Method for simultaneously identifying components of radix rehmanniae and radix rehmanniae preparata and application thereof in quality evaluation of traditional Chinese medicine compound preparation

Abstract

The present application belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a method for simultaneously identifying components of radix rehmanniae recen and radix rehmanniae preparata and application thereof in quality evaluation of traditional Chinese medicine compound preparation. The present application simultaneously identifies three components of stachydrine, raffinose and manninotriose in the components of radix rehmanniae recen and radix rehmanniae preparata by adopting thin layer chromatography, uses isopropyl alcohol-water-methanol liquid with a volume ratio of 3:2:2 as developing agent, develops on a silica gel G thin layer plate, develops color, and performs identification. The thin layer chromatography developed by the present application realizes simultaneous identification of radix rehmanniae recen and radix rehmanniae preparata, and can be further used in angelica six rehmannia decoction or other traditional Chinese medicine compound preparation containing components of radix rehmanniae recen and radix rehmanniae preparata. After thin layer chromatography analysis, the spot develops color clearly, and is simple, fast, efficient, stable and good in repeatability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a method for simultaneously identifying components of radix rehmanniae and radix rehmanniae praeparata and application thereof in quality evaluation of traditional Chinese medicine compound preparation. BACKGROUND

[0002] Danggui Liu Huang decoction is a decoction from Jin Dynasty Li Dongyuan's Lan Shi Mi Cang, and the medicinal materials used are all from the Pharmacopoeia: Angelica sinensis, Radix Rehmanniae, Radix Rehmanniae Praeparata, Coptis chinensis, Scutellaria baicalensis, Phellodendri chinensis, Astragalus membranaceus, a total of 7 medicines, and its efficacy is mainly for: nourishing yin and purging fire, consolidating the exterior and stopping sweating, treating yin deficiency with excessive fire and night sweat, fever, night sweat, flushed face, heart palpitations, dry mouth, constipation, and yellowish urine. Traditional Chinese medicine decoction has complex components and many interference factors, and effective means should be used to reflect the authenticity and stability of its quality and provide rich identification information. Patent document CN117420253A discloses a thin layer identification method for Danggui Liu Huang decoction and its compound preparation, which takes Astragalus membranaceus as the control medicinal material, takes astragaloside in it as the control sample, and takes the volume ratio of ethyl acetate, n-propanol and water as 5-10:1-8:1-6 as the developing agent for identification. However, Danggui Liu Huang decoction contains 7 medicines, and it is difficult to comprehensively analyze Danggui Liu Huang decoction by using astragaloside as the identification, and at the same time, Danggui Liu Huang decoction contains Radix Rehmanniae and Radix Rehmanniae Praeparata, and the two have many similar components, so the method cannot accurately identify Radix Rehmanniae and Radix Rehmanniae Praeparata in Danggui Liu Huang decoction. In addition, the identification method of Radix Rehmanniae Praeparata in Chinese Pharmacopoeia only records the identification method of Radix Rehmanniae Praeparata, which uses verbascoside as the control sample, ethyl acetate-methanol-formic acid (16:0.5:2) as the developing agent, and uses 0.1% 2,2-diphenyl-1-pyrrolidinol ethanol solution for color development and air drying, which cannot simultaneously identify Radix Rehmanniae and Radix Rehmanniae Praeparata in Danggui Liu Huang decoction, and it is also difficult to comprehensively evaluate Danggui Liu Huang decoction.

[0003] At present, the quality of Danggui Liu Huang decoction is mainly evaluated by high performance liquid chromatography, and the quality markers are mainly arvidic acid, astragaloside, catalpol, wogonin, rehderiside D, baicalin, phellodendrine, wogonoside, and berberine, and at present, it is difficult to quickly identify Radix Rehmanniae and Radix Rehmanniae Praeparata in Danggui Liu Huang decoction. Therefore, it is of great significance to establish a rapid and simple method for identifying Radix Rehmanniae and Radix Rehmanniae Praeparata in Danggui Liu Huang decoction for the comprehensive and scientific identification of Danggui Liu Huang decoction and the quality evaluation of traditional Chinese medicine compound preparation containing Radix Rehmanniae and Radix Rehmanniae Praeparata. SUMMARY

[0004] In view of the problems in the prior art, the application provides a method for simultaneously identifying components of radix rehmanniae and radix rehmanniae praeparata and application thereof in evaluation of traditional Chinese medicine compound preparations, which can simultaneously qualitatively identify stachydrine, raffinose and manninotriose in radix rehmanniae and radix rehmanniae praeparata, and lays a foundation for research on reference substances and preparations of traditional Chinese medicine compound preparations.

[0005] In a first aspect, the application provides a thin layer chromatography method for simultaneously identifying stachydrine, raffinose and manninotriose, wherein the steps of the thin layer chromatography method include: taking stachydrine, raffinose and manninotriose reference substances respectively, adding methanol to prepare a mixed solution, taking the mixed solution and spotting it on a thin layer plate, using isopropyl alcohol-water-methanol as a developing agent to develop, taking out, air-drying, developing color, and observing.

[0006] Further, the developing color is any one of 1,3-naphthalenediol and 1-naphthol.

[0007] Further, the mixed solution contains 1 mg of stachydrine, 1 mg of raffinose and 1 mg of manninotriose per 1 mL of the mixed solution.

[0008] Further, the volume ratio of the developing agent is 3:2:2.

[0009] In a second aspect, the application provides a method for simultaneously identifying components of radix rehmanniae and radix rehmanniae praeparata, which includes the following steps:

[0010] S1, preparation of a test sample solution

[0011] Preparation method of a corresponding decoction: 2.33 g of angelica, radix rehmanniae, radix rehmanniae praeparata, radix scrophulariae, radix scutellariae and cortex phellodendri, and 4.66 g of radix astragali are taken and placed in a 1 L electric ceramic medicine decocting pot, 300 mL of water is added, and decoction is performed until 150 mL of liquid is obtained, and then filtration is performed to obtain a corresponding decoction of angelica six radix scrophulariae.

[0012] About 8 g of the corresponding decoction of angelica six radix scrophulariae is taken, methanol is added, ultrasonic treatment is performed, and filtration is performed to obtain a test sample solution;

[0013] S2, preparation of a reference solution

[0014] The corresponding decoction of radix rehmanniae is taken, methanol is added, ultrasonic treatment is performed, and filtration is performed to obtain a corresponding reference solution of the decoction of radix rehmanniae;

[0015] The corresponding decoction of radix rehmanniae praeparata is taken, methanol is added, ultrasonic treatment is performed, and filtration is performed to obtain a corresponding reference solution of the decoction of radix rehmanniae praeparata;

[0016] According to the thin layer chromatography method for simultaneously identifying stachydrine, raffinose and manninotriose in claim 1, stachydrine, raffinose and manninotriose reference substances are taken respectively, methanol is added to prepare a reference mixed solution of the reference substances;

[0017] S3, thin layer chromatography identification

[0018] The solutions are taken respectively and spotted on the same thin layer plate, and isopropanol-water-methanol is used as the developing agent to develop, take out, dry, color, and observe.

[0019] Further, the amount of methanol added in the step S1 is 2-5 times of the amount of the actual decoction.

[0020] Further, 1 mg of stachyose, 1 mg of raffinose and 1 mg of manninotriose are contained in each 1 mL of the reference mixed solution prepared in the step S2.

[0021] Further, the thin layer plate in the step S3 includes a silica gel G thin layer plate (Qingdao Haoyang), a silica gel G thin layer plate (Yantai Huanghai), and a silica gel G thin layer plate (Merck).

[0022] Preferably, the thin layer plate in the step S3 is the silica gel G thin layer plate (Merck).

[0023] Preferably, the developing agent in the step S3 is isopropanol, water, and methanol with a volume ratio of 3:2:2.

[0024] Further, any one of 1,3-naphthalenediol and 1-naphthol is used as a color developing agent for color development in the step S3.

[0025] In a third aspect, the application provides a method for simultaneously identifying the components of raw rehmannia and prepared rehmannia, and application of the method to a traditional Chinese medicine compound preparation containing raw rehmannia and prepared rehmannia.

[0026] Further, the traditional Chinese medicine compound preparation includes Dangui Liuhuang Decoction, Shengyu Decoction, and Daru Decoction, but is not limited to the above listed compound preparations, and other compound preparations not listed in the traditional Chinese medicine compound preparation containing raw rehmannia and prepared rehmannia are also applicable.

[0027] Compared with the prior art, the application has the following beneficial effects:

[0028] The application develops a thin layer chromatography method for simultaneously identifying stachyose, raffinose, and manninotriose, using isopropanol-water-methanol liquid with a volume ratio of 3:2:2 as the developing agent, silica gel G thin layer plate for development, color development, identification of raw rehmannia and prepared rehmannia, compared with the prior art, the application first develops a thin layer chromatography detection method for raw rehmannia and prepared rehmannia, further, the developed thin layer chromatography method realizes the simultaneous identification of raw rehmannia and prepared rehmannia, which can be further used in Dangui Liuhuang Decoction containing raw rehmannia and prepared rehmannia, after thin layer chromatography analysis, the spot color is clear, non-tailing, and no cross, simple, fast, efficient, good stability and repeatability, and the qualitative and quantitative techniques of Dangui Liuhuang Decoction are further improved. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Thin layer chromatogram of the sample of Example 1.

[0030] Figure 2 Thin layer chromatogram of the sample of Example 2.

[0031] Figure 3 Thin layer chromatogram of the sample of Comparative Example 1.

[0032] Figure 4 Thin layer chromatogram of the sample of Comparative Example 2.

[0033] Figure 5 Thin layer chromatogram of the sample of Comparative Example 3.

[0034] Figure 6 Thin layer chromatogram of the sample of Comparative Example 4.

[0035] Figure 7 Thin layer chromatogram of the sample of Comparative Example 5.

[0036] Figure 8 Thin layer chromatogram of the sample of Comparative Example 6. DETAILED DESCRIPTION

[0037] The experimental methods in the following examples of the present application, unless otherwise specified, are usually carried out according to the conventional conditions, or according to the conditions suggested by the manufacturers. The various common chemical reagents used in the examples are commercially available products.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0039] As used in the description of the application and the appended claims, the singular forms "a", "an" and "the" are used. Plural references herein include singular forms, unless otherwise stated. It will be further understood that the terms "comprises" and "comprising", when used in this specification, take their broadest meaning from the context where they are used induced to encompass the terms "consisting of" and "consisting essentially of" and vice versa.

[0040] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the specific embodiments. It should be understood that the description is only exemplary, but not to limit the scope of the present application. In addition, in the following description, the description of well-known structures and techniques is omitted to avoid unnecessary obscurity of the concept of the present application.

[0041] All other embodiments obtained by a person of ordinary skill in the art without making creative efforts based on the embodiments of the present application fall within the protection scope of the present application. The following embodiments further describe the present application, but the embodiments are not used to limit the protection scope of the present application.

[0042] Information of the test sample, the control sample and the control medicinal material used in the embodiment of the identification method of the present application:

[0043] The corresponding physical decoction of Angelica Six Huang Decoction (batch number: DL191030-1);

[0044] The preparation method of the corresponding physical decoction of Angelica Six Huang Decoction is as follows: 2.33 g of Angelica sinensis, 2.33 g of Radix Rehmanniae, 2.33 g of Radix Rehmanniae Praeparata, 2.33 g of Scutellaria baicalensis, 2.33 g of Coptis chinensis, 2.33 g of Phellodendri, and 4.66 g of Astragalus membranaceus are placed in a 1 L electric ceramic decoction pot, 300 mL of water is added, and decoction is performed until 150 mL of liquid is obtained. Filtration is performed, and the corresponding physical decoction of Angelica Six Huang Decoction is obtained.

[0045] The corresponding physical decoction of Angelica Six Huang Decoction with negative Radix Rehmanniae (batch number: DL191015Y-DH);

[0046] The corresponding physical decoction of Angelica Six Huang Decoction with negative Radix Rehmanniae Praeparata (batch number: DL191015Y-SD);

[0047] The corresponding physical decoction of Radix Rehmanniae single formula (batch number: DL191014D-DH);

[0048] The corresponding physical decoction of Radix Rehmanniae Praeparata single formula (batch number: DL191014D-SD).

[0049] The preparation methods of the physical decoctions are consistent with the preparation method of the corresponding physical decoction of Angelica Six Huang Decoction.

[0050] Embodiment 1

[0051] The method and steps for simultaneously identifying Radix Rehmanniae and Radix Rehmanniae Praeparata by using Angelica Six Huang Decoction in this embodiment are as follows:

[0052] S1, preparation of the test sample solution

[0053] About 8 g of the corresponding physical decoction of Angelica Six Huang Decoction (batch number: DL191030-1) is taken, 4 times the amount of methanol is added, ultrasonic treatment is performed for 10 minutes, and filtration is performed, to obtain the test sample solution of Angelica Six Huang Decoction;

[0054] The corresponding physical decoction of Angelica Six Huang Decoction with negative Radix Rehmanniae (batch number: DL191015Y-DH) is taken, and the negative solution of Radix Rehmanniae is prepared by the same method;

[0055] The corresponding physical decoction of Angelica Six Huang Decoction with negative Radix Rehmanniae Praeparata (batch number: DL191015Y-SD) is taken, and the negative solution of Radix Rehmanniae Praeparata is prepared by the same method;

[0056] S2, Preparation of Reference Solution

[0057] Take the corresponding real decoction of Shengdihuang single formula (batch number: DL191014D-DH) and prepare the corresponding real reference solution of Shengdihuang single formula by the same method;

[0058] Take the corresponding real decoction of Shushugan single formula (batch number: DL191014D-SD) and prepare the corresponding real reference solution of Shushugan single formula by the same method;

[0059] Take the appropriate amount of viosugar, raffinose and manninotriose reference substances, and add methanol to prepare a reference mixture solution containing 1 mg of viosugar, 1 mg of raffinose and 1 mg of manninotriose per 1 mL;

[0060] S3, Identification by Thin Layer Chromatography

[0061] Take 5 μL of the above solution and spot it on the same silica gel G thin layer plate. Use isopropyl alcohol-water-methanol (3:2:2) as the developing agent, develop, take out, air dry, spray with 1,3-naphthalenediol test solution, heat until the spots develop clearly, and observe under daylight;

[0062] The results of the determination according to the above identification method are shown in Figure 1 The results show that the test sample chromatogram of Danggui Liuhuang Decoction (i.e., the compound corresponding to the real object in the figure) shows the same color spots at the corresponding positions of the negative solution, the single formula corresponding to the real object reference solution and the reference mixture solution chromatogram.

[0063] Example 2

[0064] The method and procedure for simultaneously identifying Shengdihuang and Shushugan by Danggui Liuhuang Decoction in this example are as follows:

[0065] S1, Preparation of Test Sample Solution

[0066] Take about 8 g of the corresponding real decoction of Danggui Liuhuang Decoction (batch number: DL191030-1), add 4 times the amount of methanol, and ultrasonically treat for 10 minutes. Filter to obtain the test sample solution of Danggui Liuhuang Decoction;

[0067] Take the appropriate amount of Danggui Liuhuang Decoction Shengdihuang negative corresponding real decoction (batch number: DL191015Y-DH), and prepare the Shengdihuang negative solution by the same method;

[0068] Take the appropriate amount of Danggui Liuhuang Decoction Shushugan negative corresponding real decoction (batch number: DL191015Y-SD), and prepare the Shushugan negative solution by the same method;

[0069] S2, Preparation of Reference Solution

[0070] Take the corresponding real decoction of Shengdihuang single formula (batch number: DL191014D-DH) and prepare the corresponding real reference solution of Shengdihuang single formula by the same method;

[0071] Take the corresponding real decoction of Shuganliu single prescription (batch number: DL191014D-SD) as appropriate, and prepare the corresponding real reference solution of Shuganliu single prescription by the same method;

[0072] S3, thin layer chromatography identification

[0073] Take 5 μL of the above solution and spot it on the same silica gel G thin layer plate. Use isopropyl alcohol-water-methanol (3:2:2) as the developing agent, develop, take out, air dry, spray with 1-naphthol test solution, heat until the spots are clearly colored, and observe under daylight;

[0074] The results of the above identification method are shown in Figure 2 The results show that the test sample chromatogram of Danggui Liuhuang Decoction (i.e., the compound corresponding to the real object in the figure) shows the same colored spots at the corresponding positions of the chromatograms of the single prescription corresponding to the real object reference solution and the reference mixed solution.

[0075] Comparative Example 1

[0076] The method and steps for simultaneously identifying Shudihuang and Shuganliu in Danggui Liuhuang Decoction are as follows:

[0077] S1, preparation of test sample solution

[0078] Take about 8 g of the corresponding real decoction of Danggui Liuhuang Decoction (batch number: DL191030-1) and extract it with an equal volume of water-saturated n-butanol for 3 times. Combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 2 mL of methanol, and filter to obtain the test sample solution of Danggui Liuhuang Decoction;

[0079] Take an appropriate amount of the Shudihuang negative corresponding real decoction of Danggui Liuhuang Decoction (batch number: DL191015Y-DH) and prepare the Shudihuang negative solution by the same method;

[0080] Take an appropriate amount of the Shuganliu negative corresponding real decoction of Danggui Liuhuang Decoction (batch number: DL191015Y-SD) and prepare the Shuganliu negative solution by the same method;

[0081] S2, preparation of reference solution

[0082] Take an appropriate amount of the corresponding real decoction of Shudihuang single prescription (batch number: DL191014D-DH) and prepare the corresponding real reference solution of Shudihuang single prescription by the same method;

[0083] Take an appropriate amount of the corresponding real decoction of Shuganliu single prescription (batch number: DL191014D-SD) and prepare the corresponding real reference solution of Shuganliu single prescription by the same method;

[0084] Weigh 10 mg of verbascoside and accurately weigh it into a 50 mL volumetric flask. Add methanol solution and dilute to the mark, shake well, and you get it.

[0085] S3, Thin Layer Chromatography Identification

[0086] Take 10 μL of the above solution and spot on the same polyamide film, use methanol-glacial acetic acid-water (2:1:7) as developing agent, develop, take out, dry, and observe under UV light (365 nm).

[0087] The determination results by the above identification method are shown in Figure 3 The results show that: the developing effect of this method is not good, and the test sample chromatographic characteristic spots cannot be shown (the compound corresponding to the real object is the test sample solution of Danggui Liuhuang Decoction), and the method is not applicable.

[0088] Comparative Example 2

[0089] The method and procedure for simultaneously identifying Rehmanniae Radix and Rehmanniae Radix Praeparata by Danggui Liuhuang Decoction are as follows:

[0090] S1, Preparation of Test Sample Solution

[0091] Take about 8 g of Danggui Liuhuang Decoction corresponding to the real object (batch number: DL191030-1) and place it in a separatory funnel. Add ethyl acetate and shake to extract 2 times, 25 mL each time. Collect the ethyl acetate liquid, evaporate in a water bath, dissolve the residue in 2 mL of methanol, and filter to obtain the test sample solution of Danggui Liuhuang Decoction.

[0092] Take an appropriate amount of Rehmanniae Radix negative decoction corresponding to the real object of Danggui Liuhuang Decoction (batch number: DL191015Y-DH) and prepare the Rehmanniae Radix negative solution by the same method.

[0093] Take an appropriate amount of Rehmanniae Radix praeparata negative decoction corresponding to the real object of Danggui Liuhuang Decoction (batch number: DL191015Y-SD) and prepare the Rehmanniae Radix praeparata negative solution by the same method.

[0094] S2, Preparation of Reference Solution

[0095] Take an appropriate amount of Rehmanniae Radix single formula decoction corresponding to the real object (batch number: DL191014D-DH) and prepare the Rehmanniae Radix single formula decoction corresponding to the real object reference solution by the same method.

[0096] Take an appropriate amount of Rehmanniae Radix praeparata single formula decoction corresponding to the real object (batch number: DL191014D-SD) and prepare the Rehmanniae Radix praeparata single formula decoction corresponding to the real object reference solution by the same method.

[0097] S3, Thin Layer Chromatography Identification

[0098] Take 15 μL of the above solution and spot on the same silica gel G thin layer plate, use toluene-ethyl acetate (2:2) as developing agent, develop, take out, dry, and observe under UV light (365 nm).

[0099] The determination results by the above identification method are shown in Figure 4The results show that: (1) The fluorescence spots shown in the chromatogram of the test sample correspond to the spots in the chromatogram of the negative solution. No fluorescence spots corresponding to the single formula corresponding physical object were found, which lacks characteristics; (2) The chromatogram of the single formula corresponding physical object of Rehmanniae Radix and Radix Rehmanniae Preparata shows fewer spots, and there is no obvious difference between the two graphs, so it is not suitable.

[0100] Comparative Example 3

[0101] The method and procedure for simultaneously identifying Rehmanniae Radix and Radix Rehmanniae Preparata with Danggui Liu Huang Tang are as follows:

[0102] S1, Preparation of test solution

[0103] Take about 8g of Danggui Liu Huang Tang corresponding physical decoction (batch number: DL191030-1) in an evaporating dish, evaporate to dryness on a water bath, dissolve the residue in an appropriate amount of methanol, filter, evaporate the filtrate to dryness, add 15mL of water to the residue, extract with water-saturated n-butanol twice, 15mL each time, combine the n-butanol layers, wash twice with 15mL of ammonia solution each time, discard the ammonia solution, adjust to neutral with dilute hydrochloric acid, evaporate to dryness, dissolve the residue in 2mL of methanol, and filter.

[0104] Take an appropriate amount of Rehmanniae Radix negative corresponding physical decoction of Danggui Liu Huang Tang (batch number: DL191015Y-DH), and prepare the Rehmanniae Radix negative solution by the same method;

[0105] Take an appropriate amount of Radix Rehmanniae Preparata negative corresponding physical decoction of Danggui Liu Huang Tang (batch number: DL191015Y-SD), and prepare the Radix Rehmanniae Preparata negative solution by the same method;

[0106] S2, Preparation of reference solution

[0107] Take an appropriate amount of Rehmanniae Radix single formula corresponding physical decoction (batch number: DL191014D-DH), and prepare the Rehmanniae Radix single formula corresponding physical reference solution by the same method;

[0108] Take an appropriate amount of Radix Rehmanniae Preparata single formula corresponding physical decoction (batch number: DL191014D-SD), and prepare the Radix Rehmanniae Preparata single formula corresponding physical reference solution by the same method;

[0109] S3, Thin layer chromatography identification

[0110] Take 5μL of the above solutions and apply them to the same silica gel G thin layer plate, develop with chloroform-methanol-ammonia water (9.5:1:0.5), remove, dry, and observe under ultraviolet light (365nm);

[0111] The results of the determination according to the above identification method are shown in Figure 5 The results show that the single formula spots of Rehmanniae Radix and Radix Rehmanniae Preparata are not obvious, and there are many negative interference spots, so this method is not considered.

[0112] Comparative Example 4

[0113] The reference example adopts the method and procedure of simultaneously identifying Shengdihuang and Shudihuang by Danggui Liuhuang Decoction as follows:

[0114] S1, Preparation of test solution

[0115] Take about 8g of Danggui Liuhuang Decoction corresponding to the actual decoction (batch number: DL191030-1), evaporate in an evaporating dish in a water bath, and dry. Add an appropriate amount of methanol to dissolve the residue, filter, evaporate the filtrate, and add 15ml of water to the residue. Extract with water-saturated n-butanol twice, 15ml each time, combine the n-butanol layers, wash with ammonia solution twice, 15ml each time, discard the ammonia solution, adjust to neutral with dilute hydrochloric acid, evaporate, and add 2ml of methanol to dissolve the residue. Filter to obtain the solution.

[0116] Take an appropriate amount of Danggui Liuhuang Decoction Shengdihuang negative corresponding to the actual decoction (batch number: DL191015Y-DH), and prepare Shengdihuang negative solution by the same method;

[0117] Take an appropriate amount of Danggui Liuhuang Decoction Shudihuang negative corresponding to the actual decoction (batch number: DL191015Y-SD), and prepare Shudihuang negative solution by the same method;

[0118] S2, Preparation of reference solution

[0119] Take an appropriate amount of Danggui Liuhuang Decoction Shengdihuang negative corresponding to the actual decoction (batch number: DL191015Y-DH), and prepare Shengdihuang negative solution by the same method;

[0120] Take an appropriate amount of Danggui Liuhuang Decoction Shudihuang negative corresponding to the actual decoction (batch number: DL191015Y-SD), and prepare Shudihuang negative solution by the same method;

[0121] S3, Thin layer chromatography identification

[0122] Take 5μL of the above solution and apply it to the same silica gel G thin layer plate. Use chloroform-methanol (10:1) as the developing agent, develop, remove, dry, and observe under ultraviolet light (365nm);

[0123] The results of the determination according to the above identification method are shown in Figure 6 , which shows that under the above conditions, the spots are not fully developed and all accumulate at the bottom, so this method is not considered.

[0124] Reference Example 5

[0125] The reference example adopts the method and procedure of simultaneously identifying Shengdihuang and Shudihuang by Danggui Liuhuang Decoction as follows:

[0126] S1, Preparation of test solution

[0127] Take the corresponding real decoction of Angelica Six Huang decoction (batch number: DL191030-1) about 8g, placed in separatory funnel, add ethyl acetate and shake 2 times, 25ml each time, separate the ethyl acetate liquid, water bath dry, the residue is dissolved in 2ml of methanol, filter, the test solution is obtained;

[0128] Take the corresponding real decoction of Angelica Six Huang decoction (batch number: DL191030-1) about 8g, placed in separatory funnel, add ethyl acetate and shake 2 times, 25ml each time, separate the ethyl acetate liquid, water bath dry, the residue is dissolved in 2ml of methanol, filter, the test solution is obtained;

[0129] Take the corresponding real decoction of Angelica Six Huang decoction (batch number: DL191030-1) about 8g, placed in separatory funnel, add ethyl acetate and shake 2 times, 25ml each time, separate the ethyl acetate liquid, water bath dry, the residue is dissolved in 2ml of methanol, filter, the test solution is obtained;

[0130] S2, reference solution preparation

[0131] Take the corresponding real decoction of Angelica Six Huang decoction (batch number: DL191030-1) about 8g, placed in separatory funnel, add ethyl acetate and shake 2 times, 25ml each time, separate the ethyl acetate liquid, water bath dry, the residue is dissolved in 2ml of methanol, filter, the test solution is obtained;

[0132] Take the corresponding real decoction of Angelica Six Huang decoction (batch number: DL191030-1) about 8g, placed in separatory funnel, add ethyl acetate and shake 2 times, 25ml each time, separate the ethyl acetate liquid, water bath dry, the residue is dissolved in 2ml of methanol, filter, the test solution is obtained;

[0133] S3, thin layer chromatography identification

[0134] Take the above solution 15ul, respectively, on the same silica gel G thin layer plate, with toluene-ethyl acetate (6:6) as developing agent, development, take out, dry, spray with 2,4-dinitrophenylhydrazine test solution, and observe under daylight.

[0135] The results of the above identification method are shown in Figure 7 , the results show that the negative and mature Rehmannia single prescription appear yellow spots in the same position, indicating that the spot belongs to Rehmannia, and Rehmannia has no, so the condition can be used as the identification method of Rehmannia and Rehmannia. But the color of the spot and the background color are yellow, which is not conducive to observation.

[0136] Comparative example 6

[0137] The method and steps of this example for identifying Rehmannia and Rehmannia in Angelica Six Huang decoction are as follows:

[0138] S1, preparation of test solution

[0139] Take the corresponding real decoction of Angelica Six Huang decoction (batch number: DL191030-1) about 8g, placed in separatory funnel, add ethyl acetate and shake 2 times, 25ml each time, separate the ethyl acetate liquid, water bath dry, the residue is dissolved in 2ml of methanol, filter, the test solution is obtained;

[0140] Take Angelica Six Huang decoction Shengdihuang negative corresponding real liquid (batch number: DL191015Y-DH) appropriate amount, made Shengdihuang negative solution;

[0141] Take Angelica Six Huang decoction Shudihuang negative corresponding real liquid (batch number: DL191015Y-SD) appropriate amount, made Shudihuang negative solution;

[0142] S2, reference solution preparation

[0143] Take Shengdihuang single corresponding real decoction liquid (batch number: DL191014D-DH) appropriate amount, made Shengdihuang single corresponding real reference solution by the same method;

[0144] Take Shudihuang single corresponding real decoction liquid (batch number: DL191014D-SD) appropriate amount, made Shudihuang single corresponding real reference solution by the same method;

[0145] S3, thin layer chromatography identification

[0146] 5 μL of the above solution was taken and spotted on the same silica gel G thin layer plate, with toluene-ethyl acetate (6:6) as the developing agent, developed, removed, dried, sprayed with 2,4-dinitrophenylhydrazine test solution, and observed under daylight.

[0147] The results of the determination according to the above identification method are shown in Figure 8 , which shows that: Figure 8 and Figure 7 are basically the same, that is, the sample spotted with n-butanol layer extraction, after spraying the color developing agent, the effect of the sample spot extracted with ethyl acetate is basically the same.

[0148] Obviously, the above examples are only examples for the purpose of clarity, and do not limit the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for simultaneously identifying the components of Rehmannia glutinosa and Rehmannia glutinosa Libosch, characterized in that, It comprises the following steps: S1, preparation of test sample solution Take the corresponding real decoction of Danggui Liuhuang Decoction, add methanol, ultrasonic treatment, filter, and obtain the test sample solution of Danggui Liuhuang Decoction; S2, preparation of reference solution Take the corresponding real decoction of Shengdihuang Single Formula, add methanol, ultrasonic treatment, filter, and obtain the corresponding real reference solution of Shengdihuang Single Formula; Take the corresponding real decoction of Shu-dihuang Single Formula, add methanol, ultrasonic treatment, filter, and obtain the corresponding real reference solution of Shu-dihuang Single Formula; Take the corresponding real decoction of Shu-dihuang Single Formula, add methanol, ultrasonic treatment, filter, and obtain the corresponding real reference solution of Shu-dihuang Single Formula; S3, thin layer chromatography identification Take each solution and point it on the same thin layer plate, use isopropyl alcohol-water-methanol as developing agent, take out, dry, color, and observe; The thin layer plate in step S3 is a silica gel G thin layer plate; The developing agent in step S3 is isopropyl alcohol, water, and methanol with a volume ratio of 3:2:2; The color in step S3 is developed by using any one of 1,3-naphthalenediol and 1-naphthol as color developing agent.

2. The method for simultaneously identifying the components of Rehmannia glutinosa and Rehmannia glutinosa Libosch by claim 1, characterized in that, The amount of methanol added in step S1 is 2-5 times the amount of the corresponding real decoction of Danggui Liuhuang Decoction.

3. The method for simultaneously identifying the components of Rehmannia glutinosa and Rehmannia glutinosa Libosch by claim 1, characterized in that, Each 1 mL of the reference mixed solution prepared in step S2 contains 1 mg of stachyose, 1 mg of raffinose, and 1 mg of manninotriose.

4. The method for simultaneously identifying the components of Shengdihuang and Shu-dihuang according to claim 1 is applied to a traditional Chinese medicine compound preparation containing Shengdihuang and Shu-dihuang, and the traditional Chinese medicine compound preparation is Danggui Liuhuang Decoction.

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