Method for constructing HPLC (High Performance Liquid Chromatography) characteristic chromatograms of Chinese mahonia leaf medicinal material, decoction pieces, standard decoction and formula granules thereof

Abstract

The invention provides a Chinese mahonia leaf medicinal material, decoction pieces, standard decoction and formula granule HPLC characteristic chromatogram construction method, which comprises: A) extracting Chinese mahonia leaves by using a solvent to obtain a liquid to be detected; b) determining the liquid to be detected by adopting a high performance liquid chromatography to obtain an HPLC characteristic chromatogram of the Chinese mahonia leaf raw material; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 column; a mobile phase A is acetonitrile, a mobile phase B is 0.2% phosphoric acid and 0.2% triethylamine, and gradient elution is carried out. According to the method, high performance liquid chromatography is adopted, acetonitrile-0. 2% phosphoric acid-0. 2% triethylamine is selected as a mobile phase for gradient elution, berberine hydrochloride is used as a reference substance, palmatine hydrochloride is used as a reference substance, the HPLC characteristic chromatograms of the Chinese mahonia leaf medicinal materials, decoction pieces, standard decoction and formula granules thereof are established, repeatability and precision are good, the method is stable and reliable, and the method is suitable for industrial production. The quality of Chinese mahonia leaf medicinal materials, decoction pieces, standard decoction and formula granules thereof can be controlled.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for constructing an HPLC characteristic spectrum of a Mahonia lauraceae medicinal material, a decoction piece, a standard decoction and formula particles thereof. Background Art

[0002] Mahonia bealei is the dried leaf of the Berberidaceae plant Mahonia bealei (Fort.) Carr. It has the effects of clearing away heat, drying dampness and detoxifying. It is commonly used in clinical practice for pulmonary tuberculosis, hemoptysis, bone steaming, hot flashes, dizziness, tinnitus, soreness of waist and knees, damp-heat jaundice, dysentery, red and swollen eyes, carbuncle, ulcer, leucorrhea, and wind-heat cold. Mahonia bealei (Mahonia bealei) of the Berberidaceae family was first recorded in "Ben Cao Zai Xin" and has been used to this day. It is a commonly used medicine for the clinical treatment of pulmonary tuberculosis and joint pain in traditional Chinese medicine.

[0003] The existing quality control methods for the slices and preparations of Mahonia suffruticosa mainly focus on the determination of moisture, ash, thin layer identification, total flavonoids and alkaloid components. The fingerprint and characteristic spectrum technology of traditional Chinese medicine can characterize the characteristics of the main chemical components of the tested samples in an overall and macroscopic manner, and is currently recognized as one of the most suitable methods for the quality control of the intrinsic substance groups of traditional Chinese medicine and natural medicine.

[0004] However, the characteristic spectrum of the Mahoniae oleracea disclosed in the prior art is only for the characteristic spectrum of the medicinal material, while there is no detection method for the slices, standard decoctions and granules. Chinese medicine has complex ingredients and is mostly used as decoctions. Therefore, the research and development of the HPLC characteristic spectrum for the simultaneous determination of the Mahoniae oleracea medicinal material, slices, standard decoctions and their formula granules is very necessary. 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 HPLC characteristic spectra of Mahonia laurel medicinal materials, decoction pieces, standard decoctions and their formula granules. The HPLC characteristic spectra method of Mahonia laurel medicinal materials, decoction pieces, standard decoctions and their formula granules constructed by the present invention is stable and reliable, and can control the quality of Mahonia laurel medicinal materials, decoction pieces, standard decoctions and their formula granules.

[0006] The present invention provides a method for constructing HPLC characteristic spectrum of Mahonia suffruticosa medicinal materials, decoction pieces, standard decoctions and their formula particles, comprising:

[0007] A) extracting Mahonia truncatula leaves with a solvent to obtain a test solution;

[0008] B) the test solution is measured by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the Mahonia laurel leaf 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 0.2% phosphoric acid-0.2% triethylamine, and the gradient elution is performed;

[0010] The gradient elution is specifically:

[0011] 0-3min, phase A: 12%→14%, phase B: 88%→86%;

[0012] 3-19 min, phase A: 14%→18%, phase B: 86%→82%;

[0013] 19-28 min, phase A: 18% → 29%, phase B: 82% → 71%;

[0014] 28-48 min, phase A: 29%→30%, phase B: 71%→70%;

[0015] 48-55min, phase A: 30%→35%, phase B: 70%→65%.

[0016] The invention provides a method for constructing HPLC characteristic spectrum of a Mahonia laurel medicinal material, decoction pieces, standard decoction and its formula particles. First, the Mahonia laurel raw material is taken and solvent extraction is performed to obtain a test solution. The solvent is preferably hydrochloric acid-30% methanol (1:100).

[0017] According to the present invention, the raw materials of the Mahoniae oleifera are the medicinal materials, decoction pieces, standard decoctions and formula granules thereof.

[0018] According to the present invention, the preparation of the test solution of the Mahonia leaf medicinal material / decoction piece is specifically as follows: dissolving the Mahonia leaf with hydrochloric acid-30% methanol, heating and refluxing for extraction, cooling, shaking, and filtering to obtain the solution;

[0019] The heating reflux time is 30 minutes; wherein the volume ratio of hydrochloric acid to 30% methanol is 1:100.

[0020] The extraction solvent of the present invention is hydrochloric acid-30% methanol (1:100), that is, 1 ml of hydrochloric acid is added to 100 ml of 30% methanol.

[0021] The ratio of the mass g of the Mahonia laurel raw material to the volume mL of the solvent is 0.5:20-30, preferably 0.5:25.

[0022] The preparation of the standard decoction of Mahonia suffruticosa leaves is specifically as follows: dissolving the Mahonia suffruticosa leaves in hydrochloric acid-30% methanol (1:100), ultrasonic extraction, cooling, shaking, filtering, and obtaining the decoction; the ultrasonic power is 600W and the frequency is 40kHz; the volume ratio of hydrochloric acid-30% methanol is 1:100; the ratio of the mass g of the Mahonia suffruticosa leaves raw material to the volume mL of the solvent is 0.2:20-30; preferably 0.2:25.

[0023] The preparation of the mahonia leaf formula granules is specifically as follows: dissolving the mahonia leaf in hydrochloric acid-30% methanol, ultrasonic extraction, cooling, shaking, filtering, and obtaining the mahonia leaf; the ultrasonic power is 600W, the frequency is 40kHz; the volume ratio of hydrochloric acid-30% methanol is 1:100. The ratio of the mass g of the mahonia leaf raw material to the volume mL of the solvent is 0.2:20-30, preferably 0.2:25.

[0024] The invention adopts the hydrochloric acid-30% methanol extraction solvent, which has large chromatographic peak information, good peak shape of each characteristic peak, moderate separation, good extraction effect, and good extraction time of 30 minutes.

[0025] The Mahoniae leaf raw material of the present invention is Mahoniae leaf medicinal material, Mahoniae leaf decoction pieces, Mahoniae leaf standard decoction or Mahoniae leaf formula granules. The present invention does not limit it, and the above raw materials can all be subjected to quality control and qualitative detection by the method of the present invention.

[0026] The present invention also includes preparing reference substances and reference medicinal material reference solutions:

[0027] Take berberine hydrochloride reference substance and palmatine hydrochloride reference substance respectively, dissolve them in methanol to obtain reference substance solutions.

[0028] In some specific embodiments, the concentration of the berberine hydrochloride reference substance is 60 μg / mL; the concentration of the palmatine hydrochloride reference substance is 20 μg / mL;

[0029] Take the reference medicinal material of Mahonia oleifera and dissolve it in hydrochloric acid-30% methanol (1:100), heat and reflux to extract, cool it, filter it, and obtain the reference medicinal material solution;

[0030] The reference substance and reference medicinal material reference substance solutions are determined by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference substance, respectively; and based on the chromatograms of the reference substance and the reference medicinal material reference substance, the components of the HPLC characteristic spectra of the Mahonia leaf medicinal material, decoction pieces, standard decoction and its formula granules are qualitatively determined.

[0031] The mobile phase A of the present invention is acetonitrile, the mobile phase B is 0.2% phosphoric acid-0.2% triethylamine, and the gradient elution is performed; the gradient elution of the present invention is preferably as follows:

[0032] 0 The gradient elution is specifically:

[0033] 0-3min, phase A: 12%→14%, phase B: 88%→86%;

[0034] 3-19 min, phase A: 14%→18%, phase B: 86%→82%;

[0035] 19-28 min, phase A: 18% → 29%, phase B: 82% → 71%;

[0036] 28-48 min, phase A: 29%→30%, phase B: 71%→70%;

[0037] 48-55min, phase A: 30%→35%, phase B: 70%→65%.

[0038] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0039] The chromatographic column is a C18 column with specifications of 5.0 μm and 4.6×250 mm; the column temperature is 20° C.

[0040] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above 20°C condition.

[0041] The mobile phase flow rate is preferably 0.8 ml / min.

[0042] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 0.8 ml / min, which was the most preferred solution.

[0043] The detection wavelength of the present invention is preferably 346 nm.

[0044] The inventors found that at 346 nm, the chromatographic information was rich, each component had good absorption, the response value was moderate, the peak separation was good, and the baseline was stable.

[0045] The injection volume of the present invention is preferably 5 μL.

[0046] The beneficial effect of the present invention is that under one liquid chromatography condition, the substance groups of Mahonia laurel L. medicinal materials, decoction pieces, standard decoctions and their formula granules are controlled by fingerprints, and berberine hydrochloride and bamipine hydrochloride fingerprints are used for positioning; the cost of detection can be greatly reduced and qualitative detection can be achieved.

[0047] The similarity of HPLC characteristic spectra of Mahonia laurel L. medicinal materials, decoction pieces, standard decoctions and their formula granules was evaluated by using the Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic spectra of Mahonia laurel L. medicinal materials, decoction pieces, standard decoctions and their formula granules consisting of 6 characteristic peaks were obtained, among which peak 5 was bamipine hydrochloride and peak 6 was berberine hydrochloride.

[0048] In the characteristic spectrum of the standard decoction of Mahonia latifolia, berberine hydrochloride 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: 0.15 (peak 1), 0.18 (peak 2) 0.44 (peak 3), and 0.66 (peak 4).

[0049] Quality judgment standard: Take samples of Mahoniae laurel leaves, decoction pieces, standard decoctions and their formula granules, and operate according to the same method as above to obtain the characteristic spectra of Mahoniae laurel leaves, decoction pieces, standard decoctions and their 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 and sample characteristic spectra of Mahoniae laurel leaves, decoction pieces, standard decoctions and their formula granules, and the similarity is greater than 0.90.

[0050] The method provided by the present invention can effectively monitor the quality of different batches of Mahoniae oleifera medicinal materials, decoction pieces, standard decoctions and formula granules thereof, so as to stabilize the quality. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensively monitoring the quality of products.

[0051] The characteristic spectrum of Mahonia lauraceae medicinal materials, decoction pieces, standard decoctions and formula granules established by the present invention takes psoralen as a reference, focuses on the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the products, and the method is scientific and reliable.

[0052] The newly developed characteristic spectrum method of the present invention can detect components with greater polarity in Mahoniae 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 Mahoniae and its preparations. The authenticity, quality consistency and stability of Mahoniae and its preparations can be effectively detected and controlled. It provides a basis for effectively controlling and more comprehensively evaluating the quality of Mahoniae standard decoction. It ensures the uniformity and stability of the quality of Mahoniae and its standard decoction.

[0053] The invention provides a method for constructing an HPLC characteristic spectrum of a Mahonia leaf medicinal material, a decoction piece, a standard decoction and a formula granule thereof, comprising: A) extracting the Mahonia leaf with a solvent to obtain a test liquid; B) determining the test liquid with a high performance liquid chromatography method to obtain an HPLC characteristic spectrum of the Mahonia leaf raw material; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; the mobile phase A is acetonitrile, and the mobile phase B is 0.2% phosphoric acid-0.2% triethylamine, and gradient elution. The invention adopts high performance liquid chromatography, selects acetonitrile-0.2% phosphoric acid-0.2% triethylamine as the mobile phase for gradient elution, takes berberine hydrochloride reference substance and bamipine hydrochloride as reference substances, and establishes an HPLC characteristic spectrum of the Mahonia leaf medicinal material, a decoction piece, a standard decoction and a formula granule thereof, with good repeatability and precision, and the method is stable and reliable, and the quality of the Mahonia leaf medicinal material, a decoction piece, a standard decoction and a formula granule thereof can be controlled. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Investigation of solvents for extracting Mahonia suffruticosa L.

[0055] Figure 2 Investigation of the amount of hydrochloric acid added;

[0056] Figure 3 Extraction time investigation;

[0057] Figure 4 Sampling volume investigation;

[0058] Figure 5 Extraction method inspection and investigation;

[0059] Figure 6 Chromatographic peak identification;

[0060] Figure 7 Surveys with different instruments;

[0061] Figure 8 Investigation of different chromatographic columns;

[0062] Fig. 9 Characteristics of Mahonia suffruticosa L. (broad-leaved Mahonia suffruticosa L.) medicinal material;

[0063] Fig.10 Characteristics of Mahonia suffruticosa (broad-leaved Mahonia suffruticosa) medicinal pieces;

[0064] Figure 11-1 Comparative characteristic map of medicinal materials;

[0065] Figure 11-2 Characteristic spectrum of medicinal pieces;

[0066] Fig.12 Investigation of solvents for extracting Mahonia suffruticosa decoction;

[0067] Fig.13 Investigation of extraction methods;

[0068] Fig.14 Extraction time investigation;

[0069] Fig.15 Investigation of solvent addition amount;

[0070] Fig.16 Investigation of solvent addition amount;

[0071] Fig.17 This is the chromatographic peak identification diagram;

[0072] Fig.18 Surveys with different instruments;

[0073] Fig.19 Investigation of different chromatographic columns;

[0074] Fig. 20 Characteristic spectrum of the standard decoction of Bauhinia bark;

[0075] Fig.21 Control feature map;

[0076] Fig. 22 3D chromatogram of Mahonia leaf formula granules;

[0077] Fig.23 Chromatograms of different wavelengths of Mahonia leaf formula granules;

[0078] Fig.24 Column temperature investigation;

[0079] Fig.25 Flow rate investigation;

[0080] Fig.26 Delayed inspection;

[0081] Fig. 27 Investigation of extraction solvents;

[0082] Fig.28 Investigation of the amount of hydrochloric acid added;

[0083] Fig.29 Extraction time investigation;

[0084] Fig.30 Sampling volume investigation;

[0085] Fig.31 Extraction method inspection and investigation;

[0086] Fig.32 Chromatographic peak identification;

[0087] Fig.33 Spectrum of reference substance berberine hydrochloride;

[0088] Fig.34 Sample berberine hydrochloride spectrum;

[0089] Fig.35 Spectrum of reference substance Bamipine hydrochloride;

[0090] Fig.36 Sample berberine hydrochloride spectrum;

[0091] Fig.37 Surveys with different instruments;

[0092] Fig.38 Investigation of different chromatographic columns;

[0093] Fig.39 Characteristics of Mahonia leaf formula granules;

[0094] Fig.40 Control feature map;

[0095] Fig.41 Characteristic spectrum of Comparative Example 1. DETAILED DESCRIPTION

[0096] The present invention provides a method for constructing HPLC characteristic spectra of Mahonia suffruticosa medicinal materials, decoction pieces, standard decoctions and their formula particles. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0097] To further illustrate the present invention, a method for constructing an HPLC characteristic spectrum of a Mahonia lauraceae medicinal material, decoction pieces, standard decoction and its formula granules provided by the present invention is described in detail below in conjunction with examples.

[0098] Example 1 HPLC characteristic spectrum of medicinal materials and decoction pieces

[0099] 1 Experimental instruments and materials

[0100] High performance liquid chromatography: waters, Agilent;

[0101] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0102] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0103] Ultrasonic cleaner: KQ5200DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0104] Chromatographic columns: Agilent 5TC-C18 (4.6 mm × 250 mm, 5 μm), ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm), C18(2)100A (4.6mm×250mm, 5μm).

[0105] 2. Reagents and test drugs

[0106] Methanol, phosphoric acid, and triethylamine were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade.

[0107] Bamipine hydrochloride (China Food and Drug Inspection Institute, batch number: 110732-201913, purity: 85.7%)

[0108] Berberine hydrochloride (China Food and Drug Inspection Institute, batch number: 110713-202316, purity: 85.9%)

[0109] Mahonia leaf (Mahonia macrophylla leaf) control medicinal material (Mahonia macrophylla leaf (Mahonia macrophylla leaf)) (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTYS018801)

[0110] Mahonia leaf medicinal material (from Sichuan New Green Pharmaceutical Co., Ltd. batch number: YC-1, YC-2, YC-3, YC-4, YC-5, YC-6, YC-7, YC-8, YC-9, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17.)

[0111] Mahonia leaf slices (from Sichuan New Green Pharmaceutical Co., Ltd. batch number: YP-1, YP-2, YP-3, YP-4, YP-5, YP-6, YP-7, YP-8, YP-9, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19.)

[0112] 3. Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid-0.2% triethylamine was used as mobile phase B, gradient elution was performed according to the provisions in the following table, the flow rate was 0.8 ml per minute; the column temperature was 20°C; the detection wavelength was 346 nm. The theoretical plate number calculated based on the berberine hydrochloride peak should not be less than 3000.

[0113]

[0114] Preparation of reference solution Take 0.5 g of Mahonia laurel leaves (broad-leaved Mahonia laurel leaves) as a reference medicinal material, place it in a stoppered conical flask, add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh the weight, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake well, filter, and take the filtrate as a reference solution for the reference medicinal material.

[0115] Take appropriate amount of berberine hydrochloride reference substance and palmatine hydrochloride reference substance, weigh accurately, add methanol to prepare solutions containing 60 μg of berberine hydrochloride and 20 μg of palmatine hydrochloride per 1 ml, respectively, to obtain the reference substance solutions.

[0116] Preparation of test solution: Take about 0.5 g of medicinal material powder (passed through No. 3 sieve), add 25 ml of hydrochloric acid-30% methanol (1:100) mixed solution, weigh the weight, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with hydrochloric acid-30% methanol (1:100) mixed solution, shake well, filter, and take the filtrate to obtain the test solution.

[0117] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0118] 4 Preparation of test solution

[0119] 4.1 Investigation of extraction solvent

[0120] Take 7 portions of this medicinal material (batch number: YC-1), each about 0.5 g, put them in a stoppered conical flask, and examine the extraction solvents of methanol: hydrochloric acid (100:1), 30% methanol: hydrochloric acid (100:1), 50% methanol: hydrochloric acid (100:1), 70% methanol: hydrochloric acid (100:1), ethanol: hydrochloric acid (100:1), water: hydrochloric acid (100:1), and 30% methanol, 25 ml each. Seal the flask, heat and reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product. Figure 1 Investigation of solvents for extracting Mahonia suffruticosa L.

[0121] The results showed that when the extraction solvent was 30% methanol, the extraction rate of berberine hydrochloride and palmatine hydrochloride was low. When it was 30% methanol: hydrochloric acid (100:1), the peak shapes of the characteristic peaks were good and the separation was moderate. The extraction solvent was temporarily set to 30% methanol: hydrochloric acid (100:1).

[0122] 4.2 Investigation of the proportion of hydrochloric acid added

[0123] Take the powder of this medicinal material (batch number: YC-1) (pass through No. 3 sieve), take three portions, each about 0.5g, put them in a stoppered conical bottle, add 25ml of hydrochloric acid-30% methanol (0.5:100), hydrochloric acid-30% methanol (1:100), and hydrochloric acid-30% methanol (3:100) respectively for inspection, seal, heat and reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Figure 2 . Figure 2 Investigation of the amount of hydrochloric acid added.

[0124] The results showed that the extraction effect was better when hydrochloric acid-30% methanol (1:100) was added to the test sample, so the amount of hydrochloric acid added to the test sample was 1.0.

[0125] 4.3 Extraction time investigation

[0126] Take the powder of medicinal material (batch number: YC-1) (pass through No. 3 sieve), take three portions, about 0.5g each, put them into a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100), stopper, heat and reflux, and observe the extraction time of the test sample at 20 minutes, 30 minutes, and 40 minutes respectively, cool, shake well, filter, and take the filtrate to obtain. Figure 3 . Figure 3 Extraction time investigation.

[0127] The results showed that the effect difference was better when the extraction time of the test sample was 30 minutes, so the extraction time of the test sample was determined to be 30 minutes.

[0128] 4.4 Sampling volume investigation

[0129] Take the powder of this medicinal material (batch number: YC-1) (pass through No. 3 sieve), take three portions, about 0.2g, 0.5g and 0.8g each, put them in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100) for inspection, seal it, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Figure 4 . Figure 4 Sampling volume investigation; the results show that when the sampling volume is 0.5g, the peak shape and separation of each chromatographic peak are better, so the sampling volume is selected as 0.5g.

[0130] 4.5 Investigation of extraction methods

[0131] Take the powder of this medicinal material (batch number: YC-1) (pass through No. 3 sieve), take two portions, each about 0.5g, put them in a stoppered conical bottle, add 25ml of hydrochloric acid-30% methanol (1:100) for inspection, seal it, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, reflux extraction for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Figure 5 . Figure 5The extraction method is investigated.

[0132] The results showed that reflux extraction had better effect, so reflux extraction was selected.

[0133] In summary, the preparation method of the test solution of the characteristic spectrum of the medicinal material Mahonia odorifera (broad-leaved Mahonia odorifera) is determined as follows: take about 0.5 g of the medicinal material powder (passed through a No. 3 sieve), add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh the weight, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake well, filter, and take the filtrate to obtain the product.

[0134] 5 Methodological investigation

[0135] 5.1 Chromatographic peak identification

[0136] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Mahonia laurel (broad-leaved Mahonia laurel leaf).

[0137] Preparation of reference solution: Take 0.5 g of Mahonia laurel leaves (broad-leaved Mahonia leaves) as a reference medicinal material, place it in a stoppered conical flask, add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh the weight, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake well, filter, and take the filtrate as a reference solution for the reference medicinal material.

[0138] Take appropriate amount of berberine hydrochloride reference substance and palmatine hydrochloride reference substance, weigh accurately, add methanol to prepare solutions containing 60 μg of berberine hydrochloride and 20 μg of palmatine hydrochloride per 1 ml, respectively, to obtain the reference substance solutions.

[0139] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution of the medicinal material Mahonia latifolia (broad-leaved Mahonia latifolia) was prepared.

[0140] Locate the characteristic spectrum peaks of Mahonia suffruticosa (broad-leaved Mahonia suffruticosa). Figure 6 . Figure 6 Chromatographic peak identification. The results showed that there were 6 characteristic peaks in the test solution, of which peak 5 was bamipine hydrochloride and peak 6 was berberine hydrochloride. In summary, in the following methodological investigation, the 6 characteristic peaks in the test sample were investigated.

[0141] 5.2 Precision investigation

[0142] Take the test solution of Mahonia oleracea (broad-leaved Mahonia oleracea) medicinal material (batch number: YC-1), inject 6 times continuously according to the proposed experimental method, 5ul each time, and calculate the relative retention time of each characteristic peak. See Table 1.

[0143] Table 1 Precision investigation - relative retention time

[0144]

[0145] The results showed that the RSD range of the relative retention time of each characteristic peak of the sample was 0.3% to 4.3%, and the instrument had good precision.

[0146] 5.3 Repeatability study

[0147] Six samples of the Mahonia oleracea (broad-leaved Mahonia oleracea) medicinal material (batch number: YC-1) were prepared and tested according to the proposed experimental method. See Table 3.

[0148] Table 2 Repeatability study - relative retention time

[0149]

[0150] The results showed that the RSD range of the relative retention time of the six samples was 0.1% to 3.1%, indicating that the method had good repeatability.

[0151] 5.4 Investigation with different instruments

[0152] Based on the above experimental conditions, two portions of Mahonia oleracea (broad-leaved Mahonia oleracea) medicinal materials (batch number: YC-1) were weighed to prepare the test solution, which was measured on Agilent and Waters liquid chromatographs, respectively. See Table 3, Figure 7 . Figure 7 Different instrument surveys.

[0153] Table 3 Instrument durability investigation - relative retention time

[0154]

[0155] The results showed that when the two instruments were used to detect the test samples, the RSD range of the relative retention time of each characteristic peak was 0.5% to 17.4%, indicating that the instruments had good durability.

[0156] 5.5 Inspection by different personnel and time

[0157] Based on the experimental conditions proposed above, two portions of Mahonia oleracea (broad-leaved Mahonia oleracea) medicinal materials (batch number: YC-1) were weighed by different personnel (A, B) at different times (T1, T2) to prepare test samples for determination. See Table 4.

[0158] Table 4 Personnel and time inspection-relative retention time

[0159]

[0160] The results showed that when different personnel measured the same sample at different times, the RSD range of the relative retention time of each characteristic peak was 2.4%, and the method had good stability.

[0161] 5.6 Investigation of different chromatographic columns

[0162] Based on the above experimental conditions, the reverse phase C18 column 1 Agilent 5TC-C18 (4.6 mm × 250 mm, 5 μm), reverse phase C18 column 2 ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm), reverse phase C18 column 3 C18(2)100A (4.6mm×250mm, 5μm) was used for analysis and investigation, and the relative retention time of each characteristic peak was calculated. The results are shown in Table 5. Figure 8 . Figure 8 Investigation of different chromatographic columns.

[0163] Table 5 Column durability investigation - relative retention time

[0164]

[0165] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 1.4% and 17.7% when the three chromatographic columns were used to detect the samples, indicating that the chromatographic columns had good durability.

[0166] 5.7 Stability Study

[0167] Based on the above experimental conditions, take the same test solution and measure it at 0h, 2h, 6h, 10h, 12h and 24h respectively. See Table 6.

[0168] Table 6 Stability Study - Retention Time

[0169]

[0170] The results showed that the RSD of the characteristic peak retention time was between 0.2% and 3.8%, and the sample solution was stable within 24 hours.

[0171] 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 6 characteristic peaks were included in the subsequent investigation.

[0172] 6. Verification of Mahonia Lour. medicinal materials and decoction pieces and establishment of HPLC content determination method

[0173] 6.1 Verification results of 15 batches of Euonymus japonicus leaves and 17 batches of decoction pieces.

[0174] Fig. 9Characteristic spectrum of Mahonia suffruticosa (broad-leaved Mahonia suffruticosa) medicinal material; among them, peak 5: palmatine hydrochloride; peak 6 (S): berberine hydrochloride. (S1-S17 are: YC-1, YC-2, YC-3, YC-4, YC-5, YC-6, YC-7, YC-8, YC-9, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17.)

[0175] Table 7 Relative retention time of characteristic spectra of Mahonia suffruticosa (broad-leaved Mahonia suffruticosa) medicinal materials

[0176]

[0177] 19 batches of Mahonia leaves (broad-leaved Mahonia leaves) were tested, and the results are shown in Fig.10 , Table 7. Fig.10 Characteristic spectrum of Mahonia leaf (broad-leaved Mahonia leaf) slices; Peak 5: Bamipine hydrochloride; Peak 6 (S): Berberine hydrochloride (S1-S19 are: YP-1, YP-2, YP-3, YP-4, YP-5, YP-6, YP-7, YP-8, YP-9, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19)

[0178] Table 8 Relative retention time of characteristic spectra of decoction pieces

[0179]

[0180]

[0181] 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 6 peaks with good repeatability were selected as characteristic peaks. The relative retention time RSD of the 6 characteristic peaks of 17 batches of Mahonia leaves (broad-leaved Mahonia leaves) were all less than 2.0%.

[0182] Final Regulation:

[0183] The test sample chromatogram should show 6 characteristic peaks, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 5 and peak 6 should correspond to the retention time of the reference peaks of berberine hydrochloride and berberine hydrochloride, respectively. The peak corresponding to the berberine hydrochloride reference is the S peak. The relative retention time of peak 1, peak 2, peak 3, peak 4 and S peak is calculated. The relative retention time of peak 3 and peak 4 should be within the range of ±10% of the specified value, and the specified value is: 0.44 (peak 3) and 0.66 (peak 4). The relative retention time of peak 1 and peak 2 should be within the specified value range, and the specified value is 0.11-0.20 (peak 1) and 0.14-0.24 (peak 2).

[0184] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 17 batches of Mahonia leaves (broad-leaved Mahonia leaves) medicinal materials, and a reference characteristic spectrum of the characteristic spectrum of Mahonia leaves (broad-leaved Mahonia leaves) medicinal materials was established. Figure 11-1 It is the reference characteristic spectrum of medicinal materials; among them, Peak 5: Bamipine hydrochloride; Peak 6 (S): berberine hydrochloride; Figure 11-2 Characteristic spectrum of the medicinal piece, where Peak 5: Bamipine hydrochloride; Peak 6 (S): berberine hydrochloride.

[0185] Conclusion: The characteristic spectrum method of Mahonia laurel leaves medicinal materials and decoction pieces can effectively detect Mahonia laurel leaves medicinal materials and decoction pieces, which proves that the method is feasible.

[0186] 7 Determination of the characteristic spectrum method of Mahonia suffruticosa L.

[0187] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid-0.2% triethylamine was used as mobile phase B, gradient elution was performed according to the provisions in the following table, the flow rate was 0.8 ml per minute; the column temperature was 20°C; the detection wavelength was 346 nm. The theoretical plate number calculated based on the berberine hydrochloride peak should be no less than 3000.

[0188]

[0189] Preparation of reference solution Take 0.5g of Mahonia leaf (broad-leaved Mahonia leaf) as a reference medicinal material, place it in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100) mixed solution, weigh it, heat and reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with hydrochloric acid-30% methanol (1:100) mixed solution, shake it well, filter it, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of berberine hydrochloride reference substance and palmatine hydrochloride reference substance, weigh them accurately, add methanol to make solutions containing 60μg berberine hydrochloride and 20μg palmatine hydrochloride per 1ml, and obtain them as the reference solution of the reference substance.

[0190] Preparation of test solution: Take about 0.5 g of medicinal material powder (passed through No. 3 sieve), add 25 ml of hydrochloric acid-30% methanol (1:100) mixed solution, weigh the weight, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with hydrochloric acid-30% methanol (1:100) mixed solution, shake well, filter, and take the filtrate to obtain the test solution.

[0191] The determination method is to accurately aspirate 5μl of the reference solution and the test solution, respectively, inject them into the liquid chromatograph, and determine them. There should be 6 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material reference. Peak 5 and peak 6 should correspond to the retention time of the reference peaks of the reference substance of the hydrochloride palmatine and the reference substance of the hydrochloride berberine. The peak corresponding to the peak of the hydrochloride reference substance is the S peak. The relative retention time of peak 1, peak 2, peak 3, peak 4 and S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified value is: 0.15 (peak 1), 0.18 (peak 2) 0.44 (peak 3), 0.66 (peak 4). See Figure 11.

[0192] Example 2 Standard decoction HPLC characteristic spectrum

[0193] 1 Experimental instruments and materials

[0194] Chromatographic columns: Agilent 5TC-C18 (4.6 mm × 250 mm, 5 μm), ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm), C18(2)100A (4.6mm×250mm, 5μm).

[0195] Standard decoction of Mahonia suffruticosa L. (from Sichuan New Green Pharmaceutical Batch No.: BT-1, BT-2, BT-3, BT-4, BT-5, BT-6, BT-7, BT-8, BT-9, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19.)

[0196] The rest is the same as in Example 1.

[0197] 3. Investigation of chromatographic conditions

[0198] 3.1 Proposed chromatographic conditions

[0199] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid-0.2% triethylamine solution was used as mobile phase B, gradient elution was performed according to the provisions in the table below, the flow rate was 0.8 ml per minute; the column temperature was 20°C; the detection wavelength was 346 nm. The theoretical plate number calculated based on the berberine hydrochloride peak should be no less than 3000.

[0200]

[0201] Preparation of reference solution Take 0.5g of the reference medicinal material of Mahonia suffruticosa L., place it in a stoppered conical flask, add 25ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh it, heat and reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake it well, filter it, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of berberine hydrochloride reference substance and palmatine hydrochloride reference substance, weigh them accurately, add methanol to make solutions containing 60μg of berberine hydrochloride and 20μg of palmatine hydrochloride per 1ml, and obtain them as the reference solution of the reference substance.

[0202] Preparation of test solution: Take an appropriate amount of standard decoction, take about 0.2 g, place in a stoppered conical flask, add 25 ml of hydrochloric acid-30% methanol (1:100) mixed solution, weigh the weight, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with hydrochloric acid-30% methanol (1:100) mixed solution, shake well, filter, and take the filtrate to obtain.

[0203] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0204] 4 Preparation of test solution

[0205] 4.1 Investigation of extraction solvent

[0206] Take 0.2 g of this product (batch number: BT-1) and place it in a stoppered conical flask. The extraction solvents for the test sample are methanol: hydrochloric acid (100:1), 30% methanol: hydrochloric acid (100:1), 50% methanol: hydrochloric acid (100:1), 70% methanol: hydrochloric acid (100:1), ethanol: hydrochloric acid (100:1), water: hydrochloric acid (100:1), and 30% methanol. 25 ml each was tested. Seal the flask, treat with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product. Fig.12 The extraction solvent of Mahonia leaf decoction was investigated; the results showed that when the extraction solvent was 30% methanol, the extraction rate of berberine hydrochloride and palmatine hydrochloride was low, and when it was 30% methanol: hydrochloric acid (100:1), the peak shapes of the characteristic peaks were good and the separation was moderate. The extraction solvent was temporarily set to 30% methanol: hydrochloric acid (100:1).

[0207] 4.2 Investigation of the proportion of hydrochloric acid added

[0208] Take an appropriate amount of this product (batch number: BT-1), take about 0.2g, place it in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (0.5:100), hydrochloric acid-30% methanol (1:100), and hydrochloric acid-30% methanol (3:100), seal it, ultrasonicate for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Fig.13 The extraction method was investigated; the results showed that the extraction effect was better when hydrochloric acid-30% methanol (1:100) was added to the test sample, and the ultrasonic method was fast and simple, so the ratio of hydrochloric acid to 30% methanol in the test sample was temporarily set as hydrochloric acid-30% methanol (1:100).

[0209] 4.3 Extraction time investigation

[0210] Take an appropriate amount of this product (batch number: BT-1), take about 0.2g, put it in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100), plug it tightly, and treat it with ultrasound (power 600W, frequency 40kHz). Observe the extraction time of the test product at 20 minutes, 30 minutes, and 40 minutes respectively, cool it, shake it well, filter it, and take the filtrate to obtain it. Fig.14 . Fig.14 Extraction time investigation;

[0211] The results showed that the effect difference was better when the extraction time of the test sample was 30 minutes, so the extraction time of the test sample was determined to be 30 minutes.

[0212] 4.4 Sampling volume investigation

[0213] Take an appropriate amount of this product (batch number: BT-1), take about 0.1g, 0.2g and 0.3g respectively, put it in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100) for inspection, seal it, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain it. Fig.15 . Fig.15 Investigation of solvent addition amount;

[0214] The results show that when the sampling amount is 0.2g, the peak shape and separation of each chromatographic peak are better, so the sampling amount is 0.2g.

[0215] 4.5 Investigation of extraction methods

[0216] Take appropriate amount of this product (batch number: BT-1), take two portions of 0.2g, put them in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100) for inspection, seal, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, reflux extraction for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Fig.16 . Fig.16 Investigation of solvent addition amount;

[0217] The results showed that there was little difference in the characteristic spectra between ultrasonic extraction and reflux extraction. Considering the ease of operation, ultrasonic extraction was finally selected.

[0218] In summary, the preparation method of the test solution of the characteristic spectrum of the standard decoction of Mahonia is determined as follows: take an appropriate amount of the standard decoction, take about 0.2 g, put it in a stoppered conical flask, add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh the weight, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake well, filter, and take the filtrate to obtain.

[0219] 5 Methodological investigation

[0220] 5.1 Chromatographic peak identification

[0221] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Mahonia suffruticosa standard decoction.

[0222] Preparation of reference solution: Take 0.5 g of Euonymus japonicus leaves as the accompanying reference medicinal material, place it in a stoppered conical flask, add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh the weight, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake well, filter, and take the filtrate as the accompanying reference medicinal material reference solution.

[0223] Take appropriate amount of berberine hydrochloride reference substance and palmatine hydrochloride reference substance, weigh accurately, add methanol to prepare solutions containing 60 μg of berberine hydrochloride and 20 μg of palmatine hydrochloride per 1 ml, respectively, to obtain the reference substance solutions.

[0224] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of the standard decoction of Mahonia lactiflora leaves.

[0225] Position the characteristic spectrum peaks of the standard decoction of Mahonia suffruticosa L. Fig.17 . Fig.17 The figure is a chromatographic peak identification diagram; the results show that there are 6 characteristic peaks in the test solution, of which peak 5 is bamipine hydrochloride and peak 6 is berberine hydrochloride. In summary, in the following methodological investigation, the 6 characteristic peaks in the test sample were investigated.

[0226] 5.2 Precision investigation

[0227] Take the test solution of Mahonia oleifera standard decoction (batch number: BT-1), inject 6 times continuously according to the proposed experimental method, 5 μl each time, and calculate the retention time of each characteristic peak. See Table 9.

[0228] Table 9 Precision investigation - retention time

[0229]

[0230] The results showed that the RSD range of the retention time of each characteristic peak was 0.1% to 3.2%, indicating that the instrument had good precision.

[0231] 5.3 Repeatability study

[0232] Six portions of the standard decoction of Mahonia suffruticosa L. (batch number: BT-1) were accurately weighed, prepared and measured according to the proposed experimental method, and the relative retention time 0 of each characteristic peak was calculated. See Table 10.

[0233] Table 10 Repeatability study - relative retention time

[0234]

[0235]

[0236] The results showed that the RSD range of the relative retention time of the six samples was 0.2-3.0%, indicating that the method had good repeatability.

[0237] 5.4 Investigation with different instruments

[0238] Based on the experimental conditions proposed above, two portions of the standard decoction of Mahonia suffruticosa L. (batch number: BT-1) were accurately weighed to prepare the test solution, which was measured on Shimadzu and Waters liquid chromatographs, respectively, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. See Table 11, Fig.18 . Fig.18 Different instrument surveys.

[0239] Table 11 Instrument durability study - relative retention time

[0240]

[0241] The results showed that when the two instruments were used to detect the test samples, the RSD range of the relative retention time of each characteristic peak was 0.4% to 12.5%, indicating that the instruments had good durability.

[0242] 5.5 Different people and different times

[0243] Based on the experimental conditions proposed above, two portions of the standard decoction of Mahonia suffruticosa (batch number: BT-1) were accurately weighed by different personnel (A, B) at different times (T1, T2) to prepare the test sample, conduct the determination, and calculate the relative retention time of each characteristic peak. See Table 12.

[0244] Table 12 Personnel and time inspection-relative retention time

[0245]

[0246] The results showed that when different personnel measured the same sample at different times, the RSD range of the relative retention time of each characteristic peak was 0.1% to 0.4%, and the method had good stability.

[0247] 5.6 Investigation of different chromatographic columns

[0248] Based on the above experimental conditions, the reverse phase C18 column 1 Agilent 5TC-C18 (4.6 mm × 250 mm, 5 μm), reverse phase C18 column 2 ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm), reverse phase C18 column 3 C18(2)100A (4.6mm×250mm, 5μm) was used for analysis and investigation, and the relative retention time of each characteristic peak was calculated. The results are shown in Table 13. Fig.19 . Fig.19 Investigation of different chromatographic columns.

[0249] Table 13 Column durability investigation - relative retention time

[0250]

[0251] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 1.7% and 15.4% when the three chromatographic columns were used to detect the samples, indicating that the chromatographic columns had good durability.

[0252] 5.6 Stability Study

[0253] Based on the above proposed experimental conditions, take the same test solution and measure it at 0h, 2h, 6h, 10h, 12h and 24h respectively. See Table 14.

[0254] Table 14 Stability Study-Retention Time

[0255]

[0256]

[0257] The results showed that the RSD of the characteristic peak retention time was between 0.1% and 0.9%, and the sample solution was stable within 24 hours.

[0258] 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 6 characteristic peaks were included in the subsequent investigation.

[0259] 6. Validation of the standard decoction of Mahonia suffruticosa leaves and establishment of HPLC content determination method

[0260] 6. The verification results of batch 119 of standard decoction of Euonymus japonicus leaves are shown in Table 15.

[0261] Using this method, 19 batches of samples were analyzed for characteristic spectra and relative retention times were calculated. Fig. 20 , Table 15. Fig. 20 Characteristic spectrum of standard decoction of Mahonia suffruticosa L. (S1-S21 are: BT-1, BT-2, BT-3, BT-4, BT-5, BT-6, BT-7, BT-8, BT-9, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19)

[0262] Table 15 Relative retention time of Mahonia suffruticosa standard decoction

[0263]

[0264] 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 6 peaks with good durability were selected as characteristic peaks. According to the results of the methodological investigation and the verification results of 19 batches of standard soup, the theoretical plate number calculated based on the berberine hydrochloride peak should be no less than 5000.

[0265] The relative retention time of each characteristic peak is stable and within the range of ±10% of the average value, so the relative retention time of each peak is temporarily set at ±10%.

[0266] Final regulations: There should be 6 characteristic peaks in the chromatogram of the test sample, and the retention times of the 6 characteristic peaks in the chromatogram of the control medicinal material reference material should correspond. Peak 5 and Peak 6 should correspond to the retention times of the reference material peaks of bamipine hydrochloride and berberine hydrochloride, respectively. The peak corresponding to the berberine hydrochloride reference material is the S peak. The relative retention times of Peak 1, Peak 2, Peak 3, Peak 4 and Peak S are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.15 (Peak 1), 0.18 (Peak 2) 0.44 (Peak 3), 0.66 (Peak 4).

[0267] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 19 batches of standard decoctions of Mahonia suffruticosa leaves, and a comparison spectrum of the characteristic spectrum of standard decoctions of Mahonia suffruticosa leaves was established. Fig.21 Control characteristic spectrum; Peak 5: Palmatine hydrochloride; Peak 6 (S): Berberine hydrochloride. Conclusion: The characteristic spectrum method of Mahonia suffruticosa standard decoction can effectively detect Mahonia suffruticosa standard decoction, proving that the method is feasible.

[0268] 7 Determination of characteristic spectrum of standard decoction of Mahonia suffruticosa L.

[0269] [Characteristic spectrum] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0270] Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid-0.2% triethylamine solution (pH=2.3) was used as mobile phase B, gradient elution was performed according to the provisions in the following table, the flow rate was 0.8 ml per minute; the column temperature was 20°C; the detection wavelength was 346 nm. The theoretical plate number calculated based on the berberine hydrochloride peak should be no less than 3000.

[0271]

[0272] Preparation of reference solution Take 0.5g of Mahonia suffruticosa leaf as a reference medicinal material, place it in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100) mixed solution, weigh it, heat and reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with hydrochloric acid-30% methanol (1:100) mixed solution, shake it well, filter it, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amount of berberine hydrochloride reference substance and palmatine hydrochloride reference substance, weigh them accurately, add methanol to make solutions containing 60μg berberine hydrochloride and 20μg palmatine hydrochloride per 1ml, and then use them as reference solution.

[0273] Preparation of test solution: Take an appropriate amount of the product, take about 0.2 g, place in a stoppered conical flask, add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh the weight, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake well, filter, and take the filtrate to obtain the solution.

[0274] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0275] There should be 6 characteristic peaks in the chromatogram of the test sample, and the retention times of the 6 characteristic peaks in the chromatogram of the control medicinal material reference material should correspond. Among them, peak 5 and peak 6 should correspond to the retention times of the reference material peaks of bamipine hydrochloride and berberine hydrochloride, respectively. The peak corresponding to the berberine hydrochloride reference material is the S peak. The relative retention times of peaks 1, 2, 3, 4 and S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.15 (peak 1), 0.18 (peak 2) 0.44 (peak 3), 0.66 (peak 4).

[0276] Example 3 Formula Granule HPLC Characteristic Spectrum

[0277] 1 Experimental instruments and materials

[0278] Mahonia leaf formula granules (from Sichuan New Green Pharmaceutical, batch numbers: KL-1, KL-2, KL-3.)

[0279] The rest is the same as in Example 2.

[0280] 3. Investigation of chromatographic conditions

[0281] 3.1 Proposed chromatographic conditions

[0282] Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid-0.2% triethylamine was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.8 ml per minute; the column temperature was 20°C; the detection wavelength was 346 nm. The theoretical plate number calculated based on the berberine hydrochloride peak should be no less than 3000.

[0283]

[0284] 3.2 Determination of detection wavelength

[0285] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the test solution, and the chromatograms of the test solution at wavelengths of 220nm, 250nm, 280nm, 320nm, 346nm, and 360nm were extracted respectively. Figure 22-Figure 23 . Fig. 22 3D chromatogram of Mahonia leaf formula granules; Fig.23 Chromatograms of Mahonia leaf formula granules at different wavelengths.

[0286] The results showed that when the detection wavelength was 346 nm, the chromatographic peak had more information and the chromatogram baseline was more stable, so the detection wavelength was determined to be 346 nm.

[0287] 3.3 Column temperature investigation

[0288] Based on the experimental conditions proposed above, the column temperatures of 20℃, 25℃ and 30℃ were investigated. Fig.24 . See Tables 16-17. Fig.24 Column temperature inspection.

[0289] Table 16 Column temperature investigation-retention time

[0290]

[0291] Table 17 Column temperature investigation-relative retention time

[0292]

[0293] The results showed that when the column temperature was 20℃, the chromatogram peak shape was more symmetrical, the separation was better, and the peaks were more complete. Therefore, the column temperature was determined to be 20℃.

[0294] 3.4 Flow rate investigation

[0295] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Fig.25 , Table 18 to Table 19. Fig.25 Flow rate inspection.

[0296] Table 18 Flow rate investigation-retention time

[0297]

[0298] Table 19 Flow rate investigation - relative retention time

[0299]

[0300] The results show that when the flow rate is 0.8 ml / min, the chromatogram peak shape is better and the separation is moderate. Therefore, the flow rate is determined to be 0.8 ml / min.

[0301] 3.5 Delay Investigation

[0302] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Fig.26 . Fig.26 Delay investigation: The results showed that the sample had almost no main chromatographic peak after 55 minutes, so the sample detection time was set at 55 minutes.

[0303] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of the Mahonia leaf formula granules are determined as follows: octadecylsilane bonded silica gel is used as the filler (column length 250mm, inner diameter 4.6mm, particle size 5μm); acetonitrile is used as mobile phase A, 0.2% phosphoric acid-0.2% triethylamine solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; the flow rate is 0.8ml per minute; the column temperature is 20℃; the detection wavelength is 346nm. The theoretical plate number calculated based on the berberine hydrochloride peak should not be less than 3000.

[0304]

[0305] 4 Preparation of test solution

[0306] 4.1 Investigation of extraction solvent

[0307] Take 0.2g of the formula granules (batch number: KL-1), grind them into powder, put them into a stoppered conical flask, and test the extraction solvents of the test sample, methanol: hydrochloric acid (100:1), 30% methanol: hydrochloric acid (100:1), 50% methanol: hydrochloric acid (100:1), 70% methanol: hydrochloric acid (100:1), ethanol: hydrochloric acid (100:1), water: hydrochloric acid (100:1), and 30% methanol, 25ml each, seal the flask, and treat with ultrasound (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Fig. 27 . Fig. 27 Investigation of extraction solvents.

[0308] The results showed that when the extraction solvent was 30% methanol, the extraction rate of berberine hydrochloride and palmatine hydrochloride was low. When it was 30% methanol: hydrochloric acid (100:1), the peak shapes of the characteristic peaks were good and the separation was moderate. The extraction solvent was temporarily set to 30% methanol: hydrochloric acid (100:1).

[0309] 4.2 Investigation of the proportion of hydrochloric acid added

[0310] Take 0.2g of the formula granules (batch number: KL-1), grind them into powder, put them into a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (0.5:100), hydrochloric acid-30% methanol (1:100), and hydrochloric acid-30% methanol (3:100), seal it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain the product. Fig.28 . Fig.28 Investigation of the amount of hydrochloric acid added.

[0311] The results showed that the extraction solvent investigation results showed that the chromatogram of hydrochloric acid-30% methanol (1:100) was better than that of 30% methanol; the results of the investigation of different proportions of hydrochloric acid addition showed that the chromatogram of adding 1ml hydrochloric acid to 100ml 30% methanol was better.

[0312] 4.3 Extraction time investigation

[0313] Take 0.2g of the formula granules (batch number: KL-1), grind them into powder, put them into a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100), stopper it tightly, and treat it with ultrasound (power 600W, frequency 40kHz). The extraction time of the test sample is 20 minutes, 30 minutes, and 40 minutes respectively. Cool it, shake it well, filter it, and take the filtrate to obtain the product. Fig.29 . Fig.29 Extraction time investigation: The results showed that the effect difference was better when the extraction time of the test sample was 30 minutes, so the extraction time of the test sample was determined to be 30 minutes.

[0314] 4.4 Sampling volume investigation

[0315] Take 0.1g, 0.2g and 0.3g of the formula granules (batch number: KL-1), grind them into powder, put them into a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100), seal it, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain the product. Fig.30 . Fig.30 Sampling volume investigation; the results show that when the sampling volume is 0.2g, the peak shape and separation of each chromatographic peak are better, so the sampling volume is selected as 0.2g.

[0316] 4.4 Investigation of extraction methods

[0317] Take appropriate amount of formula granules (batch number: KL-1), grind them finely, take two portions of 0.2g, weigh them accurately, put them in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100), seal it tightly, inspect it, seal it tightly, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, reflux extraction for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain it. Fig.31 . Fig.31 The extraction method is investigated.

[0318] The results showed that there was little difference in the characteristic spectra between ultrasonic extraction and reflux extraction. Considering the ease of operation, ultrasonic extraction was finally selected.

[0319] In summary, the preparation method of the test solution of the characteristic spectrum of the formula granules of Mahonia leaf is determined as follows: take an appropriate amount of the product, grind it into powder, take 0.2g, put it in a stoppered conical flask, add 25ml of a mixed solution of hydrochloric acid-30% methanol (1:100), stopper it, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake it well, filter it, and take the filtrate to obtain it.

[0320] 5 Methodological investigation

[0321] 5.1 Chromatographic peak identification

[0322] Preparation of test solution: According to the experimental conditions determined above, prepare the test solution of Mahonia leaf formula granules.

[0323] Preparation of reference solution: Take 0.5 g of Mahonia leaf (broad-leaved Mahonia leaf) reference medicinal material, place it in a stoppered conical flask, add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh the weight, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake well, filter, and take the filtrate as the reference solution of the (broad-leaved Mahonia leaf) reference medicinal material.

[0324] Take appropriate amount of berberine hydrochloride reference substance and palmatine hydrochloride reference substance, weigh accurately, add methanol to prepare solutions containing 60 μg of berberine hydrochloride and 20 μg of palmatine hydrochloride per 1 ml, respectively, to obtain the reference substance solutions.

[0325] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of the Mahonia lactiflora formula granules.

[0326] Locate the characteristic spectrum peaks of the granules of the ten great magnolia leaves formula. Fig.32 ; Fig.32 Chromatographic peak identification; Fig.33 Spectrum of reference substance berberine hydrochloride; Fig.34 Spectrum of reference substance Bamipine hydrochloride; Fig.35 The spectrum of the sample berberine hydrochloride; Fig.36 Sample Bamipine hydrochloride spectrum.

[0327] The results showed that there were 6 characteristic peaks in the test solution, of which peak 5 was bamipine hydrochloride and peak 6 was berberine hydrochloride. In summary, in the following methodological investigation, the 6 characteristic peaks in the test sample were investigated.

[0328] 5.2 Precision investigation

[0329] Take the test solution of the formula granules of the ten great magnolia leaves (batch number: KL-1), inject 6 times continuously according to the proposed experimental method, 5 μl each time, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Table 20.

[0330] Table 20 Precision investigation - relative retention time

[0331]

[0332] The results showed that the RSD range of the relative retention time of each characteristic peak of the sample was 0.00% to 0.29%, indicating that the method had good injection precision.

[0333] 5.3 Repeatability study

[0334] Six portions of the Mahonia oleifera formula granules (batch number: KL-1) were prepared and tested according to the proposed experimental method. See Table 21.

[0335] Table 21 Repeatability study - relative retention time

[0336]

[0337] The results showed that the RSD range of the relative retention time of the six samples was 0.00% to 3.10%, indicating that the method had good repeatability.

[0338] 5.4 Investigation with different instruments

[0339] Based on the experimental conditions proposed above, two portions of the formula granules of the ten great magnolia leaves (batch number: KL-1) were weighed to prepare the test solution, which was measured on Shimadzu and Waters liquid chromatographs respectively. Fig. 22 , Table 22. Fig.37 Survey with different instruments.

[0340] Table 22 Instrument durability study - relative retention time

[0341]

[0342] The results showed that when the test samples were detected using the above two instruments, the RSD of the relative retention time of each characteristic peak was 0.41% to 17.35%. Since the relative retention time values ​​of peaks 1 and 2 were small and slight fluctuations resulted in a larger RSD, it was considered that the method had good instrument precision after comprehensive consideration.

[0343] 5.5 Different people and time

[0344] Based on the experimental conditions proposed above, different personnel (A, B) weighed two portions of the formula granules of the magnolia officinalis leaves (batch number: KL-1) at different times (T1) to prepare the test samples for determination. See Table 23.

[0345] Table 23 Personnel and time inspection - relative retention time

[0346]

[0347] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00-1.75, indicating that the intermediate precision was good.

[0348] 5.5.2 Investigation of different chromatographic columns

[0349] Based on the above experimental conditions, the reverse phase C18 column 1 Agilent 5TC-C18 (4.6 mm × 250 mm, 5 μm), reverse phase C18 column 2 ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm), reverse phase C18 column 3 C18(2)100A (4.6mm×250mm, 5μm) was used for analysis and investigation, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. Fig.38 , Table 24. Fig.38 Investigation of different chromatographic columns.

[0350] Table 24 Column durability investigation - relative retention time

[0351]

[0352] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.00% and 17.98% when the samples were detected using the three chromatographic columns mentioned above.

[0353] 5.6 Stability Study

[0354] Based on the above experimental conditions, take the same test solution and measure it at 0h, 2h, 6h, 10h, 12h and 24h respectively. See Table 25.

[0355] Table 25 Stability Study - Retention Time

[0356]

[0357]

[0358] The results showed that the RSD of the characteristic peak retention time was between 0.00% and 0.25%, indicating that the sample solution was stable within 24 hours.

[0359] 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 6 characteristic peaks are included in the subsequent investigation

[0360] 5.7 Determination of characteristic peaks and establishment of reference spectrum

[0361] 5.7.1 Verification results of three batches of Mahonia suffruticosa formula granules

[0362] Using this method, characteristic spectra were analyzed on three batches of samples and relative retention times were calculated. Fig.39 , Table 26. Fig.39 Characteristic spectrum of Mahonia leaf formula granules.

[0363] Table 26 Relative retention time of Mahonia leaf formula granules

[0364]

[0365] Based on the principle of stable relative retention time and the fact that all batches of samples can be detected and the peaks are relatively stable, a total of 6 peaks with good durability were selected as characteristic peaks, namely Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, and Peak 6. According to the results of the methodological investigation and the verification results of 3 batches of particles, the theoretical plate number calculated based on the berberine hydrochloride peak should be no less than 3000.

[0366] The summary of the methodology investigation items and verification results is shown in Table 27:

[0367] Table 27 Summary of RSD% results of each item of the methodology - relative retention time / retention time

[0368]

[0369] The relative retention time of each chromatographic characteristic peak is stable, among which the relative retention time of chromatographic peaks 3, 4, and 5 is within the range of ±10% of the average value, so the relative retention time of each peak is temporarily set at ±10%. The relative retention time values ​​of peaks 1 and 2 are small, and their ±10% range is narrow, so the relative retention time of peaks 1 and 2 is specified in the range according to the verification of the characteristic spectra of medicinal materials, decoction pieces, and standard decoctions and the methodological investigation. Since the retention time of chromatographic peaks 5 and 6 varies greatly under different chromatographic columns and on different instruments, a reference substance is used to determine the two chromatographic peaks.

[0370] Final Regulation:

[0371] The test sample chromatogram should show 6 characteristic peaks, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 5 and peak 6 should correspond to the retention time of the reference peaks of berberine hydrochloride and berberine hydrochloride, respectively. The peak corresponding to the berberine hydrochloride reference is the S peak. The relative retention time of peak 1, peak 2, peak 3, peak 4 and S peak is calculated. The relative retention time of peak 3 and peak 4 should be within the range of ±10% of the specified value, and the specified value is: 0.44 (peak 3) and 0.66 (peak 4). The relative retention time of peak 1 and peak 2 should be within the specified value range, and the specified value is 0.11-0.20 (peak 1) and 0.14-0.24 (peak 2).

[0372] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize three batches of magnolia officinalis granules, and a comparison spectrum of the characteristic spectrum of magnolia officinalis granules was established. Fig.40 .

[0373] Fig.40 The characteristic spectrum of the control, where peak 5: palmatine hydrochloride; peak 6 (S): berberine hydrochloride. Conclusion: The characteristic spectrum method of the mahonia leaf formula granules can effectively detect the mahonia leaf formula granules, proving that the method is feasible.

[0374] 6 Determination of characteristic spectrum method of Mahonia leaf formula granules

[0375] [Characteristic spectrum] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0376] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid-0.2% triethylamine solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.8 ml per minute; the column temperature was 20°C; the detection wavelength was 346 nm. The theoretical plate number calculated based on the berberine hydrochloride peak should not be less than 3000.

[0377]

[0378] Preparation of reference solution Take 0.5 g of Mahonia oleracea (broad-leaved Mahonia oleracea) as a reference medicinal material, place it in a stoppered conical flask, add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), heat and reflux for 30 minutes, cool, shake well, filter, and take the filtrate as a reference solution of Mahonia oleracea (broad-leaved Mahonia oleracea). Separately take the reference solution under Example 1 as a reference solution.

[0379] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2 g, put it in a stoppered conical flask, add 25 ml of a mixed solution of hydrochloric acid-30% methanol (1:100), weigh it, treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with a mixed solution of hydrochloric acid-30% methanol (1:100), shake it well, filter it, and take the filtrate to obtain the product.

[0380] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0381] The test sample chromatogram should show 6 characteristic peaks, and the retention time should correspond to the 6 characteristic peaks in the reference drug chromatogram. Peak 5 and peak 6 should correspond to the reference peak retention time of the corresponding reference substance, and the peak corresponding to the berberine hydrochloride reference substance is the S peak. The relative retention time of peak 1, peak 2, peak 3, peak 4 and S peak is calculated. The relative retention time of peak 3 and peak 4 should be within the range of ±10% of the specified value, and the specified value is: 0.44 (peak 3) and 0.66 (peak 4). The relative retention time of peak 1 and peak 2 should be within the specified value range, and the specified value is 0.11-0.20 (peak 1) and 0.14-0.24 (peak 2).

[0382] Comparative Example 1

[0383] The present invention uses acetonitrile-0.2% potassium dihydrogen phosphate for detection. Fig.41 shown.

[0384] Take an appropriate amount of granules (batch number: KL-1), grind finely, take about 0.2g, put it in a stoppered conical flask, add 25ml of hydrochloric acid-30% methanol (1:100) mixed solution, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with hydrochloric acid-30% methanol (1:100) mixed solution, shake well, filter, and take the filtrate to obtain.

[0385] One part uses acetonitrile as mobile phase A and 0.2% phosphoric acid-0.2% triethylamine solution as mobile phase B;

[0386] One part uses acetonitrile as mobile phase A and 0.2% potassium dihydrogen phosphate solution as mobile phase B;

[0387] Gradient elution was performed as specified in the following table; the flow rate was 0.8 ml per minute; the column temperature was 20°C; and the detection wavelength was 346 nm.

[0388]

[0389] The results showed that under the same chromatographic conditions, in the acetonitrile-0.2% potassium dihydrogen phosphate mobile phase elution system, the berberine hydrochloride and bamipine hydrochloride chromatographic peaks had low peak heights and poor peak shapes, and the other chromatographic peaks showed varying degrees of reduced separation, affecting the overall detection effect of the characteristic spectrum. In the 0.2% phosphoric acid-0.2% triethylamine system, the target peak had a moderate peak height, good peak shape, better separation, and more abundant chromatographic peaks, which could more clearly reflect the chromatographic peak information in the test sample.

[0390] 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 HPLC characteristic spectra of Mahonia suffruticosa medicinal materials, decoction pieces, standard decoctions and their formula particles, comprising: A) extracting Mahonia truncatula leaves with a solvent to obtain a test solution; B) the test solution is measured by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the Mahonia laurel leaf raw material; 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 0.2% phosphoric acid-0.2% triethylamine, and the gradient elution is performed; The gradient elution is specifically: 0-3min, phase A: 12%→14%, phase B: 88%→86%; 3-19 min, phase A: 14%→18%, phase B: 86%→82%; 19-28 min, phase A: 18% → 29%, phase B: 82% → 71%; 28-48 min, phase A: 29%→30%, phase B: 71%→70%; 48-55min, phase A: 30%→35%, phase B: 70%→65%.

2. The method according to claim 1, characterized in that It also includes the preparation of reference substances and reference medicinal material solutions: Take berberine hydrochloride reference substance and palmatine hydrochloride reference substance respectively, dissolve them in methanol to obtain reference substance solutions; the concentration of the berberine hydrochloride reference substance is 60 μg / mL; the concentration of the palmatine hydrochloride reference substance is 20 μg / mL; Take the reference medicinal material of Mahonia oleifera and dissolve it in hydrochloric acid-30% methanol (1:100), heat and reflux to extract, cool it, filter it, and obtain the reference medicinal material solution; The reference substance and reference medicinal material reference substance solutions are determined by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference substance, respectively; and the components of the HPLC characteristic spectra of snake exuviae medicinal materials, decoction pieces, standard decoctions and their formula granules are qualitatively determined based on the chromatograms of the reference substance and the reference medicinal material reference substance.

3. The method according to claim 1, characterized in that The chromatographic column is a C18 column with specifications of 5.0 μm and 4.6×250 mm; the column temperature is 20° C.

4. The method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8 mL / min; the detection wavelength is 346 nm; the theoretical plate number calculated based on the berberine hydrochloride peak should be no less than 3000, and the injection volume is 5 μL.

5. The method according to claim 1, characterized in that The similarity of HPLC characteristic spectra of Mahonia laurel L. medicinal materials, decoction pieces, standard decoctions and their formula granules was evaluated by using the Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic spectra of Mahonia laurel L. medicinal materials, decoction pieces, standard decoctions and their formula granules consisting of 6 characteristic peaks were obtained, among which peak 5 was bamipine hydrochloride and peak 6 was berberine hydrochloride.

6. The method according to claim 5, characterized in that In the characteristic spectrum of the standard decoction of Mahonia latifolia, berberine hydrochloride 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: 0.15 (peak 1), 0.18 (peak 2) 0.44 (peak 3), and 0.66 (peak 4).

7. The method according to claim 1, characterized in that Step A) The raw materials of Mahonia suffruticosa L. are materials, decoction pieces, standard decoction and formula granules thereof.

8. The method according to claim 7, characterized in that The preparation of the test solution of the Mahonia leaf medicinal material / cooking piece is as follows: dissolving the Mahonia leaf in hydrochloric acid-30% methanol, heating under reflux for extraction, cooling, shaking, filtering, and obtaining the solution; The heating reflux time is 30 minutes; wherein the volume ratio of hydrochloric acid to 30% methanol is 1:

100.

9. The method according to claim 7, characterized in that: The preparation of the standard decoction of Mahonia suffruticosa leaves is as follows: dissolving Mahonia suffruticosa leaves in hydrochloric acid-30% methanol (1:100), ultrasonic extraction, cooling, shaking, filtering, and obtaining the decoction; the ultrasonic power is 600W, the frequency is 40kHz; wherein the volume ratio of hydrochloric acid-30% methanol is 1:100; The preparation of the Mahonia leaf formula granules is specifically as follows: dissolving the Mahonia leaf in hydrochloric acid-30% methanol, ultrasonically extracting, cooling, shaking, filtering, and obtaining the granules; the ultrasonic power is 600W, the frequency is 40kHz; and the volume ratio of hydrochloric acid-30% methanol is 1:

100.

10. The method according to claim 1, characterized in that The ratio of the mass g of the Mahonia laurel raw material to the volume mL of the solvent is 0.2-0.5:20-30.

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