CN115932151A - Detection method of traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis syndrome - Google Patents

Abstract

The invention discloses a detection method of a traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis syndrome, wherein the traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 5-15 parts of red peony root, 10-20 parts of glabrous greenbrier rhizome, 3-9 parts of zedoary, 10-20 parts of glabrous sarcandra herb and 5-15 parts of dark plum fruit, wherein the detection method comprises the steps of identifying the red peony root, the glabrous greenbrier rhizome, the glabrous sarcandra herb and the dark plum fruit in the traditional Chinese medicine preparation by adopting thin-layer chromatography and determining the content of paeoniflorin in the red peony root by adopting high performance liquid chromatography. The invention has the characteristics of simple, convenient, stable and reliable detection method and capability of effectively ensuring the product quality.

Description

Detection method of traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis syndrome

Technical Field

The invention relates to a detection method of a traditional Chinese medicine preparation, in particular to a detection method of a traditional Chinese medicine preparation for treating psoriasis vulgaris and blood stasis.

Background

The traditional Chinese medicine has a long history of treating psoriasis, the psoriasis has a plurality of disease names recorded in ancient books in the past, such as psoriasis and lonicera crazy, and the current disease name is recognized to be the psoriasis. Blood stasis runs through psoriasis, so that the therapeutic rule of promoting blood circulation to remove blood stasis and treating psoriasis is established, and a psoriasis treating prescription is created in the prior art. The original psoriasis prescription is large in prescription, multiple in components, difficult in quality control and high in development difficulty, and the inventor utilizes 3 animal models closely related to the pathogenesis link and clinical expression of psoriasis to carry out drug effect screening and evaluation, optimizes the psoriasis prescription and obtains the Pao Ling prescription.

The 'Shao Ling Fang' is prepared with red peony root, glabrous greenbrier rhizome, zedoary, black plum and glabrous sarcandra herb and through extraction, concentration, drying and pressing. In the formula, the red paeony root and the glabrous greenbrier rhizome are used as monarch drugs, the red paeony root is sour, bitter and cold in property and enters liver channels, the function of removing blood stasis, cooling blood and removing ecchymoses is realized, the glabrous greenbrier rhizome is sweet and mild in property and enters the liver and stomach channels, the function of tonifying spleen and eliminating dampness is realized, and the skin dampness and toxicity are removed, so that the itching is relieved, and the red paeony root and the glabrous greenbrier rhizome are used as monarch drugs together. The zedoary is pungent, bitter and warm in flavor, belongs to the liver and spleen channels, can enter blood and break blood, is fragrant and warm in flavor, is beneficial to promoting qi circulation, can treat both the fruit of blood stasis and the cause of blood stasis, is a special medicine for treating blood stasis, is a ministerial medicine for assisting the blood stasis and the speckle removal of the red paeony root and restricting the cold and cool property of the red paeony root; the dark plum is sour and astringent, enters liver, spleen, lung and large intestine channels, and is used as an assistant monarch and ministerial drug to solve the secondary symptoms of dryness, pruritus and the like caused by the unsmooth blood stasis and dryness of psoriasis; sarcandra glabra, named as sarcandra glabra, is bitter and pungent in taste, has mild nature, enters heart and liver channels, and has the effects of promoting blood circulation, removing ecchymoses and dredging collaterals. The herb is good at entering blood system to activate blood and remove macula, while its bitter and pungent taste can remove exterior, so it is used as a guiding drug to direct the herbs to skin diseases. The whole formula has the effects of promoting blood circulation, removing blood stasis, removing speckles, removing dampness, detoxifying and relieving itching, has the characteristics of 'removing blood stasis without consuming blood, nourishing blood without assisting dampness' as a formula, and is suitable for patients with psoriasis vulgaris.

The Paeonia lactiflora and poria cocos formula is mainly used for treating moderate psoriasis vulgaris, and can improve and relieve skin damage symptoms, affected areas and pruritus symptoms of a patient; in terms of safety, no adverse event occurred in the medical examination of hematuria routine, liver function, kidney function, blood coagulation of patients, and no serious adverse event related to the drug was found in the analysis of adverse events. Therefore, the 'Pao ling fang' has good effects of improving psoriasis skin lesions and relieving inflammation states on an imiquimod psoriasis model, the action links relate to multiple links of resisting keratinocyte hyperproliferation, resisting inflammation, regulating immunity and the like, and the 'Pao ling fang' selectively inhibits Th1 cells under the condition of equivalent drug effects, and prompts that the 'Pao ling fang' has smaller toxic and side effects compared with the traditional immunosuppressant; compared with the prepared Chinese patent medicine such as the radix paeoniae alba and poria cocos tablets on the market, the radix paeoniae alba and poria cocos tablets aim at the core pathological link of blood stasis, do not contain toxic medicines, and have better curative effect, and are safer and more reliable.

However, at present, the 'Shao Ling Fang' does not have scientific and reasonable quality standard, the prescription composition is complex, and the quality of the medicinal materials seriously affects the curative effect of the compound preparation. How to quickly identify the quality of medicinal materials in the compound preparation has an important effect on improving the quality of products.

Disclosure of Invention

The invention aims to provide a detection method of a traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis. The invention has the characteristics of simple, convenient, stable and reliable detection method and capability of effectively ensuring the product quality.

The technical scheme of the invention is as follows: a detection method of a traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis syndrome comprises the following raw materials in parts by weight: 5-15 parts of red peony root, 10-20 parts of glabrous greenbrier rhizome, 3-9 parts of zedoary, 10-20 parts of glabrous sarcandra herb and 5-15 parts of dark plum fruit, wherein the detection method comprises the steps of identifying the red peony root, the glabrous greenbrier rhizome, the glabrous sarcandra herb and the dark plum fruit in the traditional Chinese medicine preparation by adopting thin-layer chromatography and determining the content of paeoniflorin in the red peony root by adopting high performance liquid chromatography.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the identification method of the red peony root comprises the following steps: taking 1g of the ground Chinese medicinal preparation, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, evaporating filtrate to dryness, dissolving residue in water, adding the residue on a polyamide column, washing with water until effluent is colorless, mixing the eluate with the filtrate, evaporating to dryness, and dissolving residue in 1ml of ethanol to obtain a sample solution; eluting polyamide column with ethanol, collecting eluate 20ml, evaporating to dryness, dissolving residue with ethanol 1ml, and collecting solution; adding ethanol 10ml into radix Paeoniae Rubra reference material 0.5g, shaking for 5 min, filtering, evaporating filtrate, and dissolving residue with ethanol 2ml to obtain reference solution; performing thin layer chromatography test, sucking sample solution and control solution 4 μ l each, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-formic acid as developing agent at volume ratio of 7: 2: 0.5, taking out, air drying, spraying 5% vanillin sulfuric acid solution, and heating until the spots are clearly developed.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris and blood stasis, the identification method of the glabrous greenbrier rhizome comprises the following steps: taking 1g of rhizoma Smilacis Glabrae as reference material, adding 20ml of ethanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate, and dissolving residue with 1ml of ethanol to obtain reference solution. According to the thin-layer chromatography test, 5 mul of the test solution and 5-10 mul of the glabrous greenbrier rhizome reference solution are respectively spotted on the same silica gel G thin-layer plate according to the volume ratio of 3: 6: 2:1, petroleum ether, ethyl acetate, methanol and formic acid are used as developing agents, the developing agents are developed, taken out, dried, sprayed with an aluminum trichloride test solution, placed for 5 minutes and then placed under an ultraviolet lamp for inspection.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the identification method of glabrous sarcandra herb is specifically as follows: taking 2g of glabrous sarcandra herb as a reference medicinal material, adding 30ml of petroleum ether, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating to dryness, and dissolving residues in 1ml of ethanol to obtain a reference solution; performing thin-layer chromatography, sucking 2-4 μ l of each of the reference solution and the sample solution, respectively dropping on the same silica gel G thin-layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent at volume ratio of 6: 2:1, taking out, air drying, and inspecting under ultraviolet lamp.

In the aforementioned detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the sample solutions of glabrous greenbrier rhizome and glabrous sarcandra herb are both standby solutions for identification of red peony root, or the preparation method of the sample solutions of glabrous greenbrier rhizome and glabrous sarcandra herb is as follows: taking 1g of the ground traditional Chinese medicine preparation, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, evaporating filtrate to dryness, dissolving residues in water, adding the residues on a polyamide column, washing with water until effluent is colorless, eluting with ethanol, collecting 20ml of eluent, evaporating to dryness, and dissolving residues in 1ml of ethanol to obtain a sample solution.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the identification method of the dark plum comprises the following steps: taking 1g of ground traditional Chinese medicine preparation, adding 15ml of water, carrying out ultrasonic treatment for 15 minutes, centrifuging, taking supernatant, concentrating to 9ml, adding 1ml of formic acid, mixing uniformly, adding ethyl acetate into the solution, shaking and extracting twice, 10ml each time, combining ethyl acetate solutions, evaporating to dryness, and adding 1ml of ethanol into residues to dissolve the residues to obtain a sample solution; adding 25ml of water into 1g of dark plum control medicinal material, heating and refluxing for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residue with 2ml of ethanol to obtain a control solution. Performing thin layer chromatography test, sucking sample solution 5 μ l and reference solution 2 μ l, respectively dropping on the same silica gel G thin layer plate, taking the upper layer solution of butyl acetate-formic acid-water at volume ratio of 4: 2 as developing agent, spreading, taking out, air drying, spraying with 0.04% bromophenol blue ethanol solution, and inspecting in sunlight.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the content determination method of paeoniflorin in red paeony root comprises the following steps: performing gradient elution by using paeoniflorin as a reference, octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and 0.1% phosphoric acid solution as a mobile phase B, wherein the mobile phase A is 14-20% and the mobile phase B is 86-80% within 0-20 min; within 20-30 min, the mobile phase A is from 20% to 80%, and the mobile phase B is from 86% to 20%; within 30-40 min, the proportion of the mobile phase A is 80 percent, and the proportion of the mobile phase B is 20 percent; the detection wavelength is 230nm; precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the preparation method of the reference substance solution comprises the following steps: collecting paeoniflorin control dried in phosphorus pentoxide vacuum dryer for 36 hr, and adding methanol to obtain solution containing 0.3mg per 1 ml; the preparation method of the test solution comprises the following steps: taking 0.5g of the ground traditional Chinese medicine preparation, adding 30ml of 80% methanol, sealing, weighing,heating and refluxing for 1 hr, cooling, weighing, adding 80% methanol to make up for the lost weight, shaking, filtering, and collecting the filtrate. Each tablet of the product contains paeoniflorin (C) as main ingredient ₂₃ H ₂₈ O ₁₁ ) Calculated, not less than 4mg.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 15 parts of red peony root, 20 parts of glabrous greenbrier rhizome, 9 parts of zedoary, 20 parts of glabrous sarcandra herb and 15 parts of dark plum fruit.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris and blood stasis, the traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 9 parts of red peony root, 15 parts of glabrous greenbrier rhizome, 6 parts of zedoary, 15 parts of glabrous sarcandra herb and 6 parts of dark plum fruit.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the preparation method of the traditional Chinese medicine preparation comprises the following steps: extracting the rhizoma smilacis glabrae with 50% ethanol twice by heating, adding ethanol which is 10 times of the rhizoma smilacis glabrae for the first time, adding ethanol which is 8 times of the rhizoma smilacis glabrae for the second time, extracting for 1 hour each time, filtering, combining ethanol solutions, recovering ethanol under reduced pressure, concentrating to obtain clear paste with the relative density of 1.12-1.14, filtering, and keeping the clear paste and dregs for later use; decocting radix Paeoniae Rubra, curcumae rhizoma, herba Pileae Scriptae and mume fructus in water twice, adding 10 times of water for the first time, soaking for 1 hr, and decocting for 1 hr; mixing the second time of the medicinal residues with the rhizoma smilacis glabrae after alcohol extraction, adding 10 times of water, decocting for 1 hour, filtering, mixing the filtrates, concentrating under reduced pressure to obtain clear paste with the relative density of 1.12-1.14, filtering, mixing with the rhizoma smilacis glabrae clear paste, adding silicon dioxide, uniformly mixing, and spray drying to obtain dry paste powder; adding pregelatinized starch and sodium carboxymethyl starch into the dry extract powder, mixing, granulating with 90% ethanol, drying at 70 deg.C, grading, and adding pregelatinized starch, sodium carboxymethyl starch, silicon dioxide and magnesium stearate.

In the detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis, the traditional Chinese medicine preparation can be prepared into any conventional preparation, including tablets, pills, capsules, granules, oral liquid, sprays, injections, suspensions and microsphere preparations.

Compared with the prior art, the invention has the beneficial effects that:

the poria cocos prescription provided by the invention comprises red peony root, glabrous greenbrier rhizome, zedoary, glabrous sarcandra herb and dark plum fruit, can treat psoriasis vulgaris blood stasis, and limits and optimizes the identification method of the red peony root, the glabrous greenbrier rhizome, the glabrous sarcandra herb and the dark plum fruit and the content determination method of the red peony root. The identification method of the red peony root, the glabrous greenbrier rhizome, the glabrous sarcandra herb and the dark plum fruit has the advantages of high accuracy, high precision, high recovery rate, high stability, strong specificity, good repeatability, no interference and the like; the method for measuring the content of the red paeony root has the advantages of simple operation, low cost, short detection time, good separation effect, sensitivity, accuracy and the like. The invention can effectively detect whether the product is qualified or not and whether the quality is good or bad, ensures the consistency of the drug effect and the stability of the quality, and further ensures the stability of the product quality and the safety and effectiveness of clinical medication.

Drawings

FIG. 1 shows UV absorption of paeoniflorin control by HPLC;

FIG. 2 is an HPLC chart of a paeoniflorin control;

FIG. 3 is an HPLC plot of a red peony root-lacking negative control sample;

FIG. 4 is a diagram of the effect of a specificity investigation test for thin-layer identification of radix Paeoniae Rubra;

FIG. 5 is a diagram showing the identification effect of thin layers of radix Paeoniae Rubra of prefabricated thin-layer plates;

FIG. 6 is a diagram showing the identification effect of the thin layer of radix Paeoniae Rubra with a hand-laid thin layer plate;

FIG. 7 is a graph showing the identification effect of thin layer of radix Paeoniae Rubra at 10 deg.C;

FIG. 8 is a graph showing the identification effect of thin layer of radix Paeoniae Rubra at 25 deg.C;

FIG. 9 is a graph of the identification effect of thin layer of radix Paeoniae Rubra under the condition of humidity of 18%;

FIG. 10 is a graph of the identification effect of thin layer of radix Paeoniae Rubra under the condition of 88% humidity;

FIG. 11 is a diagram showing the effect of the color development condition and specificity investigation test for identification of rhizoma Smilacis Glabrae thin layer;

FIG. 12 is a diagram showing the identification effect of the thin layer of Smilax glabra of the prefabricated thin layer plate;

FIG. 13 is a graph showing the identification effect of rhizoma Smilacis Glabrae thin layer on a hand-laid thin layer plate;

FIG. 14 is a graph showing the identification effect of rhizoma Smilacis Glabrae thin layer at 10 deg.C;

FIG. 15 is a graph showing the identification effect of rhizoma Smilacis Glabrae thin layer at 25 deg.C;

FIG. 16 is a graph of the identification effect of rhizoma Smilacis Glabrae thin layer under the humidity of 18%;

FIG. 17 is a graph of the identification effect of thin layer of radix Paeoniae Rubra under the condition of 88% humidity;

FIG. 18 is a diagram showing the color development conditions and specificity of thin-layer differentiation of glabrous sarcandra herb;

FIG. 19 is a graph showing the effect of thin-layer differentiation of the herb of the prefabricated thin-layer plate;

FIG. 20 is a graph showing the effect of thin-layer characterization of glabrous sarcandra herb on a hand-laid thin-layer sheet;

FIG. 21 is a graph showing the identification effect of thin glabrous sarcandra herb at 10 deg.C;

FIG. 22 is a graph showing the identification effect of thin glabrous sarcandra herb at 25 deg.C;

FIG. 23 is a graph showing the identification effect of thin glabrous sarcandra herb layer under the condition of 18% humidity;

FIG. 24 is a graph showing the effect of thin-layer differentiation of glabrous sarcandra herb under a humidity of 88%;

FIG. 25 is a diagram showing the color development conditions and specificity of thin-layer identification of Prunus mume;

FIG. 26 is a diagram showing the discrimination of thin ebony layers in a prefabricated thin layer plate;

FIG. 27 is a diagram showing the effect of identifying the thin layer of Japanese apricot in the case of manually laying the thin layer plate;

FIG. 28 is a graph showing the identification effect of thin layers of Japanese apricot at 10 ℃;

FIG. 29 is a graph showing the identification effect of thin mume fructus layers at 25 deg.C;

FIG. 30 is a graph showing the identification effect of thin layers of Japanese apricot under the condition of humidity of 18%;

FIG. 31 is a graph showing the identification effect of thin layers of Japanese apricot under the condition of 88% humidity.

Detailed Description

The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.

Example (b):

a Chinese medicinal preparation (Poria formula) for treating psoriasis vulgaris with blood stasis syndrome is prepared from: 600g of red peony root, 1000g of glabrous greenbrier rhizome, 400g of zedoary, 1000g of glabrous sarcandra herb and 400g of dark plum fruit.

The preparation method of the tuckahoe prescription comprises the following steps: taking the five medicines, adding 50% ethanol into rhizoma smilacis glabrae, heating and extracting twice, adding 10 times of ethanol into the rhizoma smilacis glabrae for the first time, adding 8 times of ethanol into the rhizoma smilacis glabrae for the second time, extracting for 1 hour each time, filtering, combining ethanol solutions, recovering ethanol under reduced pressure, concentrating to obtain clear paste with the relative density of 1.12-1.14 (60 ℃), filtering, and reserving the clear paste and dregs for later use. Decocting the rest four materials such as radix Paeoniae Rubra with water twice, adding 10 times of water for the first time, soaking for 1 hr, and decocting for 1 hr; mixing the second time with the dregs after the alcohol extraction of the glabrous greenbrier rhizome, adding 10 times of water, decocting for 1 hour, filtering, mixing the filtrates, concentrating under reduced pressure to obtain clear paste with the relative density of 1.12-1.14 (60 ℃), filtering, mixing with the glabrous greenbrier rhizome clear paste, adding a proper amount of silicon dioxide, mixing uniformly, and performing spray drying to obtain dry paste powder. Adding appropriate amount of pregelatinized starch and carboxymethyl starch sodium into dry extract powder, mixing, granulating with 90% ethanol, drying at 70 deg.C, grading, adding appropriate amount of pregelatinized starch, carboxymethyl starch sodium, silicon dioxide and magnesium stearate, mixing, pressing into 1000 tablets, and coating with film coat.

The detection method of the tuckahoe prescription comprises the following steps:

(1) The identification method of red peony root is: taking a proper amount of the product, grinding, taking 1g, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, drying the filtrate by distillation, dissolving the residue in a proper amount of water, adding the mixture on a treated polyamide column (80-100 meshes, 5g, the inner diameter of the column is 1cm, and the column is filled with water by a wet method), washing the column with water until the effluent is colorless, combining the eluate with the passing solution, drying by distillation, and dissolving the residue in 1ml of ethanol to obtain a sample solution. Eluting polyamide column with ethanol, collecting eluate 20ml, evaporating to dryness, dissolving residue with ethanol 1ml, and collecting solution. Adding 0.5g of radix Paeoniae Rubra as control material, adding 10ml of ethanol, shaking for 5 min, filtering, evaporating the filtrate, and dissolving the residue with 2ml of ethanol to obtain control solution. Performing thin layer chromatography (general rule 0502 of the four parts of the 2020 edition of Chinese pharmacopoeia), sucking sample solution and control solution 4 μ l each, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-formic acid (7: 2: 0.5) as developing agent, taking out, air drying, spraying 5% vanillin sulfuric acid solution, and heating until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

(2) The identification method of the glabrous greenbrier rhizome comprises the following steps: taking a proper amount of the product, grinding, taking 1g, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, drying the filtrate by distillation, dissolving the residue in a proper amount of water, adding the mixture on a treated polyamide column (80-100 meshes, 5g, the inner diameter of the column is 1cm, and the column is filled in a wet method by water) and washing with water until the effluent is colorless, then eluting with ethanol, collecting 20ml of the eluate, drying by distillation, and dissolving the residue in 1ml of ethanol to obtain a sample solution. Or taking the solution for standby in the identification method of red peony root as a test solution.

Taking 1g of rhizoma Smilacis Glabrae as reference material, adding 20ml of ethanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate, and dissolving residue with 1ml of ethanol to obtain reference solution. Performing thin-layer chromatography (China pharmacopoeia 2020 edition four-part general rules 0502) test, sucking 5 μ l of test solution, and 5-10 μ l of rhizoma Smilacis Glabrae reference solution, respectively dropping on the same silica gel G thin-layer plate, developing with petroleum ether, ethyl acetate, methanol, formic acid (3: 6: 2). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.

(3) The method for identifying the glabrous sarcandra herb comprises the following steps: taking a proper amount of the product, grinding, taking 1g, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, drying the filtrate by distillation, dissolving the residue in a proper amount of water, adding the mixture on a treated polyamide column (80-100 meshes, 5g, the inner diameter of the column is 1cm, and the column is filled in a wet method by water) and washing with water until the effluent is colorless, then eluting with ethanol, collecting 20ml of the eluate, drying by distillation, and dissolving the residue in 1ml of ethanol to obtain a sample solution. Or taking the standby solution in the identification method of the red paeony root as a test solution.

Taking 2g of herba Pileae Scriptae control medicinal material, adding 30ml of petroleum ether, performing ultrasonic treatment for 30min, filtering, evaporating to dryness, and dissolving the residue with 1ml of ethanol to obtain a control solution. Performing thin layer chromatography (China pharmacopoeia 2020 edition four-part general rules 0502) test, sucking control solution and sample solution 2-4 μ l respectively, dropping on the same silica gel G thin layer plate, developing with cyclohexane-ethyl acetate-formic acid (6: 2: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.

(4) The identification method of the dark plum comprises the following steps: taking a proper amount of the product, grinding, taking 1g, adding 15ml of water, carrying out ultrasonic treatment for 15 minutes, centrifuging, taking supernatant, concentrating to 9ml, adding 1ml of formic acid, mixing uniformly, adding ethyl acetate into the solution, shaking and extracting twice, 10ml each time, combining ethyl acetate solutions, evaporating to dryness, and dissolving residues in 1ml of ethanol to obtain a sample solution. Adding 25ml of water into 1g of dark plum control medicinal material, heating and refluxing for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residue with 2ml of ethanol to obtain a control solution. Performing thin layer chromatography (2020 version of Chinese pharmacopoeia, general rules of the four parts 0502), sucking 5 μ l of test solution and 2 μ l of control solution, respectively dropping on the same silica gel G thin layer plate, developing with butyl acetate-formic acid-water (4: 2) upper layer solution as developing agent, taking out, air drying, spraying with 0.04% bromophenol blue ethanol solution, and inspecting in sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

(5) The method for detecting the content of paeoniflorin in red paeony root comprises the following steps: the content is determined according to high performance liquid chromatography (China pharmacopoeia 2020 edition four-part general regulation 0512).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength was 230nm. The number of theoretical plates is not less than 3000 calculated according to paeoniflorin peak;

preparation of control solutions: collecting appropriate amount of penoniflorin control which is dried in phosphorus pentoxide reduced pressure drier for 36 hr, precisely weighing, and adding methanol to obtain solution containing 0.3mg per 1ml to obtain control solution;

preparation of a test solution: grinding the product with different weights, weighing 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 30ml of 80% methanol, sealing, weighing, heating and refluxing for 1 hr, cooling, weighing again, supplementing the weight loss with 80% methanol, shaking, filtering, and collecting the subsequent filtrate to obtain the sample solution;

the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Each tablet of the product contains paeoniflorin (C) as main ingredient ₂₃ H ₂₈ O ₁₁ ) Calculated, not less than 4mg.

In order to ensure the scientific, reasonable and feasible detection method, the detection method is researched and investigated.

1. Investigating the content determination methodology of red peony root:

the red paeony root is the monarch drug in the paeoniflorin tablet (the preparation of the invention), paeoniflorin is the main effective component of the red paeony root, so paeoniflorin is selected as the index component of the internal quality of the preparation, and high performance liquid chromatography is adopted to carry out sample determination.

1. Test materials

Agilent technologies1200series high performance liquid chromatograph, one hundred thousand electronic balance (mettler-toledo instruments (shanghai) ltd.), methanol (analytical grade 20130411, shanghai shenbo chemical ltd.), paeoniflorin reference (lot number 110736-201337/110736-201438), provided by the chinese food and drug assay institute. All reagents are analytically pure. Pao ling pian (20160306, 20160310, 20160316, 20190118, 20190123, 20190126).

2. Chromatographic conditions were prepared according to the conditions in the examples.

3. Preparation of control solutions

Prepared in the manner of the examples.

4. Preparation of test solution

Prepared in the manner of the examples.

5. Preparation of negative control solution (lacking red peony root): taking the other prescription medicinal materials except the red peony root, preparing a negative sample according to the preparation process method, and preparing a negative control solution without the red peony root according to the preparation method of the test solution.

6. Methodology validation

6.1, performing a system adaptability test according to the chromatographic conditions and the sample treatment method, wherein paeoniflorin is separated from other components at a baseline, and the peak shape is good; the test result of the negative sample shows that other components do not interfere with the determination of the paeoniflorin. The results are shown in FIGS. 1 to 3.

6.2, examination of linearity and range of paeoniflorin: precisely sucking a proper amount of paeoniflorin reference substance solution (C =0.51246 mg/ml), respectively adding methanol to dilute into solutions with the concentrations of 0.128mg/ml, 0.192mg/ml, 0.256mg/ml, 0.320mg/ml and 0.384mg/ml, respectively and precisely sucking 10 mul, injecting into a liquid chromatograph, measuring peak area integral values, and drawing a standard curve by taking the concentration of the reference substance solution as an abscissa and the peak area integral values as ordinates, wherein the regression equation is as follows: y = (2E + 07) x-51326, r =0.9998. The result shows that paeoniflorin has good linear relation in the range of 0.128-0.384 mg/ml.

6.3 precision test

Precision test of paeoniflorin: the same sample (lot number 20160223) was sampled at 3 different concentrations, namely low, medium and high (80%, 100% and 120% respectively), and 3 test solutions were prepared for each concentration, and the samples were measured by different liquid chromatographs by different operators, and the results of the 9 samples were evaluated to show that the instrument precision was good at an RSD of 1.76%.

6.4 repeatability test

The same batch of samples (batch number 20160223) were taken, 3 different concentrations of low, medium and high (concentrations 80%, 100% and 120% respectively) were sampled, 3 test solutions were prepared for each concentration for measurement, and the RSD was 0.88% as evaluated by the measurement results of 9 samples, indicating that the method had good reproducibility.

6.5 stability test

Respectively and precisely sucking 10 mu l of the test solution to be tested, injecting samples for 0, 2, 4, 8 and 12 hours, measuring the peak area integral value, wherein the RSD value is 2.21 percent, which shows that the paeoniflorin in the test solution has good stability within 12 hours.

6.6 sample application recovery test

Taking the same batch of samples (batch number 20160223, the content is 11.20 mg/g), designing high, medium and low concentrations (3 concentrations are 80%, 100% and 120% respectively), controlling the ratio of the added amount of the paeoniflorin reference substance with the medium concentration to the amount of the component to be measured in the sample to be measured at 1: 1, preparing 3 sample solutions respectively, measuring, evaluating by using 9 measurement results, and calculating the recovery rate. The results are shown in Table 1.

TABLE 1 Paeoniflorin sample application recovery test results (n = 9)

Analysis of the results in table 1 shows that the sample recovery mean value is 98.84% and the RSD is 1.85%, indicating that the method has high accuracy.

6.7 durability test

And (3) selecting the comparison of different chromatographic columns and column temperatures in the experiment, processing according to the processing methods under 3 and 4 items, and respectively inspecting the factors.

6.7.1 investigation of different chromatography columns

2 portions of the same batch of samples (batch 20160223) were taken and analyzed by assay using A: TECIC 18 (250 mm. Times.4.6 mm,5 μm, column number: 1000-15), B: eilide C18 (250 mm. Times.4.6 mm,5 μm, column number E2622802), C: sepaxC18 (250 mm. Times.4.6 mm,5 μm, column number: 12301470737) was used in the column assay of three brands, and the results are shown in Table 2.

TABLE 2 measurement results of different chromatographic columns

As shown by the results in Table 2, there was no significant difference in the results when measured using three brands of columns.

6.7.2 investigation of different column temperatures

Taking the same batch of samples, preparing a test solution according to a content determination method, and respectively performing determination at the column temperature of 30 ℃, 20 ℃ and 10 ℃, wherein the results are shown in Table 3.

TABLE 3 investigation of different column temperatures

The results in Table 3 show that the column temperature is 30-10 deg.C, and the determination results have no obvious difference.

6.8 and 6 batches of samples have content measurement results: the content of paeoniflorin in the six samples was determined according to the above content determination method, and the results are shown in table 4.

TABLE 4 determination results of paeoniflorin content in Paeoniflorin tablets of 6 batches

As analyzed from the results in Table 4, the average paeoniflorin content in the above six samples was 6.48 mg/tablet, and considering the loss problem in mass production, it was tentatively determined that each tablet of this product contains paeoniflorin (C) in an amount of 70% of the average paeoniflorin content ₂₃ H ₂₈ O ₁₁ ) Calculated, not less than 4mg.

2. Identification of red peony root:

radix Paeoniae Rubra is dried root of Paeoniauveitetchillynch belonging to Ranunculaceae, and is recorded in 2020 edition Chinese pharmacopoeia (part one). The experiment selects red peony root as reference medicine, firstly adopts ultrasonic extraction, then uses polyamide column to elute and remove impurity to extract the characteristic component of red peony root in the peony root-poria tablet, and makes thin-layer identification on the red peony root medicine, and examines the influence of specificity, different thin-layer plates, different temperatures and different humidities on the thin-layer chromatography of the red peony root medicine in the preparation. The test result shows that: the red peony root reference medicinal material is taken as a reference substance, trichloromethane-methanol-formic acid (7: 2: 0.5) is taken as an expansion system, the thin-layer identification characteristic of the red peony root medicinal material is obvious, the specificity is strong, and the red peony root reference medicinal material can be taken as a thin-layer identification method of the red peony root medicinal material in the peony root-poria tablet, so the red peony root reference medicinal material is listed in the detection project of the invention.

2.1 instruments, reagents and samples

The preparation method comprises the following steps of providing a KQ-250DB type numerical control ultrasonic cleaner (ultrasonic instruments, inc. of Kunshan), a DGG-9246A electric heating constant temperature blast drying box (Shanghai Qixin scientific instruments, inc.), a one hundred thousand electronic balance (Mettler-Tooliduo instruments, shanghai) and reagents which are analytically pure, wherein the red peony root control medicinal materials (Chinese medicine biological product institute, with the number of 121092-201) and the peony-poria sheets (20160223, 20160306, 20160310 and 20160316 are provided by pharmaceutical companies of Bailing enterprises, guizhou). Thin layer chromatography plate source: (1) Self-made thin-layer plate thin-layer chromatography silica gel G (Qingdao sea wave silica gel desiccant factory, batch number: 1506011), chemically pure; sodium carboxymethylcellulose (CMC-Na, chemical reagent division of Wuxi medicine instrument procurement supply station in Jiangsu province, batch number: 20110112); distilled water: and (4) self-making. G-0.5% CMC.Na plate, specification: 10X 20cm, thickness about 0.3mm. (2) precast slab: silica gel G thin layer plate (Qingdao sea wave silica gel desiccant Co., ltd., batch No.: 20150312), specification: 10X 20cm, and a thickness of about 0.2-0.25mm.

2.2 preparation of test solutions

Taking a proper amount of the product, grinding, taking 1g, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, drying the filtrate by distillation, adding a proper amount of water into residues for dissolving, adding the residues onto a treated polyamide column (80-100 meshes, 5g, the inner diameter of the column is 1cm, and the column is filled by a water wet method), washing the polyamide column with water until the effluent is colorless, combining the passing solution and the eluent, drying by distillation, and adding 1ml of ethanol into the residues for dissolving to obtain a sample solution.

2.3 preparation of control solutions

Taking 0.5g of radix Paeoniae Rubra as reference material, adding 10ml of ethanol, shaking for 5 minutes, filtering, evaporating filtrate to dryness, and dissolving residue with 2ml of ethanol to obtain reference solution.

2.4 preparation of negative samples

Preparing a negative sample without red peony root according to the formula proportion and the preparation process and the same method of '2.2 items'.

2.5 deployment System

Chloroform-methanol-formic acid (7: 2: 0.5).

2.6 color-developing agent

5% vanillin sulfuric acid solution.

2.7 examination of conditions of thin layer chromatography

2.7.1 specialization examination

Sucking 4 μ l of the above solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-formic acid (volume ratio of 7: 2: 0.5) as developing agent, taking out, air drying, spraying 5% vanillin sulfuric acid solution, and heating until the spots are clearly developed. In the chromatogram of the test solution, spots with the same color appear at the positions corresponding to the chromatogram of the control solution, and no interference of the negative control solution on identification is observed. The results are shown in FIG. 4.

As can be seen from FIG. 4, the test solution was not separated by the polyamide film sheet; the silica gel G plate has good separation effect and round spots, and spots with the same color appear at the corresponding positions of the chromatogram of the solution of the reference substance, which shows that the method has strong specificity and the spots are clear in color development. And a methodological validation was performed.

2.8 methodological validation

2.8.1 durability test different thin layer plates were compared, and different temperatures and humidity were used as the variation factors, and the test was performed according to the above-mentioned identification method.

2.8.1.1 different thin layer plate tests

Taking a self-made silica gel G-CMC-Na plate and a prefabricated silica gel G plate, and respectively testing according to a proposed method. The results are shown in FIGS. 5 to 6. As a result, the separation effect of the self-made silica gel G-CMC-Na plate is not obviously different from that of the prefabricated silica gel G plate. The result shows that the thin layer identification chart of the red paeony root in the Shao Ling tablet has good main spot separation effect, round spots, little influence of different thin layer plates on the main spots and better repeatability.

2.8.2.2 different temperatures

Taking the thin layer plate after sample application, developing at 10 deg.C and 25 deg.C respectively, and inspecting. The result shows that the test in the temperature range of 10-25 ℃ has no obvious influence on the identification. The test patterns are shown in FIGS. 7 to 8.

2.8.2.3 different humidity test

The spotted thin layer plates were developed in chromatography jars with relative humidity adjusted to 18% and 88% with sulfuric acid, respectively, and examined. The result shows that the test at the relative humidity of 18-88% has no obvious influence on the identification. The test patterns are shown in FIGS. 9 to 10.

The experimental spectrums with different temperatures and relative humidities are examined, so that fluorescent spots with the same color are displayed in the positions, corresponding to the positions of the chromatogram of the reference substance, of the sample chromatogram, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control is free of interference. It is thus understood that the temperature and relative humidity vary within the normal environmental range, and do not affect the effectiveness of the identification method.

The results of the above methodology verification tests show that: the separation effect of the hand-laid silica gel G-CMC-Na plate on the aspect of the thin-layer plate is not obviously different from that of a prefabricated silica gel G plate; the temperature and humidity change has little influence on the unfolding effect. Therefore, the durability of the method is considered to be good.

3. Identification of rhizoma smilacis glabrae:

rhizoma Smilacis Glabrae is dried rhizome of Smilaxglabra Roxb. Collected in the 2020 edition of Chinese pharmacopoeia (part I). Rhizoma smilacis glabrae is used as a monarch drug of a recipe of the peony lingua tablets, in the experiment, rhizoma smilacis glabrae reference medicinal material is selected as an index component to perform an experiment, the rhizoma smilacis glabrae reference medicinal material is used as a reference to perform thin-layer identification, and the influence of specificity, different thin-layer plates, different temperatures and different humidities on the thin-layer chromatography of the rhizoma smilacis glabrae medicinal material in the peony lingua tablets is examined. The test result shows that the thin layer identification characteristic of the rhizoma smilacis glabrae medicinal material is obvious and the specificity is strong when the rhizoma smilacis glabrae reference medicinal material is used as a reference and petroleum ether, ethyl acetate, methanol and formic acid (3: 6: 2. Therefore, the method can be used as a thin-layer identification method for the rhizoma smilacis glabrae medicinal material of the Paeonia lactiflora pall, poria cocos slice and the like, and is listed as a detection item of the invention.

3.1 instruments, reagents and samples

The invention discloses a KQ-250DB type numerical control ultrasonic cleaner (ultrasonic instruments, inc. of Kunshan), a DGG-9246A electric heating constant temperature air blast drying box (Shanghai Qixin scientific instruments, inc.), a one hundred thousand electronic balance (Mettler-Tooliduo instruments, shanghai) and reagents are analytically pure, glabrous greenbrier rhizome control medicinal materials (Chinese food and drug testing research institute for content determination, numbered 121439-201102) and 20160223, 20160306, 20160310 and 20160316.

3.2 preparation of test solutions

Taking a proper amount of the product, grinding, taking 1g, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, drying the filtrate by distillation, dissolving the residue in a proper amount of water, adding the mixture on a treated polyamide column (80-100 meshes, 5g, the inner diameter of the column is 1cm, and the column is filled in a wet method by water) and washing with water until the effluent is colorless, then eluting with ethanol, collecting 20ml of the eluate, drying by distillation, and dissolving the residue in 1ml of ethanol to obtain a sample solution.

3.3 preparation of control solutions

Taking 1g of rhizoma Smilacis Glabrae as reference material, adding 20ml of ethanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate, and dissolving residue with 1ml of ethanol to obtain reference solution.

3.4 preparation of negative samples

Preparing a negative sample without the glabrous greenbrier rhizome medicinal material according to the formula proportion and the preparation process, and preparing a negative sample solution according to the same method of '3.2 items'.

3.5 deployment System

Petroleum ether, ethyl acetate, methanol and formic acid (volume ratio is 3: 6: 2.

3.6 color-developing agent

And (3) testing the aluminum trichloride solution.

3.7 examination of color development conditions

Sucking 5 μ l of the above solutions, respectively dropping on the same silica gel G thin layer plate, developing, taking out, air drying, spraying with aluminum trichloride solution, standing for 5 min, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution. See fig. 11.

As can be seen from FIG. 11, the color of the spot is directly related to the temperature and time after the glabrous greenbrier rhizome is sprayed with the aluminum trichloride test solution, if the temperature is high, the spot is yellow after being placed for 3-5 minutes, the temperature is low, the spot is brown, and the color development effect is better after the glabrous greenbrier rhizome is sprayed with the aluminum trichloride test solution and heated for 3-5 minutes at 105 ℃.

3.8 methodological validation

3.8.1 specialization examination

Sucking 5 μ l of each of the above solutions, respectively dropping on the same silica gel G thin layer plate, developing with petroleum ether, ethyl acetate, methanol and formic acid (volume ratio is 3: 6: 2: 1) as developing agent, taking out, air drying, spraying with aluminum trichloride test solution, standing for 5 min, and inspecting under ultraviolet lamp (365 nm). The results are shown in FIG. 11.

As can be seen from fig. 11, the negative sample was free of interference; the spot with the same color is clear and round at the position corresponding to the chromatogram of the reference substance, and the separation effect is good. The method is proved to have strong specificity and clear spot color.

3.8.2 durability tests different thin layer panels were compared separately, with different temperatures and humidity as the variation factors, and the experiments were performed as described above for the identification method.

3.8.2.1 different thin layer plate test

Taking a self-made silica gel G-CMC-Na plate and a prefabricated silica gel G plate, and respectively testing according to a proposed method. The results are shown in FIGS. 12 to 13.

The results of fig. 12 to fig. 13 show that the thin layer identification chart of smilax glabra in the paeoniflorin tablets has good spot separation effect, the spots are round, the separation effect of the self-made silica gel G-CMC-Na plate is not obviously different from that of the prefabricated silica gel G plate, and the repeatability is better.

3.8.2.2 different temperature tests

Taking the thin layer plate after sample application, respectively developing at 10 deg.C and 25 deg.C, and inspecting. The test patterns are shown in FIGS. 14 to 15.

The results of fig. 14 to 15 show that the test in the range of 10 to 25 ℃ has no obvious influence on the identification.

The experimental spectra of different temperatures and relative humidities show that in the chromatogram of the test sample, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram of the reference sample, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control is free of interference. It is thus understood that the temperature and relative humidity vary within the normal environmental range, and do not affect the effectiveness of the identification method.

3.8.2.2 different humidity test

The spotted thin layer plates were developed in chromatography jars with relative humidity adjusted to 18% and 88% with sulfuric acid, respectively, and examined. The test patterns are shown in FIGS. 16 to 17.

The results of FIGS. 16-17 show that the test at 18-88% relative humidity has no significant effect on the identification. The experimental spectra of different temperatures and relative humidities show that in the chromatogram of the test sample, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram of the reference sample, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control is free of interference. It is thus understood that the temperature and relative humidity vary within the normal environmental range, without affecting the effectiveness of the identification method. Therefore, the durability of the method is considered to be good.

4. Identification of glabrous sarcandra herb

In the 2020 edition of the Chinese pharmacopoeia (part I). Herba sarcandrae is used as a messenger drug of the Paeonia suffruticosa tablet prescription, in the experiment, herba sarcandrae reference medicinal materials are selected as a reference, petroleum ether is adopted to fully perform ultrasonic extraction on characteristic components, thin-layer identification is performed on the herba sarcandrae medicinal materials, and the influence of different sample amounts, thin-layer plates of different manufacturers, different temperatures and different humidities on the thin-layer chromatography of the herba sarcandrae medicinal materials in the preparation is investigated. The test result shows that: the glabrous sarcandra herb reference medicinal material is used as a reference substance, cyclohexane-ethyl acetate-formic acid (the volume ratio is 6: 2: 1) is used as an expansion system, the thin-layer identification characteristic of the glabrous sarcandra herb medicinal material is obvious, the specificity is strong, and the thin-layer identification method can be used as the thin-layer identification method of the glabrous sarcandra herb medicinal material in the peony and poria tablet, so the thin-layer identification method is listed in the detection project of the invention.

4.1 instruments, reagents and samples

The preparation method comprises the following steps of providing a KQ-250DB type numerical control ultrasonic cleaner (ultrasonic instruments, inc. in Kunshan), a DGG-9246A electric heating constant temperature air blast drying box (Shanghai Qixin scientific instruments, inc.), a one hundred thousand electronic balance (Mettler-Tooliduo instruments, shanghai) and reagents which are analytically pure, glabrous sarcandra herb control medicinal materials (Chinese food and drug testing institute for content determination, numbered 121048-201205), and Paeonia cocos sheets (20160223, 20160306, 20160310 and 20160316).

4.2 preparation of test solutions

Taking a proper amount of the product, grinding, taking 1g, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, drying the filtrate by distillation, dissolving the residue in a proper amount of water, adding the mixture on a treated polyamide column (80-100 meshes, 5g, the inner diameter of the column is 1cm, and the column is filled in a wet method by water) and washing with water until the effluent is colorless, then eluting with ethanol, collecting 20ml of the eluate, drying by distillation, and dissolving the residue in 1ml of ethanol to obtain a sample solution.

4.3 preparation of control solutions

Taking 2g of herba Pileae Scriptae control medicinal material, adding 30ml of petroleum ether, performing ultrasonic treatment for 30min, filtering, evaporating to dryness, and dissolving the residue with 1ml of ethanol to obtain a control solution.

4.4 preparation of negative samples

Preparing a negative sample without sarcandra glabra medicinal material according to the prescription proportion and the preparation process, and preparing a negative sample solution according to the same method of '4.2 items'.

4.5 deployment System

Cyclohexane-ethyl acetate-formic acid (volume ratio 6: 2: 1).

4.6 examination of the color development Condition

Sucking 2-4 μ l of the above solutions, respectively dropping on the same silica gel G thin layer plate, developing, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The test chromatogram shows the same color of fluorescent spot at the corresponding position of the control chromatogram, as shown in FIG. 18.

4.7 methodological validation

4.7.1 specialization examination

Sucking 2-4 μ l of the above solutions, respectively dropping on the same silica gel G thin layer plate, developing with cyclohexane-ethyl acetate-formic acid (volume ratio of 6: 2: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm), as shown in FIG. 18.

As can be seen in fig. 18, the negative sample was not perturbed; the color of the spot is the same as that of the reference substance at the corresponding position of the chromatogram, the spot is round, and the separation effect is good. The method is proved to have strong specificity and clear spot color.

4.8.2 durability test

And respectively comparing different thin layer plates, taking different temperatures and humidity as variation factors, and carrying out experiments according to the identification method.

4.8.2.1 different thin layer plate test

Taking a self-made silica gel G-CMC-Na plate and a prefabricated silica gel G plate, and respectively testing according to a proposed method. The results are shown in FIGS. 19 to 20.

The results of fig. 19-20 show that the thin layer identification of smilax glabra in the paeoniflorin tablets have good spot separation effect, the spots are round, the separation effect of the self-made silica gel G-CMC-Na plate is not obviously different from that of the prefabricated silica gel G plate, and the repeatability is better.

4.8.2.2 different temperature tests

Taking the thin layer plate after sample application, developing at 10 deg.C and 25 deg.C respectively, and inspecting. The test patterns are shown in FIGS. 21 to 22. The results of FIGS. 21 to 22 show that the test at 10 to 25 ℃ has no obvious influence on the identification.

The experimental spectra of different temperatures and relative humidities show that in the chromatogram of the test sample, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram of the reference sample, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control is free of interference. It is thus understood that the temperature and relative humidity vary within the normal environmental range, without affecting the effectiveness of the identification method.

4.8.2.2 differential humidity test

The spotted thin layer plates were developed in chromatography jars with relative humidity adjusted to 18% and 88% with sulfuric acid, respectively, and examined. The test patterns are shown in FIGS. 23 to 24.

The results of FIGS. 23-24 show that the test at 18-88% relative humidity has no significant effect on the identification. The experimental spectra of different temperatures and relative humidities show that in the chromatogram of the test sample, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram of the reference sample, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control is free of interference. It is thus understood that the temperature and relative humidity vary within the normal environmental range, and do not affect the effectiveness of the identification method. Therefore, the durability of the present method is considered to be good.

5. Identification of dark plum

In the 2020 edition of the Chinese pharmacopoeia (part I). The experiment selects dark plum reference medicinal materials as reference, adopts ultrasonic to extract characteristic components, uses ethyl acetate to shake and extract the characteristic components of dark plum in the Shao ling tablets after formic acid, performs thin layer identification on the dark plum medicinal materials, and inspects the influence of thin layer plates of different manufacturers, different temperatures and different humidity on the thin layer chromatography of the dark plum medicinal materials in the preparation. The test result shows that: the dark plum reference medicinal material is taken as a reference substance, the butyl acetate-formic acid-water (volume ratio is 4: 2) upper layer solution is taken as an expansion system, the thin layer identification characteristic of the swollen dark plum medicinal material is obvious, the specificity is strong, and the dark plum reference substance can be taken as a thin layer identification method of the dark plum medicinal material in the Shao Ling tablet, so the dark plum reference medicinal material is listed as a detection project of the invention.

5.1 instruments, reagents and samples

The invention discloses a KQ-250DB type numerical control ultrasonic cleaner (ultrasonic instruments, inc. of Kunshan), a DGG-9246A electric heating constant temperature blast drying box (Shanghai Qixin scientific instruments, inc.), a one hundred thousand electronic balance (Mettler-Tooliduo instruments, shanghai) and reagents are analytically pure, dark plum control medicinal materials (Chinese food and drug testing research institute for content determination, with lot numbers of 121208-201305) and Paeonia lactiflora slice (20160223, 20160306, 20160310 and 20160316).

5.2 preparation of test solutions

Taking a proper amount of the product, grinding, taking 1g, adding 15ml of water, carrying out ultrasonic treatment for 15 minutes, centrifuging, taking supernatant, concentrating to 9ml, adding 1ml of formic acid, mixing uniformly, adding ethyl acetate into the solution, shaking and extracting twice, 10ml each time, combining ethyl acetate solutions, evaporating to dryness, and dissolving residues in 1ml of ethanol to obtain a sample solution.

5.3 preparation of control solutions

Taking 1g of mume fructus as reference material, adding 25ml of water, heating and refluxing for 30min, filtering, evaporating filtrate to dryness, and dissolving residue with 2ml of ethanol to obtain reference solution.

5.4 preparation of negative samples

Preparing a negative sample without dark plum medicinal materials according to the formula proportion and the preparation process, and preparing a negative sample solution according to the same method of '5.2 items'.

5.5 deployment System

Butyl acetate-formic acid-water (volume ratio 4: 2) upper layer solution.

5.6 examination of color development conditions

Sucking sample solution 5 μ l and control solution 2 μ l, respectively dropping on the same silica gel G thin layer plate, developing with butyl acetate-formic acid-water (volume ratio of 4: 2) upper layer solution as developing agent, taking out, air drying, spraying with 0.04% bromophenol blue ethanol solution, and inspecting under sunlight. Spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution, as shown in FIG. 25.

5.7 color developing Agents

0.04% bromophenol blue in ethanol.

5.8 methodological validation

5.8.1 specialization examination

Sucking sample solution 5 μ l and control solution 2 μ l, respectively dropping on the same silica gel G thin layer plate, developing with butyl acetate-formic acid-water (volume ratio of 4: 2) upper layer solution as developing agent, taking out, air drying, spraying with 0.04% bromophenol blue ethanol solution, and inspecting in sunlight as shown in FIG. 25.

As can be seen in fig. 25, the negative sample was not perturbed; the spot with the same color is clear and round at the position corresponding to the chromatogram of the reference substance, and the separation effect is good. The method is proved to have strong specificity and clear spot color.

5.8.2 durability test

And respectively comparing different thin layer plates, taking different temperatures and humidity as variation factors, and carrying out experiments according to the identification method.

5.8.2.1 different thin layer plate test

Taking a self-made silica gel G-CMC-Na plate and a prefabricated silica gel G plate, and respectively testing according to a proposed method. The results are shown in FIGS. 26 to 27.

The results of fig. 26-27 show that the thin layer identification chart of the dark plums in the paeoniflorin tablets has good spot separation effect, the spots are round, the separation effect of the self-made silica gel G-CMC-Na plate is not obviously different from that of the prefabricated silica gel G plate, and the repeatability is better.

5.8.2.2 different temperature test

Taking the thin layer plate after sample application, developing at 10 deg.C and 25 deg.C respectively, and inspecting. The test patterns are shown in FIGS. 28 to 29. The results of FIGS. 28 to 29 show that the test at 10 to 25 ℃ has no obvious influence on the identification.

The experimental spectra of different temperatures and relative humidities show that in the chromatogram of the test sample, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram of the reference sample, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control is free of interference. It is thus understood that the temperature and relative humidity vary within the normal environmental range, and do not affect the effectiveness of the identification method.

5.8.2.2 differential humidity test

The spotted thin layer plates were developed in chromatography jars adjusted to 18% relative humidity and 88% relative humidity with sulfuric acid, respectively, and examined. The test patterns are shown in FIGS. 30 to 31.

The results of FIGS. 30-31 show that the test at 18-88% relative humidity has no significant effect on the identification. The experimental spectrums with different temperatures and relative humidities show that spots with the same color are displayed on the positions, corresponding to the positions of the chromatogram of the reference substance, of the chromatogram of the test substance, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control is free of interference. It is thus understood that the temperature and relative humidity vary within the normal environmental range, and do not affect the effectiveness of the identification method. Therefore, the durability of the present method is considered to be good.

Claims (10)

1. A detection method of a traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis syndrome is characterized by comprising the following steps: the traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 5-15 parts of red peony root, 10-20 parts of glabrous greenbrier rhizome, 3-9 parts of zedoary, 10-20 parts of glabrous sarcandra herb and 5-15 parts of dark plum fruit, wherein the detection method comprises the steps of identifying the red peony root, the glabrous greenbrier rhizome, the glabrous sarcandra herb and the dark plum fruit in the traditional Chinese medicine preparation by adopting thin-layer chromatography and determining the content of paeoniflorin in the red peony root by adopting high performance liquid chromatography.

2. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 1, characterized in that: the identification method of the red paeony root comprises the following steps: taking 1g of the ground traditional Chinese medicine preparation, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, evaporating filtrate to dryness, dissolving residues in water, adding the residues on a polyamide column, washing with water until effluent is colorless, combining the eluate with a passing solution, evaporating to dryness, and dissolving residues in 1ml of ethanol to obtain a sample solution; eluting polyamide column with ethanol, collecting eluate 20ml, evaporating to dryness, dissolving residue with ethanol 1ml, and collecting solution; adding 0.5g of radix Paeoniae Rubra as control material into 10ml of ethanol, shaking for 5 min, filtering, evaporating the filtrate, and dissolving the residue with 2ml of ethanol to obtain control solution; performing thin layer chromatography test, sucking sample solution and control solution 4 μ l each, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-formic acid as developing agent at volume ratio of 7: 2: 0.5, taking out, air drying, spraying 5% vanillin sulfuric acid solution, and heating until the spots are clearly developed.

3. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 2, characterized in that: the identification method of the glabrous greenbrier rhizome comprises the following steps: taking 1g of rhizoma Smilacis Glabrae as reference material, adding 20ml of ethanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate, and dissolving residue with 1ml of ethanol to obtain reference solution. According to the thin-layer chromatography test, 5 mul of the test solution and 5-10 mul of the glabrous greenbrier rhizome reference solution are respectively spotted on the same silica gel G thin-layer plate according to the volume ratio of 3: 6: 2:1, petroleum ether, ethyl acetate, methanol and formic acid are used as developing agents, the developing agents are developed, taken out, dried, sprayed with an aluminum trichloride test solution, placed for 5 minutes and then placed under an ultraviolet lamp for inspection.

4. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 3, characterized in that: the method for identifying the glabrous sarcandra herb comprises the following steps: taking 2g of glabrous sarcandra herb as a reference medicinal material, adding 30ml of petroleum ether, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating to dryness, and dissolving residues in 1ml of ethanol to obtain a reference solution; performing thin-layer chromatography, sucking 2-4 μ l of each of the reference solution and the sample solution, respectively dropping on the same silica gel G thin-layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent at volume ratio of 6: 2:1, taking out, air drying, and inspecting under ultraviolet lamp.

5. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 4, characterized in that: the test solution for the glabrous greenbrier rhizome and the glabrous sarcandra herb is a standby solution for identifying red paeony root, or the preparation method of the test solution for the glabrous greenbrier rhizome and the glabrous sarcandra herb comprises the following steps: taking 1g of the ground traditional Chinese medicine preparation, adding 20ml of ethanol, carrying out ultrasonic treatment for 15 minutes, filtering, evaporating filtrate to dryness, dissolving residues in water, adding the residue on a polyamide column, washing with water until effluent is colorless, eluting with ethanol, collecting eluent, 20ml, evaporating to dryness, and dissolving residues in 1ml of ethanol to obtain a sample solution.

6. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 1, characterized in that: the identification method of the dark plum comprises the following steps: taking 1g of ground traditional Chinese medicine preparation, adding 15ml of water, carrying out ultrasonic treatment for 15 minutes, centrifuging, taking supernatant, concentrating to 9ml, adding 1ml of formic acid, mixing uniformly, adding ethyl acetate into the solution, shaking and extracting twice, 10ml each time, combining ethyl acetate solutions, evaporating to dryness, and adding 1ml of ethanol into residues to dissolve the residues to obtain a sample solution; taking 1g of dark plum as a reference material, adding 25ml of water, heating and refluxing for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residues in 2ml of ethanol to obtain a reference solution. Performing thin layer chromatography test, sucking sample solution 5 μ l and reference solution 2 μ l, respectively dropping on the same silica gel G thin layer plate, taking the upper layer solution of butyl acetate-formic acid-water at volume ratio of 4: 2 as developing agent, spreading, taking out, air drying, spraying with 0.04% bromophenol blue ethanol solution, and inspecting in sunlight.

7. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 1, characterized in that: the method for measuring the content of paeoniflorin in red paeony root comprises the following steps: performing gradient elution by using paeoniflorin as a reference, octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and 0.1% phosphoric acid solution as a mobile phase B, wherein the mobile phase A is 14-20% and the mobile phase B is 86-80% within 0-20 min; within 20-30 min, the mobile phase A is from 20% to 80%, and the mobile phase B is from 86% to 20%; within 30-40 min, the proportion of the mobile phase A is 80 percent, and the proportion of the mobile phase B is 20 percent; the detection wavelength is 230nm; precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

8. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 7, characterized in that: the preparation method of the reference substance solution comprises the following steps: collecting paeoniflorin control dried in phosphorus pentoxide vacuum dryer for 36 hr, and adding methanol to obtain solution containing 0.3mg per 1 ml; the preparation method of the test solution comprises the following steps: taking 0.5g of the ground traditional Chinese medicine preparation, adding 30ml of 80% methanol, sealing, weighing, heating and refluxing for 1 hour, cooling, weighing again, complementing the weight loss with 80% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine preparation.

9. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 1, characterized in that: the traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 15 parts of red peony root, 20 parts of glabrous greenbrier rhizome, 9 parts of zedoary, 20 parts of glabrous sarcandra herb and 15 parts of dark plum fruit.

10. The detection method of the traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis according to claim 1, characterized in that: the preparation method of the traditional Chinese medicine preparation comprises the following steps: extracting the rhizoma smilacis glabrae with 50% ethanol twice by heating, adding ethanol which is 10 times of the rhizoma smilacis glabrae for the first time, adding ethanol which is 8 times of the rhizoma smilacis glabrae for the second time, extracting for 1 hour each time, filtering, combining ethanol solutions, recovering ethanol under reduced pressure, concentrating to obtain clear paste with the relative density of 1.12-1.14, filtering, and keeping the clear paste and dregs for later use; decocting radix Paeoniae Rubra, curcumae rhizoma, herba Pileae Scriptae and mume fructus in water twice, adding 10 times of water for the first time, soaking for 1 hr, and decocting for 1 hr; mixing the second time of the medicinal residues with the rhizoma smilacis glabrae after alcohol extraction, adding 10 times of water, decocting for 1 hour, filtering, mixing the filtrates, concentrating under reduced pressure to obtain clear paste with the relative density of 1.12-1.14, filtering, mixing with the rhizoma smilacis glabrae clear paste, adding silicon dioxide, uniformly mixing, and spray drying to obtain dry paste powder; adding pregelatinized starch and sodium carboxymethyl starch into the dry extract powder, mixing, granulating with 90% ethanol, drying at 70 deg.C, grading, and adding pregelatinized starch, sodium carboxymethyl starch, silicon dioxide and magnesium stearate.

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