A method for characterizing the decoction of Rhizoma Cynomorii and Rhizoma Cynomorii charcoal slices, standard decoctions and formula granules and their identification

Abstract

The present invention provides a method and identification of a standard decoction characteristic spectrum of Guanzhong and Guanzhong charcoal slices, standard decoctions, and formula granules, including: A) dissolving Guanzhong charcoal raw materials with a solvent to obtain a test liquid; B) determining the test liquid with high performance liquid chromatography to obtain a UPLC characteristic spectrum of the Guanzhong charcoal raw materials; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is 0.1% formic acid, and the gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.1% formic acid as the mobile phase for gradient elution, takes protocatechuic acid as a reference, establishes Guanzhong charcoal slices, standard decoctions, and formula granules UPLC characteristic spectrum, has good repeatability and precision, is stable and reliable, and can control the quality of Guanzhong charcoal slices, standard decoctions, and formula granules.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method and identification of a standard decoction characteristic spectrum of Rhizoma Cynomorii and Rhizoma Cynomorii charcoal decoction pieces, a standard decoction and formula granules. Background Art

[0002] Guanzhong is the dried rhizome of Woodwardia unigemmata (Makino.) Nakai., a plant of the Pteridaceae family. It is commonly known as "Guoji Guanzhong". It is dug in spring and autumn, the petioles are cut off, the fibrous roots and soil are removed, washed, and dried in the sun; or sliced ​​while fresh and dried in the sun. It has the effects of clearing heat and detoxifying, stopping bleeding, and healing sores and promoting tissue regeneration. Guanzhong standard decoction is a freeze-dried powder made from the processed medicinal materials according to a fixed preparation process; Guanzhong formula granules are formula granules made from the processed Guanzhong medicinal materials according to the main quality indicators of the standard decoction. Guanzhong is a commonly used Chinese medicine. It was first recorded in the "Shennong Bencao Jing" as a medicine. It has the effects of clearing heat and detoxifying, and expelling insects. The morphology, habitat, and origin of Guanzhong recorded in the herbal books of various dynasties indicate that there are many varieties of Guanzhong original plants. Literature records that there are as many as 11 families, 18 genera, and 58 species of original plants used as Guanzhong in my country. The survey results of Ai Tiemin and others show that in the 1980s, 27 plants were used as the source plants of Guanzhong. The "Chinese Medicine Dictionary" records that there are 7 original plants of Guanzhong. Yang Chunyu and others have sorted out 38 original plants of Guanzhong through investigation, of which 18 are widely purchased as Guanzhong for medicinal purposes. Even the "Chinese Pharmacopoeia" has changed the regulations on the original plants of Guanzhong many times. The 1977 edition included two species, Mianma Guanzhong and Ziqi Guanzhong. After the 1995 edition, only Mianma Guanzhong was included. Ziqi Guanzhong was added in the 2010 edition. It can be seen that the Guanzhong class of medicinal materials is extremely complex.

[0003] Guanzhong charcoal is made by frying the Guanzhong medicinal material according to the stir-frying method (General Rule 0213) until the outer surface is black and the inside is brown; it enhances the hemostatic effect. Guanzhong charcoal standard decoction is a freeze-dried powder made from the medicinal material after processing according to a fixed preparation process; Guanzhong charcoal formula granules are formula granules made from the Guanzhong medicinal material after processing according to the main quality indicators of the standard decoction. In order to ensure the uniformity and stability of the quality of Guanzhong charcoal slices, standard decoctions, and formula granules.

[0004] Therefore, a new characteristic spectrum method was established to control the quality of Guanzhong charcoal, and it can clearly distinguish Guanzhong charcoal from Guanzhong and its preparations. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing UPLC characteristic spectra of Guanzhong charcoal slices, standard decoctions, and formula granules. The UPLC characteristic spectra method of Guanzhong charcoal slices, standard decoctions, and formula granules constructed by the present invention is stable and reliable, and can control the quality of Guanzhong charcoal slices, standard decoctions, and formula granules.

[0006] A method for constructing UPLC characteristic spectrum of radix guanylodis macrophyllae slices, standard decoctions, and formula granules, comprising:

[0007] A) dissolving the carbon raw material with a solvent to obtain a test solution;

[0008] B) the test solution is measured by high performance liquid chromatography to obtain a UPLC characteristic spectrum of the charcoal raw material;

[0009] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is 0.1% formic acid, and the elution is gradient.

[0010] The method for constructing UPLC characteristic spectrum of charcoal slices, standard decoctions and formula particles provided by the present invention firstly takes charcoal raw materials, dissolves them in solvent, and obtains a test solution. The solvent is preferably 70% methanol.

[0011] The present invention adopts the above extraction solvent, the chromatographic peak information volume is large and the effect is good.

[0012] Specifically, the charcoal raw material is boiled in water, filtered, the filter residue is dissolved in a solvent, ultrasonically extracted, shaken, filtered, and the product is obtained.

[0013] The extraction method of the present invention is ultrasonic extraction after boiling or direct ultrasonic extraction;

[0014] The specific parameters of ultrasound after boiling are: the mass ratio of the charcoal to water is preferably 1:50; the boiling time is preferably 20 to 30 minutes; the ultrasonic power is preferably 600W, and the frequency is preferably 40kHz; the extraction time is preferably 15 to 45 minutes; more preferably 15 to 30 minutes;

[0015] The specific parameters of direct ultrasonic extraction are: using 70% methanol for ultrasonication for 10 to 15 minutes.

[0016] Medicinal materials, decoction pieces and control medicinal materials were extracted by ultrasonic method after boiling in water, while standard decoctions and formula granules were extracted by direct ultrasonic method.

[0017] The ratio of the mass g of the carbon raw material to the volume mL of the solvent is preferably (0.1-1):(10-50); more preferably 0.2:25.

[0018] The raw materials of the smilax china charcoal are smilax china medicinal materials, smilax china decoction pieces, standard decoctions, and formula granules. The present invention does not limit them, and the above raw materials can all be subjected to quality control and qualitative detection by the method of the present invention.

[0019] The present invention also includes preparing a reference solution: taking protocatechuic acid and 5-hydroxymethylfurfural respectively, adding water to boil, filtering, dissolving the filter residue with 70% methanol to obtain a reference solution; the extraction method of the present invention is ultrasonic extraction after boiling or direct ultrasonic extraction; the mass ratio of the Guanzhong medicinal material and water is preferably 1:50; the boiling time is preferably 20 to 30 minutes; the ultrasonic power is preferably 600W, and the frequency is preferably 40kHz; the extraction time is preferably 15 to 45 minutes; more preferably 15 to 30 minutes. Wherein, the ratio of the mass g of the Guanzhong charcoal reference medicinal material to the volume mL of the solvent is preferably (0.1 to 1): (10 to 50); more preferably 0.2:25.

[0020] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the UPLC characteristic spectra of the charcoal slices, standard decoction, and formula granules are qualitatively measured based on the chromatogram of the reference.

[0021] Mobile phase A was acetonitrile, mobile phase B was 0.1% formic acid, and gradient elution was performed.

[0022] The gradient elution of the present invention is preferably specifically:

[0023] 0-8min, phase A: 1%, phase B: 99%;

[0024] 8-18min, phase A: 1%-15%, phase B: 99%-85%;

[0025] 18-35min, phase A: 15%-25%, phase B: 85%-75%;

[0026] 35-50min, phase A: 25%-26%, phase B: 75%-74%.

[0027] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0028] 1.6μm, 2.1×150mm; column temperature 35℃.

[0029] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above 35°C condition.

[0030] The mobile phase flow rate is preferably 0.3 ml / min.

[0031] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 0.3 ml / min, which was the most preferred solution.

[0032] The detection wavelength of the present invention is preferably 250 nm.

[0033] The inventors found that at the above wavelength, the chromatographic information is rich, each component has good absorption, the response value is moderate, the peak separation is good, and the baseline is stable.

[0034] The injection volume of the present invention is 1 μL.

[0035] The beneficial effect of the present invention is that under one liquid chromatography condition, the substance groups of the radix guanzhong charcoal slices, standard decoction and formula granules are controlled by fingerprints, and the fingerprints are positioned by protocatechuic acid; the cost of detection can be greatly reduced and qualitative detection can be achieved.

[0036] The similarity of the UPLC characteristic spectra of Guanzhongtan slices, standard decoction and formula granules was evaluated by the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, and the UPLC standard characteristic spectra of Guanzhongtan slices, standard decoction and formula granules were obtained, which consisted of 13 characteristic peaks, among which peak 1 was hydroxymethylfurfural and peak 3 was protocatechuic acid.

[0037] In the characteristic spectrum of the standard decoction of Guanzhong Carbon, protocatechuic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.51 (peak 4), 2.75 (peak 5), 2.81 (peak 6), 3.34 (peak 7), 3.43 (peak 8), 3.50 (peak 9), 3.67 (peak 10), 4.57 (peak 11), 4.72 (peak 12), 4.77 (peak 13);

[0038] In the characteristic spectrum of the Guanzhong Charcoal Slices, protocatechuic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.86 (peak 2), 1.48 (peak 4), 2.67 (peak 5), 2.72 (peak 6), 3.24 (peak 7), 3.32 (peak 8), 3.39 (peak 9), 3.55 (peak 10), 4.44 (peak 11), 4.57 (peak 12), 4.61 (peak 13);

[0039] In the characteristic spectrum of the Guanzhong charcoal formula particles, protocatechuic acid is used as the reference peak S peak to calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.50 (peak 4), 2.72 (peak 5), 2.77 (peak 6), 3.30 (peak 7), 3.39 (peak 8), 3.46 (peak 9), 3.62 (peak 10), 4.53 (peak 11), 4.65 (peak 12), and 4.70 (peak 13).

[0040] Through testing, the invention shows that the cyperus rotundus and its preparation have only 11 peaks, which are less than the peak 1 and the peak 2 of the cyperus rotundus charcoal, and the cyperus rotundus ... charcoal can be clearly identified.

[0041] Quality judgment standard: Take samples of Guanzhongtan slices, standard decoctions, and formula granules, and operate according to the same method as above to obtain the characteristic spectra of Guanzhongtan slices, standard decoctions, and formula granules. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) of the National Pharmacopoeia Committee to analyze the standard characteristic spectra of Guanzhongtan slices, standard decoctions, and formula granules and the sample characteristic spectra, and the similarity is greater than 0.90.

[0042] The method provided by the present invention can effectively monitor the quality of different batches of Guanzhong carbonized slices, standard decoctions, and formula granules to stabilize their quality. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensive monitoring of product quality.

[0043] The characteristic spectra of the charcoal slices, standard decoction and formula granules established by the present invention take protocatechuic acid and 5-hydroxymethylfurfural as references, focus on the order of each characteristic peak and the correlation with medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the products, and the method is scientific and reliable.

[0044] The newly-created characteristic spectrum method of the present invention can detect the components with greater polarity in Rhizoma Cyrthospermi and its standard decoction. The test sample preparation method is simple and easy to operate, and relatively more characteristic peaks can be identified. Accurate and reliable characteristic spectrum detection can be performed on Rhizoma Cyrthospermi and its preparations. The authenticity, quality consistency and stability of Rhizoma Cyrthospermi and its preparations can be effectively detected and controlled. It provides a basis for effectively controlling and comprehensively evaluating the quality of Rhizoma Cyrthospermi standard decoction. It ensures the uniformity and stability of the quality of Rhizoma Cyrthospermi and its standard decoction.

[0045] The present invention provides a method for constructing UPLC characteristic spectrum of Guanzhongtan decoction pieces, standard decoctions, and formula granules, comprising: A) dissolving Guanzhongtan raw materials with a solvent to obtain a test solution; B) determining the test solution with high performance liquid chromatography to obtain a UPLC characteristic spectrum of the Guanzhongtan raw materials; the chromatographic conditions of the high performance liquid chromatography are: a chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid, and gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.1% formic acid as the mobile phase for gradient elution, takes protocatechuic acid as a reference, and establishes UPLC characteristic spectrum of Guanzhongtan decoction pieces, standard decoctions, and formula granules, which has good repeatability and precision, and the method is stable and reliable, and can control the quality of Guanzhongtan decoction pieces, standard decoctions, and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1-1 The reference characteristic spectrum of the Guanzhongcharoan decoction pieces disclosed in Example 1 of the present invention;

[0047] Figure 1-2 It is the comparative characteristic spectrum of the Chinese medicine Guanzhong under the same determination conditions;

[0048] Figure 2 This is the result of the investigation on the extraction method of the charcoal slices of Guanzhong;

[0049] Figure 3 This is the chromatogram of different solvent extraction of Guanzhong carbonized slices;

[0050] Figure 4 This is the chromatogram of different extraction times of Guanzhongtan decoction pieces;

[0051] Figure 5 Chromatograms for different extraction solvent amounts;

[0052] Figure 6 This is the result diagram of chromatographic peak identification;

[0053] Figure 7 The following are the results of investigations with different instruments;

[0054] Figure 8 This is the result diagram of different chromatographic columns;

[0055] Figure 9-1 This is the characteristic spectrum of Guanzhongtan decoction pieces;

[0056] Figure 9-2 This is the characteristic spectrum of Guanzhongtan decoction pieces;

[0057] Figure 9-3 This is the characteristic spectrum of the medicinal material of Rhizoma Cynomorii;

[0058] Figure 9-4 This is the characteristic spectrum of the medicinal material of Rhizoma Cynomorii;

[0059] Figure 9-5 This is a comparative characteristic spectrum of the medicinal material of Rhizoma Cynomorii;

[0060] Fig.10 To investigate the extraction method of Guanzhong carbonization decoction;

[0061] Fig.11 Investigation of solvent for extracting Guanzhong carbonization decoction;

[0062] Fig.12 Chromatograms at different extraction times;

[0063] Fig.13 Chromatograms of different extraction solvent amounts;

[0064] Fig.14 Chromatographic peak identification;

[0065] Fig.15 Surveys with different instruments;

[0066] Fig.16 Investigation of different chromatographic columns;

[0067] Fig.17 Characteristic spectrum of the standard decoction of Guanzhongtan;

[0068] Fig.18 This is the characteristic spectrum of the standard decoction of Guanzhongtan;

[0069] Fig.19 This is the reference characteristic spectrum of the standard decoction of Rhizoma Cynomorii;

[0070] Fig. 20 Investigation of mobile phase types;

[0071] Fig.21 Column temperature inspection chromatogram;

[0072] Fig. 22 Flow rate investigation;

[0073] Fig.23 Delayed inspection;

[0074] Fig.24 Chromatograms of different extraction methods;

[0075] Fig.25 Chromatograms of extraction with different solvents;

[0076] Fig.26 Chromatograms at different extraction times;

[0077] Fig. 27 Chromatograms of different extraction solvent amounts;

[0078] Fig.28 Chromatographic peak identification;

[0079] Fig.29 Surveys with different instruments;

[0080] Fig.30 Investigation of different chromatographic columns;

[0081] Fig.31 Verification diagram of characteristic spectra of 3 batches of Guanzhong formula granules;

[0082] Fig.32 This is the reference characteristic spectrum of the Guanzhong carbon formula particles;

[0083] Fig.33 This is the characteristic spectrum of the medicinal material of Rhizoma Cynomorii;

[0084] Fig.34 This is the characteristic spectrum of the medicinal material of Rhizoma Cynomorii;

[0085] Fig.35 This is a comparative characteristic spectrum of the medicinal material of Rhizoma Cynomorii;

[0086] Fig.36 This is the characteristic spectrum of the standard decoction of Rhizoma Cynomorii;

[0087] Fig.37 This is the characteristic spectrum of the standard decoction of Rhizoma Cynomorii;

[0088] Fig.38 This is the reference characteristic spectrum of the standard decoction of Rhizoma Cynomorii;

[0089] Fig.39 This is the characteristic spectrum of Guanzhong formula granules;

[0090] Fig.40 This is the reference characteristic spectrum of Guanzhong Formula Granules;

[0091] Fig.41 This is the result diagram of different gradient conditions of Comparative Example 4. DETAILED DESCRIPTION

[0092] To further illustrate the present invention, a method for constructing a UPLC characteristic spectrum of a charcoal slice, a standard decoction, and a formula granule provided by the present invention is described in detail below in conjunction with examples.

[0093] High performance liquid chromatograph: Agilent 1290; Shimadzu LC-40B X3; Thermo Fisher Vanquish Flex;

[0094] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0095] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0096] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0097] Chromatographic column: T3 2.1×150mm,1.6μm; Phenomenex OmegaPS C18100A2.1×150mm,1.6μm; ACQUITY BEH Shield RP18 2.1×150mm,1.7μm

[0098] Methanol and acetonitrile were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0099] Protocatechuic acid reference substance (China Food and Drug Inspection Institute, batch number: 110809-201906),

[0100] 5-Hydroxymethylfurfural reference substance (China Food and Drug Inspection Institute, batch number: 111626-202215)

[0101] Rhizoma Cynomorii (Sichuan Institute of Drug Control, batch number: NO.SCZD003-201905);

[0102] Guanzhong Tan decoction pieces batch number (YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP -10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, YP-20);

[0103] Freeze-dried powder of standard decoction of Guanzhong carbon (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20).

[0104] Carbonaceous guanzhong formula granules (batch numbers: KL-01, KL-001, KL-002, KL-003).

[0105] Example 1 Screening of Chromatographic Conditions for Medicinal Materials and Decoction Pieces of Rhizoma Cynomorii

[0106] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A; 0.1% formic acid solution as mobile phase B, gradient elution as specified in the table below; column temperature 35°C; flow rate 0.3 ml / min; detection wavelength 250 nm. The theoretical plate number calculated based on the protocatechuic acid peak should be no less than 5000.

[0107]

[0108] Preparation of reference solution: Take 1g of Guanzhong reference medicinal material, place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate, add 25ml of 70% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 15 minutes, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of protocatechuic acid reference substance, accurately weigh it, and accurately add methanol to make a solution containing 20μg of protocatechuic acid per 1ml, which is used as the reference solution of the reference substance.

[0109] Take about 1g of the powder (passed through No. 3 sieve), put it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 15 minutes, shake well, filter, and take the filtrate to obtain the product.

[0110] Determination method: Accurately pipette 1 μl of the test solution and the reference solution, inject into the liquid chromatograph, and determine.

[0111] There should be 13 characteristic peaks in the chromatogram of the test sample. Except for peak 1 and peak 2, the retention times of the other 11 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. The peak corresponding to the protocatechuic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.77 (peak 1), 0.86 (peak 2), 1.48 (peak 4), 2.67 (peak 5), 2.72 (peak 6), 3.24 (peak 7), 3.32 (peak 8), 3.39 (peak 9), 3.55 (peak 10), 4.44 (peak 11), 4.57 (peak 12), 4.61 (peak 13). The results are shown in Figure 1. Figure 1-1 is a comparative characteristic spectrum of the Guanzhongcharoan decoction pieces disclosed in Example 1 of the present invention, Figure 1-2 The above is a comparative characteristic spectrum of the Chinese medicine Rhizoma Cyperi under the same determination conditions. The method of the present invention can well distinguish the Chinese medicine Rhizoma Cyperi charcoal from the Chinese medicine Rhizoma Cyperi.

[0112] Example 2: Investigation of extraction method

[0113] The results of ultrasonic extraction, reflux extraction and ultrasonic extraction after water decoction were compared. The chromatographic peak information of ultrasonic extraction after water decoction was consistent with that of the standard decoction of Guanzhong carbon. Finally, ultrasonic extraction after water decoction was selected. Figure 2 , Figure 2 This is a graph showing the results of an investigation into the extraction methods of the Guanzhong charcoal slices.

[0114] Example 3 Extraction solvent investigation:

[0115] Methanol, water, 50% methanol and 70% methanol were investigated as extraction solvents. The results showed that the chromatographic peaks obtained with 70% methanol as the extraction solvent had a large amount of information and good separation, so 70% methanol was selected as the extraction solvent. Figure 3 , Figure 3 This is the chromatogram of extraction of Guanzhong carbonized slices with different solvents.

[0116] Example 4: Extraction time study

[0117] The chromatograms of ultrasonic extractions for 15 minutes, 30 minutes, and 60 minutes were basically the same. Therefore, the extraction time was determined to be 15 minutes. Figure 4 , Figure 4These are chromatograms of the charcoal slices of Guanzhong at different extraction times.

[0118] Example 5: Investigation of the amount of extraction solvent

[0119] The addition amount of extraction solvent was compared with 25ml, 50ml and 100ml. When the amount of extraction solvent was 25ml, the size of the chromatographic peak was more suitable, so the amount of extraction solvent was determined to be 25ml. Figure 5 , Figure 5 The chromatograms are for different extraction solvent amounts.

[0120] Example 6 Methodological Investigation

[0121] 6.1 Chromatographic peak identification

[0122] Preparation of test solution: Prepare the test solution of Guanzhong medicinal material according to the final test sample preparation method. That is: take about 1g of the powder of this product (passed through No. 3 sieve), put it in a stoppered conical bottle, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 15 minutes, shake well, filter, and take the filtrate to obtain.

[0123] Preparation of reference substance solution: Take an appropriate amount of protocatechuic acid reference substance, weigh it accurately, and add methanol to make a reference substance solution containing 20 μg of protocatechuic acid per 1 ml.

[0124] Preparation of control medicinal material solution: Take 1 g of Guanzhong control medicinal material, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0125] Preparation of negative control solution: According to the experimental conditions proposed above, prepare negative control solution lacking the herb Guanzhong. Locate the characteristic spectrum peak of the herb Guanzhong. Figure 6 , Figure 6 This is the result diagram of chromatographic peak identification.

[0126] 6.2 Precision test

[0127] Take the test solution of Guanzhong Carbon Piece (Batch No.: YP-01), inject 6 times continuously according to the proposed experimental method, 1 μl each time, and calculate the retention time and peak area of ​​each characteristic peak. See Table 1-1.

[0128] Table 1-1 Precision investigation - retention time

[0129]

[0130] Table 1-2 Precision Investigation-Peak Area

[0131]

[0132] The results showed that the RSD of the retention time of each characteristic peak was 0.02%-0.18%, and the RSD of the peak area was 0.02%-0.18%. The instrument has good precision.

[0133] 6.3 Repeatability study

[0134] Take 6 portions of Guanzhong Charcoal Pieces (Batch No.: YP-01) and prepare and test them according to the proposed experimental method. See Tables 2-1 and 2-2.

[0135] Table 2-1 Repeatability Study - Relative Retention Time Ratio

[0136]

[0137]

[0138] Table 2-2 Repeatability study - relative peak area ratio

[0139]

[0140] The results showed that the relative retention time RSD of each characteristic peak was 0.00%-0.54%, and the relative peak area RSD was 0.00%-9.08%. This method has good repeatability.

[0141] 6.4 Intermediate precision study

[0142] 6.4.1 Different people at different times

[0143] Based on the experimental conditions proposed above, the sample solution of the charcoal slices of Guanzhong (batch number: YP-01) was prepared by different personnel and at different times for determination. See Tables 3-1 and 3-2.

[0144] Table 3-1 Intermediate precision-relative retention time ratio

[0145]

[0146] Table 3-2 Intermediate precision-relative peak area ratio

[0147]

[0148]

[0149] The results show that the method has good precision when different people measure the same sample at different times.

[0150] 6.5 Different instruments

[0151] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated respectively. Figure 7 , Figure 7 Tables 4-1 and 4-2 show the results of investigations using different instruments.

[0152] Table 4-1 Different instruments investigated - relative retention time

[0153]

[0154] Table 4-2 Different instruments investigated - relative peak areas

[0155]

[0156] The results showed that when the three instruments were used for detection, the RSD of the relative retention time of each characteristic peak was between 0.67% and 17.16%, and the RSD of the relative peak area of ​​each characteristic peak was between 0.00% and 43.84%, indicating that the durability of different instruments was poor.

[0157] 6.6 Column durability investigation

[0158] Based on the experimental conditions proposed above, chromatographic column 1, chromatographic column 2, and chromatographic column 3 were investigated respectively. Figure 8 , Figure 8 The results of the investigation of different chromatographic columns are shown in Tables 5-1 and 5-2.

[0159] Table 5-1 Chromatographic column durability inspection - relative retention time

[0160]

[0161] Table 5-2 Chromatographic column durability inspection - relative peak area

[0162]

[0163]

[0164] Depend on Figure 8 It can be seen that T3 2.1×150mm, 1.6μm (column 1) has better chromatogram separation, so it is recommended to use this column T3 2.1×150mm,1.6μm.

[0165] 6.7 Stability Study

[0166] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 3h, 6h, 9h, 12h, 18h and 24h respectively. See Table 6-1 and 6-2.

[0167] Table 6-1 Stability Study - Retention Time

[0168]

[0169] Table 6-2 Stability Study-Peak Area

[0170]

[0171] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.02% and 0.19%, and the RSD of the peak area was between 2.13% and 6.48%. The sample solution was relatively stable within 24 hours.

[0172] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 13 characteristic peaks were included in the subsequent investigation.

[0173] 6.8 Verification of the characteristic spectrum of Guanzhongtan decoction pieces

[0174] This method was used to analyze the characteristic spectra of 20 batches of Guanzhong carbonized slices, and the relative retention time and relative peak area ratio were calculated. The results are shown in Figure 9-1 and 9-2 , Tables 7-1 and 7-2.

[0175] in Figure 9-1 This is the characteristic spectrum of Guanzhongtan decoction pieces. The batch numbers from bottom to top are: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10;

[0176] Figure 9-2 This is the characteristic spectrum of Guanzhong Charcoal Pieces. The batch numbers from bottom to top are: YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, and YP-20.

[0177] Table 7-1 Relative retention time of characteristic spectra of 20 batches of Guanzhongtan decoction pieces

[0178]

[0179] Table 7-2 Relative peak areas of characteristic spectra of Guanzhong carbonized slices

[0180]

[0181]

[0182] According to the principle of stable relative retention time and the fact that each batch of samples can be detected and the peak is relatively high, a total of 13 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 3 was used as the S peak, the relative retention time RSD of the characteristic peaks of 20 batches of Guanzhong charcoal slices was between 0.00% and 0.26%, and the relative retention time RSD of the 13 characteristic peaks of 20 batches of Guanzhong charcoal slices was less than 10%.

[0183] Final regulations: There should be 13 characteristic peaks in the chromatogram of the test sample. Except for peak 1 and peak 2, the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. The peak corresponding to the peak of the protocatechuic acid reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.77 (peak 1), 0.86 (peak 2), 1.48 (peak 4), 2.67 (peak 5), 2.72 (peak 6), 3.24 (peak 7), 3.32 (peak 8), 3.39 (peak 9), 3.55 (peak 10), 4.44 (peak 11), 4.57 (peak 12), 4.61 (peak 13).

[0184] 6.9 Result of characteristic spectrum of Radix Cynomorii:

[0185] The characteristic spectra of 20 batches of Guanzhong medicinal materials are as follows Figure 9-3 and Figure 9-4 As shown, Figure 9-3 It is the characteristic spectrum of the medicinal material of Rhizoma Cynomorii ((the batch numbers from bottom to top are: XLS202107277, XLS202107278, XLS202107279, XLS202107280, XLS202107281, XLS202107282, XLS202107283, XLS202107284, XLS202107285, XLS202107286); Figure 9-4 It is the characteristic spectrum of Radix Codonopsis pilosulae (the batch numbers from bottom to top are: XLS202107287, XLS202107288, XLS202107289, XLS202107290, XLS202107291, XLS202107292, XLS202107293, XLS202107294, XLS202107295, 010176-2012001);

[0186] Figure 9-5 This is the reference characteristic spectrum of the medicinal material of Rhizoma Cyperi; Peak 1 (S): protocatechuic acid; Note: Chromatographic column: T32.1×150mm,1.6μm.

[0187] Example 7 Selection of Chromatographic Conditions for Standard Decoction of Cyathula capitata

[0188] The chromatographic conditions and system suitability test of the characteristic spectrum of the standard decoction of Guanzhong were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length was 150 mm, inner diameter was 2.1 mm, and particle size was 1.6 μm); acetonitrile was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the column temperature was 35°C; the flow rate was 0.3 ml per minute, the detection wavelength was 250 nm, and the number of theoretical plates calculated based on the protocatechuic acid peak should be no less than 5000.

[0189]

[0190] Preparation of reference solution

[0191] Take 1g of Guanzhong reference medicinal material, place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate, add 25ml of 70% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of protocatechuic acid reference substance, accurately weigh it, and accurately add methanol to make a solution containing 20μg of protocatechuic acid per 1ml, which is used as the reference solution of the reference substance.

[0192] Preparation of test solution

[0193] Take 0.2 g of the standard decoction of Guanzhong Carbon, place it in a stoppered conical flask, add 50 ml of 70% methanol, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes. Take it out, cool it, and take the filtrate to obtain the product.

[0194] Determination method

[0195] Accurately pipette 1 μl of the test solution and reference solution, inject into the liquid chromatograph, and measure to obtain the result.

[0196] Selection of wavelength, column temperature, and flow rate: The material composition of the standard soup and the finished particles is basically consistent, and the characteristic chromatographic conditions are consistent with the system adaptability and the characteristic spectrum method of the particles.

[0197] 7.1 Investigation of extraction methods

[0198] After comparing reflux extraction and ultrasonic extraction, the chromatographic peak information was roughly the same, and ultrasonic extraction was finally selected due to its easy operation. Fig.10 . Fig.10 To investigate the extraction method of Guanzhong carbonized decoction.

[0199] 7.2 Investigation of extraction solvents

[0200] Methanol, water, 50% methanol and 70% methanol were investigated as extraction solvents. The results showed that the chromatographic peaks obtained with 70% methanol as the extraction solvent had a large amount of information and good separation, so 70% methanol was selected as the extraction solvent. Fig.11 . Fig.11 To investigate the solvent for extracting Guanzhong carbonized decoction.

[0201] 7.3 Extraction time investigation

[0202] The chromatograms were basically the same after comparing the ultrasonic extraction time of 15 minutes, 30 minutes, and 60 minutes, so the extraction time was determined to be 15 minutes. Fig.12 . Fig.12 Chromatograms at different extraction times.

[0203] 7.4 Investigation of the amount of extraction solvent

[0204] The addition amount of extraction solvent was compared with 25ml, 50ml and 100ml. When the amount of extraction solvent was 25ml, the size of the chromatographic peak was more suitable, so the amount of extraction solvent was determined to be 25ml. Fig.13 . Fig.13 Chromatograms of different extraction solvent amounts.

[0205] 7.5 Finalization of the test sample preparation method

[0206] Take about 0.2 g, place in a stoppered conical flask, add 25 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, shake well, filter, and take the filtrate to obtain the product.

[0207] Example 8 Methodological Study on Standard Decoction of Rhizoma Cynomorii

[0208] 8.1 Chromatographic peak identification

[0209] Preparation of test sample solution: Prepare the test sample solution of Guanzhong Carbon Standard Decoction according to the final test sample preparation method.

[0210] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference substance, weigh it accurately, and add methanol to make a reference solution containing 20 μg of protocatechuic acid per 1 ml.

[0211] Preparation of control medicinal material solution: Take 1 g of Guanzhong control medicinal material, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0212] Preparation of negative control solution: According to the experimental conditions proposed above, prepare a negative control solution lacking the standard decoction of Guanzhong. Locate the characteristic spectrum peaks of the standard decoction of Guanzhong. Fig.14 . Fig.14 Chromatographic peak identification.

[0213] 8.2 Precision test

[0214] Take the test solution of the standard decoction of Guanzhong Carbon (batch number: BT-01), inject 6 times continuously according to the proposed experimental method, 1 μl each time, and calculate the retention time and peak area of ​​each characteristic peak. See Tables 8 and 9.

[0215] Table 8 Precision investigation - retention time

[0216]

[0217]

[0218] Table 9 Precision Investigation-Peak Area

[0219]

[0220] The results showed that the RSD of the retention time of each characteristic peak was 0.02%-0.41%, and the RSD of the peak area was 0.49%-2.18%. The instrument has good precision.

[0221] 8.3 Repeatability Study

[0222] Accurately weigh 6 portions of the standard decoction of Guanzhong Carbon (batch number: BT-01) and prepare and measure according to the proposed experimental method. See Tables 10-1 and 10-2.

[0223] Table 10-1 Repeatability study - relative retention time ratio

[0224]

[0225] Table 10-2 Repeatability study - relative peak area ratio

[0226]

[0227] The results showed that the relative retention time RSD of each characteristic peak was 0.00%-0.37%, and the relative peak area RSD was 0.00%-15.65%. This method has good repeatability.

[0228] 8.4 Intermediate precision study

[0229] 8.4.1 Inspection by different personnel and at different times

[0230] Based on the experimental conditions proposed above, the standard decoction of Guanzhong (batch number: BT-01) was accurately weighed, and the test solution was prepared by different personnel and at different times for determination. See Tables 11-1 and 11-2.

[0231] Table 11-1 Intermediate precision-relative retention time ratio

[0232]

[0233] Table 11-2 Intermediate precision-relative peak area ratio

[0234]

[0235] The results show that the method has good precision when different people measure the same sample at different times.

[0236] 8.4.2 Investigation with different instruments

[0237] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated respectively. Fig.15 , Tables 12-1 and 12-2. Fig.15 Different instrument surveys.

[0238] Table 12-1 Different instruments investigated - relative retention time

[0239]

[0240] Table 12-2 Different instruments investigated - relative peak areas

[0241]

[0242]

[0243] The results showed that when the three instruments were used for detection, the RSD of the relative retention time of each characteristic peak was between 9.21% and 16.74%, and the RSD of the relative peak area of ​​each characteristic peak was between 0.00% and 68.79%, indicating that the durability of different instruments was poor.

[0244] 8.5 Durability test

[0245] 8.5.1 Column durability test

[0246] Based on the above proposed experimental conditions, the chromatographic column 1, chromatographic column 2, and chromatographic column 3 were investigated respectively. Fig.16 , Tables 13-1 and 13-2. Fig.16 Investigation of different chromatographic columns.

[0247] Table 13-1 Chromatographic column durability inspection - relative retention time

[0248]

[0249] Table 13-2 Chromatographic column durability inspection - relative peak area

[0250]

[0251] Depend on Fig.16 It can be seen that T3 2.1×150mm, 1.6μm (column 1) has better chromatogram separation, so it is recommended to use this column T3 2.1×150mm,1.6μm.

[0252] 8.5.2 Stability Study

[0253] Based on the above experimental conditions, take the same test solution and measure it at 0h, 3h, 6h, 9h, 12h, 18h and 24h respectively. See Table 14-1 and 14-2.

[0254] Table 14-1 Stability Study ~ Retention Time

[0255]

[0256] Table 14-2 Stability Study-Peak Area

[0257]

[0258] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.02% and 0.21%, and the RSD of the peak area was between 0.27% and 5.41%. The sample solution was relatively stable within 24 hours.

[0259] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 13 characteristic peaks were included in the subsequent investigation.

[0260] Example 9 Determination of characteristic peaks and establishment of reference spectrum

[0261] 9.1 Verification results of 20 batches of standard decoction of Guanzhong Carbon This method was used to perform characteristic spectrum analysis on 20 batches of standard decoction samples and calculate the relative retention time and relative peak area ratio. Figure 17-18 , Tables 15-1 and 15-2. Fig.17 Characteristic spectrum of standard decoction of Guanzhong Carbon (the batch numbers from bottom to top are: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10); Fig.18 This is the characteristic spectrum of the standard decoction of Guanzhong Carbon (the batch numbers from bottom to top are: BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20).

[0262] Table 15-1 Relative retention time of 20 batches of standard decoction of Guanzhong carbon

[0263]

[0264]

[0265] Table 15-2 Relative peak areas of 20 batches of standard decoction of Guanzhong carbon

[0266]

[0267] According to the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 13 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 3 was used as the S peak, the relative retention time RSD of the characteristic peaks of 20 batches of Guanzhongtan standard decoction was 0.40% to 0.86%, and the relative retention time RSD of the 13 characteristic peaks of 20 batches of Guanzhongtan standard decoction was less than 10%.

[0268] Final regulations: There should be 13 characteristic peaks in the chromatogram of the test sample. Except for peak 1 and peak 2, the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. The peak corresponding to the peak of the protocatechuic acid reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.51 (peak 4), 2.75 (peak 5), 2.81 (peak 6), 3.34 (peak 7), 3.43 (peak 8), 3.50 (peak 9), 3.67 (peak 10), 4.57 (peak 11), 4.72 (peak 12), 4.77 (peak 13). Fig.19 This is the reference characteristic spectrum of the standard decoction of Guanzhong, peak 1: 5-hydroxymethylfurfural; peak 3 (S): protocatechuic acid; Note: Chromatographic column: T3 2.1×150mm,1.6μ.

[0269] Example 10 Chromatographic conditions of the charcoal formula particles

[0270] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length was 150 mm, inner diameter was 2.1 mm, particle size was 1.6 μm); acetonitrile was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed as specified in the following table; column temperature was 35°C; flow rate was 0.3 ml / min, detection wavelength was 250 nm, and the theoretical plate number calculated based on the protocatechuic acid peak should be not less than 5000.

[0271]

[0272] 10.1 Preparation of reference solution

[0273] Take 1g of Guanzhong reference medicinal material, place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate, add 25ml of 70% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of protocatechuic acid reference substance, accurately weigh it, and accurately add methanol to make a solution containing 20μg of protocatechuic acid per 1ml, which is used as the reference solution of the reference substance.

[0274] 10.2 Preparation of test solution

[0275] Take the Guanzhong carbon formula particles, grind them into powder, take about 0.2g, put it in a stoppered conical flask, add 50ml of 70% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take it out, cool it, and take the filtrate to obtain it.

[0276] 10.3 Determination method

[0277] Accurately pipette 1 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure to obtain the result.

[0278] 10.4 Wavelength Selection

[0279] The wavelength is selected as 250nm.

[0280] 10.5 Investigation of mobile phase types

[0281] Based on the experimental conditions proposed above, the mobile phases of acetonitrile-water, acetonitrile-0.1% formic acid, and acetonitrile-0.1% phosphoric acid were investigated. Fig. 20 . Fig. 20 The results showed that when the mobile phase was acetonitrile-0.1% formic acid, the chromatographic peak separation was good, so the mobile phase was acetonitrile-0.1% formic acid.

[0282] 10.6 Column Temperature Investigation

[0283] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Fig.21 . Fig.21 Column temperature investigation chromatogram.

[0284] The results showed that when the column temperature was 35℃, the chromatographic peak separation was good, and the column temperature was temporarily set at 35℃.

[0285] 10.7 Flow rate investigation

[0286] Based on the experimental conditions proposed above, the flow rates of 0.25ml / min, 0.3ml / min, and 0.35ml / min were investigated. Fig. 22 . Fig. 22 Flow rate inspection.

[0287] The results show that when the flow rates are 0.25ml / min, 0.3ml / min and 0.35ml / min, the information content of each characteristic peak chromatogram is basically the same. When the flow rate is 0.3ml / min, the chromatographic peak separation is better, so the flow rate is determined to be 0.30ml / min.

[0288] 10.8 Delay Investigation

[0289] Based on the experimental conditions proposed above, the chromatogram acquisition time was set to 2 times. Fig.23 . Fig.23 Delayed investigation: The results showed that no chromatographic peak appeared in the chromatogram after 50 minutes.

[0290] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of Guanzhong Formula Granules are determined as follows: octadecylsilane bonded silica gel is used as the filler (column length is 150mm, inner diameter is 2.1mm, particle size is 1.6μm); acetonitrile is used as mobile phase A, 0.1% formic acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; column temperature is 35℃; flow rate is 0.3ml / min, and detection wavelength is 250nm. The theoretical plate number calculated based on the protocatechuic acid peak should not be less than 5000.

[0291]

[0292] Example 11 Preferred method for pre-treatment of smilax china charcoal particles

[0293] 11.1 Investigation of extraction methods

[0294] After comparing reflux extraction and ultrasonic extraction, the chromatographic peak information was roughly the same, and ultrasonic extraction was finally selected due to its easy operation. Fig.24 . Fig.24 Chromatograms of different extraction methods.

[0295] 11.2 Investigation of extraction solvents

[0296] Methanol, water, 50% methanol and 70% methanol were investigated as extraction solvents. The results showed that the chromatographic peaks obtained with 70% methanol as the extraction solvent had a large amount of information and good separation, so 70% methanol was selected as the extraction solvent. Fig.25 . Fig.25 Chromatograms of extractions with different solvents.

[0297] 11.3 Extraction time investigation

[0298] The chromatograms of ultrasonic extractions for 15 minutes, 30 minutes, and 60 minutes were basically the same. Therefore, the extraction time was determined to be 15 minutes. Fig.26 . Fig.26Chromatograms at different extraction times.

[0299] 11.4 Investigation of the amount of extraction solvent

[0300] The extraction solvent addition amounts of 25 ml, 50 ml, and 100 ml were compared. When the extraction solvent amount was 25 ml, the chromatographic peak size was more appropriate, so the extraction solvent amount was determined to be 25 ml. Fig. 27 . Fig. 27 Chromatograms of different extraction solvent amounts.

[0301] 11.5 Finalization of the test sample preparation method

[0302] Take an appropriate amount of this product, grind it into powder, take about 0.2g, put it in a stoppered conical flask, add 25ml of 70% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 15 minutes, shake well, filter, and take the filtrate to obtain the product.

[0303] Example 12 Methodological Investigation of the Formula Granules of Cyathula capitata

[0304] 12.1 Chromatographic peak identification

[0305] Preparation of test solution: Prepare the test solution of Guanzhong carbon formula particles according to the final test sample preparation method.

[0306] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference substance, weigh it accurately, and add methanol to make a reference solution containing 20 μg of protocatechuic acid per 1 ml. Take an appropriate amount of 5-hydroxymethylfurfural reference substance, weigh it accurately, and add methanol to make a reference solution containing 50 μg of 5-hydroxymethylfurfural per 1 ml.

[0307] Take 1 g of the control medicinal material Guanzhong, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0308] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of the Glechoma longituba formula granules.

[0309] Position the characteristic spectrum peaks of Guanzhong Formula Granules, see Fig.28 . Fig.28 Chromatographic peak identification.

[0310] 12.2 Precision test

[0311] Take the test solution of Guanzhong Formula Granules (Batch No.: KL-01) and inject 6 times in succession according to the proposed experimental method, 1 μl each time, and calculate the retention time and peak area of ​​each characteristic peak. See Tables 16-1 and 16-2.

[0312] Table 16-1 Precision Investigation-Retention Time

[0313]

[0314]

[0315] Table 16-2 Precision Investigation-Peak Area

[0316]

[0317] The results showed that the RSD of the retention time of each characteristic peak was 0.02%-0.11%, and the RSD of the peak area was 0.23%-2.49%. The instrument has good precision.

[0318] 12.3 Repeatability Study

[0319] Weigh 6 portions of the formula granules of Guanzhong Carbon (batch number: KL-01) accurately and prepare and measure them according to the proposed experimental method. See Tables 17-1 and 17-2.

[0320] Table 17-1 Repeatability Study - Relative Retention Time Ratio

[0321]

[0322] Table 17-2 Repeatability Study - Relative Peak Area Ratio

[0323]

[0324]

[0325] The results showed that the relative retention time RSD of each characteristic peak was 0.00%-0.28%, and the relative peak area RSD was 0.00%-9.49%. This method has good repeatability.

[0326] 12.4 Intermediate precision

[0327] 12.4.1 Different people at different times

[0328] Based on the experimental conditions proposed above, the Guanzhong granules were accurately weighed, and the test solutions were prepared by different personnel and at different times for determination. See Tables 18-1 and 18-2.

[0329] Table 18-1 Intermediate precision-relative retention time ratio

[0330]

[0331] Table 18-2 Intermediate precision-relative peak area ratio

[0332]

[0333] The results show that the method has good precision when different people measure the same sample at different times.

[0334] 12.4.2 Instrument durability inspection

[0335] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated respectively. Fig.29 , Tables 19-1 and 19-2. Fig.29 Different instrument surveys.

[0336] Table 19-1 Chromatographic column durability inspection - relative retention time

[0337]

[0338] Table 19-2 Chromatographic column durability inspection - relative peak area

[0339]

[0340]

[0341] The results showed that when the three instruments were used for detection, the RSD of the relative retention time of each characteristic peak was between 0.67% and 17.08%, and the RSD of the relative peak area of ​​each characteristic peak was between 0.00% and 43.49%, indicating that the durability of different instruments was poor.

[0342] 12.5 Durability Test

[0343] 12.5.1 Column Durability Assessment

[0344] Based on the experimental conditions proposed above, chromatographic column 1, chromatographic column 2, and chromatographic column 3 were investigated respectively. Fig.30 , Tables 20-1 and 20-2. Fig.30 Investigation of different chromatographic columns.

[0345] Table 20-1 Chromatographic column durability inspection - relative retention time

[0346]

[0347] Table 20-2 Chromatographic column durability inspection - relative peak area

[0348]

[0349] Depend on Fig.30 It can be seen that T3 2.1×150mm, 1.6μm (column 1) has better chromatogram separation, so it is recommended to use this column T3 2.1×150mm,1.6μm.

[0350] 12.6 Stability Study

[0351] Based on the above proposed experimental conditions, take the same test solution and measure it at 0h, 3h, 6h, 9h, 12h, 18h and 24h respectively. See Table 21-1 and 21-2.

[0352] Table 21-1 Stability Study ~ Retention Time

[0353]

[0354] Table 21-2 Stability Study-Peak Area

[0355]

[0356] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.02% and 0.24%, and the RSD of the peak area was between 0.40% and 7.32%. The sample solution was relatively stable within 24 hours.

[0357] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 13 characteristic peaks were included in the subsequent investigation.

[0358] Example 13 Determination of characteristic peaks and establishment of reference spectrum

[0359] 13.1 Batch of Guanzhong Formula Granules Verification Results

[0360] The proposed method was used to measure the characteristic spectra of three batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.31 , Tables 22-1 and 22-2. Fig.31 Verification diagram of characteristic spectra of 3 batches of Guanzhong formula granules.

[0361] Table 22-1 Relative retention time of three batches of cyperus rotundus charcoal granules

[0362]

[0363] Table 22-2 Relative peak area of ​​3 batches of cyperus rotundus charcoal formula particles

[0364]

[0365] According to the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 13 peaks with good repeatability were selected as characteristic peaks. The results show that when peak 3 is used as the S peak, the relative retention time RSD of the characteristic peaks of the three batches of Guanzhong carbon formula particles is between 0.00% and 0.37%, and the relative retention time RSD of the 13 characteristic peaks of the three batches of Guanzhong carbon formula particles is less than 10%.

[0366] Final regulations: There should be 13 characteristic peaks in the chromatogram of the test sample. Except for peak 1 and peak 2, the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. The peak corresponding to the peak of the protocatechuic acid reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.50 (peak 4), 2.72 (peak 5), 2.77 (peak 6), 3.30 (peak 7), 3.39 (peak 8), 3.46 (peak 9), 3.62 (peak 10), 4.53 (peak 11), 4.65 (peak 12), 4.70 (peak 13).

[0367] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize three batches of Guanzhong carbon formula particles, and a comparison spectrum of the characteristic spectrum of Guanzhong carbon formula particles was established. Fig.32 , Fig.32 This is the reference characteristic spectrum of Guanzhong carbon formula particles. Peak 1: 5-hydroxymethylfurfural; Peak 3 (S): protocatechuic acid; Chromatographic column: T32.1mm×150mm,1.6μm.

[0368] Comparative Example 1

[0369] The same conditions as in Example 1 were used to measure the radix cyrtonemae. The results were as follows: Figure 33 to Figure 35 Said, wherein Fig.33 This is the characteristic spectrum of the medicinal material Guanzhong. Fig.34 This is the characteristic spectrum of the medicinal material Guanzhong. Fig.35 This is the reference characteristic spectrum of the medicinal material of Rhizoma Cyperi; Peak 1 (S): protocatechuic acid; Note: Chromatographic column: T3 2.1×150mm, 1.6μm. It can be seen from the above that the method of the present invention can well identify the decoction pieces of Guanzhong medicinal material and Guanzhong charcoal medicinal material decoction pieces.

[0370] Comparative Example 2

[0371] The same conditions as in Example 1 were used to measure the standard decoction of Guanzhong, and the results were as follows Figure 36 to Figure 38 Said, wherein Fig.36This is the characteristic spectrum of the standard decoction of Guanzhong (the batch numbers from bottom to top are: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10). Fig.37 This is the characteristic spectrum of the standard decoction of Guanzhong (the batch numbers from bottom to top are: BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20). Fig.38 This is the reference characteristic spectrum of the standard decoction of Guanzhong; Peak 1 (S): protocatechuic acid; Note: Chromatographic column: T32.1×150mm, 1.6μm. It can be seen from the above that the method of the present invention can well identify the standard decoction of Rhizoma Cyperi and the standard decoction of Rhizoma Cyperi charcoal.

[0372] Comparative Example 3

[0373] The same conditions as in Example 1 were used to measure the Guanzhong formula granules. The results are as follows Figure 39-40 Said, wherein Fig.39 The characteristic spectrum of Guanzhong formula granules (from bottom to top: KL-001; KL-002; KL-003, Fig.40 This is the reference characteristic spectrum of Guanzhong Formula Granules; Peak 1 (S): protocatechuic acid; Note: Chromatographic column: T32.1×150mm, 1.6μm. It can be seen from the above that the method of the present invention can well identify the Guanzhong Guanzhong formula particles and Guanzhong charcoal Guanzhong formula particles.

[0374] There are only 11 peaks for Guanzhong and its preparations, which have fewer peaks 1 and 2 than Guanzhong charcoal, and can clearly identify Guanzhong and Guanzhong charcoal.

[0375] Comparative Example 4

[0376] This comparative example adopts different gradient conditions, and the rest of the results are the same as those of Example 1:

[0377] Mobile phase gradient:

[0378] Gradient 1:

[0379]

[0380]

[0381] The results are as follows Fig.41 As shown, it can be seen from the figure that peak 1.2 is missing; peak 5 is separated, and peaks 12 and 13 have poor separation.

[0382] Gradient 2:

[0383]

[0384] The results are as follows Fig.41 As shown, it can be seen from the figure that peaks 2 and 3 are not separated, and peaks 12 and 13 have poor separation.

[0385] Gradient 3:

[0386]

[0387] The results are as follows Fig.41 As shown, it can be seen from the figure that peaks 11, 12, and 13 are missing.

[0388] It can be seen from the above that only the gradient conditions of the present invention can achieve a good peak separation effect.

[0389] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing characteristic spectra of Rhizoma Cyperi and Rhizoma Cyperi charcoal slices, standard decoctions, and formula granules, comprising: A) Boil the charcoal raw material with water, filter it, add solvent to the residue to dissolve it, perform ultrasonic extraction, shake well, and filter it; The solvent is 70% methanol; The medicinal source of the Cyperus rotundus or Cyperus rotundus charcoal is Cibotium barometz; B) The test solution is measured by high performance liquid chromatography to obtain a UPLC characteristic spectrum of the charcoal raw material; The HPLC conditions are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is 0.1% formic acid, and the elution is gradient; The gradient elution is specifically: 0-8min, phase A: 1%, phase B: 99%; 8-18min, phase A: 1%-15%, phase B: 99%-85%; 18-35min, phase A: 15%-25%, phase B: 85%-75%; 35-50min, phase A: 25%-26%, phase B: 75%-74%; The similarity of the UPLC characteristic spectra of Guanzhongtan slices, standard decoction and formula granules was evaluated by the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, and the UPLC standard characteristic spectra of Guanzhongtan slices, standard decoction and formula granules were obtained, which consisted of 13 characteristic peaks, among which peak 1 was hydroxymethylfurfural and peak 3 was protocatechuic acid.

2. The method according to claim 1, characterized in that The method also includes preparing a reference solution: taking protocatechuic acid and 5-hydroxymethylfurfural respectively, adding water to boil, filtering, and dissolving the filter residue with 70% methanol to obtain a reference solution; the concentration of the reference solution is 20 μg / mL; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the UPLC characteristic spectra of the charcoal slices, standard decoction, and formula granules are qualitatively measured based on the chromatogram of the reference.

3. The method according to claim 1, characterized in that: The chromatographic column is a C18 column with specifications of 1.6 μm and 2.1×150 mm; the column temperature is 35° C.

4. The method according to claim 3, characterized in that The flow rate of the mobile phase was 0.3 mL / min; the detection wavelength was 250 nm; and the injection volume was 1 μL.

5. The method according to claim 1, characterized in that In the characteristic spectrum of the standard decoction of Guanzhong Carbon, protocatechuic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.51 (peak 4), 2.75 (peak 5), 2.81 (peak 6), 3.34 (peak 7), 3.43 (peak 8), 3.50 (peak 9), 3.67 (peak 10), 4.57 (peak 11), 4.72 (peak 12), 4.77 (peak 13); In the characteristic spectrum of Guanzhongtan decoction pieces, protocatechuic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.86 (peak 2), 1.48 (peak 4), 2.67 (peak 5), 2.72 (peak 6), 3.24 (peak 7), 3.32 (peak 8), 3.39 (peak 9), 3.55 (peak 10), 4.44 (peak 11), 4.57 (peak 12), 4.61 (peak 13); In the characteristic spectrum of the Guanzhong carbon formula particles, protocatechuic acid is used as the reference peak S peak to calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.50 (peak 4), 2.72 (peak 5), 2.77 (peak 6), 3.30 (peak 7), 3.39 (peak 8), 3.46 (peak 9), 3.62 (peak 10), 4.53 (peak 11), 4.65 (peak 12), and 4.70 (peak 13).

6. The method according to claim 1, characterized in that Step A): the ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 15-45min.

7. The method according to claim 1, characterized in that In step A), the ratio of the mass g of the carbon raw material to the volume mL of the solvent is (0.1-1): (10-50).

8. The method according to claim 1, characterized in that Step A): the charcoal raw materials are charcoal slices, standard decoctions, and formula granules.

9. A method for identifying the characteristic spectrum of Rhizoma Cynomorii and Rhizoma Cynomorii charcoal slices, standard decoctions, and formula granules, characterized in that: By using the method described in any one of claims 1 to 8 for identification, the Guanzhong and its preparations have only 11 peaks, which are less than the peaks 1 and 2 of the Guanzhong charcoal, and the Guanzhong and Guanzhong charcoal can be clearly identified.

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