is also prepared in the same way;

[0084] c. Pipette 8 μl of the test solution and 8 μl of the reference solution onto the same high-performance silica gel G thin-layer plate. Use chloroform-ethyl acetate-methanol-formic acid (45:7:15:0.3) as the developing solvent. Develop the plate, remove the plate, dry it, spray it with vanillin sulfuric acid test solution, and heat it until the spots are clearly colored.

[0085] (3) Identification of baicalin in Qinghouyan granules by HPLC:

[0086] a. Chromatographic conditions: A 250*4.6 mm Elite column was used with a mobile phase of methanol-water-glacial acetic acid (50:50:1) and a detection wavelength of 276 nm.

[0087] b. Preparation of test solution: Grind the sample to be tested, accurately weigh 0.4 g, and place in a 100 ml flask. Accurately add 60 ml of 50% ethanol solution adjusted to pH 11 with sodium hydroxide solution, weigh the solution, and sonicate at 300 W, 33 kHz for 50 min. Allow to cool, and make up the lost weight with 50% ethanol solution. Shake well, filter, and collect the filtrate.

[0088] c. Preparation of reference solution: Prepare baicalin reference solution in the same manner as the test solution; prepare negative samples lacking baicalin in the same manner;

[0089] d. High performance liquid chromatography determination: Accurately aspirate the test solution, reference solution and negative sample solution lacking Scutellaria baicalensis into the high performance liquid chromatography instrument for determination.

[0090] Comparative Example 1

[0091] The thin layer identification of Radix Ophiopogonis in the Qinghouyan Granules of this comparative example is an existing technology, specifically:

[0092] Preparation of test solution: Take 5g of the test sample Qinghouyan Granules, add 20ml of water, add 1ml of hydrochloric acid (6mol / L), boil for 5 minutes, let cool, extract twice with chloroform, 10ml each time, combine the extracts, evaporate to dryness, add 1ml of chloroform to the residue, and dissolve it as the test solution. Preparation of control medicinal material solution: Take 1g of Ophiopogon japonicus control medicinal material, add 20ml of water, boil for 10 minutes, filter, and prepare the control medicinal material solution with the filtrate in the same way; the negative sample solution without Ophiopogon japonicus is also prepared in the same way. Thin layer chromatography is tested by thin layer chromatography. 5μl of each of the above solutions are taken and spotted on the same silica gel G thin layer plate, developed with chloroform-acetone (3:2) as the developing agent, taken out, cooled and dried, sprayed with 10% sulfuric acid ethanol solution, and dried at 105°C for about 5 minutes. Identification results are shown in Figure 2 (Qinghouyan Granules 1, Qinghouyan Granules 2, and Qinghouyan Granules 3 are medicines from different batches respectively). The graph shows that the negative result has interference, which is difficult to judge.

[0093] Comparative Example 2

[0094] The thin layer identification of the Forsythia suspensa in the Qinghouyan granules of this comparative example is an existing technology, specifically:

[0095] Weigh 10 g of the test sample Qinghouyan granule powder, add 50 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate in a water bath, and dissolve the residue in 1 mL of methanol to obtain the test solution. Take 1 g of Forsythia suspensa control medicinal material and prepare a control medicinal material solution in the same way. Take the Forsythia suspensa reference substance, add methanol to prepare a solution containing 1 mg per 1 mL, which is the reference solution; the negative sample without Forsythia suspensa is also prepared in the same way. Take the control sample prepared according to the production process without Forsythia suspensa, and prepare a negative control solution in the same way. Use a microcapillary to spot the above solutions on the same silica gel G thin layer plate, spot 20 μL of the test solution and 10 μL of the reference solution, the developing agent system is chloroform-methanol (5:1), and after development, spray with 5% vanillin sulfuric acid solution for color development, and heat at 105°C until the spots are clear. See the results. Figure 4 (Qinghouyan Granules 1, Qinghouyan Granules 2, and Qinghouyan Granules 3 are medicines from different batches, and Forsythiaside Control Medicinal Material Solution 1, Forsythiaside Control Medicinal Material Solution 2, and Forsythiaside Control Medicinal Material Solution 3 are medicinal materials from different batches, respectively). The graph shows that the negative result has a tail, which is difficult to judge.

[0096] Comparative Example 3

[0097] The identification of baicalin in the Qinghouyan granules of this comparative example is based on existing technology, as follows:

[0098] a. Chromatographic conditions: A 250*4.6 mm Elite column was used with methanol-water-glacial acetic acid (50:50:1) as the mobile phase and the detection wavelength was 276 nm.

[0099] b. Preparation of the test solution: Take about 0.5g of the sample to be tested, accurately weigh it, place it in a 100ml volumetric flask, add an appropriate amount of 50% ethanol, ultrasonically treat (power 250W, frequency 33kHz) for 30min, let it cool, add 50% ethanol to dilute to the scale, shake well, filter, and take the filtrate.

[0100] c. Preparation of reference solution: Prepare baicalin reference solution in the same manner as the test solution; prepare negative samples lacking baicalin in the same manner.

[0101] d. High performance liquid chromatography determination: Accurately aspirate the test solution, reference solution and negative sample solution lacking Scutellaria baicalensis into the high performance liquid chromatography instrument for determination. The results are shown in Figure 6 , the figure shows that the sample has many impurities and negative interference.

[0102] In summary, the present invention mainly improves the preparation method of the test solution, thereby improving the purity and concentration of the sample to be tested, and solves the problems of negative interference, negative tailing, and difficulty in judging in the prior art of identifying Ophiopogon japonicus and Forsythia suspensa in Qinghouyan Granules.

[0103] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granules, characterized in that: The steps include: (1) Thin layer identification of Radix Ophiopogonis in Qinghouyan Granules: a. Preparation of test solution: Take the sample to be tested, add methanol, add sodium hydroxide solution, heat to a boil, keep boiling, let cool, filter, extract the filtrate with chloroform by shaking, adjust the pH of the aqueous layer to 3-5, extract 2-4 times with n-butanol by shaking, combine the n-butanol layers, evaporate to dryness, dissolve the residue in methanol to obtain the test solution; b. Preparation of control herbal solution: Take Ophiopogon japonicus control herbal solution according to the preparation method of the test solution; c. Pipette the test solution and control medicinal material solution onto the same silica gel G thin layer plate, develop with n-butanol-acetic acid-water as the developing solvent, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored; the ratio of n-butanol-acetic acid-water is (4-6): (0.5-1):1; (2) Identification of Forsythia suspensa thin layer in Qinghouyan granules: a. Preparation of test solution: take the sample to be tested, add calcium carbonate, add water, heat to a boil, let cool, filter, concentrate the filtrate, add magnesium oxide, stir evenly, evaporate to dryness, reflux and extract the residue with ethanol 2-3 times, combine the ethanol solution, evaporate the residue and add methanol to dissolve it to obtain the test solution; b. Preparation of reference solution: Take the forsythiaside reference substance and add methanol to prepare a solution as the reference solution; c. Pipette the test solution and reference solution onto the same high-performance silica gel G thin layer plate, develop with chloroform-ethyl acetate-methanol-formic acid, remove, air dry, spray with vanillin sulfuric acid test solution, and heat until the spots are clearly colored; the ratio of chloroform-ethyl acetate-methanol-formic acid is (30-45):(2-7):(8-15):(0.1-0.3); (3) Identification of baicalin in Qinghouyan granules by HPLC: a. Chromatographic conditions: An Elite column was used with a mobile phase of methanol-water-acetic acid, and the detection wavelength was 276 nm. The ratio of methanol-water-acetic acid was 50:50:

1. b. Preparation of test solution: Grind the sample to be tested, accurately weigh it, place it in a flask, accurately add ethanol solution adjusted with sodium hydroxide solution to the pH, weigh it, sonicate, let it cool, add ethanol solution to make up the loss, shake well, filter, and collect the filtrate. c. Preparation of reference solution: Prepare baicalin reference solution in the same manner as the test solution; d. High performance liquid chromatography determination: Accurately aspirate the test solution and reference solution and inject them into the high performance liquid chromatography instrument for determination.

2. The method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granule according to claim 1, characterized in that: In the step (1), a. Preparation of the test solution: take 10-15 g of the sample to be tested, add 35-45 ml of 20% methanol and 4-8 ml of 20% sodium hydroxide solution in proportion, heat to boil, keep boiling for 5-10 minutes, cool, filter, and extract the filtrate by shaking with 10-30 ml of chloroform. After adjusting the pH of the water layer to 3-5, shake and extract with n-butanol 2-4 times, each time 10-30 ml, combine the n-butanol layers, evaporate to dryness, and dissolve the residue in 0.5-1.5 ml of methanol to obtain the test solution.

3. The method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granule according to claim 1, characterized in that: In the step (1), the ratio of n-butanol-acetic acid-water is 4:1:

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4. The method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granule according to claim 1, characterized in that: In the step (2), a. Preparation of the test solution: take 15-20 g of the sample to be tested, add 1-3 g of calcium carbonate in proportion, add 40-55 ml of water, heat to boil, cool, filter, concentrate the filtrate, add 2-5 g of magnesium oxide, stir evenly, evaporate to dryness, add 5-15 ml of ethanol to the residue and reflux extract 2-3 times, combine the ethanol solution, evaporate the residue to dryness and add 0.5-1.5 ml of methanol to dissolve it, and then obtain the test solution.

5. The method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granules according to claim 4, characterized in that: The filtrate was concentrated to 1 / 2 of its original volume.

6. The method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granules according to claim 1, characterized in that: In the step (2), the ratio of chloroform-ethyl acetate-methanol-formic acid is 40:5:10:0.

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7. The method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granules according to claim 1, characterized in that: In the step (3), b. Preparation of the test solution: Take the sample to be tested, grind it into powder, accurately weigh 0.2-0.4g, place it in a 100ml flask, accurately add 40-60ml of 50% ethanol solution adjusted to pH 9-11 with sodium hydroxide solution, weigh the weight, ultrasonicate, let cool, add 50% ethanol solution to make up the lost weight, shake well, filter, and take the filtrate to obtain the product.

8. The method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granule according to claim 1, characterized in that: In step (3), the specification of the chromatographic column is 250*4.6 mm.

9. The method for identifying Radix Ophiopogonis, Fructus Forsythiae and Baicalin in Qinghouyan Granules according to claim 1, characterized in that: In the step (3), the power of ultrasound is 250-300W, the frequency is 25-33kHz, and the time is 30-50min.

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