A method for identifying the raw Rehmannia glutinosa in Qinggan Zhilin Decoction

Abstract

The invention discloses a method for identifying the raw Rehmannia glutinosa in a Qinggan Zhilin Decoction, comprising the following steps: preparing a test solution using a Qinggan Zhilin Decoction sample, preparing a raw Rehmannia glutinosa sample solution lacking wine using a raw Rehmannia glutinosa sample lacking wine, preparing a raw Rehmannia glutinosa control medicinal material solution using a raw Rehmannia glutinosa sample lacking wine, and preparing a catalpol reference solution at the same time; the raw Rehmannia glutinosa sample solution lacking wine, the raw Rehmannia glutinosa control medicinal material solution and the catalpol reference solution are all control sample solutions, and the test solution and the control sample solution are spotted on the same silica gel G thin layer plate, developed with succinic acid-isopropanol-chloroform as a developing agent, and after drying, sprayed with sulfuric acid-acetic acid-ethanol solution for heating and color development, and inspected. The method of the present invention is very suitable for the identification of raw Rehmannia glutinosa in Qinggan Zhilin Decoction, with clear chromatographic spots, no obvious tailing, high accuracy, and short color development time.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine analysis, quality identification and control, and particularly relates to an identification method of raw Rehmannia glutinosa in Qinggan Zhilin Decoction. Background Art

[0002] Qingganzhilin Decoction is a traditional Chinese medicine prescription for treating leucorrhea. The specific formula is 37.30g each of white peony root (fried with vinegar), angelica root (washed with wine) and black bean, 18.65g of rehmannia root (fried with wine), 11.19g each of donkey hide gelatin (fried with white flour) and red peony root, 7.46g each of phellodendron and cyperus, 3.73g of cyperus (fried with wine), and 46g of jujube. Qingganzhilin Decoction is decocted with water and has the effects of nourishing blood, clearing liver, strengthening spleen and stopping stranguria.

[0003] Rehmannia is a perennial herbaceous plant of the Scrophulariaceae family and the Rehmannia genus. Its underground tubers are yellow-white. Generally, Rehmannia refers to raw Rehmannia. Wine-cooked Rehmannia is prepared by frying Rehmannia with wine. The function of Rehmannia fried with wine changes, and it is warm in nature. It is a good medicine for treating human anemia, qi and blood deficiency, and liver and kidney deficiency in clinical practice. In the prior art, the preparation method of wine-cooked Rehmannia is usually as follows: fry raw Rehmannia slices according to the wine-cooking method until they are slightly charred, and use 12 kg of yellow wine for every 100 kg of raw Rehmannia.

[0004] In the prior art, the identification and detection methods of Radix Rehmanniae in Chinese medicine preparations include thin layer chromatography, but the corresponding detection methods are only applicable to specific drugs. For example, the Chinese patent document with publication number CN103381217A discloses a quality control method for Liuwei Buxue Capsules, wherein the identification method of Radix Rehmanniae glutinosae includes the following steps: taking 2.5g of Radix Rehmanniae glutinosae reference medicinal material, cutting it into pieces, crushing it with a grinder, adding 10mL of 50% methanol, ultrasonically treating it for 30 minutes, filtering, evaporating the filtrate, adding 2mL of methanol to dissolve it, and using it as a reference medicinal material solution. According to the thin layer chromatography method (Appendix VIB of the first volume of the Chinese Pharmacopoeia 2010 edition), 10μL of the reference medicinal material solution and 10μL of the test sample solution are respectively taken and spotted on the same silica gel G thin layer plate, and ethyl acetate: methanol: formic acid = 16: 1: 2 is used as the developing solvent, developing, taking out, drying, impregnating with 0.5% 2,2-diphenyl-1-picrylhydrazyl anhydrous ethanol solution, and drying.

[0005] The Chinese patent document with publication number CN113009062A discloses a method for identifying Rehmannia root medicinal materials, Rehmannia root slices and finished medicines prepared from Rehmannia root slices, comprising the following steps: (1) taking a sample to be tested and preparing a test solution; (2) taking a stachyose standard to prepare a reference solution, or / and taking Rehmannia root reference medicinal materials to prepare a reference medicinal material solution; (3) aspirating the test solution and the reference solution or / and the reference medicinal material solution, spotting them on a thin layer plate, developing with a mixture of ethyl acetate, ethanol, water and ammonia water in a volume ratio of (1.5-2.5):(4.5-5.5):(3.5-4.5):(0.4-0.6) as a developing agent, drying, spraying with sulfuric acid ethanol for color development, and inspecting.

[0006] A Chinese patent document with publication number CN109709240A discloses a method for detecting a Chinese medicine composition, wherein the identification method of Radix Rehmanniae Preparata in the Chinese medicine composition comprises: (1) preparing a test solution: weighing the Chinese medicine composition, adding pure water to dissolve, extracting with n-butanol, standing, discarding the n-butanol solution, adding anhydrous ethanol to the water layer, shaking, standing, discarding the ethanol solution, adding methanol to the precipitate, ultrasonically treating, filtering, evaporating the filtrate, cooling to room temperature, and dissolving the residue in methanol solution; (2) preparing a reference solution: taking an appropriate amount of stachyose reference, adding an organic solvent to prepare a reference solution; (3) thin layer chromatography identification: taking the test solution and the reference solution, developing with ethyl acetate-methanol-water-glacial acetic acid as a developing agent, spraying with sulfuric acid ethanol solution, and heating until the spots are clearly colored.

[0007] The above methods are only applicable to the identification of Rehmannia root in specific Chinese medicine preparations, and there are problems such as interference from impurity components or serious interference caused by different compositions of Chinese medicine preparations, unclear spots, etc. The composition of Qinggan Zhilin Decoction is complex, and it is difficult to separate various components with similar structures. It is very important to develop an identification method that is easy to color, highly accurate, and suitable for wine-grown Rehmannia root in Qinggan Zhilin Decoction. Summary of the invention

[0008] The composition of Qinggan Zhilin Decoction is complex, and it is difficult to separate various components with similar structures. The present invention provides a method for identifying wine-grown Rehmannia glutinosa in Qinggan Zhilin Decoction. The method is easy to color, has high accuracy, can reduce the influence of interfering components, and has a short color development time.

[0009] The specific technical solutions adopted are as follows:

[0010] A method for identifying the raw Rehmannia glutinosa in Qinggan Zhilin Decoction comprises the following steps:

[0011] (1) Preparation of test solution

[0012] Take a sample of Qinggan Zhilin Decoction, add water, ultrasonically treat, centrifuge, and take the supernatant; shake the supernatant with ethyl acetate, discard the ethyl acetate solution, shake the water solution with n-butanol, shake the n-butanol solution with ammonia water to obtain an ammonia solution, evaporate to dryness, dissolve the residue in water and pass through a column, finally evaporate the collected eluate to dryness, dissolve the residue in an organic solvent to obtain a test solution;

[0013] (2) Preparation of control sample solution

[0014] The control sample solution is a negative sample solution and a positive sample solution, the negative sample solution is a raw Rehmannia root sample solution lacking alcohol, and the positive sample solution is a raw Rehmannia root control medicinal material solution and a catalpol reference substance solution;

[0015] (3) Identification of Rehmannia glutinosa

[0016] Spot the test solution and the control sample solution on the same silica gel G thin layer plate, develop with succinic acid-isopropanol-chloroform as the developing agent, dry, spray with sulfuric acid-acetic acid-ethanol solution, heat for color development, and inspect.

[0017] The composition of the Qinggan Zhilin Decoction sample is complex. The present invention uses catalpol as an effective ingredient to identify the wine-grown Rehmannia glutinosa therein. Catalpol is a cyclopentane glucoside compound. The present invention first uses water-saturated n-butanol to extract the saponins therein; then uses ammonia solution to wash away the acidic substances and reduce some slightly acidic glycosides; and further uses a column pass step combined with a developing agent, succinic acid-isopropanol-chloroform and a color developer, sulfuric acid-acetic acid-ethanol to reduce the influence of other interfering components in thin layer chromatography, so as to ensure that the chromatographic spots are clear and the negative is free of interference.

[0018] Preferably, the column passing step is specifically as follows: the residue is dissolved in water and passed through a D101 macroporous adsorption resin column, eluted with water and then discarded the eluent, then eluted with 30% ethanol solution, the eluent is collected and evaporated to dryness; the residue is dissolved in ethanol, passed through a neutral alumina column, eluted with ethanol and then the eluent is collected and evaporated to dryness; the residue is dissolved in water, passed through a C18 solid phase extraction column, eluted with water and 10% methanol solution in sequence, and the eluent is collected.

[0019] The D101 macroporous adsorption resin column generally has a strong adsorption capacity for non-polar organic compounds and good applicability, especially for the separation of saponins, and the purification effect is particularly good; the neutral alumina column works based on physical adsorption, and has different adsorption capacities for substances of different polarity. In the present invention, it is used to remove highly polar substances and tannins; the C18 solid phase extraction column selectively retains the target and a small amount of components with similar properties to the target through polar interactions, hydrophobic interactions or ion exchange, and then uses another solvent system to selectively elute the target, thereby achieving the purification and enrichment of the active ingredients to be tested. The present application combines the three-step column treatment of the D101 macroporous adsorption resin column-neutral alumina column-C18 solid phase extraction column, and combines suitable sample solvents and elution solvents to achieve the successful separation and enrichment of the target component catalpol, which is convenient for subsequent thin layer chromatography detection and identification.

[0020] Preferably, the amount of water corresponding to each 1g of Qinggan Zhilin Decoction sample is 12.5-25g; the ultrasonic treatment time is 20-40min; the ethyl acetate solution is discarded after shaking extraction with ethyl acetate 2-3 times; the extracts are combined after shaking extraction with n-butanol 2-3 times to obtain n-butanol solution; the extracts are combined after shaking extraction with ammonia water 2-3 times to obtain ammonia water solution; the organic solvent is ethanol, and the organic solvent corresponding to each 1g of Qinggan Zhilin Decoction sample is 0.25-0.5mL.

[0021] Specifically, the preparation method of the test solution is as follows: take 2-4 g of Qinggan Zhilin Decoction sample, add 50 mL of water, ultrasonically treat for 30 min, centrifuge for 5 minutes, take the supernatant, shake and extract twice with ethyl acetate, 20 mL each time, discard the ethyl acetate solution, shake and extract the water solution with n-butanol twice, 20 mL each time, combine the n-butanol solution, shake and extract twice with ammonia water, 20 mL each time, combine the ammonia water solution, evaporate to dryness, dissolve the residue with 10 mL of water, and pass through a D101 macroporous adsorption machine. Attach to the resin column, elute with 50 mL of water in sequence, discard the eluate, then elute with 100 mL of 30% ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 5 mL of ethanol, pass through a neutral alumina column, elute with 60 mL of ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 3 mL of water, and pass through a C18 solid phase extraction column, elute with 5 mL of water and 10% methanol in sequence, collect the eluate, combine the eluates and evaporate to dryness, dissolve the residue in 1 mL of ethanol to obtain the test solution.

[0022] In step (2), a raw Rehmannia glutinosa sample solution lacking wine is prepared using a raw Rehmannia glutinosa sample lacking wine; a raw Rehmannia glutinosa reference medicinal material solution is prepared using a raw Rehmannia glutinosa sample with wine; and a method for preparing a catalpol reference solution is as follows: a catalpol reference is taken, ethanol is added to prepare a solution with a concentration of 0.2 to 0.8 mg / mL, and the solution is used as a catalpol reference solution.

[0023] Specifically, the method for preparing the alcohol-deficient raw Rehmannia glutinosa sample solution is as follows: take 2 to 4 g of the negative sample of alcohol-deficient raw Rehmannia glutinosa, and prepare the alcohol-deficient raw Rehmannia glutinosa sample solution according to the above-mentioned method for preparing the test solution.

[0024] Specifically, the preparation method of the wine-grown Rehmannia glutinosa control medicinal material solution is as follows: take 0.5-1.5g of the wine-grown Rehmannia glutinosa control medicinal material, add 50mL of water, decoct for 30 minutes, centrifuge for 5 minutes, take the supernatant, shake and extract twice with ethyl acetate, 20mL each time, discard the ethyl acetate solution, shake and extract the aqueous solution with n-butanol twice, 20mL each time, combine the n-butanol solutions, shake and extract twice with ammonia water, 20mL each time, combine the ammonia water solutions, evaporate to dryness, dissolve the residue in 10mL of water, pass through a D101 macroporous adsorption resin column, elute with 50mL of water in turn, discard the eluate, and then elute with 100mL of 30% ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 5mL of ethanol, pass through a neutral alumina column, elute with 60mL of ethanol, collect the eluate, evaporate to dryness, and dissolve the residue in 1mL of ethanol as the wine-grown Rehmannia glutinosa control medicinal material solution. Since the raw material composition of the Jiusheng Huai Dihuang reference drug is simpler than that of Qinggan Zhilin Decoction, there is no need for further purification using a C18 solid phase extraction column.

[0025] Preferably, the inner diameter of the D101 macroporous adsorption resin column is 1.5 cm, and the column height is 10 cm. When the D101 macroporous adsorption resin column is used for elution, the elution flow rate is 1 to 3 mL / min.

[0026] Preferably, the inner diameter of the neutral alumina column is 1.5 cm, 2 g, and the filler mesh size is 100-200 meshes; when the neutral alumina column is used for elution, the elution flow rate is 1-3 mL / min.

[0027] Preferably, the specification of the C18 solid phase extraction column is 1000 mg / 6 mL, and it needs to be pre-washed with 10 mL of methanol and balanced with 20 mL of water; when eluting with the C18 solid phase extraction column, the elution flow rate is 1 to 2 mL / min.

[0028] Preferably, in step (3), the developing agent is succinic acid-isopropanol-chloroform in a volume ratio of 1:2-4:7-12; the color developer is sulfuric acid-acetic acid-ethanol solution in a volume ratio of 8-12:2-4:84-90; the heating temperature is 105-110°C, the time is 3-5 minutes; the inspection condition is inspection under 365nm ultraviolet light. When the above-mentioned developing agent and color developer are selected, the chromatographic spots are clear, without obvious tailing, and the negative has no interference; and the color development time is short.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention uses catalpol as an effective ingredient to identify the wine-grown Rehmannia root in Qinggan Zhilin Decoction, and adopts a specific extraction method and a three-step column treatment of a D101 macroporous adsorption resin column-neutral alumina column-C18 solid phase extraction column, combined with a developing agent of succinic acid-isopropanol-chloroform and a color developer of sulfuric acid-acetic acid-ethanol to reduce the influence of interfering components in thin layer chromatography, thereby achieving accurate identification of the wine-grown Rehmannia root in Qinggan Zhilin Decoction.

[0031] (2) The present invention uses succinic acid-isopropanol-chloroform with a volume ratio of 1:2-4:7-12 as a developing agent and sulfuric acid-acetic acid-ethanol solution with a volume ratio of 8-12:2-4:84-90 as a color developer. It is very suitable for the identification of Rehmannia glutinosa in Qinggan Zhilin Decoction. The chromatographic spots are clear, without obvious tailing, with high accuracy and short color development time. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a thin layer chromatography identification effect diagram of Example 1.

[0033] Figure 2 This is a thin layer chromatography identification effect diagram of Comparative Example 1.

[0034] Figure 3 This is a thin layer chromatography identification effect diagram of Comparative Example 2. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0036] In the embodiment of the present invention, the Qinggan Zhilin Decoction sample is derived from Hainan Huluwa Pharmaceutical Group Co., Ltd.

[0037] Example 1

[0038] (1) Test solution: Take 3 g of Qinggan Zhilin Decoction sample, add 50 mL of water, ultrasonically treat for 30 minutes, centrifuge for 5 minutes, take the supernatant, shake and extract twice with ethyl acetate, 20 mL each time, discard the ethyl acetate solution, shake and extract the water solution with n-butanol twice, 20 mL each time, combine the n-butanol solutions, shake and extract twice with ammonia water, 20 mL each time, combine the ammonia water solutions, evaporate to dryness, dissolve the residue in 10 mL of water, pass it through a D101 macroporous adsorption resin column (column inner diameter: 1.5 cm, column height: 10 cm, elution flow rate: 2 mL / min), elute with 50 mL of water in turn, discard the eluent, and then use 30% Elute with 100 mL of ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 5 mL of ethanol, pass through a neutral alumina column (inner diameter 1.5 cm, 2 g, 100-200 mesh, elution flow rate 2 mL / min), elute with 60 mL of ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 3 mL of water, and pass through a C18 solid phase extraction column (1000 mg / 6 mL, pre-washed with 10 mL of methanol, balanced with 20 mL of water, elution flow rate 1 mL / min), elute with 5 mL of water and 10% methanol in turn, collect the eluate, combine the eluate and evaporate to dryness, dissolve the residue in 1 mL of ethanol as the test solution;

[0039] (2) Alcohol-deficient Rehmannia glutinosa sample solution: 3 g of the negative sample of alcohol-deficient Rehmannia glutinosa was taken and prepared into the alcohol-deficient Rehmannia glutinosa sample solution according to the preparation method of the test solution; the only difference is that the Qinggan Zhilin Decoction sample is replaced with the negative sample of alcohol-deficient Rehmannia glutinosa;

[0040] (3) Wine-grown Rehmannia glutinosa control medicinal material solution: Take 1 g of wine-grown Rehmannia glutinosa control medicinal material, add 50 mL of water, decoct for 30 minutes, centrifuge for 5 minutes, take the supernatant, shake and extract twice with ethyl acetate, 20 mL each time, discard the ethyl acetate solution, shake and extract twice with n-butanol, 20 mL each time, combine the n-butanol solutions, shake and extract twice with ammonia water, 20 mL each time, combine the ammonia water solutions, evaporate to dryness, dissolve the residue in 10 mL of water, pass through a D101 macroporous adsorption resin column (column inner diameter 1. 5cm, column height 10cm, elution flow rate 2mL / min), eluted with 50mL of water in turn, discarded the eluent, and then eluted with 100mL of 30% ethanol, collected the eluent, evaporated to dryness, added 5mL of ethanol to dissolve the residue, passed through a neutral alumina column (inner diameter 1.5cm, 2g, 100-200 mesh, elution flow rate 2mL / min), added 60mL of ethanol to elute, collected the eluent, evaporated to dryness, and added 1mL of ethanol to dissolve the residue as the wine-grown Rehmannia glutinosa control medicinal material solution;

[0041] (4) The preparation method of the catalpol reference solution is as follows: take the catalpol reference substance, add ethanol to make a solution with a concentration of 0.5 mg / mL, and use it as the catalpol reference solution;

[0042] (5) Take 2 μL of the catalpol reference solution, wine-grown Rehmannia glutinosa reference medicinal material solution, test solution and wine-deficient Rehmannia glutinosa sample solution prepared in the above steps, respectively, and spot them on the same silica gel G thin layer plate, use succinic acid-isopropanol-chloroform with a volume ratio of 1:2:12 as the developing solvent, develop, take out, dry, spray with sulfuric acid-acetic acid-ethanol solution with a volume ratio of 8:2:90, heat at 105°C for 3 minutes, and immediately place under ultraviolet light (365 nm) for inspection.

[0043] The results of thin layer chromatography test were as follows Figure 1 As shown, Figure 1 The chromatographic bands in the figure are, from left to right, respectively, catalpol reference solution, wine-grown Rehmannia glutinosa reference medicinal material solution, test solution (each tested three times) and wine-deficient Rehmannia glutinosa sample solution. It can be seen from the results in the figure that the method of the present invention has high fluorescence point clarity, no obvious tailing, and no negative interference (the wine-deficient Rehmannia glutinosa sample has no spots at the same position), which can achieve accurate identification of wine-grown Rehmannia glutinosa in Qinggan Zhilin Decoction.

[0044] Example 2

[0045] The preparation methods of the catalpol reference solution, the wine-grown Rehmannia glutinosa control medicinal material solution, the test solution and the wine-deficient Rehmannia glutinosa sample solution in this embodiment are similar to those in Example 1, with the only difference being that 2 g of the Qinggan Zhilin Decoction sample is taken to prepare the test solution, 2 g of the wine-deficient Rehmannia glutinosa negative sample is taken to prepare the wine-deficient Rehmannia glutinosa sample solution, 0.5 g of the wine-grown Rehmannia glutinosa control medicinal material is taken to prepare the wine-grown Rehmannia glutinosa control medicinal material solution, and the concentration of the catalpol reference solution is 0.2 mg / mL; when preparing the wine-grown Rehmannia glutinosa control medicinal material solution, the test solution and the wine-deficient Rehmannia glutinosa sample solution, the flow rate through the D101 macroporous adsorption resin column is 1 mL / min; the flow rate through the neutral alumina column is 3 mL / min; when preparing the test solution and the wine-deficient Rehmannia glutinosa sample solution, the flow rate through the C18 solid phase extraction column is 2 mL / min.

[0046] Take 4 μL of the catalpol reference solution, wine-grown Rehmannia glutinosa reference medicinal material solution, test solution and wine-deficient Rehmannia glutinosa sample solution prepared in the above steps, respectively, and spot them on the same silica gel G thin layer plate, use succinic acid-isopropanol-chloroform with a volume ratio of 1:4:10 as the developing solvent, develop, take out, dry, spray with sulfuric acid-acetic acid-ethanol solution with a volume ratio of 12:4:84, heat at 110°C for 3 minutes, and immediately examine under ultraviolet light (365nm).

[0047] Example 3

[0048] The preparation methods of the catalpol reference solution, the wine-grown Rehmannia glutinosa control medicinal material solution, the test solution and the wine-deficient Rehmannia glutinosa sample solution in this embodiment are similar to those in Example 1, with the only difference being that 4 g of the Qinggan Zhilin Decoction sample is taken to prepare the test solution, 4 g of the wine-deficient Rehmannia glutinosa negative sample is taken to prepare the wine-deficient Rehmannia glutinosa sample solution, 1.5 g of the wine-grown Rehmannia glutinosa control medicinal material is taken to prepare the wine-grown Rehmannia glutinosa control medicinal material solution, and the concentration of the catalpol reference solution is 0.8 mg / mL; when preparing the wine-grown Rehmannia glutinosa control medicinal material solution, the test solution and the wine-deficient Rehmannia glutinosa sample solution, the flow rate through the D101 macroporous adsorption resin column is 3 mL / min; the flow rate through the neutral alumina column is 1 mL / min; when preparing the test solution and the wine-deficient Rehmannia glutinosa sample solution, the flow rate through the C18 solid phase extraction column is 2 mL / min.

[0049] Take 6 μL of the catalpol reference solution, wine-grown Rehmannia glutinosa reference medicinal material solution, test solution and wine-deficient Rehmannia glutinosa sample solution prepared in the above steps, respectively, and spot them on the same silica gel G thin layer plate, use succinic acid-isopropanol-chloroform with a volume ratio of 1:4:7 as the developing solvent, develop, take out, dry, spray with sulfuric acid-acetic acid-ethanol solution with a volume ratio of 8:2:86, heat at 105°C for 5 minutes, and immediately examine under ultraviolet light (365nm).

[0050] Comparative Example 1

[0051] The detection method of Comparative Example 1 is the same as that of Example 1, except that when the test solution and the wine-deficient Rehmannia glutinosa sample solution are prepared, the C18 solid phase extraction column operation is not performed, and the residue after the eluate of the neutral alumina column is evaporated to dryness is directly dissolved in 1 mL of ethanol as the test solution and the wine-deficient Rehmannia glutinosa sample solution; the other steps are the same as in Example 1.

[0052] The thin layer chromatography test results of this comparative example are as follows Figure 2 As shown, Figure 2 The chromatographic bands in the figure are, from left to right, respectively, catalpol reference solution, wine-grown Rehmannia glutinosa reference medicinal material solution, test solution (each tested twice) and sample solution lacking wine-grown Rehmannia glutinosa. It can be seen from the results in the figure that when the C18 solid phase extraction column is not passed through the column, although the catalpol reference solution, wine-grown Rehmannia glutinosa reference medicinal material solution and test solution all have spots of the same color at the corresponding positions, the background color of the test solution and the sample solution lacking wine-grown Rehmannia glutinosa is heavier, indicating that the C18 solid phase extraction column can achieve further separation and purification of the target components, thereby making the spots clear.

[0053] Comparative Example 2

[0054] The detection method of Comparative Example 2 is the same as that of Example 1, except that the sample solution of Rehmannia glutinosa without alcohol is not prepared, ethyl acetate-methanol-water-glacial acetic acid with a volume ratio of 5:1:2:1 is used as the developing agent, and ethanolic sulfuric acid solution is used as the color developer.

[0055] The results of thin layer chromatography test were as follows Figure 3 As shown, Figure 3 The chromatographic bands in the figure are, from left to right, respectively, catalpol reference solution, wine-grown Rehmannia glutinosa reference medicinal material solution and test solution (each tested twice). It can be seen from the results in the figure that due to the complex components of the Qinggan Zhilin Decoction in this application, there are no obvious spots on the chromatographic bands of the test solution at the corresponding positions of the chromatograms of the wine-grown Rehmannia glutinosa reference medicinal material and the catalpol reference solution, and the background color is heavy. The corresponding developing agent and color developer are not suitable for the identification of the wine-grown Rehmannia glutinosa in the Qinggan Zhilin Decoction in this application.

[0056] Sample analysis

[0057] The inspection results of Examples 1-3 and Comparative Examples 1-2 are shown in Table 1:

[0058] Table 1 Inspection results of Examples 1-3 and Comparative Examples 1-2

[0059]

[0060] From the data in Table 1, it can be seen that the test solution and the positive sample solution (catalpol reference solution or wine-grown Rehmannia glutinosa reference medicinal material solution) in the embodiment all show clear spots at the corresponding positions, while the negative sample solution shows no spots and no obvious tailing; although spots of the same color and negative interference appear at the corresponding positions in Comparative Example 1, the background color of the test solution and the negative sample solution is heavier, and there is interference from impurity components; and there are no obvious spots in Comparative Example 2, which cannot be used to identify the wine-grown Rehmannia glutinosa in Qinggan Zhilin Decoction.

[0061] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying the raw Rehmannia glutinosa in Qinggan Zhilin Decoction, characterized in that: The following steps are involved: (1) Preparation of test solution Take a sample of Qinggan Zhilin Decoction, add water, ultrasonically treat, centrifuge, and take the supernatant; shake the supernatant with ethyl acetate, discard the ethyl acetate solution, shake the water solution with n-butanol, shake the n-butanol solution with ammonia water to obtain an ammonia solution, evaporate to dryness, dissolve the residue in water and pass through a column, finally evaporate the collected eluate to dryness, dissolve the residue in an organic solvent to obtain a test solution; (2) Preparation of control sample solution The control sample solution is a negative sample solution and a positive sample solution, the negative sample solution is a raw Rehmannia root sample solution lacking alcohol, and the positive sample solution is a raw Rehmannia root control medicinal material solution and a catalpol reference substance solution; (3) Identification of wine-grown Rehmannia glutinosa Spot the test solution and the control sample solution on the same silica gel G thin layer plate, develop with succinic acid-isopropanol-chloroform as the developing agent, dry, spray with sulfuric acid-acetic acid-ethanol solution, heat to develop color, and inspect; The developing agent is succinic acid-isopropanol-chloroform in a volume ratio of 1:2~4:7~12.

2. The identification method of the wine-cured Rehmannia root in Qinggan Zhilin Decoction according to claim 1, characterized in that: The amount of water corresponding to each 1 g of Qinggan Zhilin Decoction sample is 12.5-25 g; the ultrasonic treatment time is 20-40 min; the ethyl acetate solution is discarded after shaking extraction with ethyl acetate 2-3 times; the extracts are combined after shaking extraction with n-butanol 2-3 times to obtain n-butanol solution; the extracts are combined after shaking extraction with ammonia water 2-3 times to obtain ammonia water solution; the organic solvent is ethanol, and the organic solvent corresponding to each 1 g of Qinggan Zhilin Decoction sample is 0.25-0.5 mL.

3. The identification method of wine-cured Rehmannia root in Qinggan Zhilin Decoction according to claim 1, characterized in that: In step (1), the column step is specifically as follows: the residue is dissolved in water and passed through a D101 macroporous adsorption resin column, eluted with water and discarded the eluent, then eluted with 30% ethanol solution, the eluent is collected and evaporated to dryness; the residue is dissolved in ethanol and passed through a neutral alumina column, eluted with ethanol and then the eluent is collected and evaporated to dryness; the residue is dissolved in water and passed through a C18 solid phase extraction column, eluted with water and 10% methanol solution in sequence, and the eluent is collected.

4. The method for identifying the wine-cured Rehmannia root in Qinggan Zhilin Decoction according to claim 1, characterized in that: In step (2), a raw Rehmannia glutinosa sample solution lacking alcohol is prepared using the raw Rehmannia glutinosa sample lacking alcohol; a raw Rehmannia glutinosa reference medicinal material solution is prepared using the raw Rehmannia glutinosa sample with alcohol; and the preparation method of the catalpol reference solution is as follows: take the catalpol reference, add ethanol to prepare a solution with a concentration of 0.2-0.8 mg / mL, and use it as the catalpol reference solution.

5. The identification method of the wine-cured Rehmannia root in Qinggan Zhilin Decoction according to claim 3, characterized in that: When using D101 macroporous adsorption resin column for elution, the elution flow rate is 1~3 mL / min.

6. The identification method of the wine-cured Rehmannia root in Qinggan Zhilin Decoction according to claim 3, characterized in that: The mesh number of the packing in the neutral alumina column is 100~200 mesh. When the neutral alumina column is used for elution, the elution flow rate is 1~3 mL / min.

7. The method for identifying the wine-cured Rehmannia root in Qinggan Zhilin Decoction according to claim 3, characterized in that: Before using the C18 solid phase extraction column, it needs to be pre-washed with methanol and balanced with water; when eluting with the C18 solid phase extraction column, the elution flow rate is 1~2mL / min.

8. The method for identifying the wine-cured Rehmannia root in Qinggan Zhilin Decoction according to claim 1, characterized in that: The color developer is a sulfuric acid-acetic acid-ethanol solution with a volume ratio of 8~12:2~4:84~90.

9. The method for identifying the wine-cured Rehmannia root in Qinggan Zhilin Decoction according to claim 1, characterized in that: The heating temperature is 105~110 ℃ and the time is 3~5 min.

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