A sanhua soup benchmark sample freeze-dried powder and a preparation method and quality detection method thereof

Abstract

The application provides a Sanhua decoction reference sample freeze-dried powder and a preparation method and a quality detection method thereof. The preparation method of the Sanhua decoction reference sample freeze-dried powder is optimized to prepare the freeze-dried powder, so that the modern process parameters of the classical famous prescription reference sample are obtained, and subsequent pilot production and industrialized production are facilitated. The quality of the Sanhua decoction reference sample freeze-dried powder is detected by two technical means of thin layer chromatography and high-phase liquid chromatography. The thin layer chromatography is used for identification of all medicinal materials, and according to the differences of different medicinal material components, a separate TLC detection means is formulated for the medicinal materials, and the detection of rhubarb glycoside is increased for the anti-counterfeiting of the rhubarb medicinal material. Compared with the prior art, the method is more intuitive and clear. The total content of magnolol and honokiol is determined by high-performance liquid chromatography, and a quantitative monitoring means is provided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of traditional Chinese medicine, and relates to a preparation of a classical prescription of traditional Chinese medicine Sanhua Decoction and a set of quality control methods, in particular to a Sanhua Decoction reference sample freeze-dried powder, a preparation method thereof and a quality detection method. BACKGROUND

[0002] The classical prescription is a pearl in the treasure house of Chinese medicine, and is an effective prescription verified by time. Through inheritance, screening, excavation and innovation, the theory of the classical prescription is interpreted, and the clinical efficacy, safety and economy are further clarified, so that the academic value and clinical value of the classical prescription are further highlighted and improved.

[0003] Sanhua Decoction was first recorded in Suwen Bingji Qi Yi Baomingji·Zhongfeng Lun Di Shi, and is the 55th in the State-issued Ancient Classical Prescription Catalogue (First Batch). It was created by Liu Wansu, a physician of the Jin and Yuan Dynasties, and is formed by adding Qianghuo to Xiaochengqi Decoction. It is a classical prescription for treating stroke by opening the Xuanfu. The original text is: There are six-meridian symptoms of stroke. First, use modified Jundu Xunming Decoction to treat it according to the symptoms. There is a blockage of diarrhea inside, and Sanhua Decoction is used to treat it. Composition: equal parts of Houpu, Jixing, Dahuang and Qianghuo. Grind to the size of millet. Each dose is three liang, and water is three liang. Decoction to one and a half liang, and take it all day. The degree of mildness is the standard. In the prescription, Dahuang is the monarch, which has the effects of clearing heat and resolving toxins, promoting blood circulation, purgation and purging fire; Jixing is the minister, which has the effects of breaking qi, resolving accumulation and eliminating phlegm and eliminating phlegm; Houpu is the assistant, which has the effects of treating stroke, headache and cold and heat; Qianghuo is the messenger, which has the effects of treating hemiplegia, facial paralysis and the like. The prescription can open the Xuanfu of the brain and the Xuanfu of the intestines and stomach. Through mutual influence, ascending and descending and internal and external combination, it opens the Xuanfu of the whole body, so that qi and blood are in harmony, body fluid is unobstructed, and the spirit is in harmony. Therefore, it has high clinical value.

[0004] Patent document CN108956845B discloses a Sanhua Decoction freeze-dried powder one plate four medicinal materials multi-information thin layer identification method, but the prescription dose and water amount are wrong (need to be converted into ancient prescription dynasties, not modern), the medicinal materials are not processed, that is, the medicinal materials are used incorrectly, there is no numerical value in the preparation method, the process is too rough, and the authenticity of the data and pictures is difficult to verify. Although it is a plate with multiple measurements, the medicinal materials still need to be colored or detected one by one, and the spots are too many due to the detection of multiple medicinal materials as shown in the drawing, which is not intuitive and is easy to interfere. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides a Sanhua Decoction reference sample freeze-dried powder, a preparation method thereof and a quality detection method. The preparation method can prepare the Sanhua Decoction reference sample freeze-dried powder with high content of effective components, and the quality detection method can accurately detect the product quality, which is conducive to promoting the modernization research process of the classical prescription Sanhua Decoction.

[0006] The present application is realized by the following technical solutions:

[0007] 1. A preparation method of Sanhua Decoction reference sample freeze-dried powder, comprising the following steps:

[0008] S1, 25-35 g of wine rhubarb, ginger magnolia, bran-fried bitter orange and radix angelicae pubescentis are weighed in turn, crushed, passed through a 5-mesh sieve but not a 10-mesh sieve, and placed in a decoction pot, and then soaked in 1000-2000 ml of water for 15-60 min; the proportions of wine rhubarb, ginger magnolia, bran-fried bitter orange, radix angelicae pubescentis and water are (25-35) g:(25-35) g:(25-35) g:(25-35) g:(1000-2000) ml.

[0009] Preferably, 31.0 g of each decoction piece is weighed, 1500 ml of water is added, and soaked for 45 min.

[0010] S2, the first decoction is boiled using a strong fire (150-180°C) for 15-20 min, and then boiled using a weak fire (80-120°C) for 85-100 min, and then cooled and filtered; the second decoction is boiled using a strong fire (150-180°C) for 5-10 min, and then boiled using a weak fire (80-120°C) for 15-30 min, and then filtered while hot; the two decoctions are combined, filtered using 2-4 layers of gauze, and then the filtrate is concentrated under reduced pressure at 40-70°C, and then spray freeze-dried at a freezing temperature of -100 to 0°C, to obtain the freeze-dried powder.

[0011] Preferably, the first decoction is boiled using a strong fire for 20 min, and then boiled using a weak fire for 90 min until 750 ml of liquid is obtained, and then filtered while hot; 600 ml of warm water is added, the second decoction is boiled using a strong fire for 10 min, and then boiled using a weak fire for 20 min until 300 ml of liquid is obtained, and then filtered while hot. Preferably, the two decoctions are combined, filtered using 2 layers of gauze, and then the filtrate is concentrated under reduced pressure at 60°C, and then spray freeze-dried at a freezing temperature of -50°C.

[0012] The method has an extract yield of 26.553-33.721% for the Sanhua Decoction reference sample freeze-dried powder, and the total content of magnolol and honokiol in the Sanhua Decoction reference sample freeze-dried powder is 0.656-1.608%.

[0013] 2. A method for controlling the quality of Sanhua Decoction reference sample freeze-dried powder using thin layer chromatography, comprising the following steps:

[0014] S1, identification of anthraquinones in rhubarb

[0015] (1) Preparation of the control solution: Take the control samples of aloe-emodin, rhein, emodin, chrysophanol and rheochrysidin, and add methanol to obtain a mixed control solution;

[0016] (2) Preparation of the test solution: 4.0-6.0 g of the freeze-dried powder of the Sanhua Decoction reference sample of different batches was placed in a 250 ml conical flask with a stopper, 10-50 ml of a methanol-hydrochloric acid (10:0.3) mixed solution was added, and the mixture was heated to reflux in a water bath at 80°C for 30-60 min. After cooling, the mixture was filtered to obtain the test solution;

[0017] (3) Preparation of the negative sample solution: three kinds of decoction pieces except for rhubarb were prepared into freeze-dried powder according to the above method, and an appropriate amount of the freeze-dried powder was placed in a 250 ml conical flask with a stopper. A methanol-hydrochloric acid (10:0.3) mixed solution was added, and the mixture was heated to reflux in a water bath. After cooling, the mixture was filtered to obtain the negative sample solution;

[0018] (4) Thin-layer identification method: 5-10 μl of the mixed control solution, the test solution, and the negative sample solution were spotted on a thin-layer plate, respectively. A developing agent was added, and the plate was developed, taken out, and dried. The plate was tested under a 365 nm ultraviolet lamp, and the spots were made clear by fumigation with ammonia vapor. In the test solution chromatogram, the same color spots were shown at positions corresponding to the control solution chromatogram;

[0019] As a preferred solution, in the thin-layer detection method of the anthraquinones in rhubarb described above, the preparation method of the control solution in step (1) is as follows: the control substances emodin, rheic acid, chrysophanol, chrysophanol methyl ether, and rheochrysidin were added to methanol to prepare a mixed control solution with a concentration of 1 mg / ml.

[0020] As a preferred solution, in the thin-layer detection method of the anthraquinones in rhubarb described above, the preparation method of the test solution in step (2) is as follows: 5.0 g of the freeze-dried powder of the Sanhua Decoction reference sample of 15 batches was placed in a 250 ml conical flask with a stopper, 30 ml of a methanol-hydrochloric acid (10:0.3) mixed solution was added, and the mixture was heated to reflux in a water bath at 80°C for 60 min. After cooling, the mixture was filtered to obtain the test solution;

[0021] As a preferred solution, in the thin-layer detection method of the anthraquinones in rhubarb described above, the preparation method of the negative sample solution in step (4) is as follows: three kinds of decoction pieces except for rhubarb were prepared into freeze-dried powder according to the preparation method of the freeze-dried powder of the Sanhua Decoction reference sample. 5.0 g of the freeze-dried powder was placed in a 250 ml conical flask with a stopper, 30 ml of a methanol-hydrochloric acid (10:0.3) mixed solution was added, and the mixture was heated to reflux in a water bath at 80°C for 60 min. After cooling, the mixture was filtered to obtain the negative sample solution;

[0022] As a preferred solution, in the above-mentioned thin layer detection method of anthraquinones in rhubarb, the thin layer conditions in step (5) are as follows: thin layer plate: silica gel H thin layer plate with sodium carboxymethyl cellulose as the binder; sample volume: 7 μl for the test sample solution and 10 μl for the rest; developing agent: petroleum ether (30-60°C)-ethyl formate-formic acid, in a volume ratio of (14-16):(4-6):(0.5-1);

[0023] As a preferred solution, the developing agent is petroleum ether (30-60°C)-ethyl formate-formic acid (15:5:1).

[0024] S2, identification of synephrine in Fructus Aurantii Immaturus

[0025] (1) Preparation of the control solution: synephrine control substance is taken and dissolved in methanol to prepare a 20-40 μg / ml solution, thus obtaining the control solution;

[0026] (2) Preparation of the test sample solution: 1.0-2.0 g of the freeze-dried powder of the Sanhua Decoction reference sample of different batches is placed in a 250 ml conical flask with a stopper, 10-50 ml of methanol is added, and ultrasonic treatment is performed for 10-60 min. Then, filtration is performed, thus obtaining the test sample solution.

[0027] (3) Preparation of the negative sample solution: the freeze-dried powder of three kinds of medicinal decoction pieces except Fructus Aurantii Immaturus is prepared according to the method described above, and an appropriate amount of the powder is placed in a 250 ml conical flask with a stopper, methanol is added, ultrasonic treatment is performed, and filtration is performed, thus obtaining the negative sample solution.

[0028] (4) Thin layer identification method: 5-10 μl of the synephrine control solution, the test sample solution, and the negative sample solution are spotted on a thin layer plate, a developing agent is added, the plate is taken out, dried, and sprayed with 0.5% ninhydrin ethanol solution, and color development is performed at 105°C. In the test sample chromatogram, color spots of the same color appear at positions corresponding to the control sample chromatogram.

[0029] As a preferred solution, in the above-mentioned thin layer detection method of synephrine in Fructus Aurantii Immaturus, the preparation method of the control solution in step (1) is as follows: synephrine control substance is taken and dissolved in methanol to prepare a 30 μg / ml solution, thus obtaining the control solution.

[0030] As a preferred solution, in the above-mentioned thin layer detection method of synephrine in Fructus Aurantii Immaturus, the preparation method of the test sample solution in step (2) is as follows: 1.5 g of the freeze-dried powder of the Sanhua Decoction reference sample of 15 batches is placed in a 250 ml conical flask with a stopper, 50 ml of methanol is added, ultrasonic treatment is performed for 45 min, and filtration is performed, thus obtaining the test sample solution.

[0031] As a preferred solution, the above-mentioned thin layer detection method of synephrine in Fructus Aurantii, the preparation method of the negative sample solution in step (3) is as follows: taking three kinds of decoction pieces except Fructus Aurantii, the freeze-dried powder is prepared according to the method described in the invention content 1, 1.5 g is taken and placed in a 250 ml conical flask with a plug, 50 ml of methanol is added, ultrasonic treatment is performed for 45 min, and then filtration is performed to obtain the negative sample solution;

[0032] As a preferred solution, the above-mentioned thin layer detection method of synephrine in Fructus Aurantii, the thin layer condition in step (4) is as follows: thin layer plate: silica gel G thin layer plate; sample application amount: 7 μl; developing agent: the developing agent is n-butanol-glacial acetic acid-water solution, and the volume ratio is (4-5):(1-2):(4-5);

[0033] As a preferred solution, the developing agent is n-butanol-glacial acetic acid-water solution (4:1:5);

[0034] S3, Identification of purple peucedanin in Rhizoma et Radix Notopterygii

[0035] (1) Preparation of the control solution: take the purple peucedanin control sample, add methanol to prepare a 0.3-1.0 mg / ml solution, and the control solution is obtained;

[0036] (2) Preparation of the test solution: take 1.0-2.0 g of freeze-dried powder of the Sanhua Decoction reference sample of different batches, place it in a 250 ml conical flask with a plug, add 10-50 ml of methanol, ultrasonic treatment for 10-60 min, and then filtration to obtain the test solution;

[0037] (3) Preparation of the negative sample solution: except for Rhizoma et Radix Notopterygii, the freeze-dried powder is prepared according to the method described above, and a proper amount is taken and placed in a 250 ml conical flask with a plug, methanol is added, ultrasonic treatment is performed, and then filtration is performed to obtain the negative sample solution;

[0038] (4) Thin layer identification method: take 5-7 μl of the purple peucedanin control solution, the test solution and the negative sample solution respectively, and apply them on a thin layer plate, add a developing agent, develop, take out, dry, and place it under a 365 nm ultraviolet lamp for detection. In the test solution, fluorescent spots of the same color appear at positions corresponding to the control solution;

[0039] As a preferred solution, the above-mentioned thin layer detection method of synephrine in Fructus Aurantii, the preparation method of the negative sample solution in step (3) is as follows: taking three kinds of decoction pieces except Fructus Aurantii, the freeze-dried powder is prepared according to the method described in the invention content 1, 1.5 g is taken and placed in a 250 ml conical flask with a plug, 50 ml of methanol is added, ultrasonic treatment is performed for 45 min, and then filtration is performed to obtain the negative sample solution;

[0040] As a preferred solution, the preparation method of the test solution in step (2) of the detection method of cnidii rhizoma et rhizomatis in Qiang- guai is as follows: 1.5 g of freeze-dried powder of the three kinds of decoction pieces of the reference sample of Sanhua Decoction is taken and placed in a 250 ml conical flask with a plug, 50 ml of methanol is added, and ultrasonic treatment is performed for 45 min, and then filtration is performed to obtain the test solution;

[0041] As a preferred solution, the preparation method of the test solution in step (2) of the detection method of cnidii rhizoma et rhizomatis in Qiang- guai is as follows: 1.5 g of freeze-dried powder of the three kinds of decoction pieces of the reference sample of Sanhua Decoction is taken and placed in a 250 ml conical flask with a plug, 50 ml of methanol is added, and ultrasonic treatment is performed for 45 min, and then filtration is performed to obtain the test solution;

[0042] As a preferred solution, the preparation method of the test solution in step (2) of the detection method of cnidii rhizoma et rhizomatis in Qiang- guai is as follows: 1.5 g of freeze-dried powder of the three kinds of decoction pieces of the reference sample of Sanhua Decoction is taken and placed in a 250 ml conical flask with a plug, 50 ml of methanol is added, and ultrasonic treatment is performed for 45 min, and then filtration is performed to obtain the test solution;

[0043] As a preferred solution, the preparation method of the test solution in step (2) of the detection method of cnidii rhizoma et rhizomatis in Qiang- guai is as follows: 1.5 g of freeze-dried powder of the three kinds of decoction pieces of the reference sample of Sanhua Decoction is taken and placed in a 250 ml conical flask with a plug, 50 ml of methanol is added, and ultrasonic treatment is performed for 45 min, and then filtration is performed to obtain the test solution;

[0044] S4, Magnolia officinalis in Magnolia officinalis, and Magnolia officinalis phenol identification

[0045] (1) Preparation of the control solution: Magnolia officinalis phenol, and Magnolia officinalis phenol control samples are taken and added with methanol to prepare a solution containing 30-35 μg / ml of Magnolia officinalis phenol and Magnolia officinalis phenol, and the mixed control solution is obtained;

[0046] (2) Preparation of the test solution: 1.5-2.5 g of freeze-dried powder of the three kinds of decoction pieces of the reference sample of Sanhua Decoction is taken and placed in a 250 ml conical flask with a plug, 10-50 ml of methanol is added, ultrasonic treatment is performed for 10-60 min, and then filtration is performed to obtain the test solution;

[0047] (3) Preparation of the test solution: 1.5-2.5 g of freeze-dried powder of the three kinds of decoction pieces of the reference sample of Sanhua Decoction is taken and placed in a 250 ml conical flask with a plug, 10-50 ml of methanol is added, ultrasonic treatment is performed for 10-60 min, and then filtration is performed to obtain the test solution;

[0048] (4) Thin layer identification method: 5-10 μl of the mixed control solution of Magnolia officinalis phenol and Magnolia officinalis phenol, the test solution, and the negative sample solution is respectively spotted on a thin layer plate, the developing agent is added, development is performed, and then the thin layer plate is taken out, dried, sprayed with 1% vanillin sulfuric acid solution, heated at 100°C to develop color, and the test sample chromatogram shows the same color spots at the corresponding positions of the control sample chromatogram;

[0049] As a preferred solution, the detection method of magnolol and honokiol in Magnoliae officinalis Cortex described above, the preparation method of the control solution in (1) is as follows: taking magnolol and honokiol control, adding methanol to prepare a solution of 32 μg / ml, and then a mixed control solution is obtained;

[0050] As a preferred solution, the detection method of magnolol and honokiol in Magnoliae officinalis Cortex described above, the preparation method of the test solution in (2) is as follows: taking 15 batches of freeze-dried powder of the Sanhua Decoction reference sample described above, 2.0 g is placed in a 250 ml conical flask with a plug, 25 ml of methanol is added, ultrasonic treatment is performed for 45 min, and then filtration is performed to obtain a test solution;

[0051] As a preferred solution, the detection method of magnolol and honokiol in Magnoliae officinalis Cortex described above, the preparation method of the negative sample solution in (3) is as follows: taking three kinds of decoction pieces except Magnoliae officinalis Cortex, the preparation method of the freeze-dried powder of the Sanhua Decoction reference sample described in the invention content 1 is used to prepare 2.0 g, which is placed in a 250 ml conical flask with a plug, 25 ml of methanol is added, ultrasonic treatment is performed for 45 min, and then filtration is performed to obtain a negative sample solution;

[0052] As a preferred solution, the detection method of magnolol and honokiol in Magnoliae officinalis Cortex described above, the thin layer conditions in (4) are as follows: thin layer plate: silica gel G thin layer plate; sample application amount: 5 μl of the control solution, and 8 μl for the rest; developing agent: toluene-methanol test solution, volume ratio is (16-17):(1-2);

[0053] As a preferred solution, the developing agent is toluene-methanol test solution (17:2);

[0054] S5, Inspection of Rheum emodiin in Rheum palmatum

[0055] (1) Preparation of the control solution: taking Rheum emodiin control in an appropriate amount, adding methanol to prepare a solution of 8-15 μg / ml, and then Rheum emodiin control solution is obtained;

[0056] (2) Preparation of the test solution: taking 1.5-2.5 g of freeze-dried powder of the Sanhua Decoction reference sample described above in different batches, placing it in a 250 ml conical flask with a plug, adding 10-50 ml of methanol, ultrasonic treatment for 10-60 min, and then filtration to obtain a test solution;

[0057] (3) Thin layer identification method: taking 4-5 μl of the Rheum emodiin control solution and the test solution respectively, applying them on a thin layer plate, adding a developing agent, developing, taking out, air-drying, and then testing under a 365 nm ultraviolet lamp, if the same bright blue fluorescent spots are detected in the test sample chromatogram at the positions corresponding to the control sample chromatogram, it is considered that the quality does not meet the requirements, and if the same bright blue fluorescent spots are not detected, the quality meets the requirements;

[0058] As a preferred solution, the preparation method of the reference solution in step (1) of the above-mentioned rhubarb chrysophanol inspection method is as follows: take the chrysophanol reference substance, add methanol to prepare a 10 μg / ml solution, and obtain the reference solution;

[0059] As a preferred solution, the preparation method of the test solution in step (2) of the above-mentioned rhubarb chrysophanol inspection method is as follows: take 2.0 g of the freeze-dried powder of the three kinds of soup reference sample of 15 batches, place it in a 250 ml conical flask with a plug, add 25 ml of methanol, ultrasonic treat for 45 min, filter, and obtain the test solution;

[0060] As a preferred solution, the thin layer condition in step (3) of the above-mentioned rhubarb chrysophanol inspection method is as follows: thin layer plate: polyamide film thin layer plate; sample volume: 5 μl; developing agent: toluene-ethyl acetate-acetone-methanol-formic acid test solution, volume ratio is (25-30):(4-5):(4-5):(20-25):(0.1-0.2);

[0061] As a preferred solution, the developing agent is toluene-ethyl acetate-acetone-methanol-formic acid test solution (30:5:5:20:0.1).

[0062] 3. The content of magnolol and honokiol in the three kinds of soup reference sample freeze-dried powder is determined by high performance liquid chromatography, including the following steps:

[0063] S1, preparation of the reference solution: take the magnolol and honokiol reference substance, add methanol to prepare a 55-65 μg / ml solution, and obtain the reference solution;

[0064] S2, preparation of the test solution: take 1.5-2.0 g of the freeze-dried powder of the three kinds of soup reference sample, place it in a 250 ml conical flask with a plug, add 10-50 ml of methanol, ultrasonic treat for 10-60 min, filter, and obtain the test solution;

[0065] S3, preparation of the negative sample solution: take three kinds of decoction pieces except magnolia officinalis, prepare freeze-dried powder according to the above preparation method, take an appropriate amount, place it in a 250 ml conical flask with a plug, add methanol, ultrasonic treat, filter, and obtain the test solution;

[0066] S4, determination method: precisely take 10 μl of the reference solution, the test solution, and the negative sample solution respectively, inject them into the liquid chromatograph, and record the chromatogram;

[0067] S5, according to the comparison between the three kinds of soup reference sample freeze-dried powder and the single reference substance obtained in S1, the retention time of honokiol is 7.474 min, and the retention time of magnolol is 9.581 min.

[0068] S6, the liquid phase condition in S4 is: sample amount: 5-15ul; chromatographic column: Extend-C18; mobile phase: methanol-water (78:22), isocratic elution, flow rate is 0.5-1.0ml / min; detection wavelength: 260-310nm.

[0069] As a preferred scheme, the content detection method of magnolol and honokiol in the Sanhua Decoction reference sample freeze-dried powder, the preparation method of the control solution in S1 is as follows: taking the magnolol and honokiol control samples, adding methanol to prepare 60.294ug / ml and 60.176ug / ml solutions respectively, and the control solution is obtained.

[0070] As a preferred scheme, the content detection method of magnolol and honokiol in the Sanhua Decoction reference sample freeze-dried powder, the preparation method of the test solution in S2 is as follows: taking 2.0g of the Sanhua Decoction reference sample freeze-dried powder, placing it in a 250ml conical flask with a plug, adding 25ml of methanol, ultrasonic treatment for 45min, and filtering, and the test solution is obtained.

[0071] As a preferred scheme, the content detection method of magnolol and honokiol in the Sanhua Decoction reference sample freeze-dried powder, the preparation method of the negative sample solution in S3 is as follows: taking three kinds of decoction pieces except for Magnolia officinalis, preparing freeze-dried powder according to the method, taking 2.0g and placing it in a 250ml conical flask with a plug, adding 25ml of methanol, ultrasonic treatment for 45min, and filtering, and the negative sample solution is obtained.

[0072] As a preferred scheme, the content detection method of magnolol and honokiol in the Sanhua Decoction reference sample freeze-dried powder, the liquid chromatography condition in S4 is as follows: sample amount: 10ul; chromatographic column: Extend-C18; mobile phase: methanol: water = 78:22, isocratic elution; flow rate is 0.8ml / min; detection wavelength: 294nm.

[0073] Compared with the prior art, the present application has the following beneficial effects:

[0074] The present application optimizes the preparation method of the Sanhua Decoction reference sample freeze-dried powder to prepare freeze-dried powder, including medicinal material soaking time, decocting time, decocting mode, drying mode and the like, and the best preparation method is preferred, which is a modern process parameter of the classical famous prescription reference sample, and is convenient for subsequent pilot production and industrialized production.

[0075] The application detects the quality of the freeze-dried powder of the Sanhua Decoction reference sample through two technical means of thin layer chromatography and high-phase liquid chromatography, adopts the thin layer chromatography to identify all medicinal materials in the Sanhua Decoction, formulates a separate TLC detection means according to the differences of the components of different medicinal materials, and adds the detection of rheopurgin for the anti-counterfeiting of the rhubarb medicinal material, which is more intuitive, clear and accurate compared with the prior art. The total content of magnolol and honokiol is determined by the high-performance liquid chromatography, a quantitative monitoring means is provided, and the magnolia officinalis in the Sanhua Decoction is the second important ministerial medicinal material, and the advantages of the content determination compared with the monarch medicinal material rhubarb are as follows: (1) the index components are relatively uniform and easy to detect; (2) the content is relatively high, and is still relatively stable and high after processing and decoction, and is beneficial to detection; (3) the magnolia officinalis is a key medicinal material, and the detection of the magnolia officinalis is also representative. The two technical means complement each other and constitute a complete quality control method, the detection method is comprehensive and accurate, the quality of the freeze-dried powder of the Sanhua Decoction reference sample can be more effectively controlled, and the Sanhua Decoction can be more safely used.

[0076] Further, the preparation method of the control sample and the test sample and the thin layer conditions in the thin layer chromatography are optimized, the thin layer plate has good development effect, good separation degree and high spot clarity, and the quality can be more accurately detected. BRIEF DESCRIPTION OF DRAWINGS

[0077] Figure 1 The preparation process flow chart of the freeze-dried powder of the Sanhua Decoction reference sample is described in the application.

[0078] Figure 2 The freeze-dried powder sample of the Sanhua Decoction reference sample is described in the application.

[0079] Figure 3 The particle size investigation sample of the medicinal material is described in the application, wherein A is magnolia officinalis, B is rhubarb, C is immature bitter orange and D is naringen.

[0080] Figure 4 The particle size investigation chromatogram of the medicinal material is described in the application.

[0081] Figure 5 The prescription dose investigation chromatogram is described in the application.

[0082] Figure 6 The decoction method investigation chromatogram is described in the application.

[0083] Figure 7 The drying method investigation chromatogram is described in the application.

[0084] Figure 8The preparation optimization chart for the thin layer identification of the test solution of the anthraquinones in rhubarb, wherein spot 1 and spot 4 are standard sample solutions of rhein, spot 2 and spot 5 are test solutions prepared by method 1 (5.0 g of the freeze-dried powder is heated and refluxed without being dried), spot 3 is a test solution prepared by method 2 (1.25 g of the freeze-dried powder is heated and refluxed without being dried), and spot 6 is a test solution prepared by method 3 (1.25 g of the freeze-dried powder is heated and refluxed and then dried).

[0085] Figure 9 The optimization chart for the spotting amount of the standard solution of the anthraquinones in rhubarb in the thin layer identification, wherein the spotting amount of A is 4 μl, the spotting amount of B is 7 μl, and the spotting amount of C is 10 μl.

[0086] Figure 10 The optimization chart for the spotting amount of the test solution of the anthraquinones in rhubarb in the thin layer identification, wherein the spotting amount of A is 3 μl, the spotting amount of B is 7 μl, and the spotting amount of C is 10 μl.

[0087] Figure 11 The optimization chart for the preparation of the thin layer plate for the identification of Zanghuai Zihua Qianhu glycoside, wherein A is method 1 (3% sodium acetate solution is added dropwise on the thin layer plate), B is method 2 (the thin layer plate is developed and dried in 3% sodium acetate solution), and C is method 3 (the thin layer plate is soaked in 3% sodium acetate solution).

[0088] Figure 12 The optimization chart for the developing system of the thin layer identification of magnolol and honokiol in Magnoliae officinalis Cortex, wherein A is toluene-methanol = 17:0.2 (1-mixed standard, 2-test solution); B is toluene-methanol = 17:1 (1-mixed standard, 2-3 different spotting amounts of test solution); and C is toluene-methanol = 17:2 (1-mixed standard, 2-3 different spotting amounts of test solution).

[0089] Figure 13 The thin layer chromatography identification chart of the anthraquinones in rhubarb, wherein spot 1 is a mixed standard solution (the spots from top to bottom are: chrysophanol, physcion, emodin, aloe emodin, and rhein), spot 2 is a negative sample solution of rhubarb, and spots 3-17 are freeze-dried powder solutions of 15 batches of Sanhua Decoction reference samples.

[0090] Figure 14 The thin layer chromatography identification chart of synephrine in Citri Fructus, wherein spot 1 is a synephrine control solution, spots 2 and 3 are negative control solutions of Citri Fructus, and spots 4-18 are freeze-dried powder solutions of 15 batches of Sanhua Decoction reference samples.

[0091] Figure 15The thin layer chromatogram for identifying the Radix et Rhizoma Cynanchi isaticus in Cynanchi Paniculati Radix et Rhizoma, wherein spot 1 is the Cynanchi Paniculati Radix et Rhizoma control solution, spot 2 is the Radix et Rhizoma Cynanchi isaticus negative control solution, and spots 3-17 are the freeze-dried powder solutions of the 15 batches of Sanhua Decoction reference samples.

[0092] Figure 16 The thin layer chromatogram for identifying the Magnolol and Honokiol in Magnoliae Officinalis Cortex, wherein spot 1 is the mixed standard solution of Magnolol and Honokiol (the spots from top to bottom are Magnolol and Honokiol, respectively); spot 2 is the Magnoliae Officinalis Cortex negative control solution, and spots 3-18 are the freeze-dried powder solutions of the 15 batches of Sanhua Decoction reference samples.

[0093] Figure 17 The thin layer chromatogram for checking the Chrysophanol in Rhizoma et Radix Rhei, wherein spot 1 is the standard solution, and spots 2-16 are the freeze-dried powder solutions of the 15 batches of Sanhua Decoction reference samples.

[0094] Figure 18 The high performance liquid chromatogram of the Magnolol standard solution.

[0095] Figure 19 The high performance liquid chromatogram of the Honokiol standard solution.

[0096] Figure 20 The high performance liquid chromatogram of the Magnoliae Officinalis Cortex medicinal material.

[0097] Figure 21 The high performance liquid chromatogram of the Magnoliae Officinalis Cortex negative sample solution.

[0098] Figure 22 The standard curve for determining the content of Magnolol.

[0099] Figure 23 The standard curve for determining the content of Honokiol.

[0100] Figures 24-26 The high performance liquid chromatogram of the system suitability test. The chromatographic columns are Extend-C18 (250mm×4.6mm, 5μm), Hypersil ODS C18 (250mm×4.6mm, 5μm) and Kromasil 100-5-C18 (250mm×4.6mm, 5μm) in sequence. DETAILED DESCRIPTION

[0101] In order to further understand the present application, the present application is described below in conjunction with examples, which are only used to further explain the features and advantages of the present application, and are not used to limit the claims of the present application.

[0102] The instruments and reagents used in the examples are as follows:

[0103] 1. Instruments

[0104] HH-8 digital constant water bath (Jiangnan Instrument Factory in Jintan City), rotary evaporator EYELA (Tokyo Rika Instrument Co., Ltd.), electric heating constant temperature air drying oven (Xi'an Mogina Instrument Manufacturing Co., Ltd.), decoction pot (Chaozhou Longguang Electrical Appliance Co., Ltd.), FD5-series (freeze-dried spray freeze dryer Shanghai Ur Trade Co., Ltd.), SD-Basic spray dryer (LabPlant Jia Sheng Technology Co., Ltd.), JCS-600 electronic balance (Kaifeng Group Co., Ltd.), JA2603B electronic balance (Shanghai Tianmei Balance Instrument Co., Ltd.), LE204E / 02 electronic balance (Mettler-Toledo Instrument Co., Ltd.), multifunctional pulverizer (Hefei Rongshida Small Appliance Co., Ltd.), ultraviolet analyzer (Nanjing Dawei Instrument and Equipment Co., Ltd.), Shimadzu Instrument (China) Co., Ltd. LC-16 (UV) high performance liquid chromatograph, including LC-16 liquid delivery unit, SIL-16 automatic sampler, SPD-16 ultraviolet-visible dual-wavelength detector and CTO-16 liquid storage box integrated column oven.

[0105] 2. Drugs and reagents

[0106] Synephrine reference substance (batch number 110727-202110), decursin reference substance (batch number 111821-201604), isoimperatorin reference substance (batch number 110827-201812), magnolol reference substance (batch number 110729-201915), honokiol reference substance (batch number 110730-201905), rhein reference substance (batch number 110757-201607), aloe-emodin reference substance (batch number 110795-202011), emodin reference substance (batch number 110756-201913), chrysophanol reference substance (batch number 110796-201922), and physcion reference substance (batch number 110758-201817), all of which were purchased from China Institute for Drug Control; methanol (chromatographically pure), water (ultrapure water); petroleum ether, methyl formate, formic acid, chloroform, methane, toluene, acetone, and methanol, all of which were chromatographically pure.

[0107] The preparation method of the freeze-dried powder of the Sanhua Decoction reference sample of Example 1 is optimized and includes the following steps:

[0108] S1, prescription dose investigation

[0109] The present application compares three different water amounts of 2100 ml, 2300 ml and 2500 ml, and the results show that, as the water amount increases, Figure 5 , the extraction rate also increases but the overall difference is not large, and in terms of peak output, the water amount of 2100 ml is obviously better; therefore, 2100 ml is selected as the total water amount.

[0110] S2, Granularity of decoction pieces

[0111] The granularity of the decoction pieces is investigated by two sieves of 10 mesh and 5 mesh, and the granularity of the decoction pieces is investigated as two kinds of soybean size and mung bean size, such as Figure 3 and 4 The results of the extraction rate and the peak extraction rate are not much different, and in order to reduce the loss, the soybean size is selected for decoction.

[0112] S3, soaking time investigation

[0113] The soaking and water absorption of the medicinal materials are investigated at intervals of 15 minutes to determine the optimal soaking time, and the results show that when the soaking time is 45 minutes, the water absorption rate of the decoction pieces basically reaches saturation, and therefore 45 minutes is selected as the final soaking time.

[0114] S4, decoction method investigation

[0115] The three decoction methods of one-time decoction, two-time decoction with rhubarb first, and two-time decoction with rhubarb last are compared, such as Figure 6 The extraction and peak extraction rates of the two methods are not much different, and in order to meet the requirements of traditional and modern decoction, the two-time decoction with rhubarb first is selected.

[0116] S5, drying method investigation

[0117] The spray and spray freeze-drying methods commonly used in traditional Chinese medicine and classic famous prescriptions are investigated, such as Figure 7 The results show that the extraction rates of the two methods are not much different, and in order to ensure that the bitter orange fruit and nitraria tangutorum contain volatile oil and aromatic volatile substances, the freeze spray drying method is finally selected to obtain the freeze-dried powder of the Sanhua decoction reference sample.

[0118] After determining the optimal preparation process of the freeze-dried powder of the Sanhua decoction reference sample, such as Figure 1 the optimal preparation method is obtained through a large number of experiments, which comprises the following steps:

[0119] S1, 31.0 g of rhubarb, ginger, fried bitter orange fruit, and nitraria tangutorum without rhizome are weighed, crushed, and passed through a 5-mesh sieve but not a 10-mesh sieve, and then placed in a decoction pot, 1500 ml of water is added and soaked for 45 minutes;

[0120] S2, one-time decoction for 20 minutes to boiling, gentle fire for 90 minutes to 750 ml, hot filtration, add 600 ml of warm water, two-time decoction for 10 minutes to boiling, gentle fire for 20 minutes to 300 ml, hot filtration; combine the two decoctions, and the filtrate is concentrated under reduced pressure at 60°C, and spray freeze-dried at a freezing temperature of-50°C.

[0121] The freeze-dried powder of the Sanhua decoction reference sample obtained by the present application has a sample pattern as shown inFigure 2 The yield of extract is shown in Table 1.

[0122] Table 1 Yield of extract of 15 batches of Sanjia Decoction reference sample freeze-dried powder

[0123]

[0124]

[0125] Example 2 A series of methods for controlling the quality of Sanjia Decoction reference sample freeze-dried powder by thin layer chromatography were optimized, including the following steps:

[0126] S1, Optimization of detection method of anthraquinones in Rhubarb

[0127] (1) Optimization of preparation of test sample

[0128] The present application compares different sampling amounts (1.25 g, 5.0 g) of Sanjia Decoction reference sample freeze-dried powder and whether to evaporate after heating and reflux, as shown in Table 2. Figure 8 The results show that 5.0 g of Sanjia Decoction reference sample freeze-dried powder is sampled, heated and refluxed, and not evaporated, and the sampling effect on the thin layer plate is good, so this method is selected to prepare the test sample.

[0129] (2) Optimization of sampling amount of standard solution

[0130] The present application compares different sampling amounts (4 μl, 7 μl, 10 μl) of standard solution, as shown in Table 3. Figure 9 The results show that 10 μl is the best sampling amount for clear spots, so 10 μl is selected as the sampling amount.

[0131] (3) Optimization of sampling amount of control solution

[0132] The present application compares different sampling amounts (3 μl, 7 μl, 10 μl) of control solution, as shown in Table 4. Figure 10 The results show that 7 μl is the best sampling amount for clear spots, so 7 μl is selected as the sampling amount.

[0133] S2, Optimization of detection method of decursin in Notopterygium

[0134] (1) Optimization of preparation of thin layer plate

[0135] The present application compares different preparation methods of thin layer plate (dropping 3% sodium acetate solution on the thin layer plate, placing the thin layer plate in 3% sodium acetate solution for development, drying, and soaking the thin layer plate in 3% sodium acetate solution), and the results are shown in Table 5. Figure 11, found that the thin layer plate is placed in 3% sodium acetate solution, the developed thin layer plate prepared by drying has the best developing effect, the spot is clear, and the separation degree is good, so this method is selected to prepare the thin layer plate.

[0136] S3, the optimization of the detection method of magnolol and honokiol in magnolia officinalis

[0137] (1) optimization of the developing agent

[0138] The present application compares different developing agents (toluene-methanol (17:1), toluene-methanol (17:0.2) and toluene-methanol (17:2)) through experiments, and the results are as follows Figure 12 , it is found that the separation degree of the spot is better when toluene-methanol (17:2) is used as the developing agent, so toluene-methanol (17:2) is selected as the developing agent.

[0139] Finally, the best quality detection method is determined, which comprises the following steps:

[0140] S1, identification of anthraquinones in rhubarb

[0141] (1) Preparation of the reference solution: take the reference substances aloe-emodin, rhein, emodin, chrysophanol and rheochrysidin, add methanol to prepare a mixed reference solution with a concentration of 1 mg / ml;

[0142] (2) Preparation of the test sample solution: take 5.0 g of the freeze-dried powder of the Sanhua decoction reference sample described in Example 1, and place it in a 250 ml conical flask with a stopper, accurately add 30 ml of a methanol-hydrochloric acid (10:0.3) mixed solution, place it in a 80℃ water bath for heating reflux for 60 min, cool, filter, and then the test sample solution is obtained;

[0143] (3) Preparation of the negative sample solution: take three kinds of rhubarb decoction pieces, prepare freeze-dried powder according to the method of Example 1, take 5.0 g and place it in a 250 ml conical flask with a stopper, accurately add 30 ml of a methanol-hydrochloric acid (10:0.3) mixed solution, place it in a 80℃ water bath for heating reflux for 60 min, cool, filter, and then the test sample solution is obtained;

[0144] (4) Thin layer identification method: take 10 μl of the mixed reference solution, 10 μl of the negative sample solution and 7 μl of the test sample solution respectively, and point them on the same silica gel H thin layer plate with sodium carboxymethyl cellulose as the binder, add the developing agent, develop, take out, dry, place under a 365 nm ultraviolet lamp for test, and then place in ammonia vapor for fumigation to make the spot clear, as shown in Figure 13 , in the test sample chromatogram, the same color spots are displayed at the positions corresponding to the reference sample chromatogram;

[0145] The developing agent is petroleum ether (30-60℃)-ethyl formate-formic acid test solution (15:5:1);

[0146] S2, Identification of Synephrine in Aurantii Fructus Immaturus

[0147] (1) Preparation of the control solution: Take the synephrine control sample, add methanol to prepare a 0.5 mg / ml solution, and synephrine control solution is obtained;

[0148] (2) Preparation of the test solution: Take 1.25 g of the freeze-dried powder of the Sanhua Decoction reference sample described in Example 1, place it in a 250 ml conical flask with a stopper, add 50 ml of methanol, and ultrasonically treat for 45 min. Filter to obtain the test solution;

[0149] (3) Preparation of the negative sample solution: Take the freeze-dried powder of the three kinds of decoction pieces except for Aurantii Fructus Immaturus, prepared according to the method of Example 1, take 1.25 g, place it in a 250 ml conical flask with a stopper, add 50 ml of methanol, and ultrasonically treat for 45 min. Filter to obtain the negative sample solution;

[0150] (4) Thin-layer identification method: Take 7 μl of the synephrine control solution, the test solution, and the negative sample solution respectively, and spot them on the same silica gel G thin-layer plate. Add the developing agent, develop, take out, air dry, spray with 0.5% ninhydrin ethanol solution, and heat at 105°C to develop color; if Figure 14 , in the test sample chromatogram, color spots of the same color appear at positions corresponding to the control sample chromatogram;

[0151] The developing agent is n-butanol-glacial acetic acid-water (4:1:5);

[0152] S3, Identification of Decursin in Notopterygii Rhizoma et Radix

[0153] (1) Preparation of the control solution: Take the decursin control sample, add methanol to prepare a 0.5 mg / ml solution, and decursin control solution is obtained;

[0154] (2) Preparation of the test solution: Take 1.5 g of the freeze-dried powder of the Sanhua Decoction reference sample described in Example 1, place it in a 250 ml conical flask with a stopper, add 50 ml of methanol, and ultrasonically treat for 45 min. Filter to obtain the test solution;

[0155] (3) Preparation of the negative sample solution: Take the freeze-dried powder of the three kinds of decoction pieces except for Notopterygii Rhizoma et Radix, prepared according to the method of Example 1, take 1.5 g, place it in a 250 ml conical flask with a stopper, add 50 ml of methanol, and ultrasonically treat for 45 min. Filter to obtain the negative sample solution;

[0156] (4) Thin-layer identification method: Take 7 μl of the decursin control solution, the test solution, and the negative sample solution respectively, and spot them on the same silica gel G thin-layer plate prepared by placing the thin-layer plate in 3% sodium acetate solution, drying, and developing with the developing agent. Take out, air dry, and test under a 365 nm ultraviolet lamp, ifFigure 15 The sample chromatogram shows the same color fluorescent spots at the corresponding positions of the reference chromatogram;

[0157] The developing agent is chloroform-methanol test solution (8:2);

[0158] S4, Identification of Magnolol and Honokiol in Magnoliae Officinalis Cortex

[0159] (1) Preparation of reference solution: Take Magnolol and Honokiol reference substances, add methanol to prepare a solution containing 32 μg / ml of Magnolol and Honokiol, and obtain a mixed reference solution;

[0160] (2) Preparation of sample solution: Take 2.0 g of freeze-dried powder of Sanhua Decoction reference sample described in Example 1, place it in a 250 ml conical flask with a stopper, add 25 ml of methanol, and ultrasonically treat for 45 min. Filter to obtain the sample solution;

[0161] (3) Preparation of negative sample solution: Take freeze-dried powder of three kinds of decoction pieces except Magnoliae Officinalis Cortex prepared according to the method of Example 1, take 2.0 g, place it in a 250 ml conical flask with a stopper, add 25 ml of methanol, and ultrasonically treat for 45 min. Filter to obtain the sample solution;

[0162] (4) Thin layer identification method: Take 8 μl of Magnolol and Honokiol mixed reference solution, sample solution, and negative sample solution respectively, and point them on the same thin layer plate with silica gel G. Add developing agent, develop, take out, air dry, spray 1% vanillin sulfuric acid ethanol (10%) solution, and heat at 100°C to develop color, and Figure 16 The sample chromatogram shows the same color fluorescent spots at the corresponding positions of the reference chromatogram;

[0163] The developing agent is toluene-methanol test solution (17:2);

[0164] S5, Examination of Rheoprentin in Rheum palmatum

[0165] (1) Preparation of reference solution: Take Rheoprentin reference substance, add methanol to prepare a solution of 10 μg / ml, and obtain the Rheoprentin reference solution;

[0166] (2) Preparation of sample solution: Take 2.0 g of freeze-dried powder of Sanhua Decoction reference sample described in Example 1, place it in a 250 ml conical flask with a stopper, add 25 ml of methanol, and ultrasonically treat for 45 min. Filter to obtain the sample solution;

[0167] (3) Thin layer identification method: Take 5 μl of Rheoprentin reference solution and sample solution respectively, and point them on the same polyamide film thin layer plate. Add developing agent, develop, take out, air dry, and test under 365 nm ultraviolet light, and Figure 17, in the sample chromatogram, no same bright blue fluorescent spots were detected at the positions corresponding to the control chromatogram;

[0168] The developing agent is toluene-ethyl formate-acetone-methanol-formic acid test solution (30:5:5:20:0.1).

[0169] Example 3 uses high performance liquid chromatography to determine the total content of magnolol and honokiol in the freeze-dried powder of Sanhua Decoction reference sample, including the following steps:

[0170] S1, preparation of control solution: take magnolol and honokiol control, add methanol to prepare control solution with a concentration of 60.294 μg / ml and 60.176 μg / ml, respectively;

[0171] S2, preparation of test sample solution: take 2.0 g of Sanhua Decoction reference sample freeze-dried powder of Example 1, place it in a 250 ml conical flask with a stopper, add 25 ml of methanol, ultrasonic treat for 45 min, filter, and obtain the test sample solution;

[0172] S3, preparation of negative sample solution: take three kinds of decoction pieces except for Magnolia officinalis, prepare freeze-dried powder according to the method of Example 1, take 2.0 g, place it in a 250 ml conical flask with a stopper, add 25 ml of methanol, ultrasonic treat for 45 min, filter, and obtain the negative sample solution;

[0173] S4, determination method: precisely take 10 μl of control sample solution, test sample solution, and negative sample solution, respectively, inject into the liquid chromatograph, record the chromatogram, and if Figures 18-21 ; the liquid chromatography conditions are: chromatographic column: Extend-C18; mobile phase: methanol-water (78:22), isocratic elution, flow rate is 0.8 ml / min; detection wavelength: 294 nm.

[0174] Example 4: Methodology investigation of magnolol and honokiol content determination method, including the following steps:

[0175] S1, linear relationship investigation

[0176] According to the control sample solution preparation method in Example 3, a series of concentration gradient magnolol and honokiol control sample solutions are prepared, as shown in Table 2. According to the chromatographic conditions of Example 3, the sample is analyzed, the peak area is recorded, the peak area (A) is taken as the vertical coordinate, and the sample concentration C (μg / ml) is taken as the horizontal coordinate, and the standard curve is drawn, as shown in Figure 22 and Figure 23 , and the regression equation is calculated. The regression equations of magnolol and honokiol are Y=64776X-269632 (R 2 =0.9991) and Y=71297X-28.09217 (R 2= 0.9992), indicating that both have good linear relationship in the range of 6.0-170.0 μg / ml.

[0177] Table 2 Linear relationship examination

[0178]

[0179] S2, Precision test

[0180] The magnolol and honokiol reference solution was prepared according to Example 3, and the chromatographic conditions of Example 3 were referred to. The sample was injected for 6 times in parallel, and the injection amount was 10 μl. The peak area and retention time of the sample were analyzed, and the RSD value was calculated. The results are shown in Table 3, which shows that the parallel injection precision of the equipment is good.

[0181] Table 3 Precision test results

[0182]

[0183] S3, Stability test

[0184] The sample was prepared according to the method in Example 3, and the sample was prepared from 2.0 g of the Sanhua Decoction reference sample freeze-dried powder. The sample was analyzed according to the detection method of Example 3. The sample was injected at different times of 0, 2, 6, 12, 18 and 24 h, and the injection amount was 10 μl. Magnolol and honokiol were used as reference peaks, and the peak area and retention time of the sample were analyzed, and the RSD value was calculated. The results are shown in Table 4, which shows that the chromatographic peaks of the Sanhua Decoction sample solution have almost no change within 24 h, and the stability is good.

[0185] Table 4 Stability test results

[0186]

[0187] S4, Reproducibility test

[0188] The sample solution was prepared according to the preparation method of the sample solution in Example 3. The chromatographic conditions of Example 3 were referred to. Magnolol and honokiol were used as reference peaks, and the peak area and retention time of the sample were analyzed, and the RSD value was calculated. The results are shown in Table 5, which shows that the sample chromatographic peak has good reproducibility, and the repeatability of the method is good.

[0189] Table 5 Reproducibility test results

[0190]

[0191] S5, Accuracy test

[0192] Take the recovery rate test of magnolol as an example, 1.0 g of the freeze-dried powder of the Sanhua Decoction reference sample was precisely weighed, 6 portions were prepared in conical flasks, 3 ml of the magnolol control solution (237.02 μg / ml) was precisely added, the test sample solution preparation method and the chromatographic conditions of Example 3 were referred to, magnolol and honokiol were used as reference peaks, the peak area and retention time of the sample were analyzed and the RSD value was calculated, the results were shown in Table 6, the peak area of the magnolol chromatographic peak was recorded, the average recovery rate was calculated to be 100.17%, and the RSD was 2.140%, indicating that the recovery rate of the method was good.

[0193] Table 6: Results of the magnolol recovery rate test

[0194]

[0195] The recovery rate of honokiol was measured by the same method, the average recovery rate was 100.32%, the RSD was 2.360%, and the results were shown in Table 7.

[0196] Table 7: Results of the honokiol recovery rate test

[0197]

[0198] S6: System suitability test

[0199] The same batch of samples was taken, the test sample solution was prepared according to Example 3, and the detection method of Example 3 was referred to, and the suitability of three brands of chromatographic columns Extend-C18 (250 mm x 4.6 mm, 5 μm), Hypersil ODS C18 (250 mm x 4.6 mm, 5 μm), and Kromasil 100-5-C18 (250 mm x 4.6 mm, 5 μm) was investigated. The results showed that in different chromatographic columns, magnolol and honokiol were well separated from the impurity peaks, the determination results were basically consistent, and the suitability was good. The system suitability chromatograms of magnolol and honokiol were shown in Figures 23-25 .

[0200] S7: Content determination

[0201] According to the test sample solution preparation method in Example 3, 15 batches of medicinal materials were taken to prepare test sample solutions, and the chromatographic conditions of Example 3 were referred to, and the high performance liquid chromatograph was injected for analysis, and the contents of magnolol and honokiol in the 15 batches of freeze-dried powders were calculated. The content determination results were shown in Table 8.

[0202] Table 8: Results of sample content determination

[0203]

[0204] The above experimental results show that the detection method of the Sanhua decoction reference sample freeze-dried powder has high precision, good stability, good repeatability, high sensitivity and high accuracy, can comprehensively and objectively evaluate the quality of the Sanhua decoction reference sample freeze-dried powder, and has important significance for guaranteeing clinical curative effect.

[0205] The above examples are only exemplary embodiments of the present application and are not intended to limit the present application, and the protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent replacements to the present application within the spirit and protection scope of the present application, and such modifications or equivalent replacements shall also be considered to fall within the protection scope of the present application.

Claims

1. A method for quality testing of lyophilized powder of Sanhua Tang reference sample, characterized in that, The preparation method of the freeze-dried powder of the Sanhuatang reference sample includes the following steps: S1. Take rhubarb, magnolia bark, immature bitter orange and notopterygium root slices in a mass ratio of 1:1:1:1, crush them, pass them through a 5-mesh sieve, and soak them in water for 15-60 minutes. S2. Boil at 150℃~180℃ for 15~20 min, then boil at 80℃~120℃ for 85~100 min, cool, and filter; add water to the residue, boil at 150℃~180℃ for 5~10 min, then boil at 80℃~120℃ for 15~30 min, and filter; combine the two decoctions, concentrate under reduced pressure, and spray freeze-dry to obtain the freeze-dried powder of the Sanhua Decoction reference sample; The quality testing methods include: using thin-layer chromatography to identify anthraquinones in rhubarb, to identify synephrine in immature bitter orange, to identify purslane in notopterygium root, to identify magnolol and honokiol in magnolia bark, and to examine rhein in rhubarb. The identification of anthraquinones in rhubarb using thin-layer chromatography specifically includes: (1) Preparation of reference solution: Take aloe-emodin, rhein, emodin, chrysophanol and emodin methyl ether reference standards, add methanol to obtain mixed reference solution; (2) Preparation of test solution: Take the freeze-dried powder of Sanhuatang reference sample, add a mixed solution of methanol and hydrochloric acid, heat under reflux in a water bath without evaporation, cool and filter to obtain the test solution; (3) Preparation of negative sample solution: Take Magnolia officinalis, Citrus aurantium and Notopterygium incisum slices, prepare freeze-dried powder, add a mixed solution of methanol and hydrochloric acid, heat under reflux in a water bath, cool and filter to obtain negative sample solution; (4) Thin-layer identification method: Take the mixed reference solution, test solution and negative sample solution and spot them on the thin-layer plate, add the developing solvent, develop, take out, dry, test under 365nm ultraviolet lamp, and then fumigate in ammonia vapor to make the spots clear. Observe the spots on the chromatogram of the test sample, reference and negative sample; The thin-layer conditions are: Thin-layer plate: silica gel H thin-layer plate with sodium carboxymethyl cellulose as binder; Developing solvent: petroleum ether-ethyl formate-formic acid, the volume ratio of petroleum ether, ethyl formate and formic acid is (14~16):(4~6):(0.5~1); The specific identification of pursanthin in Notopterygium incisum using thin-layer chromatography is as follows: (1) Preparation of reference solution: Take imperatorin reference standard, add methanol to obtain imperatorin reference solution; (2) Preparation of test solution: Take the lyophilized powder of Sanhuatang reference sample, add methanol, sonicate, filter, and obtain test solution; (3) Preparation of negative sample solution: Take three kinds of medicinal slices, namely rhubarb, magnolia bark and immature bitter orange, prepare the reference sample freeze-dried powder, add methanol, sonicate, filter, and obtain negative sample solution; (4) Thin-layer identification method: Spot the purpuricin reference solution, test solution and negative sample solution onto the thin-layer plate, add the developing solvent, develop, remove, air dry, and test under a UV lamp at 365nm to observe the spots on the chromatogram of the test sample, reference and negative sample; The thin-layer conditions are: thin-layer plate: silica gel G thin-layer plate; developing solvent: chloroform-methanol test solution, volume ratio of (6-8):(1-2); The thin-layer plate used is prepared by developing the silica gel G thin-layer plate in 3% sodium acetate solution and then drying it.

2. The method for quality testing of the freeze-dried powder of the Sanhua Tang reference sample according to claim 1, characterized in that, The specific method for identifying synephrine in Citrus aurantium using thin-layer chromatography is as follows: (1) Preparation of reference solution: Take synephrine reference standard, add methanol to obtain synephrine reference solution; (2) Preparation of test solution: Take the lyophilized powder of Sanhuatang reference sample, add methanol, sonicate, filter, and obtain test solution; (3) Preparation of negative sample solution: Take rhubarb, magnolia bark and notopterygium root slices, prepare the reference sample lyophilized powder, add methanol, sonicate, filter, and obtain negative sample solution; (4) Thin-layer identification method: Take synephrine reference solution, test solution and negative sample solution and spot them on the thin-layer plate, add developing solvent, take it out, dry it, spray it with 0.5% ninhydrin ethanol solution, heat and develop color; observe the spots on the chromatogram of test sample, reference and negative sample; the thin-layer conditions are: thin-layer plate: silica gel G thin-layer plate; developing solvent: the developing solvent is n-butanol-glacial acetic acid-water, and the volume ratio of n-butanol, glacial acetic acid and water is (4~5):(1~2):(4~5).

3. The method for quality testing of the lyophilized powder of the Sanhua Tang reference sample according to claim 1, characterized in that, The specific identification of magnolol and honokiol in Magnolia officinalis using thin-layer chromatography is as follows: (1) Preparation of reference solution: Take magnolol and magnolol reference standards, add methanol to obtain mixed reference solution; (2) Preparation of test solution: Take the lyophilized powder of Sanhuatang reference sample, add methanol, sonicate, filter, and obtain test solution; (3) Preparation of negative sample solution: Take three kinds of medicinal slices, namely rhubarb, immature bitter orange and notopterygium root, prepare the reference sample freeze-dried powder, add methanol, sonicate, filter, and obtain negative sample solution; (4) Thin-layer identification method: Take the mixed reference solution, test solution and negative sample solution and spot them on the thin-layer plate, add the developing solvent, develop, take out, dry, spray with 1% vanillin sulfuric acid solution, heat to develop color, and observe the spots on the chromatogram of the test sample, reference and negative sample; The thin-layer conditions are: thin-layer plate: silica gel G thin-layer plate; developing solvent: toluene-methanol test solution, the volume ratio of toluene and methanol is (16~17): (1~2).

4. The method for quality testing of the freeze-dried powder of the Sanhua Tang reference sample according to claim 1, characterized in that, The specific steps for examining rhein in rhubarb using thin-layer chromatography are as follows: (1) Preparation of reference solution: Take rhein reference standard, add methanol to obtain rhein reference solution; (2) Preparation of test solution: Take the lyophilized powder of Sanhuatang reference sample, add methanol, sonicate, filter, and obtain test solution; (3) Thin-layer identification method: Take the reference solution of rhein and the test solution and spot them on the thin-layer plate, add the developing solvent, develop, take out, dry, and test under a 365nm ultraviolet lamp to observe the spots on the chromatogram of the test sample and the reference sample; The thin-layer conditions are: thin-layer plate: polyamide film thin-layer plate; developing solvent: toluene-ethyl formate-acetone-methanol-formic acid test solution, and the volume ratio of toluene, ethyl formate, acetone, methanol and formic acid is (25~30): (4~5): (4~5): (20~25): (0.1~0.2).

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