Preparation method and detection method of Chinese mahonia leaf extract

Abstract

The invention belongs to the field of modernization of traditional Chinese medicines, and particularly relates to a preparation method and a detection method of a Chinese mahonia leaf extract. The preparation method of the Chinese mahonia leaf extract comprises the following steps: taking Chinese mahonia leaf decoction pieces, adding water, decocting, and filtering to obtain filtrate, thereby obtaining the Chinese mahonia leaf extract. The Chinese mahonia leaf extract obtained by the preparation method is high in extract yield and high in transfer rate of index components. The invention also provides a detection method and a thin-layer identification method for the total content and the total transfer rate of the index components in the Chinese mahonia leaf extract, and the content of jatrorrhizine hydrochloride, palmatine hydrochloride and berberine hydrochloride is determined through a thin-layer chromatography identification map of the Chinese mahonia leaf extract. The quality of the Chinese mahonia leaf extract can be evaluated more effectively, and meanwhile, a scientific and reasonable basis is provided for formulating process standards and quality standards of the Chinese mahonia leaf extract.

Description

Technical Field

[0001] This invention belongs to the field of modernization of traditional Chinese medicine, specifically relating to a preparation method and a detection method for an extract of Mahonia japonica leaves. Background Technology

[0002] Mahonia duclouxiana Gagnep., the dried leaf of the Mahonia plant (belonging to the Berberidaceae family), is a medicinal herb. It is bitter and cold in nature, entering the lung, liver, and kidney meridians. It has the effects of clearing deficiency heat, drying dampness, detoxifying, relieving cough and resolving phlegm, and is mainly used to treat pulmonary tuberculosis cough and night sweats. It is produced in Yunnan, Sichuan, and Guangxi provinces. It is a traditional medicine used by ethnic minorities in Guizhou Province and has a long history of use and acceptance in Chinese folk medicine. The secondary metabolites of Mahonia leaves mainly include alkaloids, phenolic acids, flavonoids, glycosides, and polysaccharides, which have various pharmacological effects such as antibacterial, anti-inflammatory, anti-allergic, antioxidant, antitumor, and hypoglycemic effects. However, at present, basic research on Mahonia japonica leaf extract is relatively weak, such as relatively few reports on chemical composition and insufficient research on pharmacological effects; moreover, there is a lot of confusion between the original medicinal material of Mahonia japonica leaf in the market, and the quality standard evaluation system of Mahonia japonica leaf extract is still imperfect, which cannot effectively evaluate its indicators and limits its application and development. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a method for preparing and detecting Mahonia japonica leaf extract.

[0004] Specifically, the present invention provides the following technical solution:

[0005] In a first aspect, the present invention provides a method for preparing an extract of Mahonia japonica leaves, comprising the following steps:

[0006] Take the leaves of Mahonia japonica, add water and decoct, filter to obtain the filtrate, and obtain Mahonia japonica leaf extract.

[0007] Preferably, the boiling process is carried out 1 to 3 times.

[0008] Preferably, the decoction is performed twice; the first decoction is performed with 10 to 18 times the amount of water; the second decoction is performed with 9 to 17 times the amount of water.

[0009] Preferably, the first decoction time is 30-120 minutes; the second decoction time is 20-80 minutes.

[0010] Preferably, the filtration is performed using a 100-300 mesh sieve.

[0011] Preferably, the filtrate is concentrated under reduced pressure.

[0012] More preferably, the concentration temperature is 55–70°C, and / or the vacuum degree is -0.080–-0.090 MPa.

[0013] Preferably, the filtrate after vacuum concentration is dried, and the drying is freeze-drying, which is divided into three stages: a. pre-freezing: the pre-freezing temperature is -50℃ to -45℃; b. primary drying: the drying temperature is -30℃ to 0℃; c. secondary drying for analysis: the drying temperature is 5℃ to 25℃.

[0014] More preferably, the pre-freezing time is 3-6 hours; the first drying time is 10-14 hours; and the second drying time is 5-7 hours.

[0015] Preferably, the filtrate after vacuum concentration is dried, and the drying is performed by vacuum drying, spray drying, microwave drying or infrared drying.

[0016] Preferably, the drying is spray drying, which includes the following steps: adding a first excipient to the extract and then drying it.

[0017] Preferably, the first excipient is maltodextrin.

[0018] Preferably, the amount of the first excipient added is 5% to 15% of the amount of Mahonia japonica leaf slices added.

[0019] Preferably, the inlet air temperature of the spray dryer is 165-175°C, and the outlet air temperature is 100-110°C.

[0020] Preferably, a second auxiliary material is added to the dried material.

[0021] Preferably, the second excipient comprises silicon dioxide and / or magnesium stearate.

[0022] Preferably, the silica accounts for a weight percentage of greater than 0 and less than or equal to 0.3% of the dried material.

[0023] More preferably, the silica accounts for 0.1% to 0.3% of the weight of the dried material.

[0024] Preferably, the magnesium stearate accounts for a weight percentage of greater than 0 and less than or equal to 0.3% of the dried material.

[0025] More preferably, the magnesium stearate accounts for 0.1% to 0.3% of the weight of the dried material.

[0026] Secondly, the present invention provides a Mahonia japonica leaf extract, which is prepared by the method described above.

[0027] Preferably, the preparation obtained from the extract of Mahonia japonica leaves is a standard decoction of Mahonia japonica leaves or a formula granule of Mahonia japonica leaves.

[0028] Thirdly, the present invention provides a method for detecting the total mass content and total transfer rate of indicative components in Mahonia japonica leaf extract, comprising the following steps:

[0029] (1) Preparation of reference solution

[0030] Weigh out the reference standards of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, respectively, and prepare solutions by adding solvents;

[0031] (2) Preparation of the test solution

[0032] Extract the leaves of Mahonia japonica and add solvent to extract;

[0033] (3) Liquid Chromatography Analysis

[0034] Using octadecylsilane-bonded silica gel as the stationary phase, with water as mobile phase A and acetonitrile as mobile phase B, the reference solution and the test solution were injected into the liquid chromatograph and analyzed using gradient elution.

[0035] Preferably, in step (1), the solvent is methanol or ethanol.

[0036] Preferably, in step (2), the preparation of the test solution includes: taking Mahonia leaf extract, adding solvent for extraction, weighing, replenishing the lost weight with extraction solvent, filtering, and taking the filtrate to obtain the test solution.

[0037] Preferably, the solvent is selected from a combination of hydrochloric acid and at least one of the following solvents: methanol, ethanol and water.

[0038] Preferably, the volume ratio of hydrochloric acid to other solvents is 1:90 to 110, and the other solvents are one or more of methanol, ethanol, and water.

[0039] Preferably, the volume ratio of hydrochloric acid to other solvents is 1:100, and the other solvents are one or more of methanol, ethanol, and water.

[0040] Preferably, the extraction solvent is selected from a combination of hydrochloric acid and methanol.

[0041] Preferably, the extraction is performed using any one of shaking, ultrasonication, or reflux.

[0042] Preferably, the extraction is performed using ultrasonic extraction.

[0043] Preferably, the extraction time is 15-60 minutes.

[0044] Preferably, in step (3), the detection wavelength of the chromatogram is 340-350 nm.

[0045] Preferably, the detection wavelength of the chromatogram is 346 nm.

[0046] Preferably, in step (3), the flow rate is 0.8 to 1.2 mL / min.

[0047] Preferably, the flow rate is 1 mL / min.

[0048] Preferably, in step (3), the column temperature is 25-35°C.

[0049] Preferably, the column temperature is 30°C.

[0050] Preferably, in step (3), the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0051] Preferably, the chromatographic column is JADE- KP-C18-AQ.

[0052] Preferably, in step (3), the mobile phase A is an aqueous solution containing potassium dihydrogen phosphate and sodium dodecyl sulfate.

[0053] Preferably, the mobile phase A is an aqueous solution containing 0.3-0.4 g of potassium dihydrogen phosphate and 0.1-0.2 g of sodium dodecyl sulfate per 100 mL of mobile phase A.

[0054] Preferably, the mobile phase A is an aqueous solution containing 0.34 g potassium dihydrogen phosphate and 0.17 g sodium dodecyl sulfate per 100 mL of mobile phase A.

[0055] More preferably, the pH of the mobile phase A is 2.0 to 4.0.

[0056] Preferably, the pH of the mobile phase A is 3.0.

[0057] Preferably, in step (3), the gradient elution procedure is as follows:

[0058] From 0 to 8 minutes, the volume percentage of mobile phase A decreased from 70% to 55%, while the volume percentage of mobile phase B increased from 30% to 45%.

[0059] Between 8 and 16.5 minutes, the volume percentage of mobile phase A decreased from 55% to 52.5%, while the volume percentage of mobile phase B increased from 45% to 47.5%.

[0060] Between 16.5 and 16.6 min, the volume percentage of mobile phase A increased from 52.5% to 70%, while the volume percentage of mobile phase B decreased from 47.5% to 30%.

[0061] 16.6–20 min, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%.

[0062] Preferably, the total mass content of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the Mahonia japonica leaf extract is 0.04 to 1.9 mg / g.

[0063] Preferably, the transfer rates of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the Mahonia japonica leaf extract are 16.2% to 45.8%.

[0064] Preferably, the transfer rate of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the Mahonia japonica leaf extract is 20% to 40%.

[0065] Preferably, the total mass content of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the standard decoction of Mahonia japonica leaves is 0.1 to 1.6 mg / g.

[0066] Preferably, the total mass content of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the Mahonia japonica leaf formula granules is 0.04 to 1.9 mg / g.

[0067] Fourthly, the present invention provides a thin-layer chromatography identification method for Mahonia japonica leaf extract, comprising the preparation of a test solution of Mahonia japonica leaf extract, thin-layer spotting, development and color development, dissolving Mahonia japonica leaf extract in methanol, sonicating, filtering to obtain a test solution of Mahonia japonica leaf extract; using toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia solution as the developing solvent.

[0068] Preferably, the volume ratio of toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia test solution is 3.5-4.5:2.5-3.5:2.5-3.5:0.7-1.7:0.1-1.

[0069] Preferably, the volume ratio of toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia solution is 4:3:3:1.2:0.5.

[0070] Preferably, 1g of Mahonia japonica leaf extract is taken, 10mL of methanol is added, and the mixture is sonicated for 60min, filtered, and the Mahonia japonica leaf extract test solution is obtained.

[0071] The beneficial effects of this invention are:

[0072] (1) This invention provides a standard decoction of Mahonia japonica leaves and its preparation method, which provides a standard for the quality of Chinese medicine formula granules and realizes the overall quality control and effective supervision of Chinese medicine formula granules.

[0073] (2) This invention uses Mahonia japonica leaves to prepare a standard decoction of Mahonia japonica leaves. The yield of the standard decoction of Mahonia japonica leaves is 11.5% to 27.8%, which is relatively high. The prepared standard decoction of Mahonia japonica leaves was analyzed by liquid chromatography. The method has good specificity, good repeatability, and good stability.

[0074] (3) The present invention provides a Chinese herbal medicine formula granule of Mahonia japonica leaf and its preparation method. The preparation method is simple, and it is prepared by decoction according to traditional methods, which is in line with the characteristics of traditional Chinese medicine.

[0075] (4) This invention can more effectively evaluate the quality of Mahonia japonica leaf formula granules by identifying the thin-layer chromatogram of Mahonia japonica leaf extract and determining the content of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride. At the same time, it provides a scientific and reasonable basis for formulating process standards and quality standards for Mahonia japonica leaf extract. Attached Figure Description

[0076] Figure 1 This is a specificity-based chromatogram for the detection method of total mass content and total transfer rate of the standard decoction of Mahonia japonica leaves.

[0077] Figure 2 The linear regression equation diagram of the total mass content and total transfer rate of the standard decoction of Mahonia japonica leaves is shown in the figure.

[0078] Figure 3 The linear regression equation of palmatine hydrochloride in the detection method of total mass content and total transfer rate of the standard decoction of Mahonia japonica leaves is shown.

[0079] Figure 4 The linear regression equation of berberine hydrochloride in the detection method of total mass content and total transfer rate of Mahonia japonica leaf standard decoction is shown in the figure.

[0080] Figure 5 This is a chromatogram showing the effect of different chromatographic columns on the determination of the content of indicative components in the standard decoction of Mahonia japonica leaves.

[0081] Figure 6 This is a chromatogram showing the effect of different chromatograms on the determination of the content of indicative components in the standard decoction of Mahonia japonica leaves.

[0082] Figure 7 This is a chromatogram showing the determination of the content of indicative components in the standard decoction of Mahonia japonica leaves at different column temperatures.

[0083] Figure 8 This is a chromatogram showing the effect of different flow rates on the determination of the content of indicative components in the standard decoction of Mahonia japonica leaves.

[0084] Figure 9This is a graph showing the effect of soaking time on the extraction content of indicative components in Mahonia japonica leaves in Example 5.

[0085] Figure 10 This is a graph showing the effect of the water addition ratio on the extraction content of the indicative components in Mahonia japonica leaves in Example 5.

[0086] Figure 11 This is a graph showing the effect of decoction time on the extraction content of key components in Mahonia japonica leaves in Example 5.

[0087] Figure 12 This is a thin-layer chromatogram of the sample quantity of the formula granules of Mahonia japonica leaf (long-column Mahonia japonica).

[0088] The following are the contents: 1. 1 μL of mixed reference standard; 2. 2 μL of mixed reference standard; 3. 4 μL of mixed reference standard; 4. 1 μL of Mahonia japonica leaf (long-column Mahonia japonica) formula granules; 5. 2 μL of Mahonia japonica leaf (long-column Mahonia japonica) formula granules; 6. 4 μL of Mahonia japonica leaf (long-column Mahonia japonica) formula granules.

[0089] Figure 13 This study focuses on the thin-layer chromatography (TLC) specificity of Mahonia japonica leaf (long-column Mahonia) formulation granules. The samples included (T: 25℃, RH: 66%; Qingdao Marine Silica G plate): 1. Negative sample; 2. Mixed reference standards (from bottom to top: berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride); 3-5. 2 μL of Mahonia japonica leaf (long-column Mahonia) formulation granules (batch number: 230901-230903).

[0090] Figure 14 This is a thin-layer chromatography (TLC) spiking confirmation of Mahonia japonica leaf (long-column Mahonia) formula granules (A is the original image, B is the image after color change). The components are: 1. Mixed reference standards (from bottom to top: berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride) 2 μL; 2. Mahonia japonica leaf (long-column Mahonia) formula granules (batch number: 230903) 2 μL; 3. Spiked solution 2 μL.

[0091] Figure 15 These are thin-layer chromatograms for the identification of Mahonia japonica leaf (long-column Mahonia) formulation granules under different humidity conditions. Among them, 1. negative sample; 2. mixed reference standard; 3-5. 2 μL of Mahonia japonica leaf (long-column Mahonia) formulation granules (batch number: 230901-230903). Test conditions for chromatogram a: T: 25℃, RH: 33%; Qingdao Marine Silica G plate. Test conditions for chromatogram b: T: 25℃, RH: 66%; Qingdao Marine Silica G plate. Test conditions for chromatogram c: T: 25℃, RH: 88%; Qingdao Marine Silica G plate.

[0092] Figure 16These are thin-layer chromatograms for the identification of Mahonia japonica leaf (long-column Mahonia) formulation granules under different temperature conditions. Among them, 1. negative sample; 2. mixed reference standard; 3-5. 2 μL of Mahonia japonica leaf (long-column Mahonia) formulation granules (batch number: 230901-230903). Test conditions for chromatogram a: T: 4℃, RH: 66%; Qingdao Marine Silica G plate. Test conditions for chromatogram b: T: 25℃, RH: 66%; Qingdao Marine Silica G plate. Test conditions for chromatogram c: T: 40℃, RH: 66%; Qingdao Marine Silica G plate.

[0093] Figure 17 These are chromatograms obtained from silica gel plates from different manufacturers. Among them, 1. negative sample; 2. mixed reference standard; 3-5. 2 μL of Mahonia japonica leaf (long column Mahonia japonica) formula granules (batch number: 230901-230903). Chromatography conditions for chromatogram a: T: 40℃, RH: 66%; Qingdao Marine silica gel G plate. Chromatography conditions for chromatogram b: T: 40℃, RH: 66%; Qingdao Marine silica gel NaOH alkaline plate. Chromatography conditions for chromatogram c: T: 40℃, RH: 66%; Sinopharm Group silica gel G plate. Chromatography conditions for chromatogram d: T: 40℃, RH: 66%; Merck silica gel G plate (Germany).

[0094] Figure 18 These are thin-layer chromatography (TLC) results for three batches of Mahonia japonica leaf (long-column Mahonia) formulation granules. The samples included: 1. negative sample; 2. mixed reference standard; 3-5. 2 μL of Mahonia japonica leaf (long-column Mahonia) formulation granules (batch numbers: 230901-230903). Test conditions: T: 25℃, RH: 66%; Qingdao marine silica gel G plate.

[0095] Figure 19 This is a specificity chromatogram for the detection method of total mass content and total transfer rate of Mahonia japonica leaf formula granules.

[0096] Figure 20 This is a peak purity chromatogram for the detection method of total mass content and total transfer rate of the Ten Great Meringue Leaf Formula Granules.

[0097] Figure 21 The linear regression equation for the detection method of total mass content and total transfer rate of Mahonia japonica leaf formula granules was investigated.

[0098] Figure 22 The linear regression equation for palmatine hydrochloride was investigated in the detection method of total mass content and total transfer rate of Mahonia japonica leaf formula granules.

[0099] Figure 23 The linear regression equation for berberine hydrochloride was investigated in the detection method of total mass content and total transfer rate of Mahonia japonica leaf formula granules.

[0100] Figure 24This is a chromatogram showing the effect of different chromatographic columns on the determination of the content of indicator components in Mahonia japonica leaf formula granules.

[0101] Figure 25 This is a chromatogram showing the effect of different chromatographic instruments on the determination of the content of indicator components in Mahonia japonica leaf formula granules.

[0102] Figure 26 This is a chromatogram showing the effect of different column temperatures on the determination of the content of indicator components in the formula granules of Mahonia japonica leaves.

[0103] Figure 27 This is a chromatogram showing the effect of different flow rates on the determination of the content of indicator components in the formula granules of Mahonia japonica leaves. Detailed Implementation

[0104] As described above, the purpose of this invention is to provide a method for preparing and detecting an extract of Mahonia japonica leaves. The Mahonia japonica leaf extract is either a standard decoction or granules of Mahonia japonica leaves. This invention standardizes the quality control and research of the standard decoction and granules of Mahonia japonica leaves, achieving overall quality control and effective supervision of both, and providing a reference for the quality control of these products.

[0105] Standard decoctions, also known as standard liquid decoctions, are a traditional and widely used form of medication in clinical practice. Standard decoctions are prepared by following the theories of traditional Chinese medicine, using standardized decoction methods, separating solids and liquids, and then concentrating or drying them using appropriate methods. The active substances in Chinese herbal formula granules are the same as those in decoctions made from Chinese herbal decoction pieces, representing a continuation of the tradition of Chinese herbal decoction pieces. Standard decoctions can serve as a benchmark for measuring whether Chinese herbal formula granules are essentially consistent with clinical decoctions.

[0106] Standardized decoctions serve as a "bridge" connecting traditional Chinese medicine (TCM) decoction pieces and modern TCM preparations. They provide a reference for controlling the quality of TCM end products, standardizing different forms of TCM administration to ensure quality uniformity and efficacy consistency, and evaluating the consistency of product quality across different manufacturers. Therefore, the establishment of quality standards for standardized TCM decoctions will provide a foundation for the development of quality standards for all final products derived from decoction pieces. However, compared to clinical applications, basic research on these decoctions is relatively weak, with fewer reports on chemical components and insufficient comprehensive research on pharmacological effects. Furthermore, the use of different raw materials for Mahonia japonica leaves is frequently misrepresented in the market. Therefore, establishing effective and reliable methods for standardizing Mahonia japonica leaf decoctions and granules is crucial for ensuring the authenticity, accuracy, and clinical efficacy of the drugs, as well as facilitating subsequent granule preparation work.

[0107] The first objective of this invention is to provide a method for preparing a standard decoction of Mahonia japonica leaves, comprising the following steps:

[0108] (1) Take the leaves of Mahonia japonica, add water and decoct, then filter to obtain the filtrate;

[0109] (2) The filtrate from step (1) is concentrated and dried, and then freeze-dried. The freeze-drying is divided into three stages: a. Pre-freezing: the pre-freezing temperature is -50℃ to -45℃; b. First drying: the drying temperature is -30℃ to 0℃; c. Second drying for analysis: the drying temperature is 5℃ to 25℃, and the standard decoction of Mahonia japonica leaves is obtained.

[0110] Preferably, in step (1), the number of times water is added and boiled is 1 to 3.

[0111] Preferably, in step (1), the decoction is performed twice, with 12 to 18 times the amount of water added for the first decoction and 10 to 14 times the amount of water added for the second decoction.

[0112] Preferably, in step (1), the first decoction time is 30-50 minutes; the second decoction time is 25-35 minutes.

[0113] Preferably, in step (1), the filtration is performed using a 100-300 mesh sieve.

[0114] Preferably, in step (2), the concentration and drying includes the following steps: concentrating the filtrate under reduced pressure to form an extract.

[0115] Preferably, in step (2), the concentration temperature is 55-65℃ and the vacuum degree is -0.080 to -0.090 MPa.

[0116] Preferably, in step (2), the pre-freezing time is 3-6 hours; the first drying time is 10-14 hours; and the second drying time is 5-7 hours.

[0117] Preferably, the yield of the standard decoction of Mahonia japonica leaves is 11.5% to 27.8%.

[0118] The second objective of this invention is to provide a method for preparing Mahonia japonica leaf granules, which includes the following steps:

[0119] (1) Take the leaves of Mahonia japonica, add water and decoct, then filter to obtain the filtrate;

[0120] (2) Concentrate the filtrate from step (1), add the first excipient and dry to obtain dry powder, then add the second excipient and granulate.

[0121] Preferably, in step (1), the decoction is performed twice, with 10 to 18 times the amount of water added for the first decoction and 9 to 17 times the amount of water added for the second decoction.

[0122] Preferably, the first decoction time is 30-120 minutes; the second decoction time is 20-80 minutes.

[0123] And / or, the filtration is performed using a 100-300 mesh screen.

[0124] Preferably, in step (2), the concentration includes the following steps: concentrating the filtrate under reduced pressure into an extract; preferably, the concentration temperature is 60-70°C and the vacuum degree is -0.080 to -0.090 MPa.

[0125] Preferably, the extract yield is 10.0% to 20.0%.

[0126] Preferably, in step (2), the drying process is selected from vacuum drying, spray drying, microwave drying, or infrared drying.

[0127] Preferably, the drying process includes the following steps: adding a first excipient to the extract and then spray drying.

[0128] Preferably, the first excipient is maltodextrin.

[0129] Preferably, the amount of the first excipient added is 5% to 15% of the amount of Mahonia japonica leaf slices added.

[0130] Preferably, the inlet air temperature is 165–175°C and the outlet air temperature is 100–110°C.

[0131] Preferably, in step (2), the second excipient comprises silicon dioxide and / or magnesium stearate.

[0132] Preferably, the percentage of silica by mass in the dry paste powder is greater than 0 and less than or equal to 0.3%.

[0133] Preferably, the percentage of magnesium stearate in the dry paste powder is greater than 0 and less than or equal to 0.3%.

[0134] The third objective of this invention is to provide a method for detecting the total mass content and total transfer rate of indicative components in the aforementioned Mahonia japonica leaf standard decoction or Mahonia japonica leaf formula granules, comprising the following steps:

[0135] (1) Preparation of reference solution

[0136] Weigh out the reference standards of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, respectively, and prepare solutions with methanol;

[0137] (2) Preparation of the test solution

[0138] Take the standard decoction or formula granules of Mahonia japonica leaves, add the extraction solvent for extraction, weigh it, replenish the lost weight with the extraction solvent, filter, and take the filtrate to obtain the test solution.

[0139] (3) Liquid Chromatography Analysis

[0140] Using octadecylsilane-bonded silica gel as the stationary phase, mobile phase A was an aqueous solution containing potassium dihydrogen phosphate and sodium dodecyl sulfate, and mobile phase B was acetonitrile. The reference solution and test solution were injected into the liquid chromatograph, and gradient elution was employed for analysis. The detection wavelength was 340–350 nm; the flow rate was 0.8–1.2 mL / min; the column temperature was 25–35 °C; the column specifications were: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; the column was a JADE- KP-C18-AQ; The gradient elution procedure is as follows:

[0141] From 0 to 8 minutes, the volume percentage of mobile phase A decreased from 70% to 55%, while the volume percentage of mobile phase B increased from 30% to 45%.

[0142] Between 8 and 16.5 minutes, the volume percentage of mobile phase A decreased from 55% to 52.5%, while the volume percentage of mobile phase B increased from 45% to 47.5%.

[0143] Between 16.5 and 16.6 min, the volume percentage of mobile phase A increased from 52.5% to 70%, while the volume percentage of mobile phase B decreased from 47.5% to 30%.

[0144] 16.6–20 min, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%.

[0145] The fourth objective of this invention is to provide a thin-layer chromatography (TLC) identification method for Mahonia japonica leaf formula granules, comprising the preparation of a sample solution of the formula granules, TLC spotting, development, and color development. 1 g of Mahonia japonica leaf formula granules are taken, 10 mL of methanol is added, and the mixture is sonicated for 60 min, then filtered to obtain the sample solution of Mahonia japonica leaf formula granules. Toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia solution (volume ratio 4:3:3:1.2:0.5) is used as the developing solvent.

[0146] The indicative components mentioned in this invention refer to berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride.

[0147] In this invention, unless otherwise specified, all reagents used in the embodiments are conventional reagents. Information on the raw materials and experimental equipment used in the embodiments is shown in Tables 1 and 2, respectively.

[0148] Table 1. Information on raw materials used in this invention.

[0149] raw material Purity / Batch Number Manufacturer / seller Phytoalkaloid hydrochloride Batch number 110733-202110, purity 90.3%. China National Institutes for Food and Drug Control Palmatine Hydrochloride Batch number 110732-201913, purity 85.7%. China National Institutes for Food and Drug Control Berberine hydrochloride Batch number 110713-202015, purity 85.9%. China National Institutes for Food and Drug Control

[0150] Table 2 Information on the experimental equipment used in this invention

[0151]

[0152]

[0153] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0154] Example 1: Preparation method of standard decoction of Mahonia japonica leaves

[0155] (1) Take 100g of Mahonia japonica leaves (long-column Mahonia japonica) slices, weigh them, place them in a clay pot, add water and decoct twice. For the first decoction, add 15 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 40 minutes. Filter the decoction through a 200-mesh sieve while hot, and record the mass of the decoction after cooling to room temperature. For the second decoction, add 13 times the amount of water, heat to a boil over high heat, then simmer over low heat for 30 minutes. Filter the decoction through a 200-mesh sieve while hot, and record the mass of the decoction after cooling to room temperature. Combine the two decoctions. The preparation method of Mahonia japonica leaf slices includes the following steps: clean the dried Mahonia japonica leaves, select and remove non-medicinal parts, impurities and deteriorated products, etc., the impurity content shall not exceed 3%, and then cut them into 0.5-1.0cm wide strips to obtain Mahonia japonica leaf slices.

[0156] (2) The decoction was transferred to a 2000mL round-bottom flask and concentrated to 100mL of concentrate (extract) using a rotary evaporator under reduced pressure. The concentration temperature was 65℃ and the vacuum degree of concentration was -0.080 to -0.090MPa. The density and yield of the concentrate were measured. The density of the concentrate was 1.01 to 1.06g / mL and the yield was 17.15%.

[0157] Under magnetic stirring, the concentrated solution was dispensed into 10mL brown vials, each with a volume of 2mL. The vials were partially stoppered and then transferred to a vacuum freeze dryer for lyophilization. The lyophilization parameters were as follows: pre-freezing temperature -45℃, pre-freezing time 240 minutes; primary drying (sublimation drying) temperature -30℃~0℃, primary drying time 840 minutes, vacuum degree 0.2mbar (specific primary drying process: drying at -30℃ for 420min; drying at -20℃ for 120min; drying at -10℃ for 120min; drying at 0℃ for 180min); secondary drying (desorption drying) temperature 5℃~25℃, secondary drying time 420 minutes, vacuum degree 0.2mbar (specific secondary drying process: drying at 5℃ for 120min; drying at 15℃ for 120min; drying at 25℃ for 180min). The resulting Mahonia japonica leaf standard decoction product was named BT2211-3.

[0158] In this invention, the method for determining the yield includes the following steps: The constant weight of the evaporating dish is recorded as m1. 10g of the concentrated liquid is placed in the constant-weight evaporating dish, dried in a water bath, and then dried in a 105℃ drying oven for 3 hours. After cooling in a desiccator for half an hour, the liquid is quickly weighed and recorded as m2. The yield is then calculated.

[0159]

[0160] Where m2 is the total weight of the dried sample and evaporating dish, g; m1 is the weight of the evaporating dish, g; the total weight of the concentrate is the total mass of the concentrate obtained in step 2, g; the amount of concentrate sampled is the amount of concentrate sampled when determining the extract yield, g; and the amount of medicinal slices sampled is the amount of medicinal slices sampled in step 1, g.

[0161] Example 2: Establishment of an analytical method for determining the content and transfer rate of Mahonia japonica leaf in a standard decoction.

[0162] 2.1 Determination of chromatographic conditions

[0163] Chromatographic conditions and system suitability test

[0164] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); mobile phase A was a buffer salt containing 0.34 g potassium dihydrogen phosphate and 0.17 g sodium dodecyl sulfate per 100 mL (pH adjusted to 3.0 with phosphoric acid); mobile phase B was acetonitrile. Gradient elution was performed according to the specifications in the table below; the flow rate was 1 mL / min; the column temperature was 30 °C; and the detection wavelength was 346 nm. The theoretical plate number, calculated based on the anthocyanin hydrochloride, should be no less than 3000.

[0165] Table 3 Gradient elution program

[0166] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0-8 70→55 30→45 8-16.5 55→52.5 45→47.5 16.5-16.6 52.5→70 47.5→30 16.6-20 70 30

[0167] The standard decoction of Mahonia japonica leaves (Mahonia longifolia) was obtained under the above chromatographic detection conditions: containing hydrochloric acid root alkaloid (C 20 H 20 ClNO4), palmatine hydrochloride (C 21 H 22 ClNO4) and berberine hydrochloride (C 20 H 18 The total amount of ClNO4 was 0.9947 mg / g. The average transfer rate of total amounts of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the standard decoction of Mahonia japonica leaves (Mahonia longifolia) was 33.02%.

[0168] The formula for calculating the total mass content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride is as follows:

[0169]

[0170] Among them, c YGJ对 A represents the concentration of cypermethrin hydrochloride in the reference standard, in μg / mL; YGJ供 A represents the peak area of ​​purslane hydrochloride in the test sample; YGJ对 c is the peak area of ​​cypermethrin hydrochloride in the reference standard; BMT对 A represents the concentration of palmatine hydrochloride in the reference standard, in μg / mL; BMT供 A represents the peak area of ​​palmatine hydrochloride in the test sample; BMT对 c is the peak area of ​​palmatine hydrochloride in the reference standard; XBJ对 A represents the concentration of berberine hydrochloride in the reference standard, in μg / mL; XBJ供 A represents the peak area of ​​berberine hydrochloride in the test sample. XBJ对 20 represents the peak area of ​​berberine hydrochloride in the reference standard; 20 is the volume of the solvent extracted during the treatment of the test sample, 20 mL; m 样 The sample weight is in μg.

[0171] The formula for calculating the total transfer rate of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride is as follows:

[0172]

[0173] 2.2 Preparation of reference solution

[0174] Take appropriate amounts of berberine hydrochloride reference standard, palmatine hydrochloride reference standard and berberine hydrochloride reference standard, accurately weigh them, and prepare a mixed reference standard solution containing 4 μg of berberine hydrochloride, 4 μg of palmatine hydrochloride and 34 μg of berberine hydrochloride per 1 mL.

[0175] 2.3 Preparation of the test solution

[0176] 2.3.1 Investigation of extraction solvent

[0177] Take an appropriate amount of standard decoction of Mahonia japonica leaves (long-columned Mahonia japonica) (BT2211-3), a total of 7 portions, with 2 parallel samples for each portion. Place each sample in a stoppered conical container and accurately add 20 mL of hydrochloric acid-methanol (volume ratio 1:100), hydrochloric acid-75% methanol (1:100), hydrochloric acid-50% methanol (1:100), hydrochloric acid-ethanol (1:100), hydrochloric acid-75% ethanol (1:100), hydrochloric acid-50% ethanol (1:100), and hydrochloric acid-water (1:100) in sequence. Weigh the sample, sonicate for 30 min (power 500W, frequency 40kHz), cool, add the appropriate solvent to make up the weight, mix well, filter, and collect the filtrate to obtain the solution of each test sample. The chromatographic analysis was performed under the above conditions, with an injection volume of 10 μL. The effects of different extraction solvents on the total content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride (total alkaloid content) were calculated to determine the optimal extraction solvent. The experimental results were calculated using the external standard one-point method. Detailed results are shown in the table below:

[0178] Table 4. Effects of different extraction solvents on the determination of total alkaloid content in standard decoction of Mahonia japonica leaves (Mahonia longifolia).

[0179]

[0180]

[0181] As shown in Table 4, different solvents have a significant effect on the total alkaloid content in the standard decoction of Mahonia japonica leaves (Mahonia longifolia). Considering the content, hydrochloric acid-methanol (volume ratio of 1:100) was selected as the extraction solvent for further research.

[0182] 2.3.2 Examination of Extraction Methods

[0183] Take an appropriate amount of the standard decoction of Mahonia japonica leaves (long-column Mahonia japonica) (BT2211-3), three times in total, with two parallel samples per sample. Place each sample in a stoppered conical container, and precisely add 20 mL of hydrochloric acid-methanol (volume ratio 1:100) sequentially. Weigh the samples, and treat them by shaking, sonication, or reflux for 30 min respectively. After cooling to room temperature, add hydrochloric acid-methanol (volume ratio 1:100) to make up the weight, mix well, filter, and collect the filtrate. Filter an appropriate amount of the filtrate through a 0.22 μm microporous membrane and place it in a high-volume sample bottle to obtain the test solutions. Perform chromatographic analysis under the above conditions, with an injection volume of 10 μL. Calculate the effect of different extraction methods on the content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, based on the total amount of these compounds, and determine the optimal extraction method. The experimental results are calculated using the external standard single-point method. Detailed results are shown in the table below.

[0184] Table 5. Effects of different extraction methods on the determination of total alkaloid content in standard decoction of Mahonia japonica leaf (Mahonia longifolia).

[0185]

[0186]

[0187] As shown in Table 5, different extraction methods have no significant effect on the total alkaloid content in the standard decoction of Mahonia japonica leaves (Mahonia longifolia). Considering the ease of operation and the differences in content, ultrasonic extraction was selected as the extraction method for further research.

[0188] 2.3.3 Examination of extraction time

[0189] Take an appropriate amount of the standard decoction of Mahonia japonica leaves (long-column Mahonia japonica) (BT2211-3), a total of 4 portions, with 2 parallel samples for each portion. Place each portion in a stoppered conical container, and accurately add 20 mL of hydrochloric acid-methanol (volume ratio 1:100) sequentially. Weigh the samples, and sonicate them (power 500W, frequency 40kHz) for 15 min, 30 min, 45 min, and 60 min respectively. After cooling to room temperature, add hydrochloric acid-methanol (volume ratio 1:100) to make up the weight, mix well, filter, and collect the filtrate. Take an appropriate amount of the filtrate and filter it through a 0.22 μm microporous membrane, and place it in a high-liquid sample bottle to obtain the test solution for each sample. Analyze the sample under the above chromatographic conditions, with an injection volume of 10 μL. Calculate the effect of different extraction times on the content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, based on the total amount of berberine hydrochloride, to determine the optimal extraction time. The experimental results were calculated using the external standard one-point method. Detailed results are shown in the table below:

[0190] Table 6. Effects of different extraction times on the determination of total alkaloid content in standard decoction of Mahonia japonica leaves (Mahonia longifolia).

[0191]

[0192] The experimental results in Table 6 show that the effects of different extraction times on the total content of the three alkaloids in the standard decoction of Mahonia japonica leaves (Mahonia japonica var. longifolia) are not significant. Considering the time cost and the content difference, ultrasonic extraction for 45 min was selected for subsequent research.

[0193] 2.3.4 Determination of the preparation method for the test solution

[0194] Based on the results of the sample pretreatment experiment, the preparation method of the test sample can be determined as follows: Take about 0.4g of standard decoction of Mahonia japonica leaves (long-column Mahonia japonica), accurately weigh it, place it in a stoppered conical flask, accurately add 20mL of hydrochloric acid-methanol (volume ratio of 1:100), weigh it, sonicate for 45min (power 500W, frequency 40kHz), cool it, make up the weight with hydrochloric acid-methanol (volume ratio of 1:100), filter and collect the filtrate, filter it through a 0.22μm microporous membrane, and place it in a high-liquid sample bottle to obtain the test sample solution.

[0195] 2.4 Validation of content determination method

[0196] 2.4.1 Specificity Examination

[0197] Accurately pipette 10 μL each of the test solution, mixed reference solution, and blank solvent of the standard decoction of Mahonia japonica leaves (long-column Mahonia japonica) (BT2211-3), and perform chromatographic analysis according to the chromatographic conditions in section 2.1. Record the chromatographic elution chromatograms. Results are shown below. Figure 1 .Depend on Figure 1 It can be seen that the chromatographic detection and analysis method is specific for berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the standard decoction of Mahonia japonica leaf (long-column Mahonia japonica).

[0198] 2.4.2 Peak Details

[0199] Accurately pipette 10 μL each of the test solution and reference solution of Mahonia japonica leaf (long-column Mahonia japonica) standard decoction (BT2211-3) and inject them into the ultra-high performance liquid chromatograph for determination. The peak purity of the target peak is then obtained. The results are shown in the table below.

[0200] Table 7 Matching values ​​for target peaks and peak purity

[0201] Target Peak Phytoalkaloid hydrochloride Palmatine Hydrochloride Berberine hydrochloride Peak purity matching value 999.98 997.15 999.99 Peak separation 30.8145 4.6180 3.3281 Theoretical number of plates 110506.6073 71165.86866 100991.08568

[0202] As shown in Table 7, the peak purity matching values ​​of the index components, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, are all >995, indicating that their peak purity meets the analytical requirements.

[0203] 2.4.3 Linear

[0204] Accurately weigh appropriate amounts of cypermethrin hydrochloride, palmatine hydrochloride, and berberine hydrochloride reference standards, place them separately in 50 mL volumetric flasks, dissolve them in methanol, dilute to the mark, and then further dilute to obtain a stock solution of reference standards containing 17.28 μg of cypermethrin hydrochloride, 18.61 μg of palmatine hydrochloride, and 139.98 μg of berberine hydrochloride per milliliter.

[0205] The stock solutions of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride reference standards were diluted by 100, 50, 20, 10, 4, 2, and 1 times, respectively, to obtain reference solutions of different concentrations of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. These solutions were then analyzed under the chromatographic conditions described in Section 2.1. The linear ranges of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride were investigated by plotting concentration on the x-axis and peak area on the y-axis. The linearity results are shown in the table below. Figures 2-4 .

[0206] Table 8. Linearity Study of Hydrochloric Acid as a Radix Alkaloid

[0207] Reference Standard Number Concentration of herbicide hydrochloride (μg / mL) Peak area (mAU) Phytoalkaloid-1 hydrochloride 0.1728 9.566 Phytoalkaloid-2 hydrochloride 0.3455 17.290 Phytoalkaloid-3 hydrochloride 0.8638 41.517 Phytoalkaloid-4 hydrochloride 1.7276 82.029 Phytoalkaloid-5 hydrochloride 4.3189 207.063 Phytoalkaloid-6 hydrochloride 8.6378 408.475 Phytoalkaloid-7 hydrochloride 17.2756 794.464

[0208] Table 9. Linearity Study of Parmatin Hydrochloride

[0209] Reference Standard Number Palmatine hydrochloride concentration (μg / mL) Peak area (mAU) Palmatine-1 Hydrochloride 0.1861 9.662 Palmatine-2 hydrochloride 0.3722 17.624 Palmatine-3 Hydrochloride 0.9305 43.117 Palmatine-4 hydrochloride 1.8610 84.910 Palmatine-5 Hydrochloride 4.6525 216.204 Palmatine-6 ​​Hydrochloride 9.3050 419.303 Palmatine-7 Hydrochloride 18.6099 825.893

[0210] Table 10 Linearity Study of Berberine Hydrochloride

[0211]

[0212]

[0213] From Tables 8-10 and Figures 2-4 The experimental results show that: the concentration of berberine hydrochloride and the peak area value have a good linear relationship in the range of 0.1728–17.2756 μg / mL, with a correlation coefficient r = 0.9998; the concentration of palmatine hydrochloride and the peak area value have a good linear relationship in the range of 0.1861–18.6099 μg / mL, with a correlation coefficient r = 0.9999; and the concentration of berberine hydrochloride and the peak area value have a good linear relationship in the range of 1.3998–139.9826 μg / mL, with a correlation coefficient r = 0.9997.

[0214] 2.4.4 Precision Test

[0215] 2.4.4.1 Instrument precision test

[0216] Accurately pipette the test solution of Mahonia japonica leaf (long-column Mahonia japonica) standard decoction (BT2211-3) and the mixed reference solution of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. Perform chromatographic analysis according to the conditions described in section 2.1, with an injection volume of 10 μL and 6 parallel injections. Calculate the RSD (%) of the target peak using the external standard single-point method, based on the total content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. See the table below for detailed results.

[0217] Table 11. Results of Instrument Precision Test for Determination of Content in Standard Decoction of Mahonia Leaf (Mahonia longifolia)

[0218]

[0219] The experimental results in Table 11 show that the RSD (%) of the total content of the target peaks is 0.57% < 3.0%, indicating that the method has good precision.

[0220] 2.4.4.2 Repeatability Test

[0221] Take approximately 0.40 g of the same batch of Mahonia japonica leaf (long-column Mahonia japonica) standard decoction (BT2211-3), accurately weigh it, and prepare 6 parallel test solutions for later use. Perform chromatographic analysis under the conditions described in section 2.1. Calculate the RSD (%) of the target peak content using the external standard single-point method, based on the total content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the test solution. See the table below for detailed results.

[0222] Table 12. Repeatability Test Results of the Standard Decoction Content Determination Method for Mahonia Leaf (Long-columned Mahonia)

[0223]

[0224] The experimental results in Table 12 show that the RSD (%) of the total content of the target peaks is 0.75% < 3.0%, indicating that the method has good repeatability.

[0225] 2.4.4.3 Intermediate Precision

[0226] Other analysts in this project team operated on different dates and under different chromatographs, taking approximately 0.40 g of the same batch of Mahonia japonica leaf (long-column Mahonia japonica) standard decoction (BT2211-3), accurately weighed, and prepared in 6 parallel portions. The test solution was prepared according to the test solution preparation method, and the chromatographic conditions in section 2.1 were used for detection. The RSD (%) value of the target peak was calculated using the external standard one-point method based on the total content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. The specific results are shown in the table below.

[0227] Table 13. Intermediate Precision Test Results of the Method for Determining the Content of Mahonia Leaf (Mahonia longifolia) in Standard Decoction

[0228]

[0229]

[0230] The experimental results in Table 13 show that the intermediate precision RSD (%) of the total alkaloid content is 2.02% < 6.0%, indicating that the intermediate precision of this method is good.

[0231] 2.4.5 Accuracy Test

[0232] Nine accurate portions of a standard decoction of Mahonia japonica leaves (Berberis longifolia) (BT2211-3, containing 0.0065% berberine hydrochloride, 0.0089% palmatine hydrochloride, and 0.0802% berberine hydrochloride) were weighed. Five mL of reference solutions containing 50%, 100%, and 150% of the tested sample content of each berberine, palmatine, and berberine hydrochloride were added to each portion, followed by 5 mL of hydrochloric acid-methanol (volume ratio 1:100). Chromatography was performed under the above conditions, with 10 μL injected into each sample. The content of the target peaks for berberine, palmatine, and berberine hydrochloride was calculated using the external standard single-point method. The recoveries and RSDs were calculated using the following formulas, and the results are shown in the table below.

[0233]

[0234] Among them, the measured amount refers to the content of the index component measured after adding the index component to the standard decoction, in μg; the spiking amount refers to the content of the index component added to the standard decoction, in μg; and the sample content refers to the content of the index component in the standard decoction, in μg.

[0235] Table 14. Determination of the content of Mahonia japonica leaf (Mahonia longifolia) in standard decoction (hydrochloric acid root alkaloid) - Results of spiking and recovery experiments.

[0236]

[0237]

[0238] Table 15. Determination of the content of Mahonia japonica leaf (Mahonia longifolia) in standard decoction (palmatine hydrochloride) by spiking and recovery test results.

[0239]

[0240] Table 16. Determination of Berberine Hydrochloride Content in Standard Decoction of Mahonia japonica Leaf (Mahonia longifolia) - Results of Spiking and Recovery Experiment

[0241]

[0242]

[0243] The experimental results in Tables 14-16 show that the recoveries of berberine hydrochloride and palmatine hydrochloride at low, medium, and high concentrations in the standard decoction are all within the range of 85%-110%; the recoveries of berberine hydrochloride at low, medium, and high concentrations in the standard decoction are all within the range of 90%-108%, indicating that the accuracy of the assay method is good.

[0244] 2.4.6 Stability Test

[0245] Take the test solution of the standard decoction of Mahonia japonica leaves (long-column Mahonia japonica) (BT2211-3), and inject it at 0, 2, 4, 6, 8, 10, 12 and 24 hours according to the chromatographic conditions in section 2.1, with an injection volume of 10 μL. Calculate the RSD (%) of the target peak content using the external standard one-point method, based on the total content of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride. The specific results are shown in the table below.

[0246] Table 17. Stability Test Results of the Method for Determining the Content of Mahonia Leaf (Long-columned Mahonia) in Standard Decoction

[0247]

[0248] The experimental results in Table 17 show that the RSD (%) of the target peak content within 24 hours is 1.24% < 3.0%, indicating that the solution has good stability within 24 hours.

[0249] 2.4.7 Durability Assessment

[0250] 2.4.7.1 Investigation of different chromatographic columns

[0251] Comparison of Taiwei Technology's JADE- The effects of three chromatographic columns—KP-C18-AQ (4.6mm×250mm, 5μm), Diamonsil C18 (4.6mm×250mm, 5μm), and Waters XSelect HSS T3 (4.6mm×250mm, 5μm)—on the peak shape and resolution of cyperine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the standard decoction of Mahonia japonica leaves (long column Mahonia japonica).

[0252] Take the standard decoction (BT2211-3) of Mahonia japonica leaves (Long-columned Mahonia japonica) as the test solution, and determine it according to the chromatographic conditions in Part 2.1. Analyze the three index components: berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. Record the chromatographic data as shown in Table 18 below. Figure 5 As shown.

[0253] Table 18. Effects of different chromatographic columns on the determination of target component content in standard decoction of Mahonia japonica leaves (long-column Mahonia japonica).

[0254]

[0255] From Table 18 and Figure 5 The experimental results show that different chromatographic columns have good durability. Considering both the target chromatographic peak shape and resolution, the JADE-1000 column was selected. KP-C18-AQ.

[0256] 2.4.7.2 Investigation of different chromatographs

[0257] Based on the existing equipment in the laboratory, Thermo ultra-high performance liquid chromatograph and Agilent ultra-high performance liquid chromatograph were selected to compare the effects of the two chromatographs on the peak shape and resolution of chromatographic peaks of purslane hydrochloride, palmatine hydrochloride and berberine hydrochloride in the standard decoction of Mahonia japonica leaves (long-column Mahonia japonica).

[0258] The standard decoction (BT2211-3) of Mahonia japonica leaves (Long-columned Mahonia japonica) was used as the test sample solution. Chromatographic analysis was performed under the conditions described above, focusing on three index components: berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. Chromatographic data were recorded. The experimental results are shown in Table 19 below. Figure 6 As shown.

[0259] Table 19. Effects of different chromatograms on the determination of target component content in standard decoction of Mahonia japonica leaf (Mahonia longifolia).

[0260]

[0261] From Table 19 and Figure 6 The experimental results show that different chromatographs have good durability, and the changes in chromatographs can meet the system adaptability requirements.

[0262] 2.4.7.3 Investigation at different column temperatures

[0263] The effects of different column temperatures (25℃, 30℃, and 35℃) on the peak shape and resolution of chromatographic peaks of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the standard decoction of Mahonia japonica leaves (long-column Mahonia japonica) were compared.

[0264] The standard decoction (BT2211-3) of Mahonia japonica leaves (Long-columned Mahonia japonica) was used as the test sample solution. Chromatographic analysis was performed under the conditions described in section 2.1, focusing on three index components: berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. Chromatographic data were recorded. The experimental results are shown in Table 20 below. Figure 7 As shown.

[0265] Table 20 Results of the determination of the content of Mahonia japonica leaf (Mahonia longifolia) in standard decoction at different column temperatures

[0266]

[0267] From Table 20 and Figure 7 The experimental results show that the peak shape and separation effect are good at all three column temperatures. At 30℃, the baseline of the chromatogram shows no drift, and the retention time is not significantly different compared to the other two temperatures. Considering the column's tolerance and the analysis time required, a column temperature of 30℃ was chosen.

[0268] 2.4.7.4 Investigation of different flow velocities

[0269] The effects of different flow rates of 0.80 mL / min, 1.00 mL / min and 1.20 mL / min on the peak shapes of cyperine, palmatine hydrochloride and berberine hydrochloride in the standard decoction of Mahonia japonica leaf (Mahonia longifolia) were compared.

[0270] The standard decoction (BT2211-3) of Mahonia japonica leaves (long-columned Mahonia japonica) was used as the test sample solution. Chromatographic analysis was performed under the conditions described in section 2.1, and the results were recorded as berberine hydrochloride, palmatine hydrochloride, and phytorepinephrine hydrochloride. The experimental results are shown in Table 21 below. Figure 8 As shown.

[0271] Table 21 Results of the determination of the content of Mahonia japonica leaf (Mahonia longifolia) in standard decoction at different flow rates

[0272]

[0273] From Table 21 and Figure 8 The experimental results show that the peak shape and separation effect are good at all three flow rates. The separation degree of each component is better and the baseline is not drifted at a flow rate of 1.00 mL / min. Therefore, the flow rate of 1.00 mL / min was selected for this experiment.

[0274] Example 3: Preparation method of standard decoction of Mahonia japonica leaves

[0275] (1) Take 100g of Mahonia japonica leaves (long-column Mahonia japonica) slices, weigh them, place them in a clay pot, add water and decoct twice. For the first decoction, add 12 times the amount of water directly, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 40 minutes. Filter the decoction through a 200-mesh sieve while hot, and record the mass of the decoction after cooling to room temperature. For the second decoction, add 10 times the amount of water, heat to a boil over high heat, then simmer over low heat for 30 minutes. Filter the decoction through a 200-mesh sieve while hot, and record the mass of the decoction after cooling to room temperature. Combine the two decoctions.

[0276] (2) The decoction was transferred to a 2000mL round-bottom flask and concentrated to 100mL under reduced pressure using a rotary evaporator. The concentration temperature was 65℃ and the vacuum degree was -0.080 to -0.090MPa. The density of the concentrated solution was measured to be 1.01 to 1.06g / mL, and the yield was 20.7%.

[0277] Under magnetic stirring, the concentrate was dispensed into 10mL brown vials, each with a volume of 2mL. The vials were partially stoppered and then transferred to a vacuum freeze dryer for freeze drying. The freeze drying parameters were the same as in Example 1. A standard decoction of Mahonia japonica leaves was obtained.

[0278] (3) Determination methods for the total mass content and total transfer rate of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the standard decoction of Mahonia japonica leaves.

[0279] The total mass content and total transfer rate of cypermethrin hydrochloride, palmatine hydrochloride and berberine hydrochloride were determined according to the detection method described in Example 2. The results were as follows: the total mass content of cypermethrin hydrochloride, palmatine hydrochloride and berberine hydrochloride was 0.357 mg / g, and the total transfer rate was 24.74%.

[0280] Example 4: Preparation method of standard decoction of Mahonia japonica leaves

[0281] (1) Take 100g of Mahonia japonica leaves (long-column Mahonia japonica) slices, weigh them, place them in a clay pot, add water and decoct twice. For the first decoction, add 18 times the amount of water directly, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 40 minutes. Filter the decoction through a 200-mesh sieve while hot, and record the mass of the decoction after cooling to room temperature. For the second decoction, add 16 times the amount of water, heat to a boil over high heat, then simmer over low heat for 30 minutes. Filter the decoction through a 200-mesh sieve while hot, and record the mass of the decoction after cooling to room temperature. Combine the two decoctions.

[0282] (2) The decoction was transferred to a 2000mL round-bottom flask and concentrated to 100mL under reduced pressure using a rotary evaporator. The concentration temperature was 65℃ and the vacuum degree was -0.080 to -0.090MPa. The density and yield of the concentrate were measured. The density of the concentrate was 1.01 to 1.06g / mL and the yield was 21.2%.

[0283] Under magnetic stirring, the concentrate was dispensed into 10mL brown vials, each with a volume of 2mL. The vials were partially stoppered and then transferred to a vacuum freeze dryer for freeze drying. The freeze drying parameters were the same as in Example 1. A standard decoction of Mahonia japonica leaves was obtained.

[0284] (3) Determination methods for the total mass content and total transfer rate of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the standard decoction of Mahonia japonica leaves.

[0285] The total mass content and total transfer rate of cypermethrin hydrochloride, palmatine hydrochloride and berberine hydrochloride were determined according to the detection method described in Example 2. The results were as follows: the total mass content of cypermethrin hydrochloride, palmatine hydrochloride and berberine hydrochloride was 0.497 mg / g, and the total transfer rate was 35.21%.

[0286] Example 5: Preparation method of Mahonia japonica leaf formula granules

[0287] (1) Extraction and Concentration: Take the leaves of Mahonia japonica (long-column Mahonia japonica) and decoct them twice with water. Soak the leaves for 30 minutes before decoction. For the first extraction, add 18 times the amount of water, heat to boiling, and maintain a gentle boil for 30 minutes. Filter (200 mesh). For the second extraction, add 17 times the amount of water, heat to boiling, and maintain a gentle boil for 20 minutes. Filter (200 mesh). The extraction temperature is 95-105℃.

[0288] Vacuum concentration was performed at a temperature range of 60–70℃ and a vacuum degree of -0.080–-0.090 MPa. The filtrate was concentrated under reduced pressure to a clear extract with a relative density of 1.01–1.06 g / mL (45±5℃).

[0289] (2) Drying and powder collection: Add 10% to 15% of the total weight of the medicinal slices to the clear paste, mix well, pass through a No. 6 sieve (100 mesh), spray dry, with an inlet air temperature of 165 to 175°C and an outlet air temperature of 100 to 110°C, collect the powder, and the dry paste powder yield is 10.0% to 20.0%.

[0290] (3) Mixing and granulation of ingredients: Add magnesium stearate and silicon dioxide of ≤0.3% by weight of intermediate (dry paste powder containing excipients), sieve (80 mesh), mix well, granulate, and make 1000g.

[0291] Table 22 Summary of Research Data Results

[0292]

[0293]

[0294] An Investigation into the Preparation Process of Mahonia japonica Leaf Formula Granules

[0295] 5.1 Selection of Extraction Process Parameters

[0296] 5.1.1 Single-factor experiment

[0297] 5.1.1.1 Examination of soaking time

[0298] Approximately 100g of medicinal slices were weighed and divided into four portions. Each portion was soaked in 15 times its volume of water for different times (0 min, 30 min, 60 min, and 90 min), then decocted for 40 min. Each portion was then decocted again in 13 times its volume of water for 30 min. The filtrates were combined to investigate the effect of soaking time on the extraction content and yield of key components in Mahonia japonica leaves (Mahonia longifolia). The results are shown in Table 23. Figure 9 As shown.

[0299] Table 23. Effects of different soaking times on the extraction content and yield of index components in Mahonia japonica leaves (Mahonia longifolia).

[0300]

[0301]

[0302] Depend on Figure 9 As shown in Table 22, the content and yield of extract are both at their maximum when the soaking time is 30 minutes, indicating the best effect. Therefore, the soaking time is set to 30 minutes.

[0303] 5.1.1.2 Investigation of the water addition ratio

[0304] Five portions of approximately 100g of medicinal slices were weighed. For the first treatment, 10, 12, 14, 16, and 18 times the amount of water were added, and the slices were soaked for 30 minutes and decocted for 40 minutes. For the second treatment, 9, 11, 13, 15, and 17 times the amount of water were added, and the slices were decocted for 30 minutes. The filtrates were combined, and the effect of the water addition ratio on the extraction content and yield of key components in Mahonia japonica leaves (Mahonia longifolia) was investigated. The results are as follows. Figure 10 See Table 24.

[0305] Table 24. Effects of different water addition ratios on the extraction content and yield of index components in Mahonia japonica leaves (Mahonia longifolia).

[0306] Factor Level Content (mg / g) Extract yield (%) (10,9) 0.3149 19.72 (12,11) 0.3569 20.74 (14,13) 0.3886 20.96 (16,15) 0.4966 21.21 (18,17) 0.4966 19.60

[0307] Depend on Figure 10 As shown in Table 23, the content increases with the increase of the water addition ratio. When the water addition ratio is (16, 15), the content extraction reaches its maximum value and then tends to level off. The yield of extract is also at its maximum when the water addition ratio is (16, 15). Therefore, the water addition ratio is set to 16 times the amount of water added for the first time and 15 times the amount of water added for the second time.

[0308] 5.1.1.3 Examination of decoction time

[0309] Approximately 100g of the medicinal slices were weighed and divided into four portions. For the first soaking, 16 times the amount of water was added, and the slices were decocted for 30 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes respectively. For the second soaking, 15 times the amount of water was added, and the slices were decocted for 20 minutes, 40 minutes, 60 minutes, and 80 minutes respectively. The filtrates were combined, and the effect of decoction time on the extraction content and yield of key components in Mahonia japonica leaves (Mahonia longifolia) was investigated. The results are as follows: Figure 11 See Table 25.

[0310] Table 25. Effects of different decoction times on the extraction content and yield of key components in Mahonia japonica leaves (Mahonia longifolia).

[0311]

[0312]

[0313] Depend on Figure 11 As shown in Table 24, the content gradually decreased and the yield of extract gradually increased with the increase of decoction time, but the overall yield of extract did not change significantly. Therefore, the decoction time was set at 30 minutes for the first decoction and 20 minutes for the second decoction.

[0314] 5.1.2 Orthogonal Experiment

[0315] 5.1.2.1 Orthogonal Experimental Design and Results

[0316] Based on the single-factor experiments, three factors were selected: soaking time (A), water addition ratio (B), and decoction time (C), as shown in the table below. The experiment was conducted according to the orthogonal experimental design table. Using the comprehensive weighted scoring method, the relative deviation (RD) and absolute value (SR) of the extract yield (%) and content (mg / g) were calculated. The entropy weight method was used to assign different weight coefficients to different index values ​​of the extraction process. Nine orthogonal experiments were designed, with a maximum score of 100. The scores of each index in each experiment were multiplied by the corresponding weight coefficient and then summed to obtain the comprehensive score. The orthogonal experimental results were calculated using the comprehensive score, and variance analysis was performed. The results are shown in the table below. (RD) i,j =|X i,j -S j | / S j ;SR i,j =1 - RD i,j )

[0317] Table 26 Factor Level Table

[0318]

[0319] Table 27 Orthogonal Experimental Design Table

[0320]

[0321] Table 28 Summary of Weight Calculation Results Using Entropy Method

[0322] Information entropy value e Information utility value d Weighting coefficient w Content (mg / g) 0.9978 0.0022 76.69％ Extract yield (%) 0.9993 0.0007 23.31％

[0323] Table 29 Evaluation of Orthogonal Experimental Design Results

[0324]

[0325] Table 30 Range Analysis of Orthogonal Experiments

[0326] A (Soaking time / min) B (water dilution ratio / mL) C (cooking time / min) K1 89.42 82.69 92.58 K2 89.77 88.70 84.69 K3 84.18 91.98 86.11 R 5.59 9.29 7.89

[0327] Table 31 Multifactor ANOVA Table for Orthogonal Experiments

[0328] sum of squares df Mean Square F p intercept 69367.269 1 69367.269 1747.625 0.001** A 58.803 2 29.401 0.741 0.574 B 132.979 2 66.489 1.675 0.374 C 106.163 2 53.082 1.337 0.428 residual 79.385 2 39.692

[0329] Using the comprehensive scoring results as the evaluation index, intuitive analysis and analysis of variance show that the order of factors affecting the extraction effect is: B>C>A, i.e., water addition ratio>decoction time>soaking time. After single-factor investigation, the selected soaking time, water addition ratio, and decoction time parameters had no significant effect on the extraction effect (P>0.05). Therefore, the optimal extraction process combination obtained from the entropy weight method comprehensive scoring and intuitive analysis is: A2B3C1 (orthogonal 6), i.e., the first water addition is 18 times the volume of water, soaking for 30 minutes, decoction for 30 minutes, and the second water addition is 17 times the volume of water, with a decoction time of 20 minutes.

[0330] 5.1.2.12 Verification Experiment

[0331] Table 32 Validation test results of the extraction process

[0332]

[0333] Based on the optimal extraction process parameters obtained from single-factor and orthogonal experiments, three parallel experiments were conducted to verify the results, as shown in Table 32 above. The RSD of both the content and the yield was <3.0%, indicating that the process is stable and feasible.

[0334] 5.2 Selection of Solid-Liquid Separation Method

[0335] Based on the actual situation of large-scale production, the ease of filtration of different mesh sizes of the extract was observed and compared, and the results are shown in Table 33.

[0336] Table 33 Observation Results of Filtration Difficulty of Mahonia Leaf (Long-columned Mahonia) with Different Filter Mesh Sizes

[0337]

[0338] As shown in Table 33, after the extract was filtered through 100, 200 and 300 mesh, the filtrate was relatively clear except for the 100 mesh filtration, with no obvious insoluble impurities, and all achieved good solid-liquid separation effect. However, the 300 mesh filtration was slower. Referring to the separation method of the standard soup and combined with the actual situation of large-scale production, the confirmed solid-liquid separation method was hot filtration with a 200 mesh sieve.

[0339] 5.3 Research on Drying Process

[0340] Take an appropriate amount of the concentrated extract and divide it into 4 equal portions, each weighing 1 kg (each portion is equivalent to 5 kg of medicinal slices). Select reasonable spray drying process parameters according to the design scheme in the table below and conduct an experiment to investigate the amount of spray drying excipients added. The results are shown in Table 34.

[0341] Table 34 Results of Investigation on the Dosage of Excipients for Spray Drying Concentrated Extract of Mahonia Leaf (Mahonia longifolia)

[0342]

[0343] The experimental results in Table 34 show that without excipients, the product tends to stick to the drying wall and is difficult to dry. However, adding maltodextrin (10.0%–15.0% of the raw material weight) facilitates drying, resulting in lower moisture content and better drying performance. This indicates that adding a certain amount of maltodextrin to the concentrated extract of Mahonia japonica leaves (long-column Mahonia japonica) significantly improves the spray drying effect and makes drying easier. Based on the observations of different excipient addition ratios, adding maltodextrin at 10.0%–15.0% of the raw material weight during the drying process is the most reasonable approach.

[0344] 5.4 Molding Process Research

[0345] Given that a certain proportion of maltodextrin has been added to the dry extract powder of Mahonia japonica leaf (long-column Mahonia japonica) formulation granules, and considering that the flowability of Mahonia japonica leaf (long-column Mahonia japonica) intermediate (dry extract powder) affects the efficiency of dry granulation and the effect of the prepared formulation granules on the difference in filling volume during packaging, silica and magnesium stearate are selected as flow aids to prepare traditional Chinese medicine preparations. This experiment used Mahonia japonica leaf (long-column Mahonia) intermediate to investigate the dosage of excipients and granulation process. Each time, 1.0 kg of Mahonia japonica leaf (long-column Mahonia) intermediate dry powder was taken. Excipients were added according to the experimental design ratio, thoroughly mixed, and then placed in a GZL100-30L dry granulator to investigate the effects of process parameters such as excipient dosage, roller speed, and pressure on the granulation effect. A sieve mesh of 12-40 mesh was used for granulation. The dosage of excipients and granulation process were evaluated based on indicators such as particle size, flowability, first-pass yield, bulk density, solubility, and fine powder rate. The selection of excipient dosage for the Mahonia japonica leaf (long-column Mahonia) granulation process is shown in Table 35.

[0346] Table 35. Examination of Granulation Process Parameters for Mahonia Leaf (Long Column Mahonia)

[0347]

[0348] The experimental results in Table 35 show that adding a certain amount of flow aid before dry granulation of the Mahonia japonica leaf (Mahonia longifolia) intermediate can significantly improve the flowability of the granules. Adding 0.1% and 0.3% of excipients both resulted in good granulation effects.

[0349] Example 6: Thin-layer chromatographic identification of Mahonia japonica leaf granules

[0350] 6.1 Instruments, Reagents and Chemicals

[0351] Instruments: Automated Thin-Layer Imaging System (TLC VISUALIZER2, CAMAG, Switzerland), Dual-Cavity Development Tank, Automated Thin-Layer Spotting System (AUTOMATIC TLC SAMPLER4, CAMAG, Switzerland), mL-204 Electronic Balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), Ultrasonic Cleaner (KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd.), Silica G Thin-Layer Plates (10cm×10cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20220507), Silica G Thin-Layer Plates (10cm×10cm, Sinopharm Chemical Reagent Co., Ltd., batch number: 20200110), Silica G Thin-Layer Plates (10cm×10cm, Merck AG), Silica G High-Efficiency Plates (10cm×10cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20190602)

[0352] Reagents: Toluene (Chongqing Chuandong Chemical Co., Ltd., batch number: 20230401), ethyl acetate (Chongqing Chuandong Chemical Co., Ltd., batch number: 20221001), methanol (Chongqing Chuandong Chemical Co., Ltd., batch number: 20220401), isopropanol (Chongqing Chuandong Chemical Co., Ltd., batch number: 2023082801), concentrated ammonia (Chongqing Chuandong Chemical Co., Ltd., batch number: 20230701).

[0353] Test reagents: Three batches of Mahonia japonica leaf (Mahonia longifolia) formula granules (batch numbers: 230901, 230902 and 230903, formula granules prepared in Example 5); negative sample of Mahonia japonica leaf (Mahonia longifolia) formula granules (maltodextrin, MD15 type: 210201); reference standards berberine hydrochloride (batch number 110733-202110, purity 90.3%), palmatine hydrochloride (batch number 110732-201913, purity 85.7%), and berberine hydrochloride (batch number 110713-202015, purity 85.9%), all purchased from the China National Institutes for Food and Drug Control.

[0354] 6.2 Solution Preparation

[0355] 6.2.1 Preparation of the test solution

[0356] Take 1g of Mahonia japonica leaf (long-column Mahonia japonica) formula granules, grind them into a fine powder, add 10mL of methanol, sonicate for 60 minutes, filter, and use as the test solution.

[0357] 6.2.2 Preparation of reference solution

[0358] Take the reference standards berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride respectively, add methanol to prepare a mixed reference solution of 0.025 mg / mL.

[0359] 6.2.3 Preparation of negative sample solution

[0360] Take 0.5g of negative sample of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (excluding Mahonia japonica leaf medicinal material), grind it into a fine powder, and treat it with the test solution in the same way to prepare the negative sample solution.

[0361] 6.3 Thin-layer chromatography conditions

[0362] Thin-layer plate: Silicone G thin-layer plate

[0363] Developing solvent: toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia (volume ratio 4:3:3:1.2:0.5)

[0364] Sample volume: 2 μL

[0365] Development method: A double-slot development tank is used, which is placed inside an ammonia vapor saturated development tank, with a development distance of about 8cm.

[0366] Inspection: Examine under ultraviolet light (365nm).

[0367] 6.4 Sampling Quantity Investigation

[0368] The test solution and the mixed reference solution of Mahonia japonica leaf (long-column Mahonia japonica) granules were separately spotted onto the same silica gel G thin-layer plate. Developed under the thin-layer chromatographic conditions described in section 6.3, the plate was then removed, air-dried, and examined under ultraviolet light (365 nm). The thin-layer chromatogram is shown below. Figure 12 .from Figure 12 It can be seen that when the sample volume is 2 μL, the main spot in the test sample at the corresponding position of the control herb is clear and there is no other interference. Therefore, the sample volume of 2 μL was selected.

[0369] 6.5 Specificity Study of the Formulation Granules of Mahonia japonica Leaf (Long-columnar Mahonia)

[0370] Take 2 μL each of the test solution, mixed reference solution, and negative sample of Mahonia japonica leaf (long-column Mahonia japonica) formula granules and spot them on the same silica gel G thin-layer plate. Develop according to the thin-layer chromatographic conditions in section 6.3. Remove the plate, air dry, and examine under ultraviolet light (365 nm). The thin-layer chromatogram is shown in [Figure 1]. Figure 13 .

[0371] Spike confirmation: Add the mixed reference solution to the test solution of Mahonia japonica leaf (long-column Mahonia japonica) formula granules as the spiked solution. Spot 2 μL each of the Mahonia japonica leaf (long-column Mahonia japonica) formula granule test solution, the mixed reference solution of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, and the spiked solution onto the same silica gel G thin-layer plate. Develop under the above-described thin-layer chromatographic conditions. Remove the plate, air dry, and examine under ultraviolet light (365 nm). The thin-layer chromatogram is shown below. Figure 14 .

[0372] As shown in the figure, the main spots of the test solution, the mixed reference standard, and the spiked solution are clear and of consistent color at their respective positions. This indicates that the three main spots in the test solution, from bottom to top, are berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, respectively.

[0373] 6.6 Investigation of different humidity levels

[0374] Two μL samples of the Mahonia japonica leaf (long-column Mahonia) formula granules test solution, a mixed reference solution of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, and a negative sample of Mahonia japonica leaf (long-column Mahonia) formula granules were spotted onto the same silica gel G thin-layer plate. The plates were developed under different humidity conditions according to the thin-layer chromatographic conditions described in section 6.3. The plates were then removed, air-dried, and examined under ultraviolet light (365 nm). The thin-layer chromatograms are shown in the figure. Figure 15 .Depend on Figure 15 It is evident that different humidity conditions significantly affect the chromatograms of the Mahonia japonica leaf granule test sample and the mixed reference solution. High humidity conditions hinder development, while low humidity conditions result in poor separation of the compounds. However, at 66% humidity, the test sample and reference solution show spots of the same color at corresponding positions, with clear color development, good separation, no tailing, and no background interference. Therefore, it is recommended to perform thin-layer chromatography at 66% humidity.

[0375] 6.7 Investigation at different temperatures

[0376] Two μL samples of the Mahonia japonica leaf (long-column Mahonia) formula granules test solution, the mixed reference solution of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, and the negative sample of Mahonia japonica leaf (long-column Mahonia) formula granules were spotted onto the same silica gel G thin-layer plate. The plates were developed under the thin-layer chromatographic conditions described in section 6.3 at different temperatures. The plates were then removed, air-dried, and examined under ultraviolet light (365 nm). The thin-layer chromatograms are shown in the figure. Figure 16 .Depend on Figure 16 It is evident that the chromatograms of the test sample and the mixed reference solution of Mahonia japonica leaf (long-column Mahonia japonica) formulation granules differ significantly under different temperature conditions. At 25℃ and 40℃, the reference and test samples show spots of the same color at corresponding positions, with clear main spots, good separation, no tailing, and no background interference. However, the chromatogram at 4℃ fails to develop well. Therefore, it is recommended to perform thin-layer chromatography at room temperature or a higher temperature.

[0377] 6.8 Investigation of Thin-Layer Boards from Different Manufacturers

[0378] Two μL samples of the test solution of Mahonia japonica leaf (long-column Mahonia) formula granules, the mixed reference solution of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, and the negative sample of Mahonia japonica leaf (long-column Mahonia) formula granules were spotted onto thin-layer plates from different manufacturers (Qingdao Marine Silica G plate, Qingdao Marine Silica NaOH alkaline plate, Sinopharm Chemical Reagent Silica G plate, and Merck plate). The plates were developed under the same temperature and humidity conditions as described in section 6.3. The plates were then removed, air-dried, and examined under ultraviolet light (365 nm). The thin-layer chromatograms are shown in [Figure 1]. Figure 17 .Depend on Figure 17 It is evident that the main spots in the chromatograms of the Mahonia japonica leaf formula granules and the mixed reference solution corresponded to each other when using silica gel thin-layer plates from different manufacturers (Qingdao Marine Silica G plate, Qingdao Marine Silica NaOH alkaline plate, Sinopharm Chemical Reagent Silica G plate, and German Merck plate), with no significant impact. This indicates that the thin-layer identification method has good durability with silica gel G plates from different manufacturers.

[0379] 6.9 Thin-layer chromatogram of Mahonia japonica leaf (long-column Mahonia japonica) formulation granules for identification

[0380] Two μL samples of different batches of Mahonia japonica leaf (long-column Mahonia) formula granules, a mixed reference solution of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, and a negative sample of Mahonia japonica leaf (long-column Mahonia) formula granules were spotted onto the same silica gel G thin-layer plate. The plate was developed under the conditions described in section 6.3. The plate was then removed, air-dried, and examined under UV light (365 nm). The thin-layer chromatogram is shown in the figure. Figure 18 .Depend on Figure 18 It is evident that the chromatograms of the test samples in the three batches of Mahonia japonica leaf (long-column Mahonia) formula granules showed the same color spots at the corresponding positions as the chromatograms of Mahonia japonica leaf (long-column Mahonia) reference medicinal material, indicating that the thin-layer identification of the three batches of Mahonia japonica leaf (long-column Mahonia) formula granules all met the requirements.

[0381] In summary, the chromatographic spots of the Mahonia japonica leaf (long-column Mahonia) formula granules showed good separation, and the corresponding positions of the Mahonia japonica leaf (long-column Mahonia) formula granules and the mixed reference solution showed spots of the same color. This method can effectively identify Mahonia japonica leaf (long-column Mahonia) formula granules that have lost their medicinal characteristics. Through thin-layer chromatography methodology evaluation, this method demonstrated good specificity and robustness, making it suitable for the thin-layer chromatographic identification of Mahonia japonica leaf (long-column Mahonia) formula granules.

[0382] Example 7: Establishment of an analytical method for determining the content and transfer rate of Mahonia japonica leaf granules.

[0383] 7.1 Instruments and Reagents

[0384] Instruments: Agilent 1290 high performance liquid chromatograph, ultra-high performance liquid chromatograph (Waters ARC); KQ-500DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 0.001 g electronic balance (model: mL204); 0.1 g electronic balance (model: XPR26DR / A).

[0385] Reagents: Methanol and acetonitrile were chromatographic grade, batch numbers 204512 and 205303 respectively, purchased from Fisher Scientific; water was ultrapure water; other reagents were analytical grade; potassium dihydrogen phosphate (batch number: 20221020, Chengdu Jinshan Chemical Co., Ltd.), sodium dodecyl sulfate (batch number: 20210426, Shanghai Wokai Pharmaceutical Co., Ltd.), and hydrochloric acid were of analytical grade.

[0386] The batch numbers of the three batches of Mahonia japonica leaf (long-column Mahonia japonica) formula granules are 230901, 230902 and 230903 respectively.

[0387] 7.2 Source and purity check of reference standards

[0388] Berberine hydrochloride (batch number 110733-202110, purity 90.3%), palmatine hydrochloride (batch number 110732-201913, purity 85.7%), and berberine hydrochloride (batch number 110713-202015, purity 85.9%) were all purchased from the China National Institutes for Food and Drug Control. They are for content determination purposes and require no pretreatment.

[0389] 7.3 Determination of chromatographic conditions

[0390] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); mobile phase A was a buffer salt containing 0.34 g potassium dihydrogen phosphate and 0.17 g sodium dodecyl sulfate per 100 mL (pH adjusted to 3.0 with phosphoric acid); mobile phase B was acetonitrile. Gradient elution was performed according to the specifications in the table below; the flow rate was 1 mL / min; the column temperature was 30 °C; and the detection wavelength was 346 nm. The theoretical plate number, calculated based on the anthocyanin hydrochloride, should be no less than 3000.

[0391] Table 36 Gradient Elution Procedure

[0392]

[0393]

[0394] 7.4 Preparation of reference solution

[0395] Accurately weigh a certain amount of cypermethrin hydrochloride, palmatine hydrochloride, and berberine hydrochloride, add them to methanol solution, and prepare a mixed reference standard. Prepare a mixed reference standard solution containing 4 μg of cypermethrin hydrochloride, 4 μg of palmatine hydrochloride, and 34 μg of berberine hydrochloride per 1 mL.

[0396] 7.5 Preparation of the test solution

[0397] 7.5.1 Investigation of different extraction solvents

[0398] Take an appropriate amount of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (batch number: 230903), a total of 7 portions, with 2 parallel samples for each portion. Place each portion in a stoppered conical container and accurately add 20 mL of hydrochloric acid-methanol (volume ratio 1:100), hydrochloric acid-75% methanol (volume ratio 1:100), hydrochloric acid-50% methanol (volume ratio 1:100), hydrochloric acid-ethanol (volume ratio 1:100), hydrochloric acid-75% ethanol (volume ratio 1:100), hydrochloric acid-50% ethanol (volume ratio 1:100), and hydrochloric acid-water (volume ratio 1:100) in sequence. Weigh the contents, sonicate for 30 min (power 500W, frequency 40kHz), cool, add the appropriate solvent to make up the weight, mix well, filter, and collect the filtrate to obtain the solution of each test sample. The chromatographic conditions described in section 7.3 were followed, with an injection volume of 10 μL. The effects of different extraction solvents on the content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride were calculated based on the total amount of these compounds, to determine the optimal extraction solvent. The experimental results were calculated using the external standard one-point method. Detailed results are shown in the table below.

[0399] Table 37. Effects of different extraction solvents on the determination of the content of three alkaloids in Mahonia japonica leaf (Mahonia longifolia) formulation granules.

[0400]

[0401]

[0402] As shown in Table 37, different solvents have a significant effect on the total content of the three alkaloids in the formula granules of Mahonia japonica leaves (long-column Mahonia japonica). Considering the differences in content, hydrochloric acid-methanol (volume ratio of 1:100) was selected as the best extraction solvent for further research.

[0403] 7.5.2 Examination of Extraction Methods

[0404] Take an appropriate amount of Mahonia japonica leaf (long-column Mahonia japonica) granules (batch number: 230903), in three portions, with two parallel samples per portion. Place each portion in a stoppered conical container, and precisely add 20 mL of hydrochloric acid-methanol (volume ratio 1:100) sequentially. Weigh the samples, and treat them by shaking, sonication, and reflux for 30 min respectively. After cooling to room temperature, add hydrochloric acid-methanol (volume ratio 1:100) to make up the weight, mix well, filter, and collect the filtrate. Take an appropriate amount of the filtrate and place it in a high-liquid sample bottle to obtain the test solution. Analyze the samples according to the chromatographic conditions in section 7.3, with an injection volume of 10 μL. Calculate the effect of different extraction methods on the content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, based on the total amount of berberine hydrochloride, and determine the optimal extraction method. The experimental results are calculated using the external standard one-point method. Detailed results are shown in the table below:

[0405] Table 38. Effects of different extraction methods on the determination of the content of three alkaloids in Mahonia japonica leaf (Mahonia longifolia) formulation granules.

[0406]

[0407] The experimental results in Table 38 show that the effects of different extraction methods on the total content of the three alkaloids in the formula granules of Mahonia japonica leaves (long-column Mahonia japonica) are not significant. Considering the differences in content, and taking into account the experimental conditions of our laboratory and the ease of operation in practice, ultrasonic extraction was selected as the extraction method for further research.

[0408] 7.5.3 Examination of extraction time

[0409] Take an appropriate amount of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (batch number: 230903), a total of 4 portions, with 2 parallel samples for each portion. Place each sample in a stoppered conical container, and precisely add 20 mL of hydrochloric acid-methanol (volume ratio 1:100) sequentially. Weigh the samples, and sonicate them (power 500W, frequency 40kHz) for 15 min, 30 min, 45 min, and 60 min respectively. After cooling to room temperature, add hydrochloric acid-methanol (volume ratio 1:100) to make up the weight, mix well, filter, and collect the filtrate. Take an appropriate amount of the filtrate and place it in a high-liquid sample bottle to obtain the test solution for later use. Analyze the samples according to the chromatographic conditions in section 7.3, with an injection volume of 10 μL. Calculate the effect of different extraction times on the content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, based on the total amount of berberine hydrochloride, and determine the optimal extraction time. The experimental results are calculated using the external standard one-point method. The specific results are shown in the table below.

[0410] Table 39. Effects of different extraction times on the determination of the content of three alkaloids in Mahonia japonica leaf (Mahonia longifolia) formulation granules.

[0411]

[0412] The experimental results in Table 39 show that the total content of the three alkaloids in the formula granules of Mahonia japonica leaf (long-column Mahonia japonica) increases with the increase of extraction time. However, the increase in content is not significant after 45 min. Considering both time cost and content differences, ultrasonic extraction for 45 min was selected for further research.

[0413] 7.5.4 Determination of the preparation method for the test solution

[0414] Based on the sample pretreatment experiment results and referring to the preparation method of the test solution of the standard decoction of Mahonia japonica leaves (long-column Mahonia japonica), the preparation method of the test sample of Mahonia japonica leaf formula granules can be determined as follows:

[0415] Take the granules of Mahonia japonica leaf formula, grind and mix them evenly, take about 0.4g, weigh accurately, place in a stoppered conical flask, add 20mL of hydrochloric acid-methanol (volume ratio of 1:100), weigh, sonicate for 45min (power 500W, frequency 40kHz), cool, replenish the lost weight with hydrochloric acid-methanol (volume ratio of 1:100), shake well, filter, and take the filtrate to obtain the product.

[0416] 7.6 Methodological Validation

[0417] 7.6.1 Specificity Examination

[0418] Accurately pipette 10 μL each of the test solution, the mixed reference solution (berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride), and the blank solvent of the Mahonia japonica leaf (long-column Mahonia japonica) granule formula (batch number: 230903), and perform chromatographic analysis according to the conditions in section 7.3. Record the chromatographic elution patterns. Results are shown below. Figure 19 .Depend on Figure 19 The chromatogram results show that this analytical method is specific for determining the content of three alkaloids in the formula granules of Mahonia japonica leaf (long-column Mahonia japonica).

[0419] 7.6.2 Peak Purity

[0420] Accurately pipette 10 μL each of the test solution and reference solution of Mahonia japonica leaf (long-column Mahonia japonica) granules (batch number: 230903) and inject them into the ultra-high performance liquid chromatograph for determination. The peak purity of the target peak is then obtained. Results are shown below. Figure 20 See Table 40.

[0421] Table 40 Matching values ​​for target peaks and peak purity

[0422] Target Peak Phytoalkaloid hydrochloride Palmatine Hydrochloride Berberine hydrochloride Peak purity matching value 999.98 997.15 999.99 Peak separation 3.8145 4.6180 3.3281 Theoretical number of plates 110506.6073 71165.86866 100991.08568

[0423] Depend on Figure 20As shown in Table 39, the peak purity matching values ​​of the components in this index are all greater than 960, indicating that the peak purity meets the analytical requirements.

[0424] 7.6.3 Examination of Linear Relationships

[0425] Accurately weigh appropriate amounts of cypermethrin hydrochloride, palmatine hydrochloride, and berberine hydrochloride reference standards, place them separately in 50 mL volumetric flasks, dissolve them in methanol, dilute to the mark, and then further dilute to obtain a stock solution of reference standards containing 17.28 μg of cypermethrin hydrochloride, 18.61 μg of palmatine hydrochloride, and 139.98 μg of berberine hydrochloride per milliliter.

[0426] The stock solutions of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride reference standards were diluted by 100, 50, 20, 10, 4, 2, and 1 times, respectively, to obtain reference solutions of different concentrations. These solutions were then analyzed under the chromatographic conditions described in Section 7.3. The linear ranges of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride were investigated by plotting concentration on the x-axis and peak area on the y-axis. The linearity results are shown in Tables 41-43 below. Figures 21-23 .

[0427] Table 41 Linearity Study of Hydrochloric Acid as a Radix Alkaloid

[0428]

[0429] Table 42 Linearity Study of Parmatin Hydrochloride

[0430]

[0431] Table 43 Linearity Study of Berberine Hydrochloride

[0432]

[0433] From Tables 41-43 and Figures 21-23 The experimental results show that: the concentration of berberine hydrochloride and the peak area value have a good linear relationship in the range of 0.1728–17.2756 μg / mL, with a correlation coefficient r = 0.9998; the concentration of palmatine hydrochloride and the peak area value have a good linear relationship in the range of 0.1861–18.6099 μg / mL, with a correlation coefficient r = 0.9999; and the concentration of berberine hydrochloride and the peak area value have a good linear relationship in the range of 1.3998–139.9826 μg / mL, with a correlation coefficient r = 0.9997.

[0434] 7.6.4 Precision Examination

[0435] Accurately pipette the test solution of Mahonia japonica leaf (long-column Mahonia japonica) granules (batch number: 230903) and the mixed reference solution of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, and perform chromatographic analysis according to the conditions in section 7.3. The injection volume is 10 μL. The RSD (%) value of the target peak is calculated using the external standard single-point method based on the total content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. Detailed results are shown in the table below.

[0436] Table 44 Precision Experiment Results of the Method for Determining the Granular Content of Mahonia Leaf (Long-columnar Mahonia) Formula

[0437]

[0438] The experimental results in Table 44 show that the RSD (%) of the total content of the target peaks is 0.60% < 4.0%, indicating that the method has good precision.

[0439] 7.6.5 Stability Test

[0440] Take the test solution of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (batch number: 230903), and inject it at 0, 2, 4, 6, 8, 10, 12 and 24 hours according to the chromatographic conditions in section 7.3, with an injection volume of 10 μL. Calculate the RSD (%) value of the target peak content using the external standard one-point method, based on the total content of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride. The specific results are shown in the table below.

[0441] Table 45. Determination of Granular Content in the Formula of Mahonia Leaf (Long-columnar Mahonia) and Stability Study of the Test Solution

[0442]

[0443] The experimental results in Table 45 show that the RSD (%) of the total content of the target peak within 24 hours is 1.49% < 4.0%, indicating that the solution has good stability within 24 hours.

[0444] 7.6.6 Repeatability Test

[0445] Take approximately 0.4 g of the test solution from the same batch of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (batch number: 230903), accurately weigh it, and prepare 6 parallel test solutions according to the above-described test solution preparation method. Perform chromatographic analysis under the conditions described in section 7.3. Calculate the RSD (%) of the target peak content using the external standard single-point method, based on the total content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the test solution. See the table below for detailed results.

[0446] Table 46. Repeatability Test Results of the Method for Determining the Granular Content of Mahonia Leaf (Long-columnar Mahonia) Formula.

[0447]

[0448] The experimental results in Table 46 show that the RSD (%) of the total content of the target peak is 1.19% < 4.0%, indicating that the method has good repeatability.

[0449] 7.6.7 Intermediate Precision Examination

[0450] Other analysts in this project team operated on different dates and under different chromatographs, taking approximately 0.4 g of the same batch of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (batch number: 230903), accurately weighed, and prepared the test solution according to the above-mentioned test solution preparation method. The test solution was then analyzed under the chromatographic conditions in section 7.3. The RSD (%) value of the target peak content was calculated using the external standard one-point method, based on the total content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the test solution. The specific results are detailed in the table below.

[0451] Table 47. Results of Intermediate Precision Experiment for Determination of Granule Content in Mahonia Leaf (Long-columnar Mahonia) Formula.

[0452]

[0453] The experimental results in Table 47 show that the intermediate precision RSD value of the total content of the three alkaloids is <8.0%, indicating that the method has good precision.

[0454] 7.6.8 Accuracy Test

[0455] Take 0.40g of Mahonia japonica leaf (long-column Mahonia japonica) granules (batch number: 230903, content of phytorepinephrine hydrochloride 0.0166%, content of palmatine hydrochloride 0.0094%, content of berberine hydrochloride 0.0159%), accurately weigh a total of 9 portions, and add 5mL each of phytorepinephrine hydrochloride, palmatine hydrochloride and berberine hydrochloride reference solutions at 50%, 100% and 150% of the content of the test sample, respectively, and then add 5mL of hydrochloric acid-methanol (volume ratio of 1:100). Perform chromatographic analysis according to the conditions in section 7.3, injecting 10μL of each sample. Calculate the content of the target peak using the external standard one-point method, based on the total content of phytorepinephrine hydrochloride, palmatine hydrochloride and berberine hydrochloride. Calculate the recovery rate and RSD according to the following formulas. The results are shown in Tables 48-50 below.

[0456]

[0457] Among them, the measured amount refers to the content of the index component measured after adding the index component to the standard decoction, in μg; the spiking amount refers to the content of the index component added to the standard decoction, in μg; and the sample content refers to the content of the index component in the standard decoction, in μg.

[0458] Table 48. Determination of the content of *Mahonia japonica* leaf (*Mahonia longifolia*) granules (hydrochloric acid herb alkaloid) by spiking and recovery test results.

[0459]

[0460] Table 49. Determination of Granular Content in Mahonia Leaf (Long-columnar Mahonia) Formula (Pamadin Hydrochloride) - Spiking and Recovery Experiment Results

[0461]

[0462]

[0463] Table 50. Determination of the content of berberine hydrochloride in the formulation of Mahonia japonica leaf (Mahonia longifolia) granules: Results of spiking and recovery experiments.

[0464]

[0465] The experimental results in Tables 48-50 show that the recovery rates of the target peaks in the granules of Mahonia japonica leaf (long-column Mahonia japonica) are all within the range of 85%-110%, and the RSDs are all less than 4.0%, indicating that the accuracy of the assay method is good.

[0466] 7.6.9 Durability Test

[0467] 7.6.9.1 Investigation of different chromatographic columns

[0468] The effects of three chromatographic columns—1-Jade-PAK KP-C18-AQ (4.6 mm × 250 mm, 5 μm), 2-Diamonsil C18 (4.6 mm × 250 mm, 5 μm), and 3-Waters XSelect HSS T3 (4.6 mm × 250 mm, 5 μm)—on the peak shape and resolution of cyperine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in Mahonia japonica leaf (long column Mahonia japonica) formulation granules were compared.

[0469] The test solution of Mahonia japonica leaf (long-column Mahonia japonica) granules (batch number: 230903) was analyzed under the chromatographic conditions described in section 7.3. Chromatin, palmatine hydrochloride, and berberine hydrochloride were calculated as phytoalkaloids, and the chromatographic data were recorded. The experimental results are as follows: Figure 24 As shown in Table 51.

[0470] Table 51 Effect of different chromatographic columns on the detection results

[0471]

[0472]

[0473] Depend on Figure 24 As shown in Table 51, the experimental results indicate that, except for Diamonsil C18, which showed poor separation performance, WatersXSelect HSS T3 and Taiwei Technology JADE- KP-C18-AQ showed good separation performance, but Taiwei Technology's JADE- The KP-C18-AQ column has superior resolution, symmetry factor, and theoretical plate number compared to the Waters XSelect HSST3 column; therefore, Taiwei Technology's JADE- KP-C18-AQ was selected as the preferred chromatographic column for this experiment.

[0474] 7.6.9.2 Investigation using different chromatographs

[0475] Based on the existing equipment in the laboratory, a Waters ultra-high performance liquid chromatograph (Water, ACQUITY H class) and an Agilent high performance liquid chromatograph (Agilent, 1290 Infinity II) were selected to compare the effects of the two chromatographs on the peak shape and resolution of chromatographic peaks of purslane hydrochloride, palmatine hydrochloride and berberine hydrochloride in the formulation granules of Mahonia japonica leaf (long column Mahonia japonica).

[0476] Take the test solution of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (batch number: 230903), and determine it according to the chromatographic conditions in section 7.3. Calculate the chromatographic data as berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. The experimental results are as follows: Figure 25 As shown in Table 52.

[0477] Table 52 Results of the investigation using different chromatographs

[0478]

[0479] Depend on Figure 25 As shown in Table 52, this analytical method exhibits good durability with different chromatographs. Variations in the chromatograph can meet the system adaptability requirements.

[0480] 7.6.9.3 Investigation at different column temperatures

[0481] The effects of different column temperatures (25℃, 30℃, and 35℃) on the peak shapes of chromatographic components of berberine, palmatine hydrochloride, and berberine hydrochloride in the formulation granules of Mahonia japonica leaf (long column Mahonia japonica) were compared.

[0482] Take the test solution of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (batch number: 230903), and determine it according to the chromatographic conditions in section 7.3. Calculate the chromatographic data as berberine, palmatine hydrochloride, and berberine hydrochloride. The experimental results are as follows: Figure 26 As shown in Table 53.

[0483] Table 53 Results of the determination of the content of Mahonia japonica leaf (long-column Mahonia japonica) granules by different column temperatures

[0484]

[0485] Depend on Figure 26 As shown in Table 53, the peak shapes and separation effects were good at all three column temperatures. At 30℃, the baseline of the chromatogram showed no drift, and the retention time was not significantly different compared to the other two temperatures. Considering the column's tolerance and the analysis time required, a column temperature of 30℃ was chosen.

[0486] 7.6.9.4 Investigation of different flow velocities

[0487] The effects of different flow rates of 0.80 mL / min, 1.00 mL / min and 1.20 mL / min on the peak shape and resolution of chromatographic peaks of purslane, palmatine hydrochloride and berberine hydrochloride in the formula granules of Mahonia japonica leaf (long-column Mahonia japonica).

[0488] Take the test solution of Mahonia japonica leaf (long-column Mahonia japonica) formula granules (batch number: 230903), and determine it according to the chromatographic conditions in section 7.3. Calculate the chromatographic data as berberine, palmatine hydrochloride, and berberine hydrochloride. The experimental results are as follows: Figure 27 As shown in Table 54.

[0489] Table 54 Results of the Determination of Granule Content in Mahonia japonica Leaf (Long-columnar Mahonia) Formula under Different Flow Rates

[0490]

[0491] Depend on Figure 27 As shown in Table 54, the peak shape and separation were good at all three flow rates. The separation of components was best at a flow rate of 1.0 mL / min, with no baseline drift. Therefore, a flow rate of 1.0 mL / min was selected for this experiment.

[0492] 7.7 Determination of particulate content in 3 batches of production samples

[0493] Take appropriate amounts of three batches of Mahonia japonica leaf (long-column Mahonia) formula granules, grind and mix them thoroughly. Take about 0.4g of each batch, and prepare two parallel samples. Prepare test solutions for the three batches of Mahonia japonica leaf (long-column Mahonia) formula granules according to the above-mentioned test solution preparation method. Under the chromatographic conditions in section 7.3, accurately pipette 10μL each of the reference solution and the test solution and inject them into the ultra-high performance liquid chromatograph for determination. The total content of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride is calculated using the external single-point method. The experimental results are shown in Table 55 below.

[0494] Table 55. Content determination results of three batches of Mahonia japonica leaf (Mahonia longifolia) granules.

[0495]

[0496] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A method for preparing an extract from Mahonia japonica leaves, characterized in that, Includes the following steps: Take the leaves of Mahonia japonica, add water and decoct, filter to obtain the filtrate, and obtain Mahonia japonica leaf extract.

2. The preparation method according to claim 1, wherein, The number of times water is added for decoction is 1 to 3; preferably, the number of times water is added for decoction is 2; the first decoction adds 10 to 18 times the amount of water; the second decoction adds 9 to 17 times the amount of water; and / or, preferably, the first decoction time is 30 to 120 minutes; the second decoction time is 20 to 80 minutes. And / or, the filtration is performed using a 100-300 mesh screen.

3. The preparation method according to claim 1 or 2, wherein, The filtrate was concentrated under reduced pressure. Preferably, the concentration temperature is 55–70°C; And / or, preferably, the vacuum degree of the concentration is -0.080 to -0.090 MPa.

4. The preparation method according to claim 3, wherein, The filtrate after vacuum concentration is further dried, and the drying is freeze-drying, which is divided into three stages: a. Pre-freezing: the pre-freezing temperature is -50℃ to -45℃; b. Primary drying for sublimation: the drying temperature is -30℃ to 0℃; c. Secondary drying for desorption: the drying temperature is 5℃ to 25℃. Preferably, the pre-freezing time is 3-6 hours; the first drying time is 10-14 hours; and / or the second drying time is 5-7 hours.

5. The preparation method according to claim 3, wherein, The filtrate after vacuum concentration is further dried, and the drying method is selected from vacuum drying, spray drying, microwave drying or infrared drying. Preferably, the drying is spray drying, which includes the following steps: adding a first excipient to the filtrate after vacuum concentration, followed by drying; More preferably, the first excipient comprises maltodextrin; More preferably, the amount of the first excipient added is 5% to 15% of the amount of Mahonia japonica leaf slices added.

6. The preparation method according to claim 5, wherein, Add a second auxiliary material to the dried material; Preferably, the second excipient comprises silicon dioxide and / or magnesium stearate; More preferably, the percentage of silica by weight of the dried material is greater than 0 and less than or equal to 0.3%; And / or, more preferably, the percentage of magnesium stearate by weight of the dried material is greater than 0 and less than or equal to 0.3%.

7. An extract of Mahonia japonica leaves, characterized in that, The Mahonia japonica leaf extract was prepared by the preparation method according to any one of claims 1-6; Preferably, the preparation obtained from the extract of Mahonia japonica leaves is a standard decoction of Mahonia japonica leaves or a formula granule of Mahonia japonica leaves.

8. A method for detecting the total mass content and total transfer rate of indicative components in the Mahonia japonica leaf extract according to claim 7, characterized in that, Includes the following steps: (1) Preparation of reference solution Weigh out the reference standards of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, respectively, and prepare solutions by adding solvents; (2) Preparation of the test solution Extract the leaves of Mahonia japonica and add solvent to extract; (3) Liquid Chromatography Analysis Using octadecylsilane-bonded silica gel as the stationary phase, with water as mobile phase A and acetonitrile as mobile phase B, the reference solution and the test solution were injected into the liquid chromatograph and analyzed using gradient elution.

9. The detection method according to claim 8, wherein, In step (2), the preparation of the test solution includes: taking the extract of Mahonia japonica leaves, adding solvent for extraction, weighing, replenishing the lost weight with the extraction solvent, filtering, and taking the filtrate to obtain the test solution. Preferably, the solvent is selected from a combination of hydrochloric acid and at least one selected from the following solvents: methanol, ethanol and water; more preferably, the extraction solvent is a combination of hydrochloric acid and methanol; And / or, preferably, the extraction is performed by any one of shaking, ultrasonication or reflux; more preferably, the extraction is performed by ultrasonication; and even more preferably, the extraction time is 15 to 60 minutes.

10. The detection method according to claim 8 or 9, wherein, In step (3), the detection wavelength of the chromatogram is 340–350 nm; And / or, in step (3), the flow rate is 0.8 to 1.2 mL / min; And / or, in step (3), the column temperature is 25–35°C; And / or, in step (3), the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; preferably, the chromatographic column is JADE- KP-C18-AQ; And / or, in step (3), the mobile phase A is an aqueous solution containing potassium dihydrogen phosphate and sodium dodecyl sulfate; preferably, the pH of the mobile phase A is 2.0 to 4.

0.

11. The detection method according to any one of claims 8-10, wherein, In step (3), the gradient elution procedure is as follows: From 0 to 8 minutes, the volume percentage of mobile phase A decreased from 70% to 55%, while the volume percentage of mobile phase B increased from 30% to 45%. Between 8 and 16.5 minutes, the volume percentage of mobile phase A decreased from 55% to 52.5%, while the volume percentage of mobile phase B increased from 45% to 47.5%. Between 16.5 and 16.6 min, the volume percentage of mobile phase A increased from 52.5% to 70%, while the volume percentage of mobile phase B decreased from 47.5% to 30%. 16.6–20 min, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%.

12. The detection method according to any one of claims 8-11, wherein, The total mass content of the extract of Mahonia japonica leaves is 0.04–1.9 mg / g, consisting of berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. And / or, the transfer rates of berberine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the Mahonia japonica leaf extract are 16.2% to 45.8%.

13. A thin-layer chromatography identification method for the Mahonia japonica leaf extract according to claim 7, comprising preparing a test solution of the Mahonia japonica leaf extract, thin-layer spotting, development, and color development, characterized in that, The extract of Mahonia japonica leaves was dissolved in methanol, sonicated, and filtered to obtain a test solution of Mahonia japonica leaf extract; toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia solution was used as the developing solvent.

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