Quality control method of houpu qiwu decoction

Abstract

The application discloses a quality control method of Houpu Qiwu Decoction, which comprises the following steps: S1, preparation of a test sample solution; S2, preparation of a control sample solution; S3, determination: respectively sucking the control sample solution and the test sample solution into a liquid chromatograph to perform high performance liquid chromatography analysis, recording chromatographic peaks, and obtaining a fingerprint spectrum of the Houpu Qiwu Decoction; high performance liquid chromatography conditions comprise using octadecylsilane bonded silica gel as a filler, using acetonitrile as a mobile phase A, and using a 0.1% phosphoric acid solution by volume fraction as a mobile phase B to perform gradient elution, and the fingerprint spectrum is determined by using double wavelengths: the detection wavelength is 320 nm at 0-32 min, and the detection wavelength is 254 nm at 32-70 min. The fingerprint spectrum of a Houpu Qiwu Decoction reference sample is established by using an ultra-high performance liquid chromatography method, and the contents of 12 kinds of index components such as glycyrrhizin are determined, so as to reflect the quality characteristics of the Houpu Qiwu Decoction reference sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality control method of Houpu Qiwu Decoction. BACKGROUND

[0002] Ancient classical prescriptions are prescriptions recorded in ancient Chinese medical books, which are widely used, have definite curative effect and obvious characteristic advantages. In April 2018, the State Administration of Traditional Chinese Medicine issued the Catalogue of Ancient Classical Prescriptions (First Batch), which identified the first batch of 100 classical prescriptions, and Houpu Qiwu Decoction ranked 27th.

[0003] Houpu Qiwu Decoction was first recorded in the book of Golden Chamber written by Zhang Zhongjing of the Eastern Han Dynasty. The book records: "disease abdominal fullness, fever for 10 days, pulse floating and rapid, diet as usual, Houpu Qiwu Decoction is used for treatment". The prescription is composed of seven medicinal materials, including Houpu, Gancao, Dahuang, Dazao, Zhishi, Gouzi and Shengjiang. In the prescription, Houpu can regulate qi and relieve fullness, guide stagnation and harmonize the abdominal qi; Dahuang can purge heat and relieve constipation, and can also lower turbid qi; Gouzi can dispel wind and cold from the muscles, and regulate the ying and wei; Zhishi can purge heat and relieve distention, and can also tonify the spleen and stomach; Gancao and Dazao can tonify qi and produce fluid, and Shengjiang can dispel cold from the exterior. The seven medicinal materials play the role of mutual complementation. Houpu Qiwu Decoction belongs to a warm and unobstructed agent, and is mainly used to treat deficiency-cold syndrome caused by cold entering the pulse and fine pulse. The main pathogenesis is that cold qi blocks the intestines and stomach, leading to undigested food and abdominal fullness. The main function of Houpu Qiwu Decoction is to regulate qi and relieve fullness, warm the middle and lower qi, attack and expel cold accumulation, and promote the replacement of old with new. At present, Houpu Qiwu Decoction is mainly used in the treatment of indigestion, abdominal pain and distension, and vomiting in modern clinical practice. It is also used in the treatment of intestinal inflammation and the control of senile stenosis caused by acute bronchial asthma.

[0004] So far, the modern research on the classical prescription Houpu Qiwu Decoction is mainly on pharmacological mechanism and clinical application. Sun Na et al. established the characteristic spectrum of Houpu Qiwu Decoction by UPLC-Q-TOF-MS, and identified 7 characteristic peaks including Gancao glycoside. Zhang Wenwei et al. optimized the water extraction process of Houpu Qiwu Decoction by orthogonal experiment. However, in general, there is less research on the quality control of Houpu Qiwu Decoction and the preparation process. SUMMARY

[0005] The main purpose of the present application is to provide a quality control method of Houpu Qiwu Decoction, which aims to solve the technical problem that the quality control and preparation process of Houpu Qiwu Decoction in the prior art are less researched, and the product quality characteristics cannot be fully reflected.

[0006] To achieve the above-mentioned purpose, the present application provides a quality control method of Houpu Qiwu Decoction, which comprises the following steps: S1. preparation of test sample solution; S2. preparation of control sample solution;

[0007] S3. Determination: the control solution and the test solution are respectively taken and injected into the ultra-high performance liquid chromatograph for high performance liquid chromatography analysis, the chromatographic peaks are recorded, and the fingerprint of the Houpu Qiwu Decoction is obtained;

[0008] The high performance liquid chromatography conditions in step S3 include: octadecylsilane-bonded silica gel as the filler, acetonitrile as the mobile phase A, and a 0.1% phosphoric acid solution by volume fraction as the mobile phase B for gradient elution, wherein the fingerprint is determined by double-wavelength: the detection wavelength is 320 nm at 0-32 min, and the detection wavelength is 254 nm at 32-70 min.

[0009] Alternatively, in step S3, the high performance liquid chromatography conditions further include: the chromatographic column is WATERS ACQUITY UPLCCSH C18, 2.1mmx100mm, 1.7um, the column temperature is 30-40℃, the injection amount is 2ul, and the flow rate is 0.25-0.35ml / min. Under the above chromatographic column, column temperature and flow rate conditions, the separation degree of the chromatographic peak is better.

[0010] Alternatively, the gradient elution conditions in step S3 are as follows: 0-5min, mobile phase A 10-12%, mobile phase B 90-88%; 5-15min, mobile phase A 12-13%, mobile phase B 88-87%; 15-20min, mobile phase A 13-15%, mobile phase B 87-85%; 20-32min, mobile phase A 15%, mobile phase B 85%; 32-35min, mobile phase A 15-28%, mobile phase B 85-72%; 35-45min, mobile phase A 28-30%, mobile phase B 72-70%; 45-50min, mobile phase A 30-35%, mobile phase B 70-65%; 50-55min, mobile phase A 35-37%, mobile phase B 65-63%; 55-60min, mobile phase A 37-50%, mobile phase B 63-50%; 60-70min, mobile phase A 50-70%, mobile phase B 50-30%.

[0011] Alternatively, step S1 includes the following steps: accurately weigh 0.25-0.5g of the Houpu Qiwu Decoction freeze-dried powder, accurately add 25-50ml of methanol in a container, weigh the mass after ultrasonic treatment for 20-30min, cool and weigh the mass again, add methanol to make up for the reduced mass and shake uniformly, centrifuge for 5-10min, filter under a 0.2-0.45um microporous filter membrane to obtain the test solution. The preparation can be prepared into corresponding freeze-dried powder or spray powder, etc.

[0012] Optionally, step S2 comprises the following steps: precisely weighing glycyrrhizin, naringin, cinnamyl aldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizinate, emodin, chrysophanol, honokiol, aloe-emodin, and honokiol reference substance, adding methanol to the above reference substance respectively, and shaking to prepare single reference stock solution containing 200-400 mg·L -1 , 800-1000 mg·L -1 , 800-1000 mg·L -1 , 80-100 mg·L -1 , 80-100 mg·L -1 , 80-100 mg·L -1 , 200-400 mg·L -1 , 200-400 mg·L -1 , 20-50 mg·L -1 , 80-100 mg·L -1 , 20-50 mg·L -1 , 80-100 mg·L -1 ; precisely measuring rhein, 6-gingerol, ammonium glycyrrhizinate, emodin, chrysophanol, honokiol, aloe-emodin, and honokiol single reference stock solution, mixing all in a container (after mixing, methanol is added for constant volume), adding different amounts of methanol and shaking to prepare mixed reference solution 1 with mass concentrations of 2-10 mg·L -1 , 2-10 mg·L -1 , 50-100 mg·L -1 , 10-50 mg·L -1 , 0.5-2 mg·L -1 , 2-10 mg·L -1 , 0.5-2 mg·L -1 , 2-10 mg·L -1 ; precisely measuring glycyrrhizin, naringin, cinnamyl aldehyde, and neohesperidin single reference stock solution, mixing all in a container, and adding different amounts of methanol and shaking to prepare mixed reference solution 2 with mass concentrations of 20-50 mg·L -1 , 200-500 mg·L -1 , 2-10 mg·L -1 , 200-500 mg·L -1 .

[0013] Optionally, the fingerprint spectrum of Houpu Qiwu Decoction includes 23 characteristic peaks, wherein, peaks No. 4, No. 20 and No. 22 are Magnolia officinalis, peaks No. 12, No. 18, No. 19, No. 21 and No. 23 are Rheum officinale, peaks No. 1, No. 2, No. 11, No. 13, No. 14, No. 16 and No. 17 are Glycyrrhiza uralensis, peaks No. 3, No. 5, No. 6, No. 9 and No. 10 are Aurantii Fructus, peak No. 7 is Cinnamomum cassia, peak No. 15 is Zingiber officinale, and peak No. 8 is a common peak.

[0014] Optionally, the method further comprises identifying at least one of Magnolia officinalis, Rheum officinale, Glycyrrhiza uralensis, Aurantii Fructus, Cinnamomum cassia and Zingiber officinale in Houpu Qiwu Decoction by thin layer chromatography.

[0015] Optionally, the thin layer identification process of Magnolia officinalis comprises: taking 1-2 g of Houpu Qiwu Decoction preparation, adding 20-30 ml of methanol and ultrasonicating for 20-30 min, filtering after shaking, evaporating the filtrate to obtain residue, dissolving the residue with 15-20 ml of water, extracting 2-3 times with diethyl ether, obtaining 15-20 ml of solution each time, combining the diethyl ether solutions, evaporating, dissolving the obtained residue with 2-3 ml of methanol to prepare a test sample solution; taking 1-2 g of Magnolia officinalis control medicinal material and Magnolia officinalis-free negative preparation respectively, adding 20-30 ml of methanol and ultrasonicating for 20-30 min respectively, filtering after shaking respectively, evaporating the filtrates to obtain residues, dissolving the residues with 15-20 ml of water respectively, extracting 2-3 times with diethyl ether respectively, obtaining 15-20 ml of solution each time, combining the diethyl ether solutions, evaporating, dissolving the obtained residues with 2-3 ml of methanol to prepare a control medicinal material solution and a negative test sample solution; taking magnolol and honokiol control samples, adding methanol to prepare a solution containing 1-2 mg per ml of each to prepare a control sample solution;

[0016] The thin layer identification process of Rheum officinale comprises: taking 1-2 g of Houpu Qiwu Decoction preparation, adding 20-30 ml of methanol and ultrasonicating for 45-60 min, filtering, evaporating 5-10 ml of the filtrate, dissolving the residue with 10-20 ml of water, adding 1-2 ml of hydrochloric acid, heating and refluxing for 30-45 min, immediately cooling, extracting 2-3 times with diethyl ether, obtaining 15-20 ml of solution each time, combining the diethyl ether solutions, evaporating, dissolving the obtained residue with 2-3 ml of methanol to prepare a test sample solution; taking 1-2 g of Rheum officinale control medicinal material and Rheum officinale-free negative preparation respectively, adding 20-30 ml of methanol and ultrasonicating for 45-60 min respectively, filtering, evaporating 5-10 ml of the filtrate respectively, dissolving the residues with 10-20 ml of water respectively, adding 1-2 ml of hydrochloric acid respectively, heating and refluxing for 30-45 min respectively, immediately cooling, extracting 2-3 times with diethyl ether respectively, obtaining 15-20 ml of solution each time, combining the diethyl ether solutions, evaporating, dissolving the obtained residues with 2-3 ml of methanol to prepare a control medicinal material solution and a negative sample solution; taking a Rheum officinale acid control sample, adding methanol to prepare a mixed solution containing 1-2 mg per ml of each to prepare a control sample solution;

[0017] The thin-layer identification process of liquorice includes: taking 1-2 g of the Houpu Qiwu Decoction preparation, adding 20-30 ml of ethanol for cold soaking for 20-30 min, filtering to obtain the filtrate, concentrating to 2-3 ml to prepare the test sample solution; taking 1-2 g of liquorice control medicinal materials and liquorice-free negative preparation, respectively adding 20-30 ml of ethanol for cold soaking for 20-30 min, respectively filtering to obtain the filtrate, respectively concentrating to 2-3 ml to prepare the control medicinal material solution and the negative sample solution; additionally taking glycyrrhizin and ammonium glycyrrhizinate control samples, adding methanol to prepare a mixed solution containing 1-2 mg / ml of each of the control sample solution;

[0018] The thin-layer identification process of Fructus Aurantii includes: taking 1-2 g of the Houpu Qiwu Decoction preparation, adding 20-30 ml of ethanol for cold soaking for 20-30 min, filtering to obtain the filtrate, concentrating to 2-3 ml to prepare the test sample solution; taking 1-2 g of Fructus Aurantii control medicinal materials and Fructus Aurantii-free negative preparation, adding 20-30 ml of ethanol for cold soaking for 20-30 min, filtering to obtain the filtrate, concentrating to 2-3 ml to prepare the control medicinal material solution and the negative sample solution; additionally taking naringin and neohesperidin control samples, adding methanol to prepare a mixed solution containing 1-2 mg / ml of each of the control sample solution;

[0019] The thin-layer identification process of Cinnamomi Cortex includes: taking 1-2 g of the Houpu Qiwu Decoction preparation, adding 20-30 ml of ethanol for cold soaking for 20-30 min, filtering to obtain the filtrate, concentrating to 2-3 ml to prepare the test sample solution; taking 1-2 g of Cinnamomi Cortex control medicinal materials and Jujubae-free negative preparation, adding 20-30 ml of ethanol for cold soaking for 20-30 min, filtering to obtain the filtrate, concentrating to 2-3 ml to prepare the control medicinal material solution and the negative sample solution; additionally taking cinnzamaldehyde control sample, adding ethanol to prepare a mixed solution containing 1-2 mg / ml of each of the control sample solution;

[0020] The thin-layer identification process of Zingiber officinale includes: taking 1-2 g of the Houpu Qiwu Decoction preparation, adding 20-30 ml of water to dissolve, using water-saturated n-butanol to extract 3-4 times, 15-20 ml each time, combining the n-butanol liquid, washing with water 2-3 times, 15-20 ml each time, discarding the water liquid, evaporating the n-butanol liquid, dissolving the residue in 2-3 ml of methanol to prepare the test sample solution; taking 1-2 g of Zingiber officinale control medicinal materials and Zingiber officinale-free negative preparation, adding 20-30 ml of water to dissolve, using water-saturated n-butanol to extract 3-4 times, 15-20 ml each time, combining the n-butanol liquid, washing with water 2-3 times, 15-20 ml each time, discarding the water liquid, evaporating the n-butanol liquid, dissolving the residue in 2-3 ml of methanol to prepare the control medicinal material solution and the negative sample solution; additionally taking 6-gingerol control sample, adding methanol to prepare a mixed solution containing 1-2 mg / ml of each of the control sample solution.

[0021] Optionally, the thin layer plate for Magnolia officinalis, Glycyrrhiza uralensis, Citrus aurantium, Cinnamomum cassia and Zingiber officinale is silica gel G thin layer plate, and the thin layer plate for Rheum officinale is silica gel H thin layer plate; the developing agent for Magnolia officinalis and Rheum officinale is cyclohexane-ethyl acetate-formic acid, the developing agent for Glycyrrhiza uralensis is ethyl acetate-methanol-glacial acetic acid-water, the developing agent for Citrus aurantium is chloroform-methanol-water, the developing agent for Cinnamomum cassia is petroleum ether-ethyl acetate, and the developing agent for Zingiber officinale is petroleum ether-chloroform-ethyl acetate; the Rheum officinale thin layer plate is observed under ultraviolet light with a wavelength of 365 nm; the color developing agent for Magnolia officinalis and Zingiber officinale is vanillin sulfuric acid solution, the color developing agent for Glycyrrhiza uralensis is sulfuric acid ethanol solution, the color developing agent for Citrus aurantium is aluminum chloride ethanol solution, and the color developing agent for Cinnamomum cassia is dinitrosophenylhydrazine ethanol solution, and all are observed under daylight after color development.

[0022] Optionally, the content determination method for adopting the Magnolia officinalis seven-ingredient decoction further comprises the following steps: preparation of a test sample solution: 0.25-0.5 g of the Magnolia officinalis seven-ingredient decoction freeze-dried powder is precisely weighed in a container, 25-50 ml of methanol is precisely added, the mass is weighed again after ultrasonic treatment for 15-20 min, and then the mass is weighed again after cooling, methanol is added to make up for the reduced mass and shaken uniformly, and centrifugal treatment is performed for 5-10 min, and then filtration is performed under an organic microporous filter membrane with a pore size of 0.2-0.45 um to obtain the test sample solution;

[0023] Preparation of a control sample solution: glycyrrhizin, naringin, cinnamyl aldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, honokiol, and aloe-emodin are precisely weighed and added into methanol to prepare single control sample stock solutions; rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, honokiol, and aloe-emodin control sample stock solutions are precisely weighed and mixed in a container, and different amounts of methanol are added and shaken to prepare a mixed control sample solution 1 with a mass concentration of 2-10 mg·L -1 , 2-10 mg·L -1 , 50-100 mg·L -1 , 10-50 mg·L -1 , 0.5-2 mg·L -1 , 2-10 mg·L -1 , 0.5-2 mg·L -1 , 2-10 mg·L -1 ; glycyrrhizin, naringin, and cinnamyl aldehyde control sample stock solutions are precisely weighed and mixed in a container, and different amounts of methanol are added and shaken to prepare a mixed control sample solution 2 with a mass concentration of 20-50 mg·L -1 , 200-500 mg·L -1 , 2-10 mg·L -1 , 200-500 mg·L -1 ;

[0024] Determination: the test sample solution and the control sample solution were respectively detected by the same UPLC method as the above fingerprint, using an ACQUITY UPLC CSH C18 column as the chromatographic column, acetonitrile as the mobile phase A, and a 0.1% phosphoric acid solution as the mobile phase B for gradient elution, at a flow rate of 0.25-0.35 ml / min.

[0025] The quality control method of the Magnoliae Officinalis Decoction of Seven Ingredients provided by the application has the following beneficial effects: based on the prior art, the application establishes the fingerprint of the Magnoliae Officinalis Decoction of Seven Ingredients reference sample by using the UPLC method, analyzes by using the similarity evaluation method and the related chemical pattern recognition method, simultaneously determines the content of 12 kinds of index components such as glycyrrhizin, and reflects the quality characteristics of the Magnoliae Officinalis Decoction of Seven Ingredients reference sample, thereby providing the reference basis for the subsequent research of the Magnoliae Officinalis Decoction of Seven Ingredients, better reflecting the correlation of the pharmacological and pharmacodynamic basis, having good specificity, good separation degree between the characteristic peaks, no obvious interference between different chemical components, and meeting the requirements of precision, stability and repeatability. The obtained fingerprint of the Magnoliae Officinalis Decoction of Seven Ingredients can comprehensively reflect the overall appearance of the multiple components in the Magnoliae Officinalis Decoction of Seven Ingredients formula, can qualitatively and quantitatively analyze the quality of the Magnoliae Officinalis Decoction of Seven Ingredients, and provides a new method for the quality control of the Magnoliae Officinalis Decoction of Seven Ingredients. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0027] Figure 1 The UPLC-PAD fingerprint of the Magnoliae Officinalis Decoction of Seven Ingredients provided by the application is shown in the following figure: Figure 2 The thin layer chromatogram of Magnoliae Officinalis provided by the application is shown in the following figure: Figure 3 The thin layer chromatogram of Rheum officinale provided by the application is shown in the following figure: Figure 4 The thin layer chromatogram of Glycyrrhiza uralensis provided by the application is shown in the following figure: Figure 5 The thin layer chromatogram of Citrus aurantium provided by the application is shown in the following figure: Figure 6 The thin layer chromatogram of Cinnamomum cassia provided by the application is shown in the following figure: Figure 7 The thin layer chromatogram of Zingiber officinale provided by the application is shown in the following figure: Figure 8 The UPLC-PAD fingerprint of the Magnoliae Officinalis Decoction of Seven Ingredients under different wavelength conditions provided by the application is shown in the following figure: Figure 9 The UPLC-PAD fingerprint of the Magnoliae Officinalis Decoction of Seven Ingredients under different mobile phase conditions provided by the application is shown in the following figure: Figure 10Comparison chart of UPLC-PAD fingerprint of Houpu Qiwu Decoction provided in the present application under different column conditions; Figure 11 Comparison chart of UPLC-PAD fingerprint of Houpu Qiwu Decoction provided in the present application under different column temperature conditions; Figure 12 Comparison chart of UPLC-PAD fingerprint of Houpu Qiwu Decoction provided in the present application under different flow rate conditions; Figure 13 Comparison chart of UPLC-PAD fingerprint of Houpu Qiwu Decoction provided in the present application under different extraction solvents; Figure 14 Comparison chart of UPLC-PAD fingerprint of Houpu Qiwu Decoction provided in the present application under different extraction methods; Figure 15 Comparison chart of UPLC-PAD fingerprint of Houpu Qiwu Decoction provided in the present application under different extraction times; Figure 16 Comparison chart of UPLC-PAD fingerprint of Houpu Qiwu Decoction provided in the present application under different solid-liquid ratios; Figure 17 Comparison chart of UPLC-PAD fingerprint of Houpu Qiwu Decoction provided in the present application under different solid-liquid ratios; Figure 18 UPLC-DAD fingerprint of 15 batches of Houpu Qiwu Decoction reference samples; Figure 19 Fingerprint of sample solution and comparative sample under the chromatographic conditions of the comparative example; Figure 20 Fingerprint of comparative sample under the chromatographic conditions of the comparative example; Figure 21 Fingerprint of comparative sample under the chromatographic conditions of the comparative example; Figure 22 Comparison chart of double-wavelength detection spectrum of sample of the present example and comparative sample under the chromatographic conditions of the present example; Figure 23 Chromatogram of sample of the present example and comparative sample under the chromatographic conditions of the present example and the comparative example.

[0028] The object, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0029] In order to make the object, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased in the market.

[0030] 1. Preparation of Houpu Qiwu Decoction material reference

[0031] The preparation process of the Houpu Qiwu Decoction reference sample is as follows: the medicinal materials of Houpu Qiwu Decoction, Magnolia officinalis 110.4 g, Radix et Rhizoma Rhei 41.4 g, Radix Glycyrrhizae 41.4 g, Fructus Aurantii 60 g, Cinnamomum cassia 27.8 g, Fructus Jujubae 30 g, and Rhizoma Zingiberis Recens 69 g, are weighed and put into a ceramic decocting pot, soaked for 30 min, and then boiled with 500 W heat, and kept boiling with 300 W heat; the lid is not opened and the mixture is not stirred during the decocting process; when about 800 mL of decoction is obtained, the mixture is filtered with a 400-mesh sieve while hot, and then cooled, vacuumized, sublimated, and dried to obtain the Houpu Qiwu Decoction reference sample freeze-dried powder.

[0032] 2. Identification of Houpu Qiwu Decoction by TLC

[0033] (1) Instruments

[0034]

[0035] (2) Materials

[0036]

[0037]

[0038] (3) Experimental content

[0039] Identification of Magnolia officinalis by TLC: 1.0 g of the freeze-dried powder of Houpu Qiwu Decoction of S1-S15 batches is added with 20 ml of methanol, tightly capped, and ultrasonically treated for 30 min, and then filtered; the filtrate is evaporated to dryness, and the residue is dissolved in 20 ml of water, and then extracted with diethyl ether for 2 times, 20 ml each time; the diethyl ether solution is combined and evaporated to dryness, and the residue is dissolved in 2 ml of methanol to obtain a test solution; 1 g of the control medicinal material of Magnolia officinalis and 1 g of the Houpu Qiwu Decoction reference sample without Magnolia officinalis are treated by the same method to obtain a control medicinal material solution and a negative test solution; 1 mg of honokiol and 1 mg of magnolol are dissolved in 1 ml of methanol to obtain a control solution. The test and control solutions are tested by TLC (General Test 0502) as follows: 5 ul of each sample, 2 ul of the control solution, and 2 ul of the control medicinal material solution are spotted on the same silica gel G thin layer plate, developed with cyclohexane-ethyl acetate-formic acid (15:5:0.2), taken out, dried, sprayed with 1% vanillin sulfuric acid solution, heated at 105°C until the spots are clear, and then observed under sunlight; the test sample chromatogram shows the same color spots at the corresponding positions of the control sample chromatogram.

[0040] Figure 2 Figure 1 is a TLC chromatogram of the thin layer plate of Houpu Qiwu Decoction, in which: 1-7 are Houpu Qiwu Decoction, S1-S7, 5 ul; 8 is the control medicinal material of Magnolia officinalis, 2 ul; 9 is honokiol, 2 ul; 10 is Houpu Qiwu Decoction without Magnolia officinalis, S1, 5 ul; 11 is magnolol, 2 ul; and 12-19 are Houpu Qiwu Decoction, S8-S15, 5 ul.

[0041] Thin-layer identification of Rhubarb: Take 1.0 g of S1-S15 batches of Magnolia Bark Seven Ingredients Decoction freeze-dried powder, add 20 ml of methanol, ultrasonic for 1 hour, filter, take 5 ml of filtrate and evaporate to dryness, add 10 ml of water to dissolve, add 1 ml of hydrochloric acid, heat reflux for 30 min, immediately cool, extract with ether 2 times, 20 ml each time, combine the ether solution, evaporate to dryness, add 2 ml of methanol to dissolve as the test solution; take 1 g of Rhubarb reference material and Rhubarb-free negative freeze-dried powder, prepare the reference material solution and negative sample solution by the same method; take Rhubarb acid control, add methanol to prepare a mixture solution containing 1 mg per 1 ml as the control solution. Test according to the thin-layer chromatography (General Rule 0502), take 2 ul of each sample and control, 10 ul of reference material, and point them on the same silica gel H thin-layer plate, develop with cyclohexane-ethyl acetate-formic acid (15:5:1) upper solution, take out and dry, observe under UV lamp 365 nm, the test sample chromatogram shows the same color spots at the corresponding positions of the control chromatogram.

[0042] Figure 3 It is the thin-layer chromatogram of Rhubarb: 1-7 is Magnolia Bark Seven Ingredients Decoction, S1-S7, 2 ul; 8 is Rhubarb reference material, 10 ul; 9 is Magnolia Bark negative, S1, 2 ul; 10 is Rhubarb acid, 2 ul; 11-18 is Magnolia Bark Seven Ingredients Decoction, S8-S15, 2 ul.

[0043] Thin-layer identification of Licorice: Take 1.0 g of S1-S15 batches of Magnolia Bark Seven Ingredients Decoction freeze-dried powder, add 20 ml of ethanol, cold soak for 20 min, filter, concentrate the filtrate to 2 ml as the test solution; take 1 g of Licorice reference material and Licorice-free negative freeze-dried powder, prepare the reference material solution and negative sample solution by the same method; take Glycyrrhizin and Ammonium Glycyrrhizinate control, add methanol to prepare a mixture solution containing 1 mg per 1 ml as the control solution. Test according to the thin-layer chromatography (General Rule 0502), take 1-2 ul of each solution, point them on the same silica gel G thin-layer plate, develop with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing agent, take out and dry, spray with 10% sulfuric acid ethanol solution, dry, heat at 105°C until the spots develop clearly, then observe under UV 365 nm, the test sample chromatogram shows the same color spots at the corresponding positions of the control chromatogram.

[0044] Figure 4 It is the thin-layer chromatogram of Licorice: 1-7 is Magnolia Bark Seven Ingredients Decoction, S1-S7, 10 ul; 8 is Licorice reference material, 10 ul; 9 is Licorice negative, S1, 10 ul; 10 is Glycyrrhizin, 5 ul; 11-18 is Magnolia Bark Seven Ingredients Decoction, S8-S15, 2 ul.

[0045] Identification of Fructus Aurantii in the TLC plate: Take 1.0 g of the freeze-dried powder of Houpu Qiwu Decoction of S1-S15 batches, add 20 ml of methanol, and ultrasonically extract for 30 min. Filter and evaporate the filtrate to dryness. Dissolve the residue in 2 ml of methanol to obtain the test solution. Take 1 g of the control medicinal material of Fructus Aurantii and the negative freeze-dried powder without Fructus Aurantii, and prepare the control medicinal material solution and the negative sample solution in the same manner. Take the control samples of naringin and neohesperidin, add methanol to prepare a mixture solution containing 1 mg of each per 1 ml as the control solution. Test according to the TLC method (General Rule 0502). Take 2 ul of each solution, and spot on the same silica gel G thin layer plate. Use the lower layer solution of chloroform-methanol-water (13:6:2) as the developing agent. Take out and dry, spray with 3% aluminum chloride ethanol solution, dry, and observe under ultraviolet light at 365 nm. In the test sample chromatogram, the same color spots appear at the corresponding positions of the control sample chromatogram.

[0046] Figure 5 The TLC chromatogram of Fructus Aurantii in the TLC plate is shown in the following table: 1-7 are Houpu Qiwu Decoction, S1-S7, 2 ul; 8 is the control medicinal material of Fructus Aurantii, 2 ul; 9 is glycyrrhizin, 2 ul; 10 is the negative sample of Fructus Aurantii, S1, 2 ul; 11 is neohesperidin, 2 ul; 12-19 are Houpu Qiwu Decoction, S8-S15, 2 ul.

[0047] Identification of Cinnamomi Cortex in the TLC plate: Take 1.0 g of the freeze-dried powder of Houpu Qiwu Decoction of S1-S15 batches, soak for 20 min, and concentrate the filtrate to 2 ml as the test solution. Take 1 g of the control medicinal material of Cinnamomi Cortex and the negative freeze-dried powder without Fructus Jujubae, and prepare the control medicinal material solution and the negative sample solution in the same manner. Take the control sample of cinnzaldehyde, add ethanol to prepare a mixture solution containing 1 mg of each per 1 ml as the control solution. Test according to the TLC method (General Rule 0502). Take 2-4 ul of each solution, and spot on the same silica gel G thin layer plate. Use petroleum ether (60-90℃)-ethyl acetate (17:3) as the developing agent. Take out and dry, spray with dinitrosophenylhydrazine ethanol solution, dry, and observe under sunlight. In the test sample chromatogram, the same color spots appear at the corresponding positions of the control sample chromatogram.

[0048] Figure 6 The TLC chromatogram of Cinnamomi Cortex in the TLC plate is shown in the following table: 1-7 are Houpu Qiwu Decoction, S1-S7, 2 ul; 8 is cinnzaldehyde, 4 ul; 9 is the negative sample of Cinnamomi Cortex, S1, 2 ul; 10 is the control medicinal material of Cinnamomi Cortex, 2 ul; 11-18 are Houpu Qiwu Decoction, S8-S15, 2 ul.

[0049] Thin layer identification of ginger: Take 1.0 g of Magnoliae officinalis seven-ingredient decoction freeze-dried powder of S1-S15 batches, respectively, add 20 ml of water to dissolve, extract with water-saturated n-butanol for 3 times, 20 ml each time, combine the n-butanol liquid, wash with water for 2 times, 20 ml each time, discard the water, evaporate the n-butanol liquid to dryness, add 2 ml of methanol to dissolve as the test solution; take 1 g of ginger control drug and ginger-free negative freeze-dried powder, respectively, prepare the control drug solution and negative sample solution by the same method; take 6-gingerol control, add methanol to prepare a mixed solution containing 1 mg per 1 ml as the control solution. Test according to the thin layer chromatography (General Rule 0502), take 10 ul of the test solution, 2-4 ul of the control drug, and 1-2 ul of the control, respectively, and point them on the same silica gel G thin layer plate, develop with petroleum ether (60-90℃)-trichloromethane-ethyl acetate (2:1:1) as the developing agent, take out and dry, spray with 1% vanillin sulfuric acid solution, heat at 105℃ until the spots develop clear color, then observe under daylight, the same colored spots appear in the corresponding positions of the control chromatogram in the test chromatogram.

[0050] Figure 7 It is the thin layer chromatogram of ginger, wherein: 1-7 is Magnoliae officinalis seven-ingredient decoction, S1-S7, 10 ul; 8 is ginger control drug, 4 ul; 9 is ginger-free negative, S1, 10 ul; 10 is 6-gingerol, 2 ul; 11-18 is Magnoliae officinalis seven-ingredient decoction, S8-S15, 10 ul.

[0051] 3. Fingerprint of Magnoliae officinalis seven-ingredient decoction

[0052] (1) Instrument

[0053]

[0054] (2) Materials

[0055] Name Batch / Grade Manufacturer Magnolol 110729-202015 China Institute for Food and Drug Control Honokiol 110730-201915 China Institute for Food and Drug Control Chrysophanol 110796-201922 China Institute for Food and Drug Control Rhein 110757-201607 China Institute for Food and Drug Control Emodin 110756-201913 China Institute for Food and Drug Control Aloin 110795-202011 China Institute for Food and Drug Control Glycyrrhizin 111610-202209 China Institute for Food and Drug Control Ammonium Glycyrrhizinate 110731-202122 China Institute for Food and Drug Control Naringin 110722-202116 China Institute for Food and Drug Control Neohesperidin 111857-201804 China Institute for Food and Drug Control Cinnamaldehyde 110710-202022 China Institute for Food and Drug Control 6-Gingerol 111833-202007 China Institute for Food and Drug Control Acetonitrile Chromatographic pure Thermo Fisher Scientific (China) Co., Ltd. Methanol Chromatographic pure Thermo Fisher Scientific (China) Co., Ltd. Water Pure water Guangzhou Watsons Food and Beverage Co., Ltd.

[0056] Note: The other reagents used in this scheme are all analytical pure.

[0057] (3) Establishment of fingerprint

[0058] Preparation of test solution: accurately weigh 0.5 g of Magnoliae officinalis seven-ingredient decoction freeze-dried powder, put it in a 50 mL conical flask with a stopper, accurately add 25 mL of methanol, weigh, ultrasonic treat (400 W, 40 kHZ) for 30 min, cool, weigh again, supplement the weight loss with methanol, shake well, centrifuge (12000 r·min -1 ) for 5 min, filter with 0.22 μm microporous filter membrane, to obtain the test solution.

[0059] Preparation of negative sample solution: The negative sample solutions of Radix Magnoliae Officinalis, Rhizoma et Radix Rhei, Radix Glycyrrhizae, Fructus Aurantii, Cortex Cinnamomi, Fructus Jujubae, Rhizoma Zingiberis Recens were prepared according to the above preparation method of test sample solution.

[0060] Preparation of reference solution: An appropriate amount of glycyrrhizin, naringin, cinnamaldehyde, neohesperidin, rhein, 6-shogaol, ammonium glycyrrhizinate, emodin, chrysophanol, honokiol, aloe-emodin, magnolol reference substances were accurately weighed, placed in different volumetric flasks, diluted to the mark with methanol, and shaken to prepare single reference stock solutions containing 420, 1032, 1066, 998.4, 104.3, 894.5, 615.2, 104.2, 52.7, 205.2, 50.1, 233.6 μg / mL respectively. -1

[0061] The reference stock solutions of glycyrrhizin, naringin, cinnamaldehyde, neohesperidin were accurately weighed, placed in the same volumetric flask, diluted to the mark with methanol, and shaken to prepare mixed reference solution 2 with mass concentrations of 16.80, 123.84, 2.13, 119.81 mg·L respectively. -1

[0062] (4) Investigation of chromatographic conditions

[0063] Selection of wavelength: According to the literature and the 2020 edition of Chinese Pharmacopoeia, the detection wavelengths of Houpu Qiwu Decoction fingerprint mainly include 237 nm, 254 nm, 280 nm, 283 nm, 290 nm, 294 nm, etc. PDA was used for full wavelength scanning (190-400 nm), and different wavelength chromatograms were compared to determine the wavelength selection criteria based on baseline, peak shape, peak number, separation degree, etc. in order to obtain maximum fingerprint information. The fingerprint of Houpu Qiwu Decoction in this scheme adopts double-wavelength elution method, the wavelength is 320 nm at 0-32 min and 254 nm at 32-70 min. According to the multi-wavelength comparison results, the double-wavelength elution method used in this scheme has more chromatographic peaks, appropriate peak height, complete peak shape, and stable baseline, which is more suitable for subsequent fingerprint chromatographic peak qualitative and quantitative research. Figure 8

[0064] ​​​Selection of mobile phase: In this scheme, 6 different mobile phases (mobile phase 1: acetonitrile-0.05% phosphoric acid; mobile phase 2: acetonitrile-0.1% formic acid; mobile phase 3: acetonitrile-0.1% acetic acid; mobile phase 4: acetonitrile-water; mobile phase 5: methanol-0.1% phosphoric acid; mobile phase 6: acetonitrile-0.1% phosphoric acid) were selected for UPLC sample comparison study to determine the appropriate mobile phase. According to the different mobile phase comparison results in the Figure 9 , it was found that when acetonitrile-phosphoric acid system was used as the mobile phase, the chromatographic peak separation was better, and acetonitrile-0.1% phosphoric acid had moderate peak time and better effect compared with acetonitrile-0.05%.

[0065] Selection of chromatographic column: In this scheme, different chromatographic column types were selected: Waters ACQUITY UPLC HSS T3 (2.1mm*100mm, 1.7μm), Waters ACQUITY UPLC BEH C 18 (2.1mm*100mm, 1.7μm), WATERS ACQUITY UPLC CSH TM C 18 (2.1mm*100mm, 1.7μm), and the test sample solution was injected according to the above determined conditions, and the UPLC fingerprint was compared to determine the most suitable chromatographic column. According to the different chromatographic column comparison results in the Figure 10 , it was found that when CSH TM C 18 column was used as the chromatographic condition, the number of chromatographic peak, the chromatographic peak separation and the response factor value were all better than the other two types of chromatographic peak, so the CSH TM C18 chromatographic column was selected as the fingerprint condition.

[0066] Selection of column temperature: In this scheme, different column temperatures (30℃, 35℃, 40℃) were selected, the test sample solution was injected according to the above determined conditions, and the UPLC fingerprint was compared to determine the most suitable column temperature. According to the different column temperature comparison results in the Figure 11 , it was found that when the column temperature was 35℃, the chromatographic peak separation was obviously better than that at other column temperatures, so the column temperature of 35℃ was selected as the most optimal fingerprint condition.

[0067] Selection of flow rate: Different flow rates (0.25mL·min -1 , 0.3mL·min -1 , 0.35mL·min -1 ) were selected, the test sample solution was injected according to the above determined conditions, and the UPLC fingerprint was compared to determine the most suitable flow rate. According to the different flow rate comparison results in the Figure 12 , it was found that when the flow rate of the mobile phase was 0.3mL·min -1At this time, the chromatographic peak separation degree is obviously better than that at other flow rates of the mobile phase, so the flow rate of the mobile phase is selected as 0.3 mL·min -1 as the optimal fingerprint condition.

[0068] In summary, the optimal chromatographic conditions determined by the scheme are as follows: the type of the high-performance liquid chromatograph is WATERS UPLC H-CLASS; the type of the chromatographic column is WATERS ACQUITY UPLC CSHTM C 18 (2.1 mm x 100 mm, 1.7 μm); the mobile phase is selected as acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B); the injection amount is 2 μL; the column temperature is 35 °C; the flow rate is 0.3 mL·min -1 ; the detection wavelength program is double-wavelength detection: 0-31 min, 320 nm; 31-70 min, 254 nm. The gradient elution conditions are as follows: 0-5 min, A 10-12%, B 90-88%; 5-15 min, A 12-13%, B 88-87%; 15-20 min, A 13-15%, B 87-85%; 20-31 min, A 15%, B 85%; 31-35 min, A 15-28%, B 85-72%; 35-45 min, A 28-30%, B 72-70%; 45-50 min, A 30-35%, B 70-65%; 50-55 min, A 35-37%, B 65-63%; 55-60 min, A 37-50%, B 63-50%; 60-70 min, A 50-70%, B 50-30%.

[0069] (5) Investigation of the preparation method of the test sample

[0070] Investigation of the extraction solvent: 0.5 g of the freeze-dried powder of the Houpu Qiwu Decoction was accurately weighed into a 50 mL conical flask with a stopper, 5 groups were weighed in total, each group was parallel twice, 25 mL of methanol, ethanol, 50% methanol, 50% ethanol and water were respectively and accurately added to each group, the mass was determined, ultrasonic treatment was performed for 30 min, the mass was determined again after cooling, the decreased mass was made up with the corresponding solvent, shaking was performed, and 0.22 μm microporous filter membrane was used for filtration, thereby obtaining the test sample solution of the corresponding solvent. Injection was performed according to the chromatographic conditions determined in the previous step, and the peak area of the characteristic peak was recorded. According to Figure 13 It is found from the comparison results of different extraction solvents in the method that when methanol is used as the extraction solvent, the obtained chromatographic peak has moderate peak time, better peak shape, higher separation degree, larger response factor value and stable baseline, so methanol is used as the test sample condition.

[0071] Investigation of extraction method: accurately weigh 0.5 g of Hoqiaoqi powder, place in a 50 mL conical flask with a stopper, a total of 3 groups, each group in parallel 2 times, each group is accurately added with 25 mL of methanol, and the mass is determined. One group is treated with ultrasonic for 30 min, the second group is refluxed for 30 min, and the third group is cold soaked for 30 min. After cooling, the mass is determined again, the reduced mass is supplemented with methanol, shaken, and filtered through a 0.22 μm microporous filter to obtain the corresponding test solution. Sample injection according to the chromatographic conditions determined in the previous step, record the peak area of the characteristic peak.

[0072] Different extraction method total peak area results

[0073]

[0074] According to the comparison results of different extraction methods in Figure 14 , it is found that the three extraction methods have little effect on the peak time and separation degree of the chromatographic peaks. The total peak area results of the chromatographic peaks with separation degree R>1.5 are calculated, and it is found that the total peak area of ultrasonic extraction is larger than that of the other two extraction methods. Therefore, ultrasonic extraction is used as the test sample extraction method for fingerprint spectrum investigation.

[0075] Investigation of extraction time: accurately weigh 0.5 g of Hoqiaoqi powder, place in a 50 mL conical flask with a stopper, a total of 4 groups, each group in parallel 2 times, each group is accurately added with 25 mL of methanol, and the mass is determined. One group is treated with ultrasonic for 15 min, the second group is treated with ultrasonic for 30 min, the third group is treated with ultrasonic for 45 min, and the fourth group is treated with ultrasonic for 60 min. After cooling, the mass is determined again, the reduced mass is supplemented with methanol, shaken, and filtered through a 0.22 μm microporous filter to obtain the corresponding test solution. Sample injection according to the chromatographic conditions determined in the previous step, record the peak area of the characteristic peak.

[0076] Different extraction time total peak area results

[0077]

[0078] According to the comparison results of different extraction methods in Figure 15 , it is found that the extraction time has little effect on the peak time and separation degree of the chromatographic peaks. The total peak area results of the chromatographic peaks with separation degree R>1.5 are calculated, and it is found that the increase of ultrasonic time has little effect on the total peak area. Considering the convenience of the experiment, ultrasonic extraction for 15 min is used as the test sample extraction time for fingerprint spectrum investigation.

[0079] The sample solution was prepared as follows: 0.5 g of the freeze-dried powder of the Hupo Qiwu Decoction was precisely weighed into a 50 mL conical flask, 25 mL of methanol was precisely added, the mass was determined again, the mass was determined again after cooling, and the lost mass was made up with methanol. The mixture was shaken and filtered through a 0.22 μm microporous filter to obtain the sample solution. The sample solution was injected into the chromatograph according to the chromatographic conditions determined in the previous step, and the peak area of the characteristic peak was recorded.

[0080] The total peak area results of different sample-to-liquid ratios

[0081]

[0082] According to the comparison results of different sample-to-liquid ratios in Figure 16 , it was found that the four sample-to-liquid ratio modes had little effect on the peak time and separation degree of the chromatographic peaks. The peak height and response factor value increased with the decrease of the sample-to-liquid ratio. Through the calculation of the total peak area results of the chromatographic peaks with a separation degree R>1.5, it was found that when converted to the same coefficient, the total peak area of the sample-to-liquid ratio of 0.5 g-25 mL was greater than that of the other three sample-to-liquid ratio modes. Therefore, the sample-to-liquid ratio of 0.5 g-25 mL was used for the investigation of the fingerprint.

[0083] In summary, the optimal sample preparation method determined in this scheme is as follows: 0.25 g of the freeze-dried powder of the Hupo Qiwu Decoction reference sample was weighed, 25 mL of methanol solution was added, the mass was determined, ultrasonic treatment was performed for 15 min, the mass was determined again after cooling, and the lost mass was made up with methanol. The mixture was shaken, centrifuged at 12000 r·min -1 for 10 min, and filtered through a 0.22 μm filter to obtain the sample solution of the Hupo Qiwu Decoction reference sample.

[0084] Comparative Example

[0085] The sample solution was prepared as follows: 0.5 g of the freeze-dried powder of the Hupo Qiwu Decoction was precisely weighed into a 50 mL conical flask, 25 mL of methanol was precisely added, the mass was determined again, the mass was determined again after cooling, and the lost mass was made up with methanol. The mixture was shaken and filtered through a 0.22 μm microporous filter to obtain the sample solution. The sample solution was injected into the chromatograph according to the chromatographic conditions determined in the previous step, and the peak area of the characteristic peak was recorded.

[0086] Chromatographic conditions: Chromatographic column type: Agilent ZORBAX Eclipse XDB-C18 chromatographic column (4.6 x 250 mm, 5 μm); mobile phase selection: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B). Gradient elution conditions: 0-15 min, A:B is 5%:95%→15%:85%; 15 min, A:B is 15%:85%; 15-45 min, A:B is 15%:85%→20%:80%; 45 min, A:B is 20%:80%; 45-70 min, A:B is 20%:80%→40%:60%; 70 min, A:B is 40%:60%; 70-90 min, A:B is 40%:60%→60%:40%; 90 min, A:B is 60%:40%; 90-110 min, A:B is 60%:40%→80%:20%. The detection wavelength is 254 nm; the flow rate is 1.0 mL / min, the column temperature is 30°C; the theoretical plate number should not be less than 3000 according to naringin peak; the injection amount is 10 ul.

[0087] The test sample and the test sample of the comparative example are injected according to the chromatographic conditions of the comparative example, as shown in the figure, A is the reference substance, B is the sample of the thick-leaf magnolia seven-ingredient decoction, and C is the sample of the comparative example. It is found from the results that the chromatographic peaks can be obtained by using the injection of the chromatographic conditions of the comparative example, but the separation effect is not good. Figure 19

[0088] The test sample and the test sample of the comparative example are injected according to the chromatographic conditions of the comparative example, as shown in the figure, A is the reference substance, B is the sample of the thick-leaf magnolia seven-ingredient decoction, and C is the sample of the comparative example. It is found from the results that the chromatographic peaks can be obtained by using the injection of the chromatographic conditions of the comparative example, but the separation effect is not good. Figure 20

[0089] The test sample and the test sample of the comparative example are injected according to the chromatographic conditions of the comparative example, as shown in the figure, A is the reference substance, B is the sample of the thick-leaf magnolia seven-ingredient decoction, and C is the sample of the comparative example. It is found from the results that the chromatographic peaks can be obtained by using the injection of the chromatographic conditions of the comparative example, but the separation effect is not good. Figure 21

[0090] The test sample and the test sample of the comparative example are injected according to the chromatographic conditions of the comparative example, as shown in the figure, A is the reference substance, B is the sample of the thick-leaf magnolia seven-ingredient decoction, and C is the sample of the comparative example. It is found from the results that the chromatographic peaks can be obtained by using the injection of the chromatographic conditions of the comparative example, but the separation effect is not good. Figure 22

[0091] The test sample and the test sample of the comparative example are injected according to the chromatographic conditions of the comparative example, as shown in the figure, A is the reference substance, B is the sample of the thick-leaf magnolia seven-ingredient decoction, and C is the sample of the comparative example. It is found from the results that the chromatographic peaks can be obtained by using the injection of the chromatographic conditions of the comparative example, but the separation effect is not good. Figure 23 ​​​​As shown, A is a comparative example, 254 nm detection; B is a comparative example, dual-wavelength detection; C is a comparative example chromatographic condition detection, as can be seen from the chromatographic results, both chromatographic conditions can complete the peak, but the peak of the condition of the present solution is faster, the number of chromatographic peaks is more, and 40 min of experimental time can be saved; By comparing the results of dual-wavelength detection method and 254 nm single-wavelength detection, it is found that when dual-wavelength detection, the chromatographic peak of 320 nm detection band has less influence on the target peak, and the target peak has better peak shape.

[0092] 4. Method validation

[0093] (1) Specificity verification

[0094] The UPLC-PDA analysis was performed on the test solution of Houpu Qiwu Decoction, the blank solvent methanol solution and the negative sample solution, and the chromatogram was recorded according to the above chromatographic conditions, as shown in Figure 17 According to the resolution and response factor value, 23 characteristic peaks were calibrated in the chromatogram of the Houpu Qiwu Decoction reference sample, and according to the comparison results with the negative chromatogram, the resolution between each characteristic peak was good, and there was no obvious interference between different chemical components, which indicated that the specificity of the fingerprint method was good.

[0095] Peak assignment and identification: The test solution of Houpu Qiwu Decoction was prepared according to the test preparation method, and the mixed reference solution was prepared according to the mixed reference preparation method, and the UPLC-PAD detection was performed according to the above chromatographic conditions, and the chromatogram was recorded, as shown in Figure 1 According to the comparison results, it is confirmed that the peaks belonging to Magnolia officinalis are peaks 4, 20 and 22, the peaks belonging to Rheum officinale are peaks 12, 18, 19, 21 and 23, the peaks belonging to Glycyrrhiza officinalis are peaks 1, 2, 11, 13, 14, 16 and 17, the peaks belonging to Citrus aurantium are peaks 3, 5, 6, 9 and 10, the peak belonging to Cinnamomum officinale is peak 7, the peak belonging to Zingiber officinale is peak 15, and peak 8 is a common peak.

[0096] Figure 1 Among them, A is Houpu Qiwu Decoction sample; B is mixed control solution 1; C is mixed control solution 2; D is methanol blank. 1 is Glycyrrhizin; 6 is Naringin; 7 is Cinnamyl Aldehyde; 10 is Neohesperidin; 12 is Rhein; 14 is 6-Gingerol; 17 is Ammonium Glycyrrhizate (S); 18 is Emodin; 19 is Chrysophanol; 20 is Honokiol; 21 is Aloe-Emodin; 22 is Magnolol; 23 is Emodin Methyl Ether.

[0097] (2) Precision verification

[0098] The test solution of Houpu Qiwu Decoction was continuously determined for 6 times according to the above chromatographic conditions, and peak No. 17 with moderate peak time and high response factor was selected as the reference peak (S) to calculate the relative retention time of each common peak. The specific results are shown in the following table. According to the results, the RSD of the relative retention time of each common peak is ≤0.26%, indicating that the instrument precision is good.

[0099] Relative retention time results of fingerprint precision investigation

[0100]

[0101] (3) Stability verification

[0102] An appropriate amount of test solution of Houpu Qiwu Decoction was taken and determined at 0, 2, 4, 8, 12, and 24 h after preparation according to the above chromatographic conditions. Peak No. 17 with moderate peak time and high response factor was selected as the reference peak (S) to calculate the relative retention time of each common peak. According to the results, the RSD of the relative retention time of each common peak is ≤0.92%, indicating that the test solution is relatively stable within 24 h. The specific results are shown in the following table.

[0103] Relative retention time results of fingerprint stability investigation

[0104]

[0105] (4) Reproducibility verification

[0106] An appropriate amount of Houpu Qiwu Decoction lyophilized powder was taken and test solution was prepared in parallel for 6 times according to the test solution preparation method. The chromatographic conditions determined in the previous step were used for determination, and peak No. 17 with moderate peak time and high response factor was selected as the reference peak (S) to calculate the relative retention time of each common peak. According to the results, the RSD of the relative retention time of each common peak is ≤0.47%, indicating that the method has good reproducibility. The specific results are shown in the following table.

[0107] Relative retention time results of fingerprint reproducibility investigation

[0108]

[0109] (5) Similarity evaluation of 15 batches

[0110] The similarity of 15 batches of fingerprint chromatograms and the generated control chromatogram was evaluated (see Figure 18 ). The similarity was 0.984, 0.989, 0.983, 0.975, 0.931, 0.971, 0.983, 0.972, 0.982, 0.990, 0.979, 0.980, 0.968, 0.979, and 0.988, respectively. The similarity results of the 15 batches were all >0.9, indicating that the similarity between the fingerprint chromatograms of the 15 batches of reference samples was good.

[0111] 15 batches of Magnoliae Officinalis Decoction Reference Sample Fingerprint Similarity Evaluation Results

[0112]

[0113]

[0114] 5. Magnoliae Officinalis Decoction Index Component Content Determination Study

[0115] Test sample preparation: accurately weigh 0.25 g of Magnoliae Officinalis Decoction freeze-dried powder, accurately add 25 mL of methanol, weigh, ultrasonic treatment (400 W, 40 kHz) for 15 min, cool, weigh again, add methanol to make up the reduced weight, shake well, centrifuge (12000 r·min-1) for 5 min, filter the centrifugate with a 0.22 μm organic microporous filter to obtain the Magnoliae Officinalis Decoction test sample solution.

[0116] Preparation of reference substances: accurately measure a certain amount of glycyrrhizin, naringin, cinnamyl aldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, and honokiol, aloe-emodin, and magnolol reference substances, respectively, and add methanol solution to prepare each single reference substance stock solution. Accurately measure a certain amount of rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, and honokiol, aloe-emodin, and magnolol reference substance stock solutions, and place them in the same volumetric flask. Dilute to the mark with methanol, shake well, and prepare a mixed reference substance solution 1 with mass concentrations of 2.09, 35.78, 24.61, 4.17, 1.05, 4.10, 1.00, and 4.67 mg·L -1 respectively. Accurately measure a certain amount of glycyrrhizin, naringin, cinnamyl aldehyde, and neohesperidin reference substance stock solutions, and place them in the same volumetric flask. Dilute to the mark with methanol, shake well, and prepare a mixed reference substance solution 2. -1

[0117] Linear relationship: take the previously prepared reference substance stock solution, dilute to the linear concentration calibration solution, inject according to the chromatographic conditions, take the peak area as the abscissa (X), and the reference substance concentration as the ordinate (Y), to draw the standard curve. The results are shown in the table below, indicating that each component has a good linear relationship in the linear range.

[0118] Linear relationship investigation results of index component content determination (n = 6)

[0119] Indicator component Linear regression equation [R 2 ]] Linear range (pg mL -1 ) Glycyrrhizin [Y = 2 * 10 -4 X -0.8865]] R 2 = 0.9997 4.20～105.00 Naringin [Y = 5 * 10 -4 X -2.3936 R 2 = 0.9993 41.28～516.00 Cinnamaldehyde [Y = 5 * 10 -5 X -0.0237 R 2 = 0.9991 0.43～10.66 Neohesperidin [Y = 5 * 10 -4 X -2.0746 R 2 = 0.9993 39.94～499.20 Rhein [Y = 6 * 10 -5 X -0.0368 R 2 = 0.9998 0.21～8.34 6-Gingerol [Y = 1.5 * 10 -4 X -0.5603 R 2 = 0.9991 8.95～143.12 Ammonium Glycyrrhizinate [Y = 2 * 10 -4 X + 0.1515 [R 2 = 0.9996 6.15～123.04 Emodin [Y = 9 * 10 -5 X - 0.695 [R 2 = 0.9994 0.52～20.84 Chrysophanol [Y = 8 * 10 -5 X + 0.1767 R 2 = 0.9994 0.26～4.22 Honokiol [Y = 6 * 10 -5 X -0.0055 [R 2 = 0.9995 1.03～24.62 Aloin [Y = 3 * 10 -5 X -0.0077 [R 2 = 0.9996 0.25～4.01 Magnolol [Y = 1 * 10 -4 X -0.0122 R 2 = 0.9996 1.17～28.03

[0120] (1) Detection limit and quantification limit

[0121] ​The control sample stock solution was diluted, and the injection was performed according to the determined chromatographic conditions, and the detection limit and the quantitative limit of each index component were determined. When the signal-to-noise ratio S / N value was 10 / 1, it was recorded as the quantitative limit, and when the signal-to-noise ratio S / N value was 3 / 1, it was recorded as the detection limit. The detection results are shown in Table 6 below.

[0122] Investigation results of the detection limit and the quantitative limit of the index components

[0123] Indicator component Detection limit pg-mL -1 ]] Quantification limit pg.m L-1 ]]> Glycyrrhizin 0.121 0.404 Naringin 0.047 0.158 Cinnamaldehyde 0.100 0.333 Neohesperidin 0.076 0.254 Rhein 0.058 0.195 6-Gingerol 0.088 0.295 Ammonium Glycyrrhizinate 0.107 0.357 Emodin 0.069 0.231 Chrysophanol 0.074 0.246 Honokiol 0.056 0.187 Aloin 0.071 0.238 Magnolol 0.066 0.221

[0124] (2) Precision

[0125] The S10 batch of Magnoliae Officinalis Decoction Reference Sample was taken as the sample, and the test sample solution was prepared according to the above conditions. The peak area RSD values of glycyrrhizin, naringin, cinnamyl aldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizinate, emodin, chrysophanol, honokiol, aloe-emodin, and magnolol were 0.67%, 1.56%, 1.78%, 1.04%, 0.81%, 1.48%, 0.65%, 1.69%, 0.61%, 0.41%, 0.51%, and 0.85%, respectively, after 6 consecutive determinations according to the optimal chromatographic conditions, indicating that the instrument precision was good.

[0126] Investigation results of the precision of the content determination of the index components (n=6)

[0127]

[0128] (3) Stability

[0129] The S10 batch of Magnoliae Officinalis Decoction Reference Sample was taken as the sample, and the test sample solution was prepared according to the above conditions. The peak area RSD values of glycyrrhizin, naringin, cinnamyl aldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizinate, emodin, chrysophanol, honokiol, aloe-emodin, and magnolol were 0.46%, 0.73%, 1.93%, 1.74%, 0.68%, 2.61%, 1.62%, 0.61%, 0.89%, 1.16%, 1.02%, and 0.67%, respectively, after 0, 2, 4, 8, 12, and 24 h of determination according to the optimal chromatographic conditions, indicating that the test sample was relatively stable within 24 h.

[0130] Investigation results of the stability of the content determination of the index components (n=6)

[0131]

[0132]

[0133] (4) Reproducibility

[0134] Take S10 batch of Magnolia officinalis seven material soup reference sample sample, according to the above optimal conditions parallel preparation of test solution 6, according to the optimal chromatographic conditions, calculate the peak area of glycyrrhizin, naringin, cinnamyl aldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizinate, emodin, chrysophanol, honokiol, aloe emodin, magnolol RSD value is 2.16%, 1.61%, 2.56%, 1.26%, 0.96%, 1.84%, 2.63%, 1.92%, 1.86%, 1.82%, 0.77%, 1.19% respectively, indicating that the method is good reproducibility.

[0135] Take the same batch of Magnolia officinalis seven material soup freeze-dried powder, precision weighing 6, preparation of test sample, calculate the content of each index component, calculate RSD value, see the table below.

[0136] Index component content determination reproducibility test results (n=6)

[0137]

[0138] (5) sample recovery

[0139] Take S10 batch of Magnolia officinalis seven material soup reference sample sample, precision weighing of test sample weight of 1 / 2 (0.125g), parallel 6, respectively according to the corresponding component content 1:1 to add reference substance solution, according to the above method preparation of test solution, according to the optimal chromatographic conditions, calculate the average sample recovery of glycyrrhizin, naringin, cinnamyl aldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizinate, emodin, chrysophanol, honokiol, aloe emodin, magnolol is 100.64%, 101.94%, 99.25%, 102.51%, 102.28%, 102.46%, 97.70%, 97.58%, 101.83%, 100.17%, 99.56%, 99.97%, RSD value is 0.75%, 1.03%, 0.82%, 1.88%, 1.05%, 0.69%, 0.46%, 0.56%, 0.85%, 0.60%, 0.89%, 0.66% respectively, indicating that the detection method is accurate and reliable.

[0140] Index component content determination sample recovery rate test results (n=6)

[0141]

[0142]

[0143] (6) content determination

[0144] Take 15 batches of Magnolia officinalis seven material soup standard sample sample, according to the above method for preparing test solution, according to the chromatographic conditions under item 2.2.1, calculate the content of each index component. The results showed that the mass fraction of glycyrrhizin, naringin, cinnamyl aldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizinate, emodin, chrysophanol, honokiol, aloe emodin, magnolol in 15 batches of standard sample were 2.41-4.87, 24.11-54.36, 0.15-1.23, 19.60-30.49, 0.09-0.58, 0.39-0.82, 3.30-7.87, 0.13-3.73, 0.03-0.05, 0.14-0.63, 0.03-0.11, 0.46-1.01 mg·g-1, the results are shown in the table below.

[0145] The mass fraction of Magnolia officinalis seven material soup index components

[0146]

[0147] The above is only the preferred embodiment of the present application, and does not limit the patent scope of the present application, any equivalent structural transformation made under the inventive concept of the present application, using the contents of the present application specification and drawings, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present application.

Claims

1. A quality control method for Houpu Qiwu Decoction, characterized in that, Includes the following steps: S1. Preparation of the test solution; Step S1 includes the following steps: accurately weigh 0.25-0.5g of the Houpu Qiwu Decoction preparation, accurately add 25-50ml of methanol to a container, weigh the sample, sonicate for 20-30min, cool and weigh the sample again, add methanol to make up the lost mass and shake well, centrifuge for 5-10min, and filter through a 0.2-0.45um microporous membrane to obtain the test solution; S2. Preparation of reference solutions; the reference standards include glycyrrhizin, naringin, cinnamaldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, honokiol, aloe-emodin and honokiol reference standards. S3. Determination: Inject the reference solution and the test solution into the ultra-high performance liquid chromatograph for high performance liquid chromatography analysis, record the chromatographic peaks, and obtain the fingerprint spectrum of Houpu Qiwu Decoction; In step S3, the high performance liquid chromatography conditions include: using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.1% (v / v) phosphoric acid solution as mobile phase B for gradient elution. The fingerprint spectrum is measured using dual wavelengths: the detection wavelength is 320 nm from 0 to 32 min, and the detection wavelength is 254 nm from 32 to 70 min. The gradient elution conditions in step S3 are as follows: 0–5 min, mobile phase A 10–12%, mobile phase B 90–88%; 5–15 min, mobile phase A 12–13%, mobile phase B 88–87%; 15–20 min, mobile phase A 13–15%, mobile phase B 87–85%; 20–32 min, mobile phase A 15%, mobile phase B 85%; 32–35 min, mobile phase A 15–28%, mobile phase B 85–72%; 35–45 min, mobile phase A 28–30%, mobile phase B 72–70%; 45–50 min, mobile phase A 30–35%, mobile phase B 70–65%; 50–55 min, mobile phase A 35–37%, mobile phase B 65–63%; 55–60 min, mobile phase A 37–50%, mobile phase B 90–88%; 50–55 min, mobile phase A 35–37%, mobile phase B 65–63%; 55–60 min, mobile phase A 37–50%, mobile phase B 90–88%; 50–55 min, mobile phase A 35–37%, mobile phase B 65–63%; 55–60 min, mobile phase A 37–50%, mobile phase B 90–88%; 50–55 min, mobile phase A 35–37%, mobile phase B 65–63%; 55–60 min, mobile phase A 37–50%, mobile phase B 90–65 ... 63-50%; 60-70 min, mobile phase A 50-70%, mobile phase B 50-30%.

2. The quality control method for Houpu Qiwu Decoction as described in claim 1, characterized in that, In step S3, the high performance liquid chromatography conditions also include: an ACQUITY UPLC CSH C18 column, 2.1 mm × 100 mm, 1.7 µm, a column temperature of 30-40 °C, an injection volume of 2 μL, and a flow rate of 0.25-0.35 mL / min.

3. The quality control method for Houpu Qiwu Decoction as described in claim 1, characterized in that, Step S2 includes the following steps: Accurately weigh glycyrrhizin, naringin, cinnamaldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, magnolol, aloe-emodin, and magnolol reference standards; add methanol to each of the above reference standards and shake well to prepare solutions with contents of 200-400 mg / L. -1 800-1000 mg·L -1 800-1000 mg·L -1 80-100 mg·L -1 80-100 mg·L -1 , 80-100 mg·L -1 200-400 mg·L -1 200-400 mg·L -1 20-50 mg·L -1 0.80-100 mg·L -1 20-50 mg·L -1 80-100 mg·L -1 A single reference standard stock solution; Accurately measure stock solutions of rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, honokiol, aloe-emodin, and honokiol, mix them thoroughly in a container, and then add different amounts of methanol and shake well to prepare solutions with mass concentrations of 2-10 mg·L⁻¹. -1 2-10 mg·L -1 50-100 mg·L -1 10-50 mg·L -1 0.5-2 mg·L -1 2-10 mg·L -1 0.5-2 mg·L -1 2-10 mg·L -1 1. Mixed reference solution; Accurately measure the stock solutions of glycyrrhizin, naringin, cinnamaldehyde, and neohesperidin, mix them thoroughly in a container, and then add different amounts of methanol and shake well to prepare solutions with mass concentrations of 20-50 mg / L. -1 200-500 mg·L -1 2-10 mg·L -1 200-500 mg·L -1 2. Mixed reference solution.

4. The quality control method for Houpu Qiwu Decoction as described in claim 1, characterized in that, The fingerprint spectrum of Houpu Qiwu Decoction includes 23 characteristic peaks, of which peaks 4, 20, and 22 are magnolia bark, peaks 12, 18, 19, 21, and 23 are rhubarb, peaks 1, 2, 11, 13, 14, 16, and 17 are licorice, peaks 3, 5, 6, 9, and 10 are immature bitter orange, peak 7 is cinnamon, peak 15 is ginger, and peak 8 is a common peak.

5. The quality control method for Houpu Qiwu Decoction as described in claim 1, characterized in that, It also includes the use of thin-layer chromatography to identify at least one of the following ingredients in Houpu Qiwu Decoction: magnolia bark, rhubarb, licorice, immature bitter orange, cinnamon, and ginger.

6. The quality control method for Houpu Qiwu Decoction as described in claim 5, characterized in that, The thin-layer chromatography identification process of Magnolia officinalis includes: taking 1-2g of Magnolia officinalis Qiwu Decoction preparation, adding 20-30ml of methanol, sonicating for 20-30min, shaking and filtering, evaporating the filtrate to dryness to obtain the residue, adding 15-20ml of water to dissolve, and extracting with ether 2-3 times, obtaining 15-20ml of solution each time, then combining the ether solutions, evaporating to dryness, and dissolving the obtained residue in 2-3ml of methanol to prepare the test solution; Take 1-2g each of Magnolia officinalis reference material and Magnolia officinalis negative test preparation, add 20-30ml of methanol and sonicate for 20-30min respectively, shake and filter, evaporate the filtrate to dryness to obtain residue, add 15-20ml of water to dissolve, extract with ether 2-3 times, each time to obtain 15-20ml of solution, combine the ether solutions, evaporate to dryness, add 2-3ml of methanol to dissolve the residue to prepare reference material solution and negative test sample solution; Separately, prepare reference solutions by adding methanol to magnolol and magnolol reference standards, each containing 1-2 mg per ml. The thin-layer chromatography identification process of rhubarb includes: taking 1-2g of Houpu Qiwu Decoction preparation, adding 20-30ml of methanol, sonicating for 45-60min, filtering, taking 5-10ml of filtrate and evaporating to dryness, dissolving the residue in 10-20ml of water, adding 1-2ml of hydrochloric acid, heating under reflux for 30-45min, cooling immediately, and extracting with ether by shaking 2-3 times, obtaining 15-20ml of solution each time, combining the ether solutions, evaporating to dryness, dissolving the obtained residue in 2-3ml of methanol to prepare the test solution; Take 1-2g each of rhubarb reference material and rhubarb-deficient negative preparation, add 20-30ml of methanol and sonicate for 45-60min respectively, filter, take 5-10ml of filtrate and evaporate to dryness, dissolve the residue in 10-20ml of water respectively, add 1-2ml of hydrochloric acid respectively, heat under reflux for 30-45min respectively, cool immediately, and extract with ether 2-3 times, obtaining 15-20ml of solution each time. Combine the ether solutions, evaporate to dryness, dissolve the residue in 2-3ml of methanol to prepare the reference material solution and negative sample solution; Separately, prepare a reference solution by adding methanol to rhein reference standard, containing 1-2 mg of each substance per ml. The thin-layer chromatography identification process for licorice includes: taking 1-2g of Houpu Qiwu Decoction preparation, adding 20-30ml of ethanol and immersing for 20-30min, filtering to obtain the filtrate, concentrating to 2-3ml, and preparing the test solution; Take 1-2g of licorice reference material and licorice-deficient negative preparation, add 20-30ml of ethanol and soak for 20-30min respectively. After filtration, obtain the filtrate and concentrate it to 2-3ml respectively to prepare the reference material solution and the negative sample solution. Separately prepare reference solutions by adding methanol to glycyrrhizin and ammonium glycyrrhizate reference standards to each of the two substances, each containing 1-2 ml of the reference standard. The thin-layer chromatography identification process of Citrus aurantium includes: taking 1-2g of Houpu Qiwu Decoction preparation, adding 20-30ml of ethanol and immersing for 20-30min, filtering to obtain the filtrate, concentrating to 2-3ml, and preparing the test solution. Take 1-2g each of the reference herb (Citrus aurantium) and the negative control preparation (Citrus aurantium lacking Citrus aurantium), add 20-30ml of ethanol and soak for 20-30min, filter to obtain the filtrate, concentrate to 2-3ml, and prepare the reference herb solution and the negative control sample solution. Separately prepare reference solutions by adding methanol to naringin and neohesperidin reference standards to each contain 1-2 mg per ml. The thin-layer chromatography identification process for cinnamon includes: taking 1-2g of Houpu Qiwu Decoction preparation, adding 20-30ml of ethanol and immersing for 20-30min, then concentrating the filtrate to 2-3ml to prepare the test solution; Take 1-2g each of cinnamon reference material and jujube-deficient negative preparation, add 20-30ml of ethanol and soak for 20-30min, then concentrate the filtrate to 2-3ml to prepare the reference material solution and negative sample solution. Separately prepare a reference solution by adding ethanol to cinnamaldehyde reference standard and making mixed solutions containing 1-2 mg of each per ml. The thin-layer chromatography identification process for ginger includes: taking 1-2g of Houpu Qiwu Decoction preparation, dissolving it in 20-30ml of water, extracting it 3-4 times with water-saturated n-butanol, 15-20ml each time, combining the n-butanol solutions, washing it 2-3 times with water, 15-20ml each time, discarding the aqueous solution, evaporating the n-butanol solution to dryness, dissolving the residue in 2-3ml of methanol to prepare the test solution; Take 1-2g each of ginger reference material and ginger-deficient negative preparation, add 20-30ml of water to dissolve, extract with water-saturated n-butanol 3-4 times, 15-20ml each time, combine the n-butanol solutions, wash with water 2-3 times, 15-20ml each time, discard the water, evaporate the n-butanol solution to dryness, dissolve the residue in 2-3ml of methanol to prepare the reference material solution and negative sample solution; Separately prepare a reference solution by adding methanol to 6-gingerol reference standard to make a mixed solution containing 1-2 mg of each substance per ml.

7. The quality control method for Houpu Qiwu Decoction as described in claim 6, characterized in that, The thin-layer plates used for Magnolia officinalis, Glycyrrhiza uralensis, Citrus aurantium, Cinnamomum cassia and Zingiber officinale are silica gel G thin-layer plates, while the thin-layer plates used for Rheum palmatum are silica gel H thin-layer plates. The developing solvents for Magnolia officinalis and Rheum palmatum are cyclohexane-ethyl acetate-formic acid; the developing solvent for Glycyrrhiza uralensis is ethyl acetate-formic acid-glacial acetic acid-water; the developing solvent for Citrus aurantium is chloroform-methanol-water; the developing solvent for Cinnamomum cassia is petroleum ether-ethyl acetate; and the developing solvent for Zingiber officinale is petroleum ether-chloroform-ethyl acetate. Rhubarb thin-layer plates were observed under ultraviolet light at a wavelength of 365 nm; the color developing agents for Magnolia officinalis and ginger were vanillin sulfuric acid solution, for licorice was sulfuric acid ethanol solution, for Citrus aurantium was aluminum trichloride ethanol solution, and for cinnamon was dinitrophenylhydrazine ethanol solution. All were observed under sunlight after color development.

8. The quality control method for Houpu Qiwu Decoction as described in claim 1, characterized in that, It also includes a method for determining the content using Houpu Qiwu Decoction, which includes the following steps: Preparation of the test solution: Accurately weigh 0.25-0.5 g of Houpu Qiwu Decoction preparation, accurately add 25-50 ml of methanol to a container, weigh the sample, sonicate for 15-20 min, cool, weigh the sample again, add methanol to make up the lost mass and shake well, centrifuge for 5-10 min, filter through an organic microporous membrane of 0.2-0.45 μm to obtain the test solution; Preparation of reference solutions: Accurately measure glycyrrhizin, naringin, cinnamaldehyde, neohesperidin, rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, and magnolol, aloe-emodin, and honokiol reference standards, and add them separately to methanol solution to prepare stock solutions of each single reference standard; accurately measure rhein, 6-gingerol, ammonium glycyrrhizate, emodin, chrysophanol, and magnolol, aloe-emodin, and honokiol reference standard stock solutions, mix them completely in a container, and then add different amounts of methanol and shake well to prepare mass concentrations of 2-10 mg·L⁻¹. -1 2-10 mg·L -1 50-100 mg·L -1 10-50 mg·L -1 0.5-2 mg·L -1 2-10 mg·L -1 0.5-2 mg·L -1 2-10 mg·L -1 Mixed reference solution 1: Accurately measure the stock solutions of glycyrrhizin, naringin, cinnamaldehyde, and neohesperidin, mix them completely in a container, then add different amounts of methanol and shake well to prepare solutions with mass concentrations of 20-50 mg / L. -1 200-500 mg·L -1 2-10 mg·L -1 200-500 mg·L -1 2. Mixed reference solution; Determination: The test solution and reference solution were analyzed by ultra-high performance liquid chromatography (UHPLC). An ACQUITY UPLCCSH C18 column was used as the chromatographic column, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B for gradient elution at a flow rate of 0.25~0.35 ml / min.

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