Quality control method of lygodium japonicum in Sanjin preparation

Abstract

The invention relates to the technical field of traditional Chinese medicines, and particularly discloses a quality control method of lygodium japonicum in a Sanjin preparation, which specifically comprises the following steps: by taking a lygodium japonicum reference extract as a reference, taking a butyl acetate-methanol-formic acid-water system or a butyl acetate-methanol-formic acid system as a developing solvent, and taking the lygodium japonicum reference extract as a reference; and carrying out qualitative identification on lygodium japonicum in the Sanjin preparation by adopting a thin layer chromatography. According to the method, a butyl acetate-methanol-formic acid-water system or a butyl acetate-methanol-formic acid system is adopted as a developing solvent, so that the migration rate and the separation degree of different polar components in lygodium japonicum are remarkably improved, spot overlapping or trailing is avoided, and it is ensured that chromatographic behaviors are clear and distinguishable. Meanwhile, a polyamide column or macroporous resin column chromatography technology is introduced to purify the test solution, interference components are effectively removed, and the definition of spots and the stability of the method are further improved.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a quality control method for Rhizoma Coptidis in Sanjin preparations. Background Art

[0002] Lygodium japonicum (Thunb.) Sw., Lygodium microphyllum (Cav.) R.Br., and Lygodium flexuosum (L.) Sw., all of the Lygodium family, are dried aerial parts. They mainly contain volatile oils, flavonoids, phenolic acids, and phenylpropanoid compounds, and are included in local medicinal material standards such as the "Guangxi Traditional Chinese Medicine Standards" and the "Guangxi Zhuang Medicine Quality Standards."

[0003] Jinsha vine is one of the raw materials used in Sanjin preparations (such as Sanjin tablets and Sanjin granules), and its quality control is crucial to the product's efficacy. Sanjin preparations, composed of golden cherry root, smilax china, ramie, Jinsha vine, and Centella asiatica, have the effects of clearing heat and detoxifying, promoting diuresis and relieving stranguria, and benefiting the kidneys. They are clinically used to treat lower-burner damp-heat syndromes, such as acute and chronic pyelonephritis, cystitis, and urinary tract infections. Currently, market demand for these preparations continues to grow, and ensuring their quality uniformity and effectiveness has become a focus of the industry.

[0004] At present, the quality detection of Jinsha Teng mainly relies on thin-layer chromatography. Chinese patent CN114720213A discloses a method for detecting the quality of Jinsha Teng preparations using a Jinsha Teng control extract as a control. This patented method uses a Jinsha Teng control extract as a control and cyclohexane-ethyl acetate-formic acid-acetone (volume ratio 15:15:0.5:1) as a developing agent for thin-layer identification. Although this method achieves preliminary identification of Jinsha Teng, it still has the problem of low matching between the developing agent and Jinsha Teng, resulting in low spot migration rate and poor separation. In addition, this method also has the problem of serious interference from impurities.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to solve the problems of poor separation effect of developing agents and serious interference of test samples in existing quality control methods of Rhizoma Coptidis, and provides a quality control method of Rhizoma Coptidis in Sanjin preparations. The method adopts butyl acetate-methanol-formic acid-water system or butyl acetate-methanol-formic acid system as developing agent for thin layer identification, which significantly improves the separation degree and spot clarity. At the same time, the method introduces polyamide column or macroporous resin column chromatography technology to purify the test sample solution, effectively removes interfering components, and further improves the spot clarity and stability of the method.

[0007] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0008] The invention provides a quality control method for Rhizoma Cibotii in a Sanjin preparation. The quality control method comprises the following steps: using a Rhizoma Cibotii control extract as a control, using a butyl acetate-methanol-formic acid-water system or a butyl acetate-methanol-formic acid system as a developing agent, and adopting thin-layer chromatography to perform qualitative identification of the Rhizoma Cibotii in the Sanjin preparation.

[0009] The present invention employs thin-layer chromatography (TLC) using a butyl acetate-methanol-formic acid-water system or a butyl acetate-methanol-formic acid system as a developing agent, and a T. cerevisiae control extract as a control, to qualitatively identify T. cerevisiae in the Sanjin preparation. The main active ingredients in T. cerevisiae include flavonoids (such as isoquercetin), phenolic acids (such as caffeic acid), terpenes, steroidal compounds, and alkaloids. These components differ significantly in polarity. Traditional developing agents (such as cyclohexane-ethyl acetate-formic acid-acetone) are unable to separate both low-polarity terpenes and high-polarity phenolic acids, resulting in low spot mobility and poor resolution.

[0010] The present invention selects a butyl acetate-methanol-formic acid-water system or a butyl acetate-methanol-formic acid system as a developing agent, and uses butyl acetate as a main solvent to provide basic elution capacity and promote the moderate migration of low-polarity components (such as terpenes); methanol has a strong polarity and can increase the polarity of the system and promote the migration of polar components (such as flavonoid glycosides); formic acid inhibits the dissociation of alkaline components through protonation and reduces tailing; water can further increase the polarity and promote the migration of high-polarity phenolic acids to the front and effectively separate them from other components; in addition, water and methanol can also increase the hydrogen bonding effect of the system, reduce the risk of solvent stratification, and ensure the stability of the system.

[0011] The present invention selects the control extract of Rhizoma Cibotii as a control, which has obvious corresponding spots, little interference, clear and definite indications, can play a good indicating role, has a unique identification effect on the Rhizoma Cibotii components in the Sanjin preparation, and improves the accuracy of this quality control method.

[0012] In the above technical solution, the volume ratio of butyl acetate-methanol-formic acid-water system when used as a developing solvent is (9-12): (2-4): (2-4): 1;

[0013] Optionally, the volume ratio of butyl acetate-methanol-formic acid-water system as the developing solvent is (10-12):(2-4):(2-4):1.

[0014] In the above technical solution, the volume ratio of butyl acetate-methanol-formic acid system when used as a developing agent is (7-9):1:1.

[0015] In the thin-layer chromatography identification method of the present invention, a butyl acetate-methanol-formic acid-water system or a butyl acetate-methanol-formic acid system is used, both of which can achieve excellent development effects. In particular, using butyl acetate-methanol-formic acid-water in a volume ratio of (9-12):(2-4):(2-4):1 or butyl acetate-methanol-formic acid in a volume ratio of (7-9):1:1 as the developing solvent achieves the best separation, clear and distinct spots, and high reproducibility, without interference from the negative control.

[0016] In the above technical solution, the quality control method comprises the following steps:

[0017] (1) Prepare the test solution;

[0018] (2) preparing a control extract solution of Caulis Spatholobi;

[0019] (3) Identification by thin layer chromatography: Spot the test sample solution and control extract solution on a thin layer plate, develop with a developing agent, and perform color development and inspection.

[0020] In the above technical solution, step (1), the preparation method of the test solution includes: taking the three gold preparations, grinding them, adding a solvent, extracting, filtering, evaporating to dryness, dissolving the residue in water, filtering and purifying through a polyamide column or a macroporous resin column, washing with water and eluting with an alcohol solution, collecting the eluate and evaporating to dryness, and dissolving the residue in an alcohol solution as the test solution.

[0021] The present invention introduces polyamide column or macroporous resin column chromatography technology to purify the test sample solution, effectively reducing the interference of other ingredients in the Sanjin preparation on the golden sand vine, further improving the spot clarity and stability of the method. The Sanjin preparation is made from the root of golden cherry, smilax china, smilax china, golden sand vine, and Centella asiatica. Among them, the ingredients such as golden cherry root tannins, steroidal saponins of smilax china, and terpenes of smilax china are similar in polarity or structure to the active ingredients in golden sand vine (such as flavonoid glycosides). They will interfere with the qualitative identification of golden sand vine using thin-layer chromatography, resulting in problems such as blurred spots and tailing, affecting the identification results.

[0022] The present invention uses a polyamide column to purify the sample solution. The amide groups form hydrogen bonds with the hydroxyl groups of flavonoids and phenolic acids, selectively retaining target components (such as isoquercetin and caffeic acid), while other components (such as rosa rosa tannins and sarsaparilla steroidal saponins) are rapidly eluted due to a lack of sufficient hydroxyl groups. Purification using a macroporous resin column, on the other hand, utilizes the hydrophobic effect of the styrene backbone to adsorb non-polar components such as steroidal saponins and terpenes, while polar components (such as flavonoid glycosides) elute with the aqueous phase.

[0023] The present invention purifies the test solution through a polyamide column or a macroporous resin column, and also avoids the influence of the auxiliary material residues in the three gold preparations on the chromatographic identification.

[0024] The present invention effectively removes interfering components through hydrogen bond adsorption of a polyamide column or polar / hydrophobic adsorption of a macroporous resin column, improves spot clarity and stability, and significantly optimizes thin-layer chromatography identification effects.

[0025] In the above technical solution, the solvent in step (1) is selected from one or more of water, alcohol solution, ethyl acetate, and acetone;

[0026] Optionally, the solvent is a methanol solution.

[0027] Optionally, the extraction method in step (1) is ultrasonic treatment or heating reflux;

[0028] Optionally, the extraction time in step (1) is 10-90 minutes;

[0029] Optionally, the extraction time in step (1) is 15-60 minutes.

[0030] In the above technical solution, the alcohol solution elution in step (1) is eluted using a methanol solution or an ethanol solution;

[0031] Alternatively, elution with ethanol solution may be used;

[0032] Optionally, elution is performed using a methanol solution with a volume fraction of 20% to 100%;

[0033] Optionally, elution is performed using an ethanol solution with a volume fraction of 20% to 100%;

[0034] Alternatively, elution may be performed using a 20% or 50% ethanol solution;

[0035] Optionally, the elution volume is 20-200 mL.

[0036] In the above technical solution, the residue is dissolved in an alcohol solution in step (1) by using methanol or ethanol;

[0037] Alternatively, dissolve in a methanol solution with a volume fraction of 30% to 100%;

[0038] Alternatively, dissolve in 50% or 100% methanol solution by volume;

[0039] Alternatively, use 2-10 mL of alcohol solution to dissolve the residue.

[0040] In the above technical solution, step (2), preparing a control extract solution of Caulis gentiana includes: taking a control extract of Caulis gentiana, dissolving it in an alcohol solution, dissolving it in an alcohol solution, shaking it, and filtering it to obtain a control extract solution;

[0041] Optionally, the alcohol solution is a methanol or ethanol solution.

[0042] In the above technical solution, the color development in step (3) is performed by spraying with a sulfuric acid methanol solution and heating until the spots are clearly colored, and the inspection is performed under ultraviolet light.

[0043] Upon inspection, if the chromatogram of the test sample shows spots of the same color at the corresponding positions in the chromatogram of the control extract, it can be determined that the test sample of the Sanjin preparation contains the ingredient of Jinsha Teng.

[0044] The quality control method provided by the present invention can accurately and effectively identify Rhizoma Cibotii in Sanjin preparations. Sanjin preparations include ingredients such as Rhizoma Cibotii root, Smilax china, Herba Centellae asiatica, and Centella asiatica. This method has broad applicability and is applicable to various dosage forms of Sanjin preparations, including pills, tablets, granules, softgels, capsules, and oral liquids. Specific products include Sanjin tablets, Sanjin granules, Sanjin capsules, and Sanjin oral liquid.

[0045] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0046] The present invention provides a quality control method for a Sanjin preparation made from the roots of Rosa rugosa, Smilax china, Herba Lycopodii, Herba Centellae asiatica, and Centella asiatica. Specifically, thin-layer chromatography is employed using a butyl acetate-methanol-formic acid-water system or a butyl acetate-methanol-formic acid system as a developing agent, and a Herba Cynoglossi control extract as a control, to achieve qualitative identification of the Herba Cynoglossi in the Sanjin preparation. Using the butyl acetate-methanol-formic acid-water system or the butyl acetate-methanol-formic acid system as a developing agent significantly improves the mobility and separation of the different polar components in Herba Cynoglossi, avoids spot overlap or tailing, and ensures clear and discernible chromatographic behavior.

[0047] The present invention introduces polyamide column or macroporous resin column chromatography technology to purify the test solution, effectively removing interfering components, and solving the cross-interference problem of multiple components in the three-gold preparation through selective adsorption or elution mechanism, further improving the spot clarity and the stability of the method.

[0048] The present invention provides an accurate and effective identification method for Rhizoma Coptidis in Sanjin preparations, which is of great significance for the quality control of Sanjin preparations and is conducive to ensuring the clinical medication effect.

[0049] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings are part of the present invention and are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an undue limitation of the present invention. Obviously, the drawings described below are only some embodiments. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without inventive effort. In the accompanying drawings:

[0051] Figure 1 This is a diagram showing the expanded effect of Example 1 of the present invention;

[0052] Figure 2 2 is a diagram showing the expanded effect of Embodiment 2-7 of the present invention;

[0053] Figure 3 This is a diagram showing the development effect of butyl acetate-methanol-formic acid (volume ratio 7:1:1) as the developing agent;

[0054] Figure 4 This is a diagram showing the development effect of cyclohexane-butyl acetate-formic acid-acetone (volume ratio 15:15:0.1:1) as the developing agent.

[0055] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but rather to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0057] Reagents and consumables:

[0058] The reagents used in the present invention (including butyl acetate, methanol, formic acid, cyclohexane, acetone, ethyl acetate, ethanol, methanolic sulfuric acid, etc.) are all commercially available conventional reagents and can be purchased or prepared by conventional methods in the art.

[0059] Polyamide column (30-100 mesh, Sinopharm Chemical Reagent Co., Ltd.)

[0060] Macroporous resin column HPD100 (Sinopharm Chemical Reagent Co., Ltd.)

[0061] The control extract of genus Jicama, Sanjin tablets, Sanjin granules and Sanjin capsules are all products of Guilin Sanjin Pharmaceutical Co., Ltd.

[0062] Example 1

[0063] Ten batches of commercially produced Sanjin tablets were tested using the following methods:

[0064] (1) Preparation of test solution: Grind three gold tablets into powder, weigh 1.5 g, add 25 mL of methanol, ultrasonicate for 45 min, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, apply the solution to a polyamide column, wash with water, and elute with 50 mL of 50% ethanol, collect the effluent and eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0065] (2) Preparation of a control extract solution of Caulis Agrimoniae: Take a control extract of Caulis Agrimoniae, add methanol to make a solution containing 10 mg per 1 mL, shake well, filter, and use it as a control extract solution.

[0066] (3) Thin layer chromatography identification: According to the thin layer chromatography method (General Rule 0502), 2 μL of each of the above two solutions were taken and spotted on the same high-performance silica gel G thin layer plate, and butyl acetate-methanol-formic acid-water (volume ratio 11:3:3:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with sulfuric acid methanol solution, heated until the spots were clearly colored, and examined under ultraviolet light. The development results were as follows: Figure 1 shown.

[0067] Example 2

[0068] Three batches of commercially produced Sanjin granules were tested using the following methods.

[0069] (1) Preparation of test solution: Take Sanjin granules, grind them into powder, weigh 1.5 g, add 25 mL of methanol, ultrasonically treat for 45 minutes, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, add the solution to a macroporous resin column, wash with water and elute with 50 mL of 50% ethanol, collect the effluent and eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0070] (2) Preparation of control extract solution: same as in Example 1.

[0071] (3) Thin layer chromatography identification: the same as in Example 1, the results are as follows Figure 2 shown.

[0072] Example 3

[0073] Three batches of commercially produced Sanjin capsules were tested using the following methods.

[0074] (1) Preparation of test solution: Take Sanjin capsules, remove the capsule shell, grind into powder, weigh 1.5 g, add 25 mL of methanol, ultrasonically treat for 45 minutes, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, add the solution to a macroporous resin column, wash with water and elute with 50 mL of 50% ethanol, collect the effluent and eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0075] (2) Preparation of control extract solution: same as in Example 1.

[0076] (3) Thin layer chromatography identification: the same as in Example 1, the results are as follows Figure 2 shown.

[0077] Example 4

[0078] Take a batch of commercial production scale Sanjin tablets and test them as follows:

[0079] (1) Preparation of test solution: Take three gold tablets, grind them into powder, weigh 2.5 g, add 15 mL of water, heat and reflux for 60 minutes, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, add the solution to a polyamide column, wash with water and elute with 100 mL of methanol, collect the effluent and eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0080] (2) Preparation of control extract solution: same as in Example 1.

[0081] (3) Thin layer chromatography identification: the same as in Example 1, the results are as follows Figure 2 shown.

[0082] Example 5

[0083] Take a batch of commercial production scale Sanjin tablets and test them as follows:

[0084] (1) Preparation of test solution: Grind three gold tablets into powder, weigh 3 g, add 50 mL of water, ultrasonically treat for 30 minutes, filter, evaporate to dryness, dissolve the residue in 50 mL of water, filter, add the solution to a polyamide column, wash with water, and elute with 100 mL of 20% ethanol, collect the effluent and eluate, evaporate to dryness, and dissolve the residue in 4 mL of methanol to prepare the test solution.

[0085] (2) Preparation of control extract solution: same as in Example 1.

[0086] (3) Thin layer chromatography identification: the same as in Example 1, the results are as follows Figure 2 shown.

[0087] Example 6

[0088] Take a batch of commercial production scale Sanjin tablets and test them as follows:

[0089] (1) Preparation of test solution: Take three gold tablets, grind them into powder, weigh 2.5 g, add 50 mL of water, ultrasonically treat for 45 minutes, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, add the solution to a polyamide column, wash with water and elute with 50 mL of ethanol, collect the effluent and eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0090] (2) Preparation of control extract solution: same as in Example 1.

[0091] (3) Thin layer chromatography identification: the same as in Example 1, the results are as follows Figure 2 As shown.

[0092] Example 7

[0093] Take a batch of commercial production scale Sanjin tablets and test them as follows:

[0094] (1) Preparation of test solution: Take three gold tablets, grind them into powder, weigh 2.5 g, add 50 mL of water, ultrasonically treat for 45 minutes, filter, evaporate to dryness, dissolve the residue in 50 mL of water, filter, add the solution to a polyamide column, wash with water and elute with 100 mL of 50% ethanol, collect the effluent and eluate, evaporate to dryness, dissolve the residue in 2 mL of 50% methanol, and use it as the test solution.

[0095] (2) Preparation of control extract solution: same as in Example 1.

[0096] (3) Thin layer chromatography identification: the same as in Example 1, the results are as follows Figure 2 shown.

[0097] result:

[0098] like Figure 1 , where point E is the control extract and points S1-S10 are the test samples of Example 1. The results show that in the chromatogram of the test sample of Example 1, spots of the same color are displayed at the corresponding positions of the chromatogram of the control extract. The spots are clear, well-separated, and have good reproducibility, proving that the quality control method provided by the present invention has good stability.

[0099] like Figure 2 , where point E is the control extract, points S1-S3 are the test samples of Example 2, points S4-S6 are the test samples of Example 3, and points S7-S10 are the test samples of Examples 4-7, respectively. The results show that the chromatograms of the test samples of Examples 2-7 all display spots of the same color at the corresponding positions in the chromatograms of the control extracts. The spots are clear and well-resolved, further demonstrating that this method is suitable for the identification and detection of the Jinshateng components in Sanjin Tablets, Sanjin Granules, and Sanjin Capsules.

[0100] Experimental Example 1 Comparison of different developing agents

[0101] The preparation process of the test solution and the control extract solution of the Herba Lycopodii was the same as that in Example 1, and different developing agents were used. Thin layer chromatography was used for detection. The detection conditions were as follows:

[0102] Chromatographic conditions: temperature: 25°C; relative humidity: 60%; spread: 8.0 cm; sample volume: 2 μL;

[0103] Thin layer plate: Thin layer chromatography silica gel precast plate.

[0104] Group 1: cyclohexane-butyl acetate-formic acid-acetone (volume ratio 15:15:0.5:1) was used as the developing solvent;

[0105] Group 2: Butyl acetate-methanol-formic acid-water (volume ratio 11:3:3:1) was used as the developing solvent;

[0106] Group 3: Butyl acetate-methanol-formic acid (volume ratio 7:1:1) was used as the developing solvent;

[0107] Group 4: Butyl acetate-methanol-formic acid-water (volume ratio 11:2:3:1) was used as the developing solvent;

[0108] Group 5: Butyl acetate-methanol-formic acid-water (volume ratio 9:3:2:1) was used as the developing solvent;

[0109] Group 6: Butyl acetate-methanol-formic acid-water (volume ratio 10:3:3:1) was used as the developing solvent;

[0110] Group 7: Butyl acetate-methanol-formic acid (volume ratio 8:1:1) was used as the developing solvent;

[0111] Group 8: Butyl acetate-methanol-formic acid (volume ratio 9:1:1) was used as the developing solvent.

[0112] Experimental results:

[0113] (1) Developing agents for different systems:

[0114] When cyclohexane-butyl acetate-formic acid-acetone (volume ratio 15:15:0.1:1) is used as a developing solvent, the developing effect is poor, far inferior to the two developing solvents of butyl acetate-methanol-formic acid-water system and butyl acetate-methanol-formic acid system. Figures 1-4 ,in, Figure 3 This is a diagram showing the development effect of butyl acetate-methanol-formic acid (volume ratio 7:1:1) as the developing agent; Figure 4 This is a diagram showing the development effect of cyclohexane-butyl acetate-formic acid-acetone (volume ratio 15:15:0.1:1) as the developing agent.

[0115] (2) Developing agents of butyl acetate-methanol-formic acid-water system in different proportions

[0116] Different proportions of butyl acetate-methanol-formic acid-water system as developing agents can all achieve effective development and can better detect the components of the golden sand vine; and the development effects of the four different proportions of butyl acetate-methanol-formic acid-water system as developing agents are equivalent, so the volume ratio of butyl acetate-methanol-formic acid-water system when used as the developing agent is selected as (9-12):(2-4):(2-4):1.

[0117] (3) Butyl acetate-methanol-formic acid system developer in different proportions

[0118] Different ratios of butyl acetate-methanol-formic acid system as developing agents can all achieve effective development and can better detect the components of Jinsha Teng; and the development effects of the three different ratios of butyl acetate-methanol-formic acid system as developing agents are comparable, so the volume ratio of butyl acetate-methanol-formic acid system is selected as (7-9):1:1 when used as the developing agent.

[0119] Experimental Example 2 Study on the preparation method of test solution

[0120] Based on the results of preliminary experimental studies, the present invention designed 10 methods for preparing test solutions. The test solutions prepared by different preparation methods were tested by thin layer chromatography. The test conditions were as follows:

[0121] Chromatographic conditions: temperature: 25°C; relative humidity: 60%; spread: 8.0 cm; sample volume: 2 μL;

[0122] Thin layer plate: Thin layer chromatography silica gel precast plate

[0123] Developing solvent: butyl acetate-methanol-formic acid (volume ratio 7:1:1)

[0124] The preparation methods of the 10 test sample solutions are as follows:

[0125] ① Grind the product into powder, take about 1.5 g, add 25 mL of methanol / 50% methanol, ultrasonically treat for 60 minutes, filter, evaporate to dryness, add 5 mL of methanol / 50% methanol to dissolve the residue, and use it as the test solution.

[0126] ② Grind the product into powder, take about 3 g, add 50 mL of methanol, ultrasonically treat for 15 minutes, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, add the solution to a polyamide column, wash with water and elute with 100 mL of methanol, collect the effluent and eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0127] ③ Grind the product into powder, take about 5 g, add 25 mL of ethyl acetate, ultrasonically treat for 30 minutes, filter, evaporate to dryness, add 1.5 mL of methanol to dissolve the residue, and use it as the test solution.

[0128] ④ Grind the product into powder, take about 2 g, add 50 mL of methanol, heat and reflux for 1 hour, cool, filter, evaporate to dryness, dissolve the residue in 25 mL of water, shake and extract with ethyl acetate twice, 25 mL each time, combine the ethyl acetate solution, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0129] ⑤ Grind about 2.5 g of this product into powder, add 25 mL of 50% methanol, heat and reflux for 30 minutes, cool, filter, evaporate to dryness, dissolve the residue in 50 mL of water, shake and extract with ethyl acetate twice, 25 mL each time, combine the ethyl acetate solution, evaporate to dryness, dissolve the residue in 5 mL of methanol, and use it as the test solution.

[0130] ⑥ Grind the product into powder, take about 3 g, add 50 mL of methanol, ultrasonically treat for 45 minutes, filter, evaporate to dryness, dissolve the residue in 20 mL of water, filter, add the solution to a macroporous resin column, wash with 50 mL of 20% ethanol / 100 mL of ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0131] ⑦ Grind the product into powder, take about 3 g, add 50 mL of methanol, ultrasonically treat for 60 minutes, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, add the solution to a polyamide column, wash with water and elute with 100 mL of ethyl acetate / 50 mL of 50% ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0132] ⑧ Grind this product into powder, take about 3 g, add 50 mL of methanol, heat and reflux for 1 hour, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, add the solution to a macroporous resin column, wash with water and elute with 100 mL of ethyl acetate, collect the eluate, evaporate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0133] ⑨ Grind this product into powder, take about 3 g, add 50 mL of methanol, heat and reflux for 1 hour, filter, evaporate to dryness, dissolve the residue in 50 mL of water, filter, add the solution to a polyamide column, wash with water and elute with 50 mL of 50% ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 4 mL of methanol, and use it as the test solution.

[0134] ⑩ Grind this product into powder, take about 3 g, add 50 mL of water / 20% methanol / 50% methanol / 70% methanol / methanol, ultrasonically treat for 45 minutes, filter, evaporate to dryness, dissolve the residue in 25 mL of water, filter, add the solution to a macroporous resin column, wash with water and elute with 100 mL of 50% ethanol, collect the effluent and eluate, evaporate to dryness, dissolve the residue in 2 mL of 50% methanol / methanol, and use as the test solution.

[0135] According to the further processing methods adopted after the samples are extracted, filtered and evaporated, the above 10 test solution preparation methods are divided into three categories, namely:

[0136] Ⅰ: Dissolve the residue in 1.5 mL or 5 mL of methanol or 5 mL of 50% methanol to prepare the test solution.

[0137] II: Dissolve the residue in 25 mL or 50 mL of water, extract twice with 25 mL of ethyl acetate by shaking, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 2 mL or 5 mL of methanol to prepare the test solution.

[0138] III: Add 20mL, 25mL or 50mL of water to the residue to dissolve it, filter it, take the solution and add it to a polyamide column or a macroporous resin column, wash it with water and elute it with 100mL of ethyl acetate or 100mL of methanol or 100mL of ethanol or 50mL of 20% ethanol or 50mL or 100mL of 50% ethanol, collect the effluent and eluate, evaporate to dryness, add 2mL of 50% methanol or 4mL of methanol to dissolve the residue as the test solution.

[0139] Among them, methods ① and ③ are Class I methods, ④ and ⑤ are Class II methods, and ②, ⑥, ⑦, ⑧, ⑨, and ⑩ are Class III methods.

[0140] Test results:

[0141] Take 1.5g, 2g, 3g, and 5g of the Sanjin preparation samples, add 15ml, 25ml, and 50ml of different amounts of water, methanol, ethyl acetate, and 50% methanol, respectively, and extract the samples by ultrasound or heating reflux method, with the extraction time being 15 minutes, 45 minutes, and 60 minutes, and the components of Jinsha Teng can be extracted.

[0142] Further analysis shows that:

[0143] The test solution prepared in methods ① and ③ of type I can detect the components of Rhizoma Coptidis, but there is obvious interference;

[0144] The test solution prepared in Class II methods ④ and ⑤ can detect the components of Jinsha Teng, but the spots corresponding to Jinsha Teng are darker and there is a little interference;

[0145] In the test solution prepared by Class III methods ⑦ and ⑧ using ethyl acetate as the eluent, the components of Jinsha Teng were detected, but the spots corresponding to Jinsha Teng were dark, indicating that the elution was insufficient and the method was not stable enough;

[0146] In Class III methods ②, ⑥, ⑦, ⑨, and ⑩, the test sample solutions prepared using methanol, ethanol, 20% ethanol, or 50% ethanol as eluents were able to clearly and significantly detect the components of the golden sand vine, indicating that the sample can be well purified after being extracted and filtered, and then passed through a polyamide column or a macroporous resin column using methanol, ethanol, 20% ethanol, or 50% ethanol as eluents. Furthermore, the elution effect was best achieved using a 20%-100% ethanol solution.

[0147] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present invention can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A quality control method for Rhizoma Coptidis in Sanjin preparations, characterized in that: The quality control method includes: The reference extract of Herba Cynanchifoliae was used as a control, and butyl acetate-methanol-formic acid-water system or butyl acetate-methanol-formic acid system was used as a developing solvent to qualitatively identify Herba Cynanchifoliaee in Sanjin preparations by thin layer chromatography.

2. The quality control method according to claim 1, characterized in that The volume ratio of butyl acetate-methanol-formic acid-water system as the developing solvent is (9-12):(2-4):(2-4):1; Preferably, the volume ratio of butyl acetate-methanol-formic acid-water system as the developing solvent is (10-12):(2-4):(2-4):

1.

3. The quality control method according to claim 1, characterized in that: The volume ratio of butyl acetate-methanol-formic acid system when used as the developing solvent is (7-9):1:

1.

4. The quality control method according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Prepare the test solution; (2) preparing a control extract solution of Caulis Spatholobi; (3) Identification by thin layer chromatography: Spot the test sample solution and control extract solution on a thin layer plate, develop with a developing agent, and perform color development and inspection.

5. The quality control method according to claim 4, characterized in that: In step (1), the preparation method of the test solution includes: taking the three gold preparations, grinding them, adding a solvent, extracting, filtering, evaporating to dryness, dissolving the residue in water, filtering and purifying through a polyamide column or a macroporous resin column, washing with water and eluting with an alcohol solution, collecting the eluate and evaporating to dryness, and dissolving the residue in an alcohol solution as the test solution.

6. The quality control method according to claim 5, characterized in that: The solvent in step (1) is selected from one or more of water, alcohol solution, ethyl acetate, and acetone; Preferably, the solvent is methanol solution; Preferably, the extraction method in step (1) is ultrasonic treatment or heating reflux; Preferably, the extraction time in step (1) is 10-90 minutes; Preferably, the extraction time in step (1) is 15-60 minutes.

7. The quality control method according to claim 5, characterized in that: The alcohol solution elution in step (1) is eluted using a methanol solution or an ethanol solution; Preferably, elution is performed using a methanol solution with a volume fraction of 20% to 100%; Preferably, elution is performed using an ethanol solution having a volume fraction of 20% to 100%; Preferably, elution is performed using an ethanol solution having a volume fraction of 20% or 50%; Preferably, the elution volume is 20-200 mL.

8. The quality control method according to claim 5, characterized in that: The residue in step (1) is dissolved in an alcohol solution by using methanol or ethanol; Preferably, a methanol solution with a volume fraction of 30%-100% is used for dissolution; Preferably, a methanol solution with a volume fraction of 50% or 100% is used for dissolution; Preferably, 2-10 mL of alcohol solution is used to dissolve the residue.

9. The quality control method according to claim 4, characterized in that: In step (2), preparing the control extract solution of the Herba Cynanchii var. cynanchii includes: taking the control extract of the Herba Cynanchii var. cynanchii, adding an alcohol solution to dissolve the extract, shaking well, filtering, and obtaining the control extract solution; Preferably, the alcohol solution is methanol or ethanol solution.

10. The quality control method according to claim 4, characterized in that: The color development in step (3) is performed by spraying with a sulfuric acid methanol solution and heating until the spots are clearly colored, and the inspection is performed under ultraviolet light.

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