Method for quality control of rehmannia glutinosa in dihuang pill type preparation

Abstract

The present application belongs to the quality control technical field of traditional Chinese medicine preparation, and particularly relates to a method for controlling the quality of rehmannia glutinosa in rehmannia pill formula preparation, which comprises the following steps: pretreating the rehmannia pill formula preparation to be tested to obtain a sample solution to be tested for qualitative identification of verbascoside component, and pretreating the rehmannia pill formula preparation to be tested to obtain a sample solution two to be tested for quantitative determination of rehmannia glycoside D component. The present application can simultaneously prepare multiple rehmannia pill formula preparation sample solutions, and realizes rapid detection by using a unified qualitative and quantitative method.

Description

TECHNICAL FIELD

[0001] The present application relates to the quality control technology field of traditional Chinese medicine preparation, and particularly relates to a method for quality control of Radix Rehmanniae in Dihuang Pills. BACKGROUND

[0002] Radix Rehmanniae is known as a “Yin drug”, which is a processed product of the tuber of Rehmannia glutinosa Libosch (Huanglian) of the Orobanchaceae family. It is one of the most representative herbs for “Yin nourishing” in traditional Chinese medicine. After being combined with other herbs, it can be used in various clinical diseases caused by liver and kidney Yin deficiency, such as type 2 diabetes, diabetic nephropathy and insulin resistance, Alzheimer's disease, Parkinson's disease and vascular dementia, rheumatoid arthritis and other osteoarthritis-related diseases, postmenopausal osteoporosis, oligomenorrhea due to kidney Yin deficiency, secondary amenorrhea due to premature ovarian failure, perimenopausal syndrome, menopausal syndrome, and the like.

[0003] Chemical composition studies have shown that Radix Rehmanniae mainly contains enoxolone, paeoniflorin, catalpol, geniposide, rehmatann A, rehmatann B, rehmatann C, rehmatann D, rehmatann A, rehmatann C, rehmatann D, and other iridoid glycosides; rhodioloside A, rhodioloside B, shikimic acid, and other ionone glycosides; trilobatin, verbascoside, pyrogallol, and other phenylethanoid compounds; in addition to sugars, nucleosides, alkaloids, phenolic acids, isoflavone glycosides, and other compounds. We once administered the five components of Radix Rehmanniae, including catalpol, rehmatann A, rehmatann D, paeoniflorin, and verbascoside, to Yin deficiency model rats, and found that they could significantly reduce the contents of T3, T4 in serum and cAMP in plasma, and the ratio of cAMP / cGMP, which suggests that the above components may be the main pharmacodynamic material basis for the Yin-nourishing and heat-clearing effects of Radix Rehmanniae.

[0004] Following the principles of safety and effectiveness, advanced technology, and economic rationality, the establishment of the standard for Radix Rehmanniae medicinal materials in the Pharmacopoeia is also a dynamic and gradual improvement process. Compared with the 2005 edition, the 2010 edition of the first volume of the Pharmacopoeia deleted the thin layer

identification

identification

content determination

[0005] Dihuang pill is a group of prescriptions with Dihuang as the main drug, which can nourish kidney essence and regulate yin and yang in the kidney. Six-Ingredient Dihuang Pill, which is a representative of the Dihuang pill, is from the Small Children's Medicine Decisions of the North Song Dynasty. It is made by removing Fuzi and Guizhi from the Kidney Qi Pill of the Golden Cabinet and replacing Gan Dihuang with Rou Dihuang. The Zhiwu Dihuang Pill of the Medical Canon of Golden Mirror is made by adding Zhimu and Huangbai to the Six-Ingredient Dihuang Pill. It is used to treat the symptoms caused by kidney yin deficiency and yin deficiency with excessive fire. The Qiju Dihuang Pill of later generations can also nourish the liver and improve eyesight. The Guishao Dihuang Pill is good at nourishing blood and regulating menstruation. The Maiwei Dihuang Pill can moisten the lungs and benefit qi. The above Dihuang pill preparations are all listed in the pharmacopoeia of past dynasties. The commonality of their prescriptions is that they are all made by adding ingredients to the Six-Ingredient Dihuang Pill. The preparation methods, specifications, and dosages are similar, forming a large category in the market of Chinese patent medicines.

[0006] The 2020 edition of Chinese Pharmacopoeia includes the standards of Six-Ingredient Dihuang Large Honeyed Pill, watered pill, concentrated pill, capsule, and soft capsule. The quality control of the prescription medicines Shanzhuyu and Mudanpi is determined by the three indicators of Morroniside, Strychnine, and Danpi Phenol. In recent years, there have been HPLC content determination of Verbascoside in Six-Ingredient Dihuang Paste, and HPLC-MS / M simultaneous determination of Verbascoside and other 10 components in Qiju Dihuang Pill. However, Verbascoside is not a legal content indicator of Dihuang in the current pharmacopoeia. The configuration of triple quadrupole mass spectrometry detector is too high to be popularized and promoted. The applicability of the entire Dihuang pill preparation is not mentioned. The quality research literature of Maiwei Dihuang Pill in recent years mainly controls the content of Wuweizi alcohol A in its preparation. No relevant reports have been found for Guishao Dihuang Pill. The pharmacopoeia standard is to calculate the content of Baishao and Mudanpi in the honeyed pill preparation with Shaoyao glycoside (C 23 H 28 O 11 ), the total amount of Morroniside (C 17 H 26 O 11 ) and Strychnine (C 17 H 26 O 10 ) of Shanzhuyu, and Danpi Phenol (C9H 10 O3) of Mudanpi. In addition to the microscopic identification of the powder of related medicines, the current pharmacopoeia standard of Zhibai Dihuang Pill identifies Mudanpi and Huangbai in the preparation with Danpi Phenol, Strychnine Hydrochloride, and Huangbai reference medicine. It regulates the content standards of Morroniside (C 17 H 26 O 10 ), Danpi Phenol (C9H 10 O3). The content determination of Smilax Saponin and Verbascoside in the preparation has also been reported in recent literature. The current standard and literature do not mention the qualitative and quantitative methods of Rou Dihuang in Gui Fuzhuan.

[0007] In summary, as the monarch drug or heavy use of medicinal herbs in Dihuang Pill, although the prescription amount accounts for more than 1 / 4 or even 1 / 3, due to the complexity of the components of the compound Chinese medicine, there are few standards and literature researches on the control of "Dihuang", especially the qualitative identification of verbascoside and the quantitative determination of Dihuang glycoside D corresponding to the official standard of Dihuang decoction pieces, and there is a lack of rapid detection technology suitable for various Dihuang pill preparations. SUMMARY

[0008] To solve the technical problem of insufficient quantitative control of the monarch drug Dihuang in the prescription, the application provides a method for quality control of Dihuang in a product containing Dihuang.

[0009] A method for quality control of Dihuang in Dihuang pill preparation, comprising the following steps:

[0010] (1) Sample pretreatment: pretreating the Dihuang pill preparation to be tested to obtain a sample solution to be tested, and pretreating the Dihuang pill preparation to be tested to obtain a sample solution to be tested;

[0011] The pretreatment one is: sequentially using methanol, water saturated n-butanol for extraction, after extraction, through a mixed column containing SP700 type macroporous adsorption resin, sequentially using water, methanol for elution, dissolving the residue with water, then through a solid phase extraction column, collecting the effluent, evaporating to dryness, dissolving the residue with methanol;

[0012] The pretreatment two is: using methanol for extraction, after cooling, using 50% methanol to make up the lost weight, then through a mixed column containing SP700 type macroporous adsorption resin, sequentially using water, methanol for elution, concentrating, dissolving the residue with methanol;

[0013] (2) Using verbascoside reference substance, Dihuang reference medicinal material and sample solution to be tested for qualitative identification of verbascoside component by thin layer chromatography;

[0014] (3) Using high performance liquid chromatography for quantitative determination of Dihuang glycoside D component in the sample solution to be tested.

[0015] Preferably, in step (1), the pretreatment one is: taking 0.5-1.0g of Dihuang pill preparation to be tested corresponding to 0.5-1.0g of Dihuang crude drug, adding 60-100% methanol, tightly sealing, dissolving, extracting for 0.5-1 hour, filtering, evaporating the filtrate to dryness, dissolving the residue with water, extracting with water saturated n-butanol for 2-4 times, combining to obtain n-butanol liquid, evaporating the n-butanol liquid to dryness, dissolving the residue with water, passing through a mixed column, eluting with water, then eluting with 30% methanol, and finally eluting with methanol, collecting the methanol eluent, recovering the solvent under reduced pressure, dissolving the residue with water after passing through a solid phase extraction column, collecting the effluent, evaporating to dryness, dissolving the residue with methanol to obtain the sample solution to be tested.

[0016] Preferably, the sample of the prepared Rehmannia glutinosa pill formula to be tested is taken, 0.5-1.0 g of which is equivalent to the amount of raw Rehmannia glutinosa, 50-100 mL of 60-100% methanol is added, the bottle is tightly capped, and the sample is shaken and vortexed to be dissolved, ultrasonic extraction is performed for 0.5-1 hour, the sample is filtered, the filtrate is evaporated to dryness, 10 mL of water is added to dissolve the residue, the water-saturated n-butanol is extracted by shaking 2-4 times, 10 mL each time, the n-butanol is combined and evaporated to dryness, 10 mL of water is added to dissolve the residue, the sample is passed through a SP700 macroporous adsorption resin column, eluted with 80 mL of water, the eluate is discarded, then eluted with 50-100 mL of 30% methanol, the eluate is discarded, and then eluted with 100-200 mL of methanol, the methanol eluate is collected, the solvent is recovered under reduced pressure, the residue is dissolved in 10 mL of water, and then passed through a C18 solid-phase extraction column, the effluent is collected, evaporated to dryness, and 2 mL of methanol is added to dissolve the residue to obtain sample solution one.

[0017] Preferably, in step (1), the method of the second pretreatment is as follows: the sample of the prepared Rehmannia glutinosa pill formula to be tested is taken, 0.5-1.0 g of which is equivalent to the amount of raw Rehmannia glutinosa, 50% methanol is added, the bottle is tightly capped, the weight is determined, extraction is performed for 0.5-1 hour, the weight is determined again after cooling, the lost weight is made up with 50% methanol, the sample is shaken and mixed, filtered, the filtrate is evaporated to dryness under reduced pressure, the residue is dissolved in water, quantitatively transferred through a mixed column, eluted with water, the eluate is discarded, then eluted with 30% methanol, the eluate is collected, concentrated, the residue is dissolved in 30-70% methanol and filtered to obtain sample solution two.

[0018] Preferably, the sample of the prepared Rehmannia glutinosa pill formula to be tested is taken, 0.5-1.0 g of which is equivalent to the amount of raw Rehmannia glutinosa, 100 mL of 50% methanol is added, the bottle is tightly capped, the weight is determined, the sample is shaken and vortexed to be dissolved, ultrasonic extraction is performed for 0.5-1 hour, the sample is cooled, the weight is determined again, the lost weight is made up with 50% methanol, the sample is shaken and mixed, filtered, 50 mL of the filtrate is taken, the solvent is recovered under reduced pressure, the residue is dissolved in 10 mL of water in portions, quantitatively transferred through a SP700 macroporous adsorption resin column, eluted with 80 mL of water, the eluate is discarded, then eluted with 50-150 mL of 30% methanol, the eluate is collected, concentrated, the residue is dissolved in 50% methanol and made up to 2 mL in a volumetric flask, shaken and mixed, filtered to obtain sample solution two.

[0019] Preferably, the inner diameter of the mixed column is 0.8-1.2 cm, the height is 10-25 cm, and the volume ratio or mass ratio of the mixed fillers in the column is diatomite:D101 macroporous adsorption resin:SP700 macroporous adsorption resin = 0-2:0-3:2-7 parts.

[0020] Preferably, in step (2), the sample solution to be tested, the control sample of verbascoside and the solution of the control medicinal material of Radix Rehmanniae Preparata are spotted on the same polyamide film plate, and methanol-glacial acetic acid-water is used as the developing agent for development, removal, air drying and observation under a UV light at a wavelength of 365 nm to see whether the sample chromatogram has the same color fluorescent spots at the positions corresponding to the control sample and the control medicinal material.

[0021] The volume ratio of methanol: glacial acetic acid: water in the developing agent is 3: 1: 7.

[0022] Preferably, in step (3), the chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX SB-C18, the mobile phase A is acetonitrile or methanol, the mobile phase B is 0.1% phosphoric acid aqueous solution or 0.1% formic acid aqueous solution, A: B = 1-10%, the volume flow rate is 1 mL·min -1 , the detection wavelength is 203 nm, and the theoretical plate number calculated according to the peak of rehmannioside D should be not less than 5000.

[0023] Preferably, the Liuwei Dihuang pill type preparation is Liuwei Dihuang pill, Maivei Dihuang pill, Guifu Dihuang pill, Zhikai Dihuang pill, Qiju Dihuang pill and Guishao Dihuang pill.

[0024] Preferably, the dosage form of the Liuwei Dihuang pill type preparation is large honeyed pill, small honeyed pill, water pill, water honeyed pill, concentrated pill, oral liquid, powder, granule, capsule and soft capsule.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] 1. The Liuwei Dihuang pill series preparation is a large category of Chinese patent medicines commonly used in clinical treatment at present, but the present application creates a standard application technology platform for Radix Rehmanniae Preparata preparation, adopts thin layer chromatography to qualitatively identify verbascoside and quantitatively determine the content of rehmannioside D, and develops a general, rapid and simple detection method for the product line, which is a further improvement on the existing quality control research system.

[0027] 2. The present application can simultaneously carry out qualitative and quantitative determination of Radix Rehmanniae Preparata in various Dihuang pill type preparations, greatly reduces the time cost of drug detection, simultaneously realizes parallel preparation of sample solutions of different manufacturers, varieties and batches and result presentation, is suitable for rapid online detection of product quality control of production enterprises and quality evaluation of Dihuang pill series products by the drug inspection department, and has a positive significance for quality improvement and process improvement. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a TLC qualitative identification and method specificity atlas of Radix Rehmanniae Preparata in Dihuang pill type preparations of the present application.

[0029] The marks in the drawings are described as follows: A is the atlas of the first to the thirteenth sample, 1-13 represent the Dihuang pill sample ZJ-1~ZJ-13, B is the spectrum of the 14th to the 28th sample, 14~28 represent the Dihuang pill sample ZJ-14~ZJ-28, R1 is verbascoside, R2 is the control medicinal material of Radix Rehmanniae Glutinosae, NC1~NC6 are six kinds of Dihuang pills of Liuwei, Maiwei, Guishao, Zhibai, Qiju, Gui Fu, and the negative preparation without Dihuang;

[0030] Figure 2 The spectrum index atlas of the control of Dihuang glycoside D is suitable for the system applicability research of the application;

[0031] The marks in the drawings are described as follows: A is the atlas of the first to the thirteenth sample, 1-13 represent the Dihuang pill sample ZJ-1~ZJ-13, B is the spectrum of the 14th to the 28th sample, 14~28 represent the Dihuang pill sample ZJ-14~ZJ-28, R1 is verbascoside, R2 is the control medicinal material of Radix Rehmanniae Glutinosae, NC1~NC6 are six kinds of Dihuang pills of Liuwei, Maiwei, Guishao, Zhibai, Qiju, Gui Fu, and the negative preparation without Dihuang;

[0032] Figure 3 The spectrum index atlas of the control of Dihuang glycoside D is suitable for the system applicability research of the application;

[0033] The marks in the drawings are described as follows: A is the atlas of the first to the thirteenth sample, 1-13 represent the Dihuang pill sample ZJ-1~ZJ-13, B is the spectrum of the 14th to the 28th sample, 14~28 represent the Dihuang pill sample ZJ-14~ZJ-28, R1 is verbascoside, R2 is the control medicinal material of Radix Rehmanniae Glutinosae, NC1~NC6 are six kinds of Dihuang pills of Liuwei, Maiwei, Guishao, Zhibai, Qiju, Gui Fu, and the negative preparation without Dihuang;

[0034] Figure 4 The spectrum index atlas of the control of Dihuang glycoside D is suitable for the system applicability research of the application;

[0035] The marks in the drawings are described as follows: A is the atlas of the first to the thirteenth sample, 1-13 represent the Dihuang pill sample ZJ-1~ZJ-13, B is the spectrum of the 14th to the 28th sample, 14~28 represent the Dihuang pill sample ZJ-14~ZJ-28, R1 is verbascoside, R2 is the control medicinal material of Radix Rehmanniae Glutinosae, NC1~NC6 are six kinds of Dihuang pills of Liuwei, Maiwei, Guishao, Zhibai, Qiju, Gui Fu, and the negative preparation without Dihuang;

[0036] Figure 5 The spectrum index atlas of the control of Dihuang glycoside D is suitable for the system applicability research of the application;

[0037] The marks in the drawings are described as follows: A is the atlas of the first to the thirteenth sample, 1-13 represent the Dihuang pill sample ZJ-1~ZJ-13, B is the spectrum of the 14th to the 28th sample, 14~28 represent the Dihuang pill sample ZJ-14~ZJ-28, R1 is verbascoside, R2 is the control medicinal material of Radix Rehmanniae Glutinosae, NC1~NC6 are six kinds of Dihuang pills of Liuwei, Maiwei, Guishao, Zhibai, Qiju, Gui Fu, and the negative preparation without Dihuang;

[0038] Figure 6 The spectrum index atlas of the control of Dihuang glycoside D is suitable for the system applicability research of the application;

[0039] The marks in the drawings are described as follows: A is the atlas of the first to the thirteenth sample, 1-13 represent the Dihuang pill sample ZJ-1~ZJ-13, B is the spectrum of the 14th to the 28th sample, 14~28 represent the Dihuang pill sample ZJ-14~ZJ-28, R1 is verbascoside, R2 is the control medicinal material of Radix Rehmanniae Glutinosae, NC1~NC6 are six kinds of Dihuang pills of Liuwei, Maiwei, Guishao, Zhibai, Qiju, Gui Fu, and the negative preparation without Dihuang;

[0040] Figure 7 The spectrum index atlas of the control of Dihuang glycoside D is suitable for the system applicability research of the application;

[0041] Figure 8 The spectrum index atlas of the control of Dihuang glycoside D is suitable for the system applicability research of the application;

[0042] Figure 9Linear relationship fitting atlas for content determination of rehymoside D in the present application;

[0043] Figure 10 HPLC content determination and specificity atlas of Radix Rehmanniae Preparata in the present application;

[0044] The marks in the drawing are described as follows: S is the rehymoside D chromatographic peak, R1 is the rehymoside D reference substance, R2 is the Radix Rehmanniae Preparata reference medicinal material, and NC1-NC6 are six kinds of Radix Rehmanniae Preparata in Liuwei Dihuang Pills, Macitong Pills, Guishao Pills, Zhiba Pills, Qiju Pills and Guifu Pills, which are negative preparations without Radix Rehmanniae Preparata;

[0045] Figure 11 HPLC quantitative determination result atlas of Radix Rehmanniae Preparata in the present application; wherein 2-28 represent Liuwei Dihuang Pills samples ZJ-2-ZJ-28. DETAILED DESCRIPTION

[0046] The specific embodiments of the present application are described in detail below, but it should be understood that the protection scope of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified.

[0047] CAMAG TLC Visualizer (Switzerland), Waters 2695 (USA, Waters 2996 PDA detector), ULUP-IV-10T ultra-pure water generator (Sichuan ULUP Ultra-pure Technology Co., Ltd.), 160DPT analytical balance (LIBROR, Japan Shimadzu Corporation), 240 analytical balance (Mettler, Switzerland), FW-100 high-speed pulverizer (Beijing Kewei Yongxing Instrument Co., Ltd.), HH-ZK6 constant temperature water bath (Zhengzhou Kaipeng Experimental Instrument Co., Ltd.), SB-5200DT ultrasonic cleaner (Ningbo Xinzhi Biological Technology Co., Ltd.); chromatographic column with storage ball layer (12*300mm*100mL*24 ports*polytetrafluoroethylene valve, Zhengzhou Xinghua Glass Instrument Factory); glass instrument airflow dryer (220V / 50Hz, 800W, Zhengzhou Kaipeng Experimental Instrument Co., Ltd.), oven (XMTD-8222, Shanghai Jinghong Experimental Equipment Co., Ltd.), thin layer quantitative capillary 1μL and 2μL (CAMAG, Switzerland), adjustable mixing instrument (MX-S, Dalongxingchuang Experimental Instrument Co., Ltd.), P-1 type thin layer chromatography development cylinder (200*100mm, Shanghai Xinyi Instrument Factory Co., Ltd.).

[0048] Analytical grade methanol, ethanol, glacial acetic acid, diatomite, etc. (Tianjin Yongda Chemical Reagent Co., Ltd.), fresh ultrapure water for liquid chromatography, chromatographic grade methanol (Fisher, USA), phosphoric acid (Tianjin Kemeluo Chemical Reagent Co., Ltd.); polyamide film (specification 20 cm x 10 cm) was purchased from Zhejiang Taizhou Lujia Biochemical Plastic Factory; macroporous adsorption resin (D101 type, Macklin) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; macroporous adsorption resin (SP700 type, Japan Mitsubishi) was purchased from Beijing Lvbaicao Technology Development Co., Ltd.; Radix Rehmanniae Preparata control drug material (batch number 121196-202007, specification 1 g / branch), verbascoside (batch number 111530-201310, specification 20 mg / branch), rehmannioside D (batch number 112063-202102, specification 20 mg / branch) were all purchased from China Institute for Drug Control.

[0049] The purchased Liuwei Dihuang Wan type decoction pieces were identified by Professor Li Gengsheng of Henan Institute of Chinese Medicine, and were all Chinese Pharmacopoeia varieties. The information of decoction pieces and prepared prescription preparation samples is shown in Table 1.

[0050] Table 1 Information of Liuwei Dihuang Wan type decoction pieces and preparation samples

[0051]

[0052]

[0053] Example 1

[0054] Detection method of Radix Rehmanniae Preparata in Liuwei Dihuang Wan

[0055] 1. Information of Liuwei Dihuang Wan

[0056]

Prescription

[0057]

Preparation method

[0058]

Characteristics

[0059] 2. Detection of Radix Rehmanniae Preparata in Liuwei Dihuang Pills

[0060] (1) Thin-layer identification of verbascoside: 2.5 g of water pills or 4 g of water pills, or 5 g of small or large honey pills, or 1.2 g of concentrated pills can be taken, finely ground, and placed in a conical flask with a stopper. In this embodiment, 5 g of large honey pills of the product were finely ground, placed in a conical flask with a stopper, and 60 mL of 80% methanol was precisely added for ultrasonic treatment (power 240 W, frequency 40 KHZ) for 30 minutes. The filtrate was evaporated to dryness, the residue was dissolved in 10 mL of water, and the water-saturated n-butanol was extracted 4 times, each time with 10 mL. The n-butanol liquid was combined and evaporated to dryness. The residue was dissolved in 10 mL of water and passed through a SP700 macroporous resin column (column inner diameter 1.2 cm, column height 20 cm). The water was eluted with 80 mL, discarded, and then 30% methanol was eluted with 80 mL, discarded, and then methanol was eluted with 150 mL. The eluate was collected, the solvent was recovered under reduced pressure, the residue was dissolved in 10 mL of water, and passed through a solid-phase extraction cartridge (1 g / 6 mL, activated with 5 mL of methanol and then balanced with 25 mL of water) with octadecylsilane-bonded silica gel as the filler. The effluent was collected, evaporated to dryness, and the residue was dissolved in 2 mL of methanol to obtain the test solution.

[0061] (2) Determination of Rehmannioside D content

[0062] Preparation of sample test solution: 2.5 g of water pills or 4 g of water pills, or 5 g of small or large honey pills, or 1.25 g of concentrated pills can be taken, finely ground, and placed in a conical flask with a stopper. In this embodiment, 5 g of large honey pills of the product were precisely weighed, placed in a conical flask with a stopper, 50 mL of 50% methanol was added, tightly sealed, and shaken and vortexed to dissolve. Ultrasonic extraction was performed for 1 hour, the solution was cooled, filtered, and the solvent was recovered to near dryness under reduced pressure. The residue was dissolved in 10 mL of water, quantitatively transferred through a SP700 macroporous resin column (column inner diameter 1.2 cm, column height 20 cm), eluted with 80 mL of water, discarded, and then eluted with 80 mL of 30% methanol. The eluate was collected, concentrated to near dryness, the residue was dissolved in 50% methanol and made up to 2 mL in a volumetric flask, shaken, filtered, and the filtrate was used as the sample test solution.

[0063] Example 2

[0064] Method for detecting Radix Rehmanniae Preparata in Maiwei Dihuang Pills

[0065] 1. Information of Maiwei Dihuang Pills

[0066] [Prescription] Ophiopogon 60g, Schisandra 40g, Rehmannia 160g, Winey Meat 80g, Peony 60g, Yam 80g, Poria 60g, Alisma 60g

[0067] [Preparation] The above eight ingredients are ground into fine powder, sieved and mixed. 35-50g of refined sugar is added to 100g of the powder with appropriate amount of water to make pills, which are dried to form water pills; or 80-110g of refined sugar is added to make small or large pills.

[0068] [Properties] The product is brown-black water pills, black-brown small or large pills; slightly sweet and sour in taste.

[0069] 2. Detection of Rehmannia in Maiwei Dihuang Pills

[0070] (1) Thin-layer identification of verbascoside: 4g of the product water pills can be ground into fine powder, or 5g of the small or large pills can be cut into pieces. The difference between this example and Example 1 is that in this example, 4g of the product water pills is ground into fine powder and placed in a conical bottle with a plug. The rest of the steps are the same as in Example 1 to prepare the sample solution.

[0071] (2) Determination of Rehmannioside D content

[0072] Preparation of sample solution: 4g of the product water pills can be ground into fine powder, or 5g of the small or large pills can be cut into pieces. The difference between this example and Example 1 is that in this example, 4g of the product water pills is ground into fine powder and accurately weighed. The rest of the steps are the same as in Example 1 to prepare the sample solution.

[0073] Example 3

[0074] Method for detecting Rehmannia in Guishao Dihuang Pills

[0075] 1. Information of Guishao Dihuang Pills

[0076] [Prescription] Angelica 40g, Winey White Peony Root 40g, Rehmannia 160g, Winey Meat 80g, Peony 60g, Yam 80g, Poria 60g, Alisma 60g

[0077] [Preparation] The above eight ingredients are ground into fine powder, sieved and mixed. 35-50g of refined sugar is added to 100g of the powder with appropriate amount of water to make pills, which are dried to form water pills; or 80-110g of refined sugar is added to make small or large pills.

[0078] [Properties] The product is brown-black water pills, black-brown small or large pills; slightly sweet and sour in taste.

[0079] 2. Detection of Rehmannia in Guishao Dihuang Pills

[0080] (1)Mucorinum thin layer identification: can take the product water pill 4g, fine; or take small pill or big pill 5g, cut, and the difference between the example 1 is that, in this example, take the product water pill 4g, put in the conical flask with plug, the rest of the steps are the same with example 1, preparation of test solution.

[0081] (2)Rehmannioside D content determination

[0082] Sample test solution preparation: can take the product water pill 4g, fine; or take small pill or big pill 5g, cut, and the difference between the example 1 is that, in this example, take the product water pill 4g, fine, accurately weighed, the rest of the steps are the same with example 1, preparation of sample test solution.

[0083] Example 4

[0084] Detection method of Radix Rehmanniae Preparata in Zhikuihuangwan

[0085] 1, Zhikuihuangwan information

[0086]

Prescription

[0087]

Preparation

[0088]

Characteristics

[0089] 2, Detection of Radix Rehmanniae Preparata in Zhikuihuangwan

[0090] (1)Mucorinum thin layer identification: can take the product 4 g, honeyed pill, fine; or take small honeyed pill or big honeyed pill 5 g, cut; or take 1.2 g, honeyed pill, fine, with the difference of example 1, take the product 4 g, honeyed pill, fine, in the conical flask with plug, the rest steps are same with example 1, preparation gets sample solution.

[0091] (2)Rehymannoside D content determination

[0092] Sample preparation: can take the product 4 g, honeyed pill, fine; or take small honeyed pill or big honeyed pill 5 g, cut; or take 1.25 g, honeyed pill, fine, with the difference of example 1, take the product 4 g, honeyed pill, fine, in the conical flask with plug, the rest steps are same with example 1, preparation gets sample solution.

[0093] Example 5

[0094] Detection method of Radix Rehmanniae in Qiju Dihuang pill

[0095] 1, Qiju Dihuang pill information

[0096]

Prescription

[0097]

Method

[0098]

Characteristics

[0099] 2, detection of Radix Rehmanniae in Qiju Dihuang pill

[0100] (1) Verbascoside thin layer identification: 4 g of the water honeyed pill can be taken, finely ground; or 5 g of the small honeyed pill or the large honeyed pill can be taken, cut into pieces; or 1.2 g of the concentrated pill can be taken, finely ground. The difference between this example and Example 1 is that, in this example, 4 g of the water honeyed pill is finely ground and placed in a conical flask with a stopper, and the rest of the steps are the same as those of Example 1, to prepare the sample solution.

[0101] (2) Rehmapicroside D content determination

[0102] Preparation of sample test solution: 4 g of the water honeyed pill can be taken, finely ground; or 5 g of the small honeyed pill or the large honeyed pill can be taken, cut into pieces; or 1.25 g of the concentrated pill can be taken, finely ground. The difference between this example and Example 1 is that, in this example, 4 g of the water honeyed pill is finely ground and accurately weighed, and the rest of the steps are the same as those of Example 1, to prepare the sample test solution.

[0103] Example 6

[0104] Method for detecting Radix Rehmanniae Preparata in Guifu Dihuang Pills

[0105] 1. Information of Guifu Dihuang Pills

[0106]

Prescription

[0107]

Preparation

[0108]

Characteristics

[0109] 2. Detection of Radix Rehmanniae Preparata in Guifu Dihuang Pills

[0110] (1) Verbascoside thin layer identification: 4 g of the water honeyed pill can be taken, finely ground; or 5 g of the small honeyed pill or the large honeyed pill can be taken, cut into pieces. The difference between this example and Example 1 is that, in this example, 4 g of the water honeyed pill is finely ground and placed in a conical flask with a stopper, and the rest of the steps are the same as those of Example 1, to prepare the sample solution.

[0111] (2) Rehmapicroside D content determination

[0112] Preparation of sample test solution: 4 g of the water honeyed pill can be taken, finely ground; or 5 g of the small honeyed pill or the large honeyed pill can be taken, cut into pieces, and accurately weighed. The difference between this example and Example 1 is that, in this example, 4 g of the water honeyed pill is finely ground and placed in a conical flask with a stopper, and the rest of the steps are the same as those of Example 1, to prepare the sample test solution.

[0113] Example 7

[0114] Method for detecting Rehmanniae Radix in Liuwei Dihuang Pills

[0115] 1. Information of Liuwei Dihuang Pills

[0116] The same as Example 1.

[0117] 2. Detection of Rehmanniae Radix in Liuwei Dihuang Pills

[0118] (1) Thin-layer identification of verbascoside: The difference between this example and Example 1 is that, in this example, the sample is extracted by 80% methanol ultrasonically and water-saturated n-butanol is shaken, 10 mL of water is added for dissolution, and then the prepared solution is passed through a mixed column (column inner diameter 1.0 cm, column height 14 cm, filler volume ratio of diatomaceous earth: D101 macroporous adsorption resin: SP700 macroporous adsorption resin = 1:1:5), and the rest of the steps are the same as those in Example 1 to prepare the test solution.

[0119] (2) Determination of Rehmannioside D content

[0120] Preparation of sample test solution: The difference between this example and Example 1 is that, in this example, the sample is extracted by 50% methanol ultrasonically, 10 mL of water is added for dissolution, and then the prepared solution is quantitatively transferred through a mixed column (column inner diameter 1.0 cm, column height 14 cm, filler volume ratio of diatomaceous earth: D101 macroporous adsorption resin: SP700 macroporous adsorption resin = 1:1:5), and the rest of the steps are the same as those in Example 1 to prepare the test solution.

[0121] When performing thin-layer identification of verbascoside in the test solutions in Examples 1-7, 1 g of Rehmanniae Radix control drug is added to 60 mL of 80% methanol to prepare a control drug solution according to the preparation method of the test solution. The verbascoside reference substance is added to methanol to prepare a solution containing 0.05 mg per 1 mL as a reference solution.

[0122] According to the test of “0502 Thin-layer chromatography” in the General Rules of the 2020 edition of Chinese Pharmacopoeia (Volume IV), 2 μL of each of the above solutions is taken and spotted on the same polyamide film, methanol-glacial acetic acid-water (3:1:7) is used as the developing agent, and the film is developed, taken out, air-dried, and observed under a UV light (365 nm).

[0123] The results are shown in Table 1: Figure 1 In the test solution of Liuwei Dihuang Pills, the same color fluorescent spots appear at the positions corresponding to the reference solution and the control drug solution.

[0124] In the test solution of Maiwei Dihuang Pills, the same color fluorescent spots appear at the positions corresponding to the reference solution and the control drug solution.

[0125] The sample chromatogram of Guishao Dihuang Pill showed the same color fluorescent spots as the control product and the control medicinal material chromatogram at the corresponding positions.

[0126] The sample chromatogram of Zhikai Dihuang Pill showed the same color fluorescent spots as the control product and the control medicinal material chromatogram at the corresponding positions.

[0127] The sample chromatogram of Qiju Dihuang Pill showed the same color fluorescent spots as the control product and the control medicinal material chromatogram at the corresponding positions.

[0128] The sample chromatogram of Guifu Dihuang Pill showed the same color fluorescent spots as the control product and the control medicinal material chromatogram at the corresponding positions.

[0129] In the content determination of rehmatannin D in the test samples in Examples 1-7, the test was performed according to the test of “0512 High Performance Liquid Chromatography” in the 2020 edition of “Chinese Pharmacopoeia” (Volume 4).

[0130] Chromatographic conditions: Agilent ZORBAX SB-C18 was used as the chromatographic column; methanol-0.1% formic acid solution (5:95) was used as the mobile phase, the volume flow rate was 1 mL·min -1 , the detection wavelength was 203 nm, and the theoretical plate number calculated according to the rehmatannin D peak should not be less than 5000.

[0131] Preparation of rehmatannin D standard solution: an appropriate amount of rehmatannin D control product was accurately weighed, dissolved and diluted with 25% methanol to obtain standard solutions with concentrations of 0.043, 0.086, 0.129, 0.172, 0.215, 0.258 and 0.301 mg / mL, respectively.

[0132] The determination results of the content of rehmatannin D are shown in Table 6.

[0133] In order to further verify the effectiveness of the present application, the following verification test was performed:

[0134] (1) Thin layer identification and method specificity of verbascoside in Dihuang Pill preparations

[0135] Preparation of Dihuang Pill negative preparation: the prescriptions of Liuwei Dihuang Pill, Maiwei Dihuang Pill, Zhikai Dihuang Pill, Guishao Dihuang Pill, Qiju Dihuang Pill and Guifu Dihuang Pill were taken out, and the decoction pieces were crushed into fine powder, sieved and mixed. Honey was heated at 100 ℃ for 10 min, and then cooled. 100 g of honey was added to 100 g of powder to prepare large honey pills, and Dihuang Pill negative preparation was obtained.

[0136] Preparation of sample and Radix Rehmanniae Praeparata control drug solution: The samples and Radix Rehmanniae Praeparata in Table 1 were treated as follows: about 5 g of Dihuang Wan Jiafang Damihewans were cut into pieces, about 4 g and 1.25 g of Shuifang Wan or concentrated pills were finely ground, and about 1 g of Radix Rehmanniae Praeparata control drug powder was precisely weighed and placed in a conical flask with a stopper. Then, 50 mL of 50% methanol was precisely added, the weight was determined, and the mixture was shaken and ultrasonically treated (power 240 W, frequency 40 KHZ) for 30 min. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 10 mL of water. The solution was extracted with water-saturated n-butanol for 4 times, 10 mL each time, and the n-butanol was combined and evaporated to dryness. The residue was dissolved in 10 mL of water and passed through a SP700 macroporous resin column (column inner diameter 1.2 cm, column height 20 cm). The column was eluted with 80 mL of water, the water was discarded, and then the column was eluted with 80 mL of 30% methanol, the 30% methanol was discarded, and then the column was eluted with 150 mL of methanol. The eluate was collected, the solvent was recovered under reduced pressure, the residue was dissolved in 10 mL of water, and the solution was passed through a solid-phase extraction cartridge (1 g / 6 mL) filled with octadecylsilane-bonded silica gel (activated with 5 mL of methanol and then balanced with 25 mL of water). The effluent was collected, evaporated to dryness, and the residue was dissolved in 2 mL of methanol to obtain the sample solution and Radix Rehmanniae Praeparata control drug solution for TLC identification.

[0137] Preparation of Radix Rehmanniae Praeparata negative control solution: 5 g of Dihuang Wan Jiafang negative preparation was prepared according to the preparation method of Dihuang Wan Jiafang sample solution to obtain the Radix Rehmanniae Praeparata negative control solution.

[0138] Preparation of control solution: about 0.5 mg of verbascoside control was dissolved in a 10 mL volumetric flask with methanol to obtain the control solution.

[0139] TLC conditions: 2 μL of each of the above sample solution, control solution, Radix Rehmanniae Praeparata control drug solution, and negative control solution was spotted on the same polyamide film, and developed with methanol-glacial acetic acid-water (3:1:7) as the developing agent. After being taken out and air-dried, the film was observed under ultraviolet light (365 nm).

[0140] Results: The TLC of Radix Rehmanniae Praeparata in Liuweidihuang Wan Jiafang preparations is shown in Figure 1 The results show that the same color fluorescent spots appear in the sample chromatogram at the corresponding positions of the control and control drug chromatogram, and the Radix Rehmanniae Praeparata negative control has no interference.

[0141] (2) System suitability test for determination of Rehmannioside D content in Dihuang Wan Jiafang preparations

[0142] Chromatographic conditions: Agilent ZORBAX SB-C18 column, column temperature 30 ℃, mobile phase methanol-0.1% phosphoric acid solution (5:95), volume flow rate 1 mL·min -1The injection volume is 10 μL.

[0143] Preparation of the reference stock solution: Take 10.75 mg of rehmannia glycoside D reference standard into a 25 mL volumetric flask, add 25% methanol to dissolve and dilute to the mark, mix well, and the solution is ready.

[0144] System suitability test: Take 10 μL each of the rehmannia glycoside D reference solution, the sample solution of the rehmannia pill-like preparation ZJ-2 prepared in Example 1, the prepared rehmannia glutinosa reference material solution, and the negative control solution of the rehmannia-deficient formula, and inject them according to the above chromatographic conditions, record the chromatogram, perform a spectral scan of the rehmannia glycoside D peak in the range of 200 nm to 400 nm, calculate the theoretical plate number and tailing factor, and perform a repeatability test.

[0145] Result: As Figures 2-7 As shown, the absorption peaks of rehmannia glutinosa (Dihuang Wan), prepared Rehmannia glutinosa (Shu Dihuang) reference material, and reference standard in the chromatograms exhibit similar spectral indices for rehmannia glutinosa (Shu Dihuang) pills. A reasonably strong absorption occurs at the shortest wavelength of the absorption curve, but no peak terminal is formed. The theoretical plate number of the rehmannia glutinosa (Shu Dihuang) peak is 7751 (USP), and the RSD of the repeatability of six responses is 1.4%. Referring to the method for the determination of content in prepared Rehmannia glutinosa (Shu Dihuang) in the current edition of the Chinese Pharmacopoeia, the detection wavelength was selected as 203 nm. The theoretical plate number of the rehmannia glutinosa (Shu Dihuang) peak should not be less than 5000, and the relative standard deviation of the peak area measurement for six consecutive injections of the reference standard solution should not exceed 2.0%. Figure 7 In the six injection results, the retention time of rehmannia glycoside D was about 35.2 min. In order to display each peak more clearly, the five spectra were moved during plotting. Specifically, the z-axis angle was set to 15 and the spacing was 5 in the chromatogram attribute parameters.

[0146] (3) Investigation on the linear relationship of the determination of rehmannia glycoside D content in Liuwei Dihuang pill-like preparations

[0147] Accurately pipette 1, 2, 3, 4, 5, 6, and 7 mL of the rehmannia glycoside D reference standard stock solution prepared in (2) into 10 mL volumetric flasks, dilute to the mark with 25% methanol solution, mix well, and inject 10 μL of the 7 different concentrations of reference standard solutions twice in sequence.

[0148] Results: The results of the linear relationship investigation for the determination of rehmannia glycoside D content are as follows: Figures 8-9 As shown, Figure 8 In the 14 injection results, the retention time of rehmannia glycoside D was approximately 36.7 min. To make each peak clearer, the chromatograms of 13 injections were shifted during plotting. Specifically, the z-axis angle was set to 15° and the spacing to 5° in the chromatogram attribute parameters. The results indicate that rehmannia glycoside D at 0.043 mg / mL... -1 ~0.301 mg·mL -1The mass concentration in the detection range showed a good linear relationship with the peak area integral value, and the regression equation was Y=6.82x10 6 X+4.51x10 3 (r=0.999432).

[0149] (4) The specificity of the HPLC method for determining the content of Radix Rehmanniae in Dihuang Pills

[0150] Preparation of sample test solution: Take 2.5 g of water pills or 4 g of water pills of Dihuang Pills, grind them finely; or take 5 g of small honey pills or large honey pills, cut them into pieces; or take 1.25 g of concentrated pills, grind them finely. Precisely weigh and place them in a conical bottle with a plug, add 50 mL of 50% methanol, tightly plug, shake and vortex to disperse, ultrasonic extraction for 1 hour, cool, filter, reduce pressure to recover the solvent to near dryness, dissolve the residue with 10 mL of water in portions, quantitatively transfer through an SP700 type macroporous resin column (column inner diameter 1.2 cm, column height 20 cm), elute with 80 mL of water, discard the water, then elute with 80 mL of 30% methanol, collect the eluate, concentrate to near dryness, dissolve the residue with 50% methanol and dilute to 2 mL in a volumetric flask, shake well, filter, and take the filtrate as the sample test solution.

[0151] Take 10 μL of each of the sample solution of Dihuang Pills, the reference substance solution of Dihuangoside D, the reference medicinal material solution of Radix Rehmanniae, and the negative control solution of Dihuang Pills without Radix Rehmanniae, inject them according to the chromatographic conditions in item (2), and record the chromatogram.

[0152] Results: In the chromatograms of each sample of Dihuang Pills and the reference medicinal material of Radix Rehmanniae, corresponding chromatographic peaks were detected at the same retention time as the reference substance of Dihuangoside D, and there was no interference from the negative control. See Figure 10 .

[0153] (5) Precision test of the HPLC method for determining the content of Radix Rehmanniae in Liuwei Dihuang Pills

[0154] Take 10 μL of the reference substance solution of Dihuangoside D and inject it 6 times in succession, record the chromatogram according to the chromatographic conditions in item (2).

[0155] Results: The RSD value of the response area integral of the chromatographic peak of Dihuangoside D in the chromatogram was 1.4%. According to the guidelines for method validation, the sample matrix is complex, the content of the components is less than 0.01%, and the acceptable range of precision RSD is <6% and can be appropriately relaxed, so it meets the requirements. See Table 2.

[0156] Table 2 Results of precision test (n=6)

[0157]

[0158]

[0159] (6) Stability test of HPLC method for determining the content of Radix Rehmanniae in Liuwei Dihuang Pills

[0160] Take about 10 g of Liuwei Dihuang Pills sample ZJ-2, prepare the sample solution for determination according to the method in (1), and place it in the automatic sample injector of the high performance liquid chromatograph. Inject 10 μL at 0, 2, 4, 6, 8, 10, 12, and 24 h, and record the peak area.

[0161] The RSD of the peak area of Radix Rehmanniae D is 1.9%, indicating that the test solution is stable at room temperature for 24 h. See Table 3.

[0162] Table 3 Results of stability test (n = 8)

[0163]

[0164] (7) Sample addition recovery test of HPLC method for determining the content of Radix Rehmanniae in Liuwei Dihuang Pills

[0165] Take about 5 g of Liuwei Dihuang Pills sample ZJ-2 with known content, accurately weigh 6 portions, and place them in 100 mL conical flasks with stoppers. Accurately add 1 mL of Radix Rehmanniae D reference substance solution with a mass concentration of 0.43 mg·mL -1 , prepare the test solution according to the sample solution preparation method in (4), inject it according to the chromatographic conditions in (2), record the peak area, and calculate the sample addition recovery rate.

[0166] Results: The average sample addition recovery rate of Radix Rehmanniae D is 97.8%, and the RSD is 2.9%, indicating that the recovery rate of the method is good. See Table 5.

[0167] Table 5 Sample addition recovery rate of Radix Rehmanniae D (n = 6)

[0168]

[0169]

[0170] (8) HPLC determination of Radix Rehmanniae in Liuwei Dihuang Pills

[0171] Take an appropriate amount of Liuwei Dihuang Pills sample from 28 batches, accurately weigh them, and prepare the test solution according to the sample solution preparation method in (4). Then, inject it according to the chromatographic conditions in (2), calculate the content of each component, repeat the determination twice, and calculate the average value. The results are shown in Table 6, Figure 11 .

[0172] Table 6 Content of Radix Rehmanniae D in Liuwei Dihuang Pills (n = 2)

[0173]

[0174] The Dihuang Pill and its similar prescriptions have complex taste compositions and numerous chemical components. The pharmacopoeia detection method of Radix Rehmanniae Praeparata is not applicable to its compound preparations. In the prior art, the preparation method of the test solution of the pill type prescription preparation usually uses boiling water to heat extract and then shakes with ethyl acetate to retain the organic solvent phase components with small polarity, or uses a certain concentration of methanol solution to ultrasonically or heat reflux extract the sample, and simply uses styrene type polar copolymer such as DA-201 type, non-polar macroporous resin such as HP20, HP21, SP700, SP825L and the like to remove impurities. However, in the Dihuang preparation, the verbascoside active component is locked as a phenylethanoid glycoside component coexisting with sugar, sugar alcohol, cyclo-oxygen ether glycoside and lignan glycoside and the like, has great polarity and is insoluble in organic solvents with small polarity. Therefore, the present application retains the components with large polarity by using n-butanol and water extraction technology after sample extraction, and effectively solves the problem of test solution multi-component interference in the analysis process by using the combined solid phase extraction technology of the mixed column or macroporous resin column.

[0175] The usual method for developing the verbascoside component in Radix Rehmanniae Praeparata and related preparations is to use 0.1% 2,2-diphenyl-1-hydrazine anhydrous ethanol solution for impregnation. However, the technical solution of the present application directly uses the fluorescence observation method. The verbascoside component has a long conjugated system and many phenolic hydroxyl groups. It is found in the test that the thin layer spot has more sensitive ultraviolet absorption characteristics than color development, and presents a more intense fluorescent spot under the 365 nm fluorescent lamp than under the 254 nm fluorescent lamp. This method effectively solves the background masking problem caused by the weak specificity of the original color development technology.

[0176] The thin layer chromatography plate used for the thin layer identification of the verbascoside component in Radix Rehmanniae Praeparata and related preparations is usually a glass plate coated with silica gel G or silica gel H. However, the method of the present application changes the silica gel plate into a polyamide film. The coating has good hydrophilic and lipophilic properties. When the sample mixture is developed with the developing agent, the mixture is separated due to the different hydrogen bond adsorption capacities of the polyamide and each polar molecule. This technology improves the thin layer separation effect, and has good method specificity without interference of the Radix Rehmanniae Praeparata negative preparation. When the content of Radix Rehmanniae Praeparata in the Dihuang Pill and its similar prescription preparations is determined by the Dihuangoside D component, in order to adapt to the complexity of the sample components, the present application uses a new mixed column pretreatment technology and optimally combines the liquid chromatography conditions to obtain good enrichment and separation of the target compound.

[0177] The Liuwei Dihuang Pill type preparation includes Liuwei Dihuang Pill, Maili Dihuang Pill, Guifu Dihuang Pill, Zhibo Dihuang Pill, Qiju Dihuang Pill, Guishao Dihuang Pill, and is not limited to the above 6 preparation formulas, and also includes other Dihuang preparation formulas added or subtracted from the basic Liuwei Dihuang Pill formula; the Liuwei Dihuang Pill type preparation dosage forms include large honeyed pills, small honeyed pills, water pills, water honeyed pills, concentrated pills, oral liquids, powders, granules, capsules and soft capsules, and are not limited to the above common dosage forms, and also include other dosage forms and sub-dosage forms classified in the 2020 edition of the Chinese Pharmacopoeia, Volume IV, “0100 General Rules for Preparations”.

[0178] It should be noted that when the present application claims involving numerical ranges, both endpoints of each numerical range and any number between the two endpoints can be selected, and in order to prevent repetition, the present application describes preferred embodiments.

[0179] Although preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0180] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. A method for detecting the mass of Rehmannia glutinosa in a Rehmannia glutinosa pill formula preparation, characterized in that, The Rehmannia Pill-type preparation is Liuwei Dihuang Pill, Maili Dihuang Pill, Guifu Dihuang Pill, Zhibai Dihuang Pill, Qiju Dihuang Pill or Guishao Dihuang Pill; The method comprises the following steps: (1) sample pretreatment: pretreating the Rehmannia Pill-type preparation to be tested to obtain a sample solution to be tested, and pretreating the Rehmannia Pill-type preparation to be tested to obtain a sample solution to be tested; The first pretreatment is as follows: taking 0.5-1.0 g of the Rehmannia Pill-type preparation sample corresponding to the amount of raw Rehmannia glutinosa, adding 60-100% methanol, tightly sealing, dissolving, extracting for 0.5-1 hour, filtering, evaporating the filtrate, dissolving the residue with water, extracting 2-4 times with water-saturated n-butanol by shaking, combining to obtain n-butanol liquid, evaporating the n-butanol liquid, dissolving the residue with water, passing through a mixed column, eluting with water, then eluting with 30% methanol, and then eluting with methanol, collecting the methanol eluate, recovering the solvent under reduced pressure, dissolving the residue with water, passing through a C18 solid-phase extraction column, collecting the effluent, evaporating, dissolving the residue with methanol, and obtaining the sample solution to be tested; The second pretreatment is as follows: extracting with methanol, dissolving the weight loss with 50% methanol after cooling, and then passing through a mixed column containing SP700 macroporous adsorption resin, and eluting with water and methanol in sequence, concentrating, and dissolving the residue with methanol; The mixed column has an inner diameter of 0.8-1.2 cm, a height of 10-25 cm, and a volume ratio or mass ratio of the mixed fillers of 0-2:0-3:2-7 parts of diatomaceous earth:D101 macroporous adsorption resin:SP700 macroporous adsorption resin; (2) using verbascoside reference substance, Rehmannia glutinosa reference medicinal material and the sample solution to be tested to qualitatively identify the verbascoside component by thin layer chromatography; (3) quantitatively determining the rehderioside D component in the sample solution to be tested by high performance liquid chromatography.

2. The method for detecting the mass of Radix Rehmanniae Preparata in a Rehmanniae Pill formula preparation according to claim 1, characterized in that, Taking 0.5-1.0 g of the Rehmannia Pill-type preparation sample corresponding to the amount of raw Rehmannia glutinosa, adding 60-100% methanol 50-100 mL, tightly sealing, oscillating and vortexing to dissolve, ultrasonic extracting for 0.5-1 hour, filtering, evaporating the filtrate, dissolving the residue with water 10 mL, extracting 2-4 times with 10 mL of water-saturated n-butanol each time, combining the n-butanol liquid, evaporating, dissolving the residue with water 10 mL, passing through an SP700 macroporous adsorption resin column, eluting with water 80 mL, discarding, eluting with 30% methanol 50-100 mL, discarding, and then eluting with methanol 100-200 mL, collecting the methanol eluate, recovering the solvent under reduced pressure, dissolving the residue with 10 mL of water, passing through a C18 solid-phase extraction column, collecting the effluent, evaporating, dissolving the residue with methanol 2 mL, and obtaining the sample solution to be tested.

3. The method for detecting the mass of Radix Rehmanniae Preparata in a Rehmanniae Pill formula preparation according to claim 1, characterized in that, In step (1), the method of the pre-treatment two is taking 0.5-1.0 g of the Rehmannia glutinosa raw drug equivalent to the Rehmannia glutinosa raw drug of the sample of the Rehmannia glutinosa pill formula preparation to be detected, adding 50% methanol, tightly closing, weighing, extracting for 0.5-1 hour, cooling, and then weighing again, supplementing the lost weight with 50% methanol, shaking, filtering, taking the continuous filtrate, recovering the solvent under reduced pressure to near dryness, dissolving the residue with water, quantitatively transferring through a mixing column, eluting with water, discarding the water, eluting with 30% methanol, collecting the eluate, concentrating, dissolving the residue with 30-70% methanol, filtering, and obtaining the sample solution two to be detected.

4. The method for detecting the mass of Radix Rehmanniae Preparata in a Rehmanniae Pill formula preparation according to claim 3, characterized in that, Take 0.5-1.0 g of the Rehmannia glutinosa raw drug equivalent to the Rehmannia glutinosa raw drug of the sample of the Rehmannia glutinosa pill formula preparation to be detected, add 50% methanol 100 mL, tightly close, weigh, shake and vortex to disperse, ultrasonic extract for 0.5-1 hour, cool, weigh again, supplement the lost weight with 50% methanol, shake, filter, take 50 mL of the continuous filtrate, recover the solvent under reduced pressure, dissolve the residue with water 10 mL, quantitatively transfer through an SP700 type macroporous adsorption resin column, elute with water 80 mL, discard the water, elute with 30% methanol 50-150 mL, collect the eluate, concentrate, dissolve the residue with 50% methanol and dilute to 2 mL in a volumetric flask, shake, filter, and obtain the sample solution two to be detected.

5. The method for detecting the mass of Radix Rehmanniae Preparata in a Rehmanniae Pill formula preparation according to claim 1, characterized in that, In step (2), the sample solution one to be detected, the verbascoside control and the Rehmannia glutinosa control medicinal material solution are spotted on the same polyamide film plate, methanol-glacial acetic acid-water is used as the developing agent, develop, take out, dry, place under the ultraviolet light, and observe whether the sample chromatogram has the same color fluorescent spots at the corresponding positions of the control and the control medicinal material chromatogram under the wavelength of 365 nm; The volume ratio of methanol: glacial acetic acid: water in the developing agent is 3:1:

7.

6. The method for detecting the mass of Radix Rehmanniae Preparata in a Rehmanniae Pill formula preparation according to claim 1, characterized in that, The chromatographic conditions in step (3) are as follows: the chromatographic column is Agilent ZORBAX SB-C18, the mobile phase A is acetonitrile or methanol, the mobile phase B is 0.1% phosphoric acid aqueous solution or 0.1% formic acid aqueous solution, A:B=1-10%, the volume flow rate is 1 mL·min -1 , and the detection wavelength is 203 nm, and the theoretical plate number calculated according to the peak of rehmannioside D should be not less than 5000.

7. The method for detecting the mass of Radix Rehmanniae Preparata in a Rehmanniae Pill formula preparation according to claim 1, characterized in that, The Rehmannia pill formula preparation dosage form is honeyed pill, water pill, water-honeyed pill, concentrated pill, oral liquid, powder, granule, capsule and soft capsule.

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