Rosa roxburghii tratt leaf extract, preparation method of preparation thereof, and content and characteristic spectrum detection method of rosa roxburghii tratt leaf extract

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to a preparation method of a roxburgh rose leaf extract, a construction method of a specific chromatogram and a detection method of brachyphyllinic acid and ellagic acid in the roxburgh rose leaf extract.The preparation method comprises the steps that roxburgh rose leaf decoction pieces are decocted twice to obtain decoction liquid, and the decoction liquid is filtered; the roxburgh rose leaf extract is prepared from roxburgh rose leaf decoction pieces, through an accelerated stability test, the content and the moisture content are relatively stable, and specific chromatograms and a content detection method are good in specificity, repeatability and stability.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a preparation method of a roxburghii leaf extract and a preparation thereof, and a content and characteristic spectrum detection method thereof. Background Art

[0002] Rosa roxburghii leaves are the dried leaves of the Rosaceae plant Rosa roxburghii Tratt. They can be picked and dried all year round. The samples to be tested are also the traditional medicinal materials used by ethnic minorities in our province. They are included in the 2019 edition of the Quality Standards for Traditional Chinese and Ethnic Chinese Medicinal Materials in Guizhou Province; they have the function of strengthening the stomach and promoting digestion, and are used to relieve food stagnation.

[0003] The content of breviole acid and ellagic acid in roxburghii leaves is relatively high. Brassinoic acid is a natural plant extract with a wide range of antibacterial effects. Studies have found that breviole acid can effectively inhibit the growth of a variety of pathogenic microorganisms, including bacteria, fungi and viruses. Ellagic acid has a variety of biologically active functions, such as antioxidant function, anti-cancer, anti-mutagenic properties, and inhibition of human immunodeficiency virus. Establishing a method for determining the content of breviole acid and ellagic acid is of great reference value for controlling the quality of roxburghii leaf slices.

[0004] Extract is a traditional form of medication widely used in clinical practice. The extract is prepared in accordance with the theory of traditional Chinese medicine, standardized by the method of clinical decoction, solid-liquid separation, and appropriate concentration or drying by a suitable method. Labeling the extract can be used as a standard reference to measure whether the Chinese medicine formula granules are basically consistent with the clinical decoction.

[0005] With the development of new dosage forms of Chinese medicine, the quality control of Chinese herbal medicine extracts and their preparations has become an urgent problem to be solved. At present, only Sichuan Province and Guizhou Province have issued standards for sea buckthorn leaf medicinal materials, and there is no reference to the standards for medicinal extracts and their preparations, which cannot provide a reference for controlling the quality of Chinese herbal medicine extract terminal products.

[0006] Therefore, the research on the extract of roxburghii leaf and its quality detection method can provide a quality control method for the product. It is an urgent problem to find a roxburghii leaf extract, a standard decoction, an extract preparation and its preparation method and quality detection method. Summary of the invention

[0007] In order to solve the above technical problems existing in the prior art, the present invention provides a preparation method of roxburghii leaf extract and a method for detecting its content and characteristic spectrum, which are as follows:

[0008] A method for preparing a roxburghii leaf extract and a preparation thereof, comprising the following steps:

[0009] (1) Take Rosa roxburghii leaf slices, add water and boil twice, filter, and combine the two decoctions after the filtrate is cooled to room temperature;

[0010] (2) concentrating the decoction under reduced pressure and at low temperature to obtain an extract concentrate, wherein the density of the extract concentrate is controlled to be between 1.15 and 1.16 g / ml;

[0011] (3) The extract is made into a desired dosage form.

[0012] Furthermore, the step (1) specifically comprises taking Rosa roxburghii leaf slices, adding water and decocting twice, directly adding the slices to 18-22 times the amount of water for the first decoction, soaking for 28-32 minutes, boiling over high heat and then keeping the mixture slightly boiling over low heat for 28-30 minutes, filtering the decoction while hot through a 300-mesh sieve, and cooling the filtrate to room temperature; adding 14-16 times the amount of water for the second decoction, boiling over high heat and then keeping the mixture slightly boiling over low heat for 18-20 minutes, filtering the decoction while hot through a 300-mesh sieve, cooling the filtrate to room temperature, and combining the two decoctions. Preferably, the step (1) comprises taking Rosa roxburghii leaf slices, adding water and decocting twice, directly adding the slices to 20 times the amount of water for the first decoction, soaking for 30 minutes, boiling over high heat and then keeping it at a low boil for 30 minutes over low heat, filtering the decoction while hot through a 300-mesh sieve, and cooling the filtrate to room temperature; adding 16 times the amount of water for the second time, boiling over high heat and then keeping it at a low boil for 20 minutes over low heat, filtering the decoction while hot through a 300-mesh sieve, cooling the filtrate to room temperature, and combining the two decoctions.

[0013] Furthermore, in step (2), the specific conditions for reduced pressure and low temperature concentration are: temperature: 65° C.; vacuum degree: -0.080 to -0.090 MPa.

[0014] Furthermore, the step (3) is to freeze-dry the obtained extract to obtain a standard decoction of roxburghii leaves; the freeze-drying parameters are: the pre-freezing temperature of the roxburghii leaf concentrate is -45°C, the pre-freezing time is 240 minutes, the sublimation drying temperature is -35°C to 0°C, the sublimation drying time is 1185 minutes, and the vacuum degree is -0.2 mbar; the analytical drying temperature is 5°C to 25°C, the analytical drying time is 445 minutes, and the vacuum degree is -0.2 mbar.

[0015] Furthermore, the step (3) is to add auxiliary materials to the obtained extract, dry it, add auxiliary materials again, mix it evenly, and granulate it to obtain the roxburghii leaf granules.

[0016] Further, the amount of the roxburghii leaf granules is 25-30%, that is, 3333-4000g of the medicinal slices are made into 1000g of granules, and the specification is that each 1g of the formula granules is equivalent to 3.3-4.0g of the medicinal slices. Preferably, the amount of the roxburghii leaf granules is 26.7%, that is, 3750g of the medicinal slices are made into 1000g of granules, and the specification is that each 1g of the formula granules is equivalent to 3.75g of the medicinal slices, and the dry extract yield range is 13.4% to 26.7%.

[0017] Furthermore, the auxiliary materials include maltodextrin, magnesium stearate, silicon dioxide, talc, microcrystalline cellulose, etc., preferably maltodextrin is added in an amount of 5%.

[0018] A method for determining the contents of multiple components in a roxburghii leaf extract and a preparation thereof, using high performance liquid chromatography for determination, using octadecylsilane bonded silica gel as a filler; using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to the provisions in the following table; the flow rate is 0.2 ml per minute; the column temperature is 20° C.; the detection wavelengths are 278 nm for shortleaf hematoxylin and 254 nm for ellagic acid:

[0019]

[0020] Furthermore, the chromatographic column used had a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

[0021] Furthermore, the test solution is prepared by the following method: take about 0.1 g of the sample to be tested, accurately weigh it, place it in a stoppered conical flask, accurately add 30 ml of 75% ethanol, weigh it, heat it under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 75% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0022] Furthermore, the reference solution is a solution prepared with methanol as solvent and contains 60 μg of brevicornulinic acid and 80 μg of ellagic acid per 1 ml.

[0023] A method for detecting characteristic spectra of a roxburghii leaf extract and a preparation thereof, wherein high performance liquid chromatography is used for determination, octadecylsilane bonded silica gel is used as a filler, acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, and gradient elution is performed according to the provisions in the following table; the flow rate is 0.2 ml per minute; the column temperature is 20° C.; the detection wavelengths are 278 nm for shortleaf hematoxylin and 254 nm for ellagic acid:

[0024]

[0025] Furthermore, the chromatographic column used had a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

[0026] Furthermore, the test solution is prepared by the following method: take about 0.1 g of the sample to be tested, accurately weigh it, place it in a stoppered conical flask, accurately add 30 ml of 75% ethanol, weigh it, heat it under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 75% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0027] Furthermore, the reference solution is a solution prepared with methanol as solvent and contains 60 μg of brevicornulinic acid and 80 μg of ellagic acid per 1 ml.

[0028] The final characteristic spectrum standard of sea buckthorn leaf extract was determined as follows: four characteristic peaks should be present in the chromatogram of the test sample, among which peak 2 should be consistent with the retention time of the ellagic acid reference peak; the peak corresponding to the ellagic acid reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value, and the specified values ​​are: 0.29 (peak 1), 1.44 (peak 3), and 1.86 (peak 4).

[0029] The invention discloses a thin layer chromatography method for extracts of roxburghii leaves and preparations thereof. The specific steps are as follows: taking 0.5 g of a sample to be tested, grinding it, adding 10 ml of ethyl acetate, ultrasonically treating it for 15 minutes, filtering, evaporating the filtrate to dryness, and adding 1 ml of methanol to dissolve the residue to prepare a test solution; taking 1 g of a roxburghii leaf control medicinal material, adding 20 ml of water, heating and refluxing for 30 minutes, filtering, extracting the filtrate twice with ethyl acetate, each time with 20 ml, combining the ethyl acetate solution, evaporating it to dryness, adding 1 ml of methanol to dissolve the residue to prepare a control medicinal material solution, and testing it according to the thin layer chromatography method, respectively taking 6 μl of the test solution and 1 μl of the control medicinal material solution and spotting them on the same silica gel G thin layer plate, using chloroform-ethyl acetate-formic acid (5:3:1) as a developing agent, developing, taking out, drying, spraying with 5% ferric chloride ethanol solution, drying, and inspecting under sunlight, and spots of the same color should appear at the corresponding positions of the chromatogram of the test sample and the chromatogram of the control medicinal material.

[0030] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0031] 1. The preparation method described in the present application can provide a standard for the quality control of the roxburghii leaf extract and its preparations such as formulated granule products, thereby achieving overall quality control and effective supervision of the roxburghii leaf extract and its preparations such as formulated granule products.

[0032] 2. The present application uses Rosa roxburghii leaf slices to prepare Rosa roxburghii leaf extract. After accelerated stability tests, the content of short-leaf scutellaria phenolic acid, ellagic acid, and water content are relatively stable, the relative retention time and relative peak area of ​​the characteristic spectrum are relatively stable, it is resistant to moisture absorption, and no impurities are produced. The prepared Rosa roxburghii leaf extract is subjected to high performance liquid chromatography and ultra-high performance liquid chromatography analysis. The method has good specificity, good repeatability, and good stability.

[0033] 3. The characteristic spectrum method established in this application uses ultra-high phase liquid chromatography, which is simple, stable, highly precise and reproducible. In addition, the characteristic spectrum of the extract obtained has many peaks, good peak shape, easy identification, and is accurate and reliable.

[0034] 4. The characteristic spectrum method established in this application is simple, time-saving, and environmentally friendly. The analysis of a batch of preparations takes only 65 minutes, which can greatly shorten the detection and analysis time, improve production efficiency, and enable large-scale production sampling and testing to quickly achieve quality control goals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a process flow chart for preparing a standard decoction of roxburghii leaves.

[0036] Figure 2 This is a photo of the YMW mechanical split medicine decoction pot.

[0037] Figure 3 It is the result of the specificity investigation map.

[0038] Figure 4 is the peak purity chromatogram.

[0039] Figure 5 It is the regression equation curve of the linear investigation of shortleaf hematoxylin phenolic acid.

[0040] Figure 6 This is the regression equation curve of the linear investigation of ellagic acid.

[0041] Figure 7 This is a graph showing the effects of different chromatographic columns on the target components in the standard decoction of roxburghii leaves.

[0042] Figure 8 This is a graph showing the effects of different chromatographic instruments on the target ingredients in the Rosa roxburghii leaf formula granules.

[0043] Fig. 9 These are the chromatogram results of the target components in the standard decoction of roxburghii leaves at different column temperatures.

[0044] Fig.10 It is the chromatogram result of the target components in the standard decoction of Rosa roxburghii leaves at different flow rates.

[0045] Fig.11 This is the DAD graph of the standard decoction of Rosa roxburghii leaves.

[0046] Fig.12 These are UPLC spectra of different mobile phase systems.

[0047] Fig.13 This is the UPLC spectrum of the characteristic spectrum of the standard decoction of Rosa roxburghii leaves in aqueous phase.

[0048] Fig.14 This is the result of investigating the characteristic spectrum of the standard decoction of sea buckthorn leaves using different extraction solvents.

[0049] Fig.15 This is a comparison chart of the extraction efficiency of different extraction solvents for the characteristic spectrum of the standard decoction of sea buckthorn leaves.

[0050] Fig.16 It is the common pattern of the characteristic spectrum of the standard decoction of Rosa roxburghii leaves (R is the reference medicinal material).

[0051] Fig.17 This is the result of investigation on the specificity of the characteristic spectrum of the standard decoction of Rosa roxburghii leaves.

[0052] Fig.18 This is the result of the overall investigation of the characteristic spectrum of the standard decoction of Rosa roxburghii leaves.

[0053] Fig.19 This is a comparison chart of UPLC using different chromatographic columns.

[0054] Fig. 20 This is a comparison chart of UPLC under different column temperatures.

[0055] Fig.21 This is a comparison chart of UPLC at different flow rates.

[0056] Fig. 22 This is the specificity result diagram of the thin layer chromatography of the roxburghii leaf formula granules, where 1. roxburghii leaf granules negative 6μl; 2. roxburghii leaf control medicinal material 1μl; 3-5. roxburghii leaf granules (231101-231103) 6μl.

[0057] Fig.23 This is the TLC identification chromatogram of the Rosa roxburghii leaf formula granules under different temperature conditions. 1. Rosa roxburghii leaf control medicinal material 1μl; 2-4. Rosa roxburghii leaf granules (231101-231103) 6μl.

[0058] Fig.24 This is the TLC identification chromatogram of the Rosa roxburghii leaf formula granules under different humidity conditions. 1. Rosa roxburghii leaf control medicinal material 1μl; 2-4. Rosa roxburghii leaf granules (231101-231103) 6μl.

[0059] Fig.25 This is the chromatogram of silica gel plates from different manufacturers. 1. Rosa roxburghii leaf control medicinal material 1μl; 2-4. Rosa roxburghii leaf granules (231101-231103) 6μl.

[0060] Fig.26 These are three batches of Rosa roxburghii leaf formula granules thin layer chromatography, including 1. Rosa roxburghii leaf control medicinal material 1μl; 2-4. Rosa roxburghii leaf granules (231101-231103) 6μl.

[0061] Fig. 27 It is the characteristic spectrum of the standard decoction of Rosa roxburghii leaves. DETAILED DESCRIPTION

[0062] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0063] Embodiment 1:

[0064] Study on the process of standard decoction of roxburghii leaves

[0065] 1. Preparation technology of standard decoction of roxburghii leaves

[0066] 1.1 Preparation method

[0067] Take 100g of Rosa roxburghii leaf slices, place in an electric ceramic pot, add water and decoct twice, add directly to 20 times of water for the first decoction, soak for 30 minutes, boil over high heat (500W) and keep boiling for 30 minutes over slow fire (200W), filter the decoction while hot through a 300-mesh sieve, and record the decoction mass after the filtrate is cooled to room temperature. Add 16 times of water for the second time, heat over high heat and boil, keep boiling for 20 minutes over slow fire, filter the decoction while hot with a 300-mesh sieve, record the decoction mass after the filtrate is cooled to room temperature, and merge the two decoctions. The decoction was transferred to a 5000ml round-bottom flask, and concentrated at low temperature and reduced pressure using a rotary evaporator (temperature: 65°C; vacuum degree: -0.080 to -0.090MPa) to 100g of extract, and its density and extract yield were measured; under magnetic stirring, it was dispensed into 5ml brown penicillin bottles, each with a dispensing volume of 2ml, and half-stoppered. After dispensing, it was transferred to a vacuum freeze dryer for freeze drying, taken out, and rolled with an aluminum cap.

[0068] 1.2 Process flow chart

[0069] See Figure 1 .

[0070] 1.3 Preparation process specifications

[0071] Table 1 Preparation process of standard decoction of roxburghii leaves

[0072]

[0073]

[0074] 2 Basis for formulating the process parameters of standard decoction of roxburghii leaves

[0075] 2.1 Instruments and Materials

[0076] 2.1.1 Instruments

[0077] Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-5205A), ceramic health pot (Huzhou Gangdian Craft Manufacturing Factory), electronic balance (Hangzhou Youheng Weighing Equipment Company, HLD-30002), one-tenth balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd., AL104), circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd., SHZ-D(III)), low-temperature coolant circulating water pump (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd., DLSB-5 / 20B), vacuum freeze dryer (Shanghai Dongfulong Technology Co., Ltd., LYO-0.5), electric constant temperature drying oven (Tianjin Test Instrument Co., Ltd., 202-2AB), electric blast constant temperature drying oven (Hunan Electric Furnace Oven Factory, 101-4A), electric constant temperature water bath (Beijing Kewei Yongxing Instrument Co., Ltd., HH-S6A), guillotine.

[0078] 2.1.2 Materials

[0079] See Table 2 below for the decoction pieces of Rosa roxburghii leaves:

[0080] Table 2 Information table of Rosa roxburghii leaf decoction pieces

[0081]

[0082] 2.2 Determination method of process optimization index

[0083] 2.2.1 Solid content determination method

[0084] Determination method of solid content of roxburghii leaf concentrate

[0085] Refer to the determination of extract content by "hot leaching method" in General Chapter 2201 Extract Determination Method of Part 4 of the 2020 edition of the "Chinese Pharmacopoeia": Accurately weigh 10 g of the concentrate, place it in an evaporating dish with constant weight, evaporate it to dryness on a water bath, dry it at 105°C for 3 hours, cool it in a dryer for 30 minutes, quickly and accurately weigh the weight, and calculate the solid content of the concentrate.

[0086] The calculation formula of the concentrate yield is: yield (%) = concentrate weight * solid content of concentrate / slice volume * 100%. The yield value of the concentrate of the standard decoction is the yield of the standard decoction.

[0087] 2.3 Preparation of decoction pieces

[0088] Take the original medicinal materials and remove impurities. See "1.3" for the preparation process.

[0089] The information of medicinal materials used in this study is shown in Table 3:

[0090] Table 3 Rosa roxburghii medicinal material information

[0091]

[0092] 2.4 Extraction process

[0093] 2.4.1 Selection of cooking utensils

[0094] According to the provisions of the National Traditional Chinese Medicine (2009) No. 3 document on decoction containers in the "Management Standards for Traditional Chinese Medicine Decoction Rooms in Medical Institutions" (decoction containers should be made of ceramic, stainless steel, copper and other materials, and iron and other corrosive utensils are prohibited) and the habit of decocting soups at home, a 5L automatic decoction ceramic pot (see Figure 2 ) as standard decoction equipment.

[0095] 2.4.2 Study on extraction process parameters

[0096] (1) Investigation of dosage of medicinal pieces

[0097] According to the recommendations under "Standard Decoction Preparation" in the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", the amount of slices used for each decoction is generally not less than 100g, and the amount of slices of flowers, leaves, etc. can be reduced as appropriate. According to the results of the preliminary test, 100g of Rosa roxburghii leaf slices are easy to operate, and the decocted dry paste is sufficient for standard decoction experimental research, so the amount of slices used in the standard decoction of Rosa roxburghii leaves is set at 100g.

[0098] (2) Investigation of water addition amount

[0099] According to the recommendations under "Standard Decoction Preparation" in the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", the amount of water added is generally appropriate to soak the medicine surface by 2 cm to 5 cm. Flower and grass Chinese medicine pieces or Chinese medicine pieces with a long decoction time can be added with water as appropriate. When 20 times the amount of water is added for the first decoction to meet the solvent dosage of "soaking the medicine surface by 2 to 5 cm", when 16 times the amount of water is added for the second decoction, the water volume has exceeded 2 cm. The final amount of water added is determined to be 20 times the amount of water for the first decoction and 16 times the amount of water for the second decoction.

[0100] (3) Soaking time investigation

[0101] According to the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", the "Standard Decoction Preparation" item recommends that the decoction pieces to be decocted should be soaked first, and the soaking time is generally not less than 30 minutes. According to the "Technical Requirements", the soaking time for the standard decoction of roxburghii leaves is determined to be 30 minutes.

[0102] (4) Decoction time

[0103] According to the recommendations under "Standard Decoction Preparation" in the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", each dose of medicine is generally decocted twice. Generally, it is boiled and then decocted for 30 minutes; antipyretic, heat-clearing, and aromatic drugs should not be decocted for a long time, and it is appropriate to boil and then decoct for 20 minutes. The decocting time of Chinese herbal medicine pieces with a harder texture can be appropriately extended; tonic Chinese herbal medicine pieces are first boiled with high heat, and then slowly decocted with low heat for about 60 minutes. The second decocting time can be appropriately shortened. Therefore, according to the "Technical Requirements", the number of decoctions of the standard decoction of roxburghii leaves is determined to be twice, and the first decoction is boiled and then decocted for 30 minutes, and the second decoction is boiled and then decocted for 20 minutes.

[0104] (5) Investigation of solid-liquid separation conditions

[0105] According to the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", the mesh size of the solid-liquid separation filter material should be above 100 mesh. In order to obtain a decoction consistent with the traditional decoction through a modern solid-liquid separation method, the filtration effect of 100 mesh, 200 mesh and 300 mesh sieves on the decoction of roxburghii leaves was mainly investigated. The results showed that when performing solid-liquid separation, 100 mesh, 200 mesh and 300 mesh sieves were all easier to filter, but the clarity of the filtrate obtained by filtering with 100 mesh and 200 mesh sieves was poor, and the amount of sediment after standing was large. The clarity of the filtrate obtained by filtering with a 300 mesh sieve was better, and the amount of sediment after standing was small. Therefore, a 300 mesh sieve was used for filtering the solid-liquid separation of the standard decoction of roxburghii leaves.

[0106] 2.4.3 Extraction process parameters to determine results

[0107] Take 100g of Rosa roxburghii leaf slices, place in an electric ceramic pot, add water and decoct twice, add 20 times of water for the first decoction, soak for 30 minutes, boil over high heat (500W) and keep boiling for 30 minutes over slow fire (200W), filter the decoction while hot through a 300-mesh sieve, and record the decoction mass after the filtrate is cooled to room temperature. Add 16 times of water for the second time, heat over high heat and boil, keep boiling for 20 minutes over slow fire, filter the decoction while hot with a 300-mesh sieve, record the decoction mass after the filtrate is cooled to room temperature, and combine the two decoctions.

[0108] 2.5 Concentration process

[0109] 2.5.1 Study on concentration process parameters

[0110] According to the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", under the item "Preparation of Standard Decoction", it is recommended to use the reduced pressure concentration method to concentrate to the specified amount of extract at low temperature. Now, by comparing different concentration temperatures, the appropriate concentration temperature is determined, and the concentration ratio is temporarily determined to be 1:1, and its concentration state is observed.

[0111] The density and extract yield of standard decoction of roxburghii leaves at different concentration temperatures were compared and determined, and the state of the concentrate was observed. The results showed that the extract yields at different concentration temperatures were basically the same, and there was not much difference between the concentration temperatures of 50℃ and 65℃. After comprehensive consideration, 65℃ was selected as the concentration temperature. When the concentration ratio was 1:1, the extract had moderate viscosity, good fluidity, and was easy to transfer. The density was between 1.15 and 1.16 g / ml, so the concentration ratio was determined to be 1:1, and other concentration ratios were not considered for the time being.

[0112] 2.5.2 Results of concentration process parameter determination

[0113] The decoction was transferred to a 2000 ml round-bottom flask and concentrated at low temperature under reduced pressure using a rotary evaporator (temperature: 65° C.; vacuum degree: -0.080 to -0.090 MPa) to 100 g of extract.

[0114] 2.7 Preparation process verification

[0115] Take 3 portions of Rosa roxburghii leaf pieces (batch number: YP2206-1, YP2206-2, YP2206-3), each 100g, and prepare Rosa roxburghii leaf standard decoction according to the Rosa roxburghii leaf standard decoction preparation process; determine the paste yield of 3 batches of Rosa roxburghii leaf standard decoction, and evaluate the stability and rationality of the standard decoction preparation process. The results are shown in Table 4 below:

[0116] Table 4 Verification of preparation method of standard decoction of roxburghii leaves

[0117]

[0118] The experimental results show that the specific gravity, solid content and paste yield of three parallel batches of standard decoction concentrate of roxburghii leaf are basically the same, indicating that the process is stable and repeatable and can be used as the preparation process of standard decoction of roxburghii leaf.

[0119] 2.6 Drying process

[0120] 2.6.1 Study on freeze-drying process parameters

[0121] According to the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", the standard decoction is generally prepared by freeze drying, which can ensure its stable quality, easy dissolution and no need to add auxiliary materials. Therefore, freeze drying is first selected as the drying method for the standard decoction of roxburghii leaves.

[0122] Under magnetic stirring, the extract was dispensed into 5 ml brown vials, with a dispensing volume of 2 ml per bottle.

[0123] Half-stopper, after subpackaging, transfer to vacuum freeze dryer for freeze drying, take out, roll on aluminum cover, and you are done. The eutectic point test result of the vacuum freeze dryer for roxburghii leaves is -43.6℃, ​​the pre-freezing temperature is -45℃, the sublimation drying temperature is -30℃~0℃, the analytical drying temperature is 5℃~25℃, and the total drying time is 28 hours. The results show that under this freeze drying condition, the standard decoction of roxburghii leaves has low moisture content and is easy to preserve.

[0124] 2.6.2 Confirmation of freeze-drying process parameters

[0125] The results of freeze-drying process parameter determination are as follows: under magnetic stirring, the product was divided into 5 ml brown penicillin bottles with a filling volume of 2 ml in each bottle, half-stoppered, and transferred to a vacuum freeze dryer for freeze drying after filling; the freeze-drying parameters were as follows: the pre-freezing temperature of the sea buckthorn leaf concentrate was -45°C, the pre-freezing time was 240 minutes, the sublimation drying temperature was -35°C to 0°C, the sublimation drying time was 1185 minutes, and the vacuum degree was -0.2 mbar; the analytical drying temperature was 5°C to 25°C, the analytical drying time was 445 minutes, and the vacuum degree was -0.2 mbar.

[0126] Example 2

[0127] Study on the Technology of Rosa Roxb. Leaf Granules

[0128] Combined with the cream yield of the standard decoction of roxburghii leaves in Example 1, in order to strictly control the cream yield of large-scale production, and based on the principle of minimum use of auxiliary materials, it is finally determined that the amount of this product is 26.7%, that is, 3750g of decoction pieces are made into 1000g of granules, and the specification is that each 1g of formula granules is equivalent to 3.75g of decoction pieces.

[0129] The production amount is determined to be 26.7%, and the -3SD to +3SD yield rate of the mean of 15 batches of standard decoctions should range from 13.5% to 32.1%. At the same time, in order to strictly control the yield rate of the roxburghii leaf formula granules in large-scale production and ensure the uniformity and stability of the quality of the roxburghii leaf formula granules, the yield rate of the dry extract of the formula granules is determined to range from 13.4% to 26.7%.

[0130] In summary, the preparation amount of the roxburghii leaf granules was determined to be 26.7%, that is, 3750g of the medicinal pieces were made into 1000g of granules, the specification was that each 1g of the formula granules was equivalent to 3.75g of the medicinal pieces, and the dry extract yield ranged from 13.4% to 26.7%.

[0131] Example 3

[0132] Analytical method for content determination of standard decoction of roxburghii leaves

[0133] 3.1 Establishment of analytical method for content determination

[0134] This product is the dried leaves of Rosa roxburghii Tratt., a plant of the Rosaceae family. "Cao Mu Bian Fang" is the earliest document that records the use of roxburghii leaves as medicine: treating scabies, carbuncle, and wounds. "Guiyang Folk Herbs" records: for treating children's heat sores, pound and apply; for external hemorrhoids, dry, grind, and apply with sesame oil. It can also be decocted. Roxburghii leaves have low environmental requirements and are easy to cultivate. Roxburghii leaves have the advantages of drought resistance, barrenness resistance, easy reproduction, and prevention of soil and water loss. In addition to Weining, it is distributed throughout Guizhou, and is also distributed in Shaanxi, Gansu, Jiangxi, Fujian, Guangxi, Hubei, Sichuan, Yunnan and other provinces and regions; mostly grown on sunny slopes, valleys, roadsides and bushes; at an altitude of 500-2500 meters. The Guizhou Provincial Traditional Chinese Medicine and Ethnic Medicinal Materials Standard (2019 Edition) records that the complete leaf sequence of this product is an odd-pinnate compound leaf, with a petiole 1.5-2.5 cm long and striped; stipules are linear, most of which are attached to the petiole, with sharp teeth and green hairs on the edge; leaflets are usually 7-11, opposite, obovate to elliptical, with fine serrations on the edge, pointed or rounded at the tip, broadly wedge-shaped at the base, glabrous on both sides, and sessile. The smell is faint and the taste is slightly sour and astringent.

[0135] The leaves of roxburghii are sweet, sour and astringent, and belong to the spleen, kidney and stomach meridians. They have the effect of digesting food and eliminating accumulation. They are mainly used to treat food accumulation, scabies, tetanus and other diseases. They are one of the ingredients of Xiaoer Xiaoshi Kaiwei Granules and Xiaopi Hewei Capsules. Modern research shows that roxburghii leaves contain a variety of chemical substances such as flavonoids, amino acids, organic acids, polyphenols, triterpenes, phytosterols and vitamin C.

[0136] Experimental studies have shown that the content of brevipeinic acid and ellagic acid in roxburghii leaves is relatively high. Brassinoic acid is a natural plant extract with a wide range of antibacterial effects. Studies have found that brevipeinic acid can effectively inhibit the growth of a variety of pathogenic microorganisms, including bacteria, fungi and viruses. Ellagic acid is a polyphenol dilactone, a dimer derivative of gallic acid, and a natural polyphenol component widely found in various soft fruits, nuts and other plant tissues. Ellagic acid has a variety of biologically active functions, such as antioxidant function, anti-cancer, anti-mutagenic properties, and inhibition of human immunodeficiency virus. In addition, ellagic acid is also an effective coagulant, which has a good inhibitory effect on a variety of bacteria and viruses, can protect the wound from bacterial invasion, prevent infection, and inhibit ulcers. At the same time, studies have found that ellagic acid also has antihypertensive and sedative effects. At present, there are few reports on the content determination of formula particles in roxburghii leaves. Therefore, this experiment carried out a quantitative determination method of the content of the standard decoction of roxburghii leaves by ultra-high performance liquid chromatography, and formulated a quantitative standard for the standard decoction, providing a scientific basis for the establishment of the quality standard of the standard decoction of roxburghii leaves. Referring to the chromatographic condition items of the characteristic spectrum of the standard decoction of roxburghii leaves, a method for determining the content of ellagic acid in the standard decoction of roxburghii leaves was established.

[0137] 3.1.1 Instruments and reagents

[0138] Agilent 1290 Infinity II, Waters Acquity UPLC RH class, 1 / 10,000 electronic balance (model: ML204), electric constant temperature water bath (model: HH-S8), methanol and acetonitrile were all chromatographically pure with batch numbers of 225331 and F22MB2201, respectively, purchased from Fisher; water was ultrapure water, and other reagents were of analytical grade. The batch numbers of the 15 batches of sea buckthorn leaf soup are: BT2206-1, BT2206-2, BT2206-3, BT2206-4, BT2206-5, BT2206-6, BT2206-7, BT2206-8, BT2206-9, BT2206-10, BT2206-11, BT2206-12, BT2206-13, BT2206-14, and BT2206-15 (provided by the laboratory process group).

[0139] 3.1.2 Reference material source and purity check

[0140] Ellagic acid (China Food and Drug Inspection Institute, batch number: 111959-201903, purity 88.8%) is used for content determination and does not require treatment before use.

[0141] 3.1.3 Determination of chromatographic conditions

[0142] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.2 ml per minute; the column temperature was 20°C; the detection wavelength was 254 nm. The theoretical plate number calculated based on the ellagic acid peak should be no less than 3000.

[0143]

[0144] 3.1.4 Preparation of reference solution

[0145] Take an appropriate amount of ellagic acid reference substance, weigh it accurately, and add methanol to make a solution containing 80 μg of ellagic acid per 1 ml, which serves as the reference substance solution.

[0146] 3.1.5 Preparation of test solution

[0147] (1) Investigation of different extraction solvents

[0148] Take an appropriate amount of the standard decoction of Rosa roxburghii leaves BT2206-1, a total of 9 portions, each with 2 parallel samples, and place them in stoppered cones respectively. Accurately add 20 ml of methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and water in sequence, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, let cool, weigh the weight again, make up the lost weight with the corresponding solvent, shake well, filter, collect the subsequent filtrate, and obtain each test solution. According to the above-mentioned chromatographic conditions, accurately aspirate 1 μl of the reference solution and the test solution, inject them into the ultra-high performance liquid chromatograph, and measure them. Based on the content of short-leaf hematoxylin and ellagic acid, calculate the effects of different extraction solvents on their content and determine the optimal extraction solvent. The experimental results were calculated using the external standard one-point method. The specific results are shown in Table 5 below:

[0149] Table 5 Effects of different extraction solvents on the content determination results of standard decoction of roxburghii leaves

[0150]

[0151] The experimental test results show that different solvents have great influence on the contents of brevicornol acid and ellagic acid in the standard decoction of roxburghii leaves. Considering the contents and chromatographic peak shapes, 75% ethanol was selected as the extraction solvent for subsequent research.

[0152] (2) Investigation of extraction methods

[0153] Take an appropriate amount of the standard decoction of sea buckthorn leaves BT2206-1, a total of 2 portions, each with 2 parallel samples, respectively placed in a stoppered cone, accurately add 20 ml of methanol in turn, weigh the weight, respectively use ultrasound and reflux treatment for 30 minutes, let cool, weigh the weight again, make up the lost weight with methanol, shake well, filter, collect the subsequent filtrate, and obtain each test solution. According to the above-mentioned chromatographic conditions, accurately aspirate 1 μl of the reference solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, and measure to obtain. Based on the content of short-leaf hematoxylin phenolic acid and ellagic acid, calculate the effects of different extraction methods on their content, and determine the best extraction method. The experimental results were calculated using the external standard one-point method. The specific results are shown in Table 6 below:

[0154] Table 6 Effects of different extraction methods on the content determination results of standard decoction of roxburghii leaves

[0155]

[0156] The experimental test results show that different extraction methods have significant effects on the contents of shortleaf scutellaria acid and ellagic acid in the standard decoction of roxburghii leaves. Considering the simplicity of operation and the difference in content, heating reflux extraction was selected as the extraction method for subsequent research.

[0157] (3) Investigation of extraction solvent volume

[0158] Take an appropriate amount of the standard decoction of roxburghii leaves BT2206-1, a total of 4 portions, each with 2 parallel samples, and place them in stoppered cones, accurately add 10ml, 20ml, 30ml, 40ml, 60ml, and 80ml of methanol in sequence, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, let cool, weigh the weight again, make up the lost weight with methanol, shake well, filter, collect the subsequent filtrate, and obtain each test solution. According to the above-mentioned chromatographic conditions, accurately aspirate 1μl of the reference solution and the test solution, inject them into the ultra-high performance liquid chromatograph, and measure them. Based on the content of short-leaf hematoxylin and ellagic acid, calculate the effects of different extraction times on their content and determine the optimal extraction time. The experimental results were calculated using the external standard one-point method, and the specific results are shown in Table 7 below:

[0159] Table 7 Effect of different extraction solvent volumes on the content determination results of standard decoction of roxburghii leaves

[0160]

[0161] The experimental test results show that the effects of different extraction solvent volumes on the contents of brevicornol acid and ellagic acid in the standard decoction of roxburghii leaves are not significant. Considering the cost and content differences, the extraction solvent volume of 30 ml was selected for subsequent research.

[0162] (4) Investigation of different extraction times

[0163] Take an appropriate amount of the standard decoction of Rosa roxburghii leaves BT2206-1, a total of 4 portions, each with 2 parallel samples, and place them in stoppered cones respectively. Accurately add 30 ml of 75% ethanol in turn, weigh the weight, heat and reflux for 0.5 hours, 1 hour, 1.5 hours, and 2 hours respectively, cool, weigh the weight again, make up the lost weight with methanol, shake well, filter, collect the filtrate, and obtain each test solution. According to the above-mentioned chromatographic conditions, accurately aspirate 1 μl of the reference solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, and measure to obtain. Based on the content of short-leaf hematoxylin and ellagic acid, calculate the effect of different extraction solvent volumes on their content, and determine the optimal extraction time. The experimental results were calculated using the external standard one-point method. The specific results are shown in Table 8 below:

[0164] Table 8 Effect of different extraction time on the content determination results of standard decoction of roxburghii leaves

[0165]

[0166] The experimental test results show that different extraction times have significant effects on the contents of shortleaf scutellaria balsamina and ellagic acid in the standard decoction of roxburghii leaves. Considering the time cost and the difference in content, heating reflux extraction for 1 hour was selected for subsequent research.

[0167] (5) Determination of the preparation method of the test solution

[0168] According to the results of sample pretreatment experiments, the preparation method of the test sample can be determined as follows: take about 0.1 g of standard decoction of roxburghii leaves, accurately weigh it, put it in a stoppered conical flask, accurately add 30 ml of 75% ethanol, weigh it, heat and reflux to extract for 1 hour, cool it, weigh it again, make up the lost weight with 75% ethanol, shake it well, filter it, and take the filtrate to obtain the product.

[0169] 3.2 Validation of content determination methodology

[0170] (1) Specificity inspection

[0171] Accurately pipette 1 μl of the test solution of standard decoction of roxburghii leaves (BT2206-1), ellagic acid reference solution and blank solvent respectively, inject them into the ultra-high performance liquid chromatography instrument, measure according to the chromatographic conditions under "1.2.3.1.3", record the chromatogram, and the results are as follows: Figure 3 shown.

[0172] The chromatogram results showed that the analytical method had good specificity for the determination of brevicol and ellagic acid in the standard decoction of Rosa roxburghii leaves.

[0173] (2) Peak purity

[0174] Accurately pipette 1 μl of the test solution and reference solution of the standard decoction of Rosa roxburghii leaves (BT2206-1) respectively, inject into the ultra-high performance liquid chromatography, and measure to obtain the peak purity of the target peak. The results are shown in Figure 4 And Table 9.

[0175] Table 9 Target peaks and peak purity matching values

[0176]

[0177] The experimental results show that the peak purity matching value of the indicator component ellagic acid is 999.90>960, and the peak purity matching value of oxidized resveratrol is 998>960, indicating that the peak purity meets the analysis requirements.

[0178] (3) Linear

[0179] Weigh an appropriate amount of brevipeinic acid and ellagic acid reference substances accurately, place them in a numbered 50ml volumetric flask, add methanol to make a solution containing 117.7960μg of brevipeinic acid and 213.1200μg of ellagic acid per 1ml, shake well, and obtain a mixed reference substance stock solution of brevipeinic acid and ellagic acid, which is placed in a refrigerator for later use. Weigh an appropriate amount of brevipeinic acid and ellagic acid reference substances accurately, place them in a numbered 25ml volumetric flask, add ethanol to make a solution containing 68.2472μg of brevipeinic acid and 138.2794μg of ellagic acid per 1ml, shake well, and obtain a mixed reference substance stock solution of brevipeinic acid and ellagic acid, which is placed in a refrigerator for later use. The mixed reference stock solution of scutellaria sutchuenenic acid and ellagic acid was diluted 1.25, 1.56, 1.95, 2.44, 3.05, 3.81, 4.77, 9.54, 19.07, 38.15, and 76.29 times to obtain reference solutions of different concentrations of ellagic acid. The chromatographic conditions were followed to perform the test on the machine, with the concentration as the abscissa and the peak area as the ordinate, to investigate the linear range of ellagic acid. The results of the linear investigation are shown in Tables 10, 11, and Figure 5-Figure 6 .

[0180] Table 10 Ellagic acid linearity study

[0181]

[0182]

[0183] Table 11 Ellagic acid linearity study

[0184]

[0185] The experimental results showed that the concentration of shortleaf hematoxylin phenolic acid and the peak area value showed a good linear relationship in the concentration range of 1.7891μg / ml to 117.7960μg / ml, and the correlation coefficient R 2 =0.9992, the linear regression equation is: y=26.036x+28.016.

[0186] The concentration of ellagic acid and the peak area value showed a good linear relationship in the range of 3.6249μg / ml to 223.9891μg / ml, and the correlation coefficient R 2 =0.9999, the linear regression equation is: y=54.042x+3.0501.

[0187] (4) Precision test

[0188] 1) Instrument precision test

[0189] 1 μl of the test solution of standard decoction of roxburghii leaves (BT2206-1) and the reference solution of brevifolia phenolic acid and ellagic acid were accurately drawn respectively, injected into the ultra-high performance liquid chromatograph, and determined to obtain the RSD (%) value of the target peak using the external standard one-point method in terms of ellagic acid. The specific results are shown in Table 12 below.

[0190] Table 12 Results of precision test for content determination method of standard decoction of roxburghii leaves

[0191]

[0192] The results show that the RSD (%) value of the target peak brevicornol phenolic acid content is 0.58% < 2%, and the RSD (%) value of the ellagic acid content is 0.58% < 1.5%, indicating that the method has good instrument precision.

[0193] 2) Repeatability test

[0194] Take about 0.1g of the same batch of standard decoction of roxburghii leaves (BT2206-1), accurately weigh, and prepare 6 parts of test solution in parallel for standby use. According to the above chromatographic conditions, the RSD (%) value of the target peak content is calculated by the external standard one-point method based on the short leaf hematoxylin and ellagic acid in the test solution. The specific results are shown in Table 13 below.

[0195] Table 13 Results of repeatability test of content determination method of standard decoction of roxburghii leaves

[0196]

[0197] The experimental results show that the RSD (%) value of the target peak brevipein phenolic acid content is 0.58% < 2%, and the RSD (%) value of the ellagic acid content is 0.58% < 1.5%, indicating that the method has good repeatability.

[0198] 3) Intermediate precision

[0199] Other analysts of this project team operated on different dates and different chromatographs, took about 0.1g of the same batch of standard decoction of roxburghii leaves (BT2206-1), accurately weighed, and paralleled 6 portions, prepared the test solution according to the test solution preparation method, and tested on the machine according to the above-mentioned chromatographic conditions, and calculated the RSD (%) value of the target peak content by the external standard one-point method based on the ellagic acid in the test solution. The specific results are shown in Table 14 below.

[0200] Table 14 Intermediate precision experimental results of the content determination method of standard decoction of roxburghii leaves

[0201]

[0202]

[0203] The experimental results show that the intermediate precision RSD (%) value of the target shortleaf sappan phenolic acid content is 1.88% <4%, and the intermediate precision RSD (%) value of the ellagic acid content is 1.74% <3%, indicating that the intermediate precision of this method is good.

[0204] (5) Accuracy test

[0205] Take 0.05 g of standard decoction of roxburghii leaves with known content (batch number BT2206-1, brevicornol acid content 8.56 mg / g, ellagic acid content 25.47 mg / g), accurately weigh a total of 6 portions, and accurately add 2 ml of brevicornol acid reference solution prepared by methanol (concentration 0.2186184 mg / ml) and 4.5 ml of ellagic acid reference solution (concentration 0.30739008 mg / ml) in sequence, evaporate to dryness, and prepare the test solution according to the test solution preparation method. Detect on the machine according to the above-mentioned chromatographic conditions, inject 1 μl respectively, calculate the content of the target peak in terms of brevicornol acid and ellagic acid, and calculate the recovery rate and RSD% according to the following formula.

[0206]

[0207] Table 15 Results of the sample recovery experiment of phenolic acid in the standard decoction of roxburghii leaves

[0208]

[0209] Table 16 Results of ellagic acid recovery test for content determination method of standard decoction of roxburghii leaves

[0210]

[0211] The specific results are shown in Tables 15 and 16. From the experimental results, it can be seen that the recovery rate of short-leaf sappanol acid in the standard decoction is within the range of 92%-105%, and the RSD% (0.91%) is less than 2%. The recovery rate of ellagic acid in the standard decoction is within the range of 95%-102%, and the RSD% (0.88%) is less than 1.5%, indicating that the accuracy of the detection method is good.

[0212] (6) Stability investigation

[0213] The test solution of the standard decoction of roxburghii leaves (batch number BT2206-1) was prepared according to the preparation method of the test solution. According to the above-mentioned chromatographic conditions, the samples were injected at 0, 2, 4, 8, 12, 16, 20, and 24 hours, with an injection volume of 1 μl. The RSD (%) value of the target peak content was calculated using the external standard one-point method, based on short-leaf scutellaria baicalenic acid and ellagic acid. The specific results are shown in Table 17 below.

[0214] Table 17 Results of stability test of content determination method of standard decoction of roxburghii leaves

[0215]

[0216] The experiment showed that the RSD(%) value of the target peak shortleaf succinic acid content within 24 hours was 0.83%<2%, and the RSD(%) value of the ellagic acid content within 24 hours was 0.83%<2%, indicating that the solution had good stability within 24 hours.

[0217] (7) Durability test

[0218] 1) Investigation of different chromatographic columns

[0219] The effects of three chromatographic columns, Endeavorsil C18-A (2.1×150mm, 1.8μm), AmethSep C18AQ (2.1*150mm, 1.8μm), and ChromCoreAQ C18 (2.1×150mm 1.8μm), on the peak shape and separation of brevicornulinic acid and ellagic acid in standard decoction of roxburghii leaves were compared.

[0220] Take the test solution of Rosa roxburghii leaf standard decoction (batch number BT2206-1) under [Assay], and measure it according to the above chromatographic conditions, and record the chromatographic data in terms of shortleaf scutellariae phenolic acid and ellagic acid. The experimental results are shown in Table 18 and Figure 7 .

[0221] Table 18 Effects of different chromatographic columns on the test results of standard decoction of roxburghii leaves

[0222]

[0223] The experimental results showed that the separation effects of the three chromatographic columns were good and could meet the requirements for the determination of the content of roxburghii leaves. However, the separation degree and theoretical plate number of the Endeavorsil C18 (2.1×150mm, 1.8μm) column were better than those of the AmethSep C18 AQ (2.1*150mm, 1.8μm) and ChromCore AQ C18 (2.1×150mm1.8μm) columns. Therefore, Endeavorsil C18 (2.1×150mm, 1.8μm) was selected as the preferred chromatographic column for this experiment.

[0224] 2) Investigation of different chromatographs

[0225] According to the existing laboratory equipment, Shimadzu ultra high performance liquid chromatograph (LC-40D XS SOLVENTDELIVRYMODULE) and Agilent high performance liquid chromatograph (Agilent, 1290Infinity II) were selected to compare the effects of the two chromatographs on the peak shape and separation of ellagic acid in the roxburghii leaf formula granules.

[0226] Take the test solution of Rosa roxburghii leaf standard decoction (batch number BT2206-1) under [Assay], and measure it according to the above chromatographic conditions, with ellagic acid as the basis, and record the chromatographic data. Figure 8 And Table 19.

[0227] Table 19 Results of investigation of different chromatographs

[0228]

[0229]

[0230] The experimental results show that the analytical method has good durability for different chromatographs and the changes of chromatographs can meet the system adaptability requirements.

[0231] 3) Investigation of different column temperatures

[0232] The effects of different column temperatures of 20℃, 25℃ and 30℃ on the chromatographic peak shape of ellagic acid in standard decoction of roxburghii leaves were compared.

[0233] Take the test solution of Rosa roxburghii leaf standard decoction (batch number BT2206-1) under [Assay], and measure it according to the above chromatographic conditions, with ellagic acid as the basis, and record the chromatographic data. Fig. 9 And Table 20.

[0234] Table 20 Detection results of the content determination method of Rosa roxburghii leaf standard soup at different column temperatures

[0235]

[0236] The results showed that the chromatographic peak shape and separation effect were good under the three column temperatures. The baseline of the chromatogram did not drift at 20°C, and there was no significant difference in retention time compared with the other two temperatures. Considering the tolerance of the chromatographic column and the time required for analysis, the column temperature was selected as 20°C.

[0237] 4) Investigation of different flow rates

[0238] The effects of different flow rates of 0.20ml / min, 0.25ml / min and 0.30ml / min on the chromatographic peak shape and separation of ellagic acid in the standard decoction of roxburghii leaves were compared.

[0239] Take the test solution of Rosa roxburghii leaf standard decoction (batch number BT2206-1) under [content determination], and determine it according to the above chromatographic conditions, with ellagic acid as the calculation, and record the chromatographic data. Fig.10 And Table 21.

[0240] Table 21 Test results of different flow rates for the content determination method of roxburghii leaf standard soup

[0241]

[0242] The results show that the peak shape and separation effect of the chromatographic peaks are good at the three flow rates. When the flow rate is 0.2 mL per minute, the separation of the target components is good and the baseline does not drift. The flow rate of 0.2 mL per minute was selected in this experiment.

[0243] Example 4

[0244] Establishment of characteristic spectrum analysis of standard decoction of Rosa roxburghii leaves

[0245] 4.1.1 Instruments and reagents

[0246] Instruments: Agilent 1290 Infinity II ultra-high performance liquid chromatograph; KQ-500DA digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 1 / 10,000 electronic balance (model: ML204); HH-4 digital constant temperature water bath (Changzhou Putian Instrument Manufacturing Co., Ltd.).

[0247] Reagents: Acetonitrile was of chromatographic grade; water was ultrapure water; other reagents were of analytical grade.

[0248] Test drug: Ellagic acid (batch number: 111959-201903, purity 88.8%) was purchased from China Food and Drug Inspection Institute. The batch numbers of 15 batches of roxburghii leaf standard soup are: BT2206-1, BT2206-2, BT2206-3, BT2206-4, BT2206-5, BT2206-6, BT2206-7, BT2206-8, BT2206-9, BT2206-10, BT2206-10, BT2206-12, BT2206-13, BT2206-14, BT2206-15 (provided by the laboratory process group).

[0249] 4.1.2 Preparation of reference solution

[0250] Take about 1.0g of the reference medicinal material of Rosa roxburghii leaf, weigh it accurately, put it in a stoppered conical bottle, accurately add 40ml of 75% ethanol, weigh it, heat and reflux for 1 hour, let it cool, weigh it again, make up the lost weight with 75% ethanol, shake it well, filter it, and take the filtrate as the reference medicinal material solution. Take an appropriate amount of ellagic acid reference substance, weigh it accurately, add methanol to make a solution containing 86μg of ellagic acid per 1ml, which is used as the reference substance solution.

[0251] 4.1.3 Determination of chromatographic conditions

[0252] (1) Determination of detection wavelength

[0253] Take the sample solution of the standard decoction of roxburghii leaves for analysis and record the absorption spectrum in the range of 190-400nm (see Fig.11 ).

[0254] The experimental results showed that at a wavelength of 260 nm, the sample solution of the standard decoction of roxburghii leaves could detect more chromatographic peak information and less baseline noise interference, so 254 nm was selected as the detection wavelength.

[0255] (2) Optimization of mobile phase

[0256] ① Consider A as the organic phase and B as the aqueous phase. The gradient is as follows:

[0257]

[0258]

[0259] Experimental results: see Fig.12 The elution ability of acetonitrile-0.1% phosphoric acid aqueous solution is stronger than that of methanol-0.1% phosphoric acid aqueous solution, so acetonitrile-0.1% phosphoric acid aqueous solution was selected for condition exploration, and various acids used in the mobile phase were investigated later.

[0260] ②Investigate the situations with and without adding acid.

[0261] Experimental results: see Fig.13 The peak shape is better when adding acid than when not adding acid. The peak shape is best when the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution system, so acetonitrile-0.1% phosphoric acid aqueous solution is selected for elution.

[0262] (3) Determination of chromatographic conditions

[0263] Octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); methanol:acetonitrile (1:1) was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.2 ml per minute; the column temperature was 20°C; the detection wavelength was 254 nm. The theoretical plate number calculated based on the ellagic acid peak should be no less than 3000.

[0264]

[0265] 4.1.4 Preparation of test solution

[0266] This experiment investigated the effects of different extraction solvents on the characteristic spectra of standard decoction of sea buckthorn leaves. Methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water were selected as extraction solvents. The characteristic spectra results of different extraction solvents were compared by temporarily determining the total peak area / sample weight of 4 chromatographic peaks and the chromatograms.

[0267] Take an appropriate amount of the standard decoction of roxburghii leaves (BT2206-1), take about 0.1g, accurately weigh, put it in a stoppered conical flask, accurately add methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol, water, 20ml each, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, cool, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. According to the chromatographic conditions determined above, inject the sample and record the chromatogram. The results of the investigation of different extraction solvents for the characteristic spectrum of the standard decoction of roxburghii leaves are shown in Fig.14 .

[0268] Experimental results: see Fig.15 When the extraction solvent is 75% ethanol, the extraction efficiency is higher, so 75% ethanol is selected as the extraction solvent.

[0269] According to the above experimental results, the sample pretreatment method of the characteristic spectrum of the standard decoction of roxburghii leaves can be determined as follows: take about 0.1g of the product, accurately weigh it, put it in a stoppered conical flask, accurately add 30ml of 75% ethanol, weigh it, heat it under reflux for 1h, cool it, weigh it again, make up the lost weight with 75% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0270] 4.1.5 Determination and identification of common peaks

[0271] The UPLC spectra of different batches of standard decoction samples of Rosa roxburghii leaves were determined, and the results were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the Chinese Pharmacopoeia Committee, and the common peaks were selected.

[0272] See Fig.16 The results showed that there were four obvious common peaks in the characteristic spectrum of the standard decoction of Rosa roxburghii leaves.

[0273] 4.1.6 Methodological validation of the characteristic profile analysis method

[0274] (1) Specificity inspection

[0275] Accurately draw 1 μl of the test solution and blank solvent under the [Characteristic Spectrum] of the standard decoction of roxburghii leaves, inject them into the liquid chromatograph, and measure them according to the chromatographic conditions under the above items. Fig.17 .

[0276] The experimental results show that the solvent has no interference on the characteristic peaks in the spectrum of the standard decoction of roxburghii leaves.

[0277] (2) Overall examination

[0278] Take the test solution under the [characteristic spectrum] item of the standard decoction of roxburghii leaves, inject it into the liquid chromatograph, extend the elution time by one time at the mobile phase ratio at the gradient end point, and analyze the characteristic spectrum.

[0279] Results Fig.18 The results showed that there was no obvious chromatographic peak after doubling the elution time under the chromatographic conditions, indicating that the chromatographic conditions basically met the principle of maximum information content.

[0280] (3) Precision inspection

[0281] Take the test solution under the [Characteristic spectrum] of the standard decoction of roxburghii leaves, repeat the injection 6 times, and the injection volume is 1μl. Temporarily calibrate 4 characteristic peaks, take peak 2 as the reference peak, calculate the relative retention time, and the experimental results are shown in Table 22 below.

[0282] Table 22 Precision results of characteristic spectrum of standard decoction of roxburghii leaf (relative retention time)

[0283]

[0284]

[0285] The experimental results show that the RSD of the relative retention time of each chromatographic peak is less than 3.0%, and the instrument has good precision.

[0286] (4) Stability test

[0287] Take the test solution under the [Characteristic spectrum] of the standard decoction of roxburghii leaves, and inject samples at 0, 2, 4, 8, 12, 16, 20, and 24 hours, respectively, with an injection volume of 1 μl. Temporarily calibrate 4 characteristic peaks, use peak 2 as the reference peak, and calculate the relative retention time. The results are shown in Table 23 below.

[0288] Table 23 Rosa roxburghii leaf standard decoction characteristic spectrum stability results (relative retention time)

[0289]

[0290] The experimental results showed that the RSD of the relative retention time of the chromatographic peak was less than 3.0%, indicating that the sample solution was relatively stable.

[0291] (5) Repeatability study

[0292] Take about 0.1g of the same batch of standard decoction of roxburghii leaves (BT2206-1), accurately weigh it, and prepare 6 parallel portions into test solution according to the preparation method of test solution under [Characteristic spectrum], and inject 1μl of each. Temporarily calibrate 4 characteristic peaks, take peak 2 as the reference peak, and calculate the relative retention time.

[0293] Table 24 Rosa roxburghii leaf standard decoction characteristic spectrum repeatability results (relative retention time)

[0294]

[0295] The experimental results showed that the relative retention time RSD of each chromatographic peak was less than 3.0%, indicating that the method had good repeatability.

[0296] (6) Durability test

[0297] ①Chromatographic column inspection

[0298] Three chromatographic columns were investigated, namely: column 1-Endeavorsil C18-A (2.1×150mm, 1.8μm); column 2-ChromCore AQ C18 (2.1×150mm 1.8μm); column 3-AmethSep C18 AQ (2.1*150mm, 1.8μm), and the effects of the three chromatographic columns on the peak elution of the characteristic spectrum of the standard decoction of Rosa roxburghii leaves were investigated.

[0299] Table 25 Results of chromatographic column investigation of characteristic spectrum of Rosa roxburghii leaf decoction (relative retention time)

[0300]

[0301] The experimental results are shown in Fig.19 As shown in Table 25, the experimental results show that different chromatographic columns have a certain influence on the peak elution. The ultra-high performance liquid chromatography column Endeavorsil C18-A (2.1×150mm, 1.8μm) is used for elution. The chromatographic peak shape is better and the separation effect is the best. Therefore, it is recommended that this method use an ultra-high performance liquid chromatography column Endeavorsil C18-A (2.1×150mm, 1.8μm).

[0302] ② Column temperature investigation

[0303] The elution conditions at different column temperatures (20℃, 25℃, 30℃) were investigated.

[0304] Table 26 Column temperature investigation results of characteristic spectrum of Rosa roxburghii leaf decoction (relative retention time)

[0305]

[0306] The experimental results are shown in Fig. 20 As shown in Table 26, the experimental results show that the column temperature has a certain influence on the peak elution, but the separation effect is best and the peak shape is better when the column temperature is 20℃, so it is recommended to use a column temperature of 20℃ for measurement.

[0307] ③ Flow rate investigation: investigate the elution conditions at different flow rates (0.20ml / min, 0.25ml / min, 0.30ml / min).

[0308] Table 27 Flow rate investigation results of characteristic spectrum of Rosa roxburghii leaf decoction (relative retention time)

[0309]

[0310] The experimental results are shown in Fig.21 As shown in Table 27, the experimental results show that the flow rate has a certain influence on the peak appearance, but the peak shape is best when the flow rate is 0.20ml / min. Therefore, it is recommended to use 0.20ml / min as the measurement flow rate.

[0311] The final characteristic spectrum should show 4 characteristic peaks, of which peak 2 should be consistent with the retention time of the ellagic acid reference peak. The peak corresponding to the ellagic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, which is: 0.29 (peak 1), 1.44 (peak 3), 1.86 (peak 4). See for details. Fig. 27 .

[0312] Example 5

[0313] Identification of Rosa roxburghii Leaf Extract

[0314] On the basis of the thin layer identification method of roxburghii leaves in the 2019 edition of the Quality Standards for Traditional Chinese and Ethnic Medicinal Materials in Guizhou Province, considering the specificity of thin layer identification of roxburghii leaves, the roxburghii leaf control medicinal material was used as a control, and chloroform-ethyl acetate-formic acid (5:3:1) was used to develop, take out, dry, spray with 5% ferric chloride ethanol solution, and dry. In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material. The results showed that spots corresponding to the control medicinal material could be detected in the samples, and the spots were clear and well separated. This method is highly operational and reproducible, so this method is included in the text of the quality standard for roxburghii leaf formula granules.

[0315] 5.1 Instruments, reagents and test drugs

[0316] Instruments: TLC VISUALIZER2 (Camag, Switzerland), double-slot developing cylinder, fully automatic TLC sampler (AUTOMATIC TLC SAMPLER4 (Camag, Switzerland), ml-204 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), ultrasonic cleaner (KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.), silica gel G thin layer plate (Qingdao Ocean Chemical Plant). All reagents were of analytical grade.

[0317] Test drugs: Rosa roxburghii leaf formula granules (batch numbers: 231101, 231102, 231103; source: Sinopharm Tongjitang (Guizhou) Pharmaceutical Co., Ltd.; Rosa roxburghii leaf reference medicinal material (121355-200401, China Food and Drug Inspection Institute).

[0318] 5.2 Solution preparation

[0319] 5.2.1 Preparation of test solution

[0320] Take 0.5 g of this product, grind it into powder, add 10 ml of ethyl acetate, treat it with ultrasound for 15 min, filter it, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0321] 5.2.2 Preparation of reference medicinal material solution

[0322] Take 1 g of Rosa roxburghii leaf as a reference medicinal material, add 20 ml of water, heat and reflux for 30 minutes, filter, extract the filtrate twice with ethyl acetate, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the reference medicinal material solution.

[0323] 5.2.3 Preparation of negative sample solution

[0324] Take 0.5 g of the negative sample of the Rosa roxburghii leaf formula granules, grind it into powder, and treat it in the same way as the test sample to serve as the negative sample solution.

[0325] 5.3 Thin layer chromatography conditions

[0326] Thin layer plate: Silicone G plate

[0327] Developing solvent: chloroform-ethyl acetate-formic acid (5:3:1)

[0328] Spotting method: spray strip spotting

[0329] Deployment method: double-slotted deployment cylinder

[0330] Inspection: Spray with 5% ferric chloride ethanol solution, let dry, and inspect under sunlight

[0331] 5.5 Investigation on the specificity of roxburghii leaf formula granules

[0332] 6 μl of the test solution and negative sample solution of the Rosa roxburghii leaf formula granules and 1 μl of the control medicinal material solution were respectively applied to the same silica gel G thin layer plate, and chloroform-ethyl acetate-formic acid (5:3:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 5% ferric chloride ethanol solution, dried, and inspected under sunlight. The experimental results are shown in Fig. 22 .

[0333] 5.6 Investigation at different temperatures

[0334] 6 μl of the test solution of the Rosa roxburghii leaf formula granules and 1 μl of the control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and developed with chloroform-ethyl acetate-formic acid (5:3:1) as the developing agent (the developing temperatures were 5°C, 25°C, and 35°C, respectively). The plate was taken out, dried, sprayed with 5% ferric chloride ethanol solution, dried, and examined under sunlight.

[0335] Depend on Fig.23 It can be seen that under different temperature conditions, the chromatogram of the test sample of the sea buckthorn leaf formula granules and the corresponding positions of the control medicinal materials show spots of the same color, with clear color development, good separation, no tailing phenomenon, and no background interference, indicating that temperature has no obvious effect on the thin layer identification of the sea buckthorn leaf formula granules, indicating that the thin layer identification method has good durability to temperature.

[0336] 5.7 Investigation at different humidity levels

[0337] 6 μl of the test solution of the Rosa roxburghii leaf formula granules and 1 μl of the control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and developed with chloroform-ethyl acetate-formic acid (5:3:1) as the developing agent (developing humidity was 33%, 66%, and 88%, respectively). The plate was taken out, dried, sprayed with 5% ferric chloride ethanol solution, dried, and inspected under sunlight.

[0338] Depend on Fig.24It can be seen that under different humidity conditions, the chromatograms of the test sample and the control medicinal materials of the sea buckthorn leaf formula granules showed spots of the same color at the corresponding positions, and the main spots were clearly colored, with good separation, no tailing phenomenon, and no background interference, indicating that changes in humidity have no obvious effect on the thin layer identification of the sea buckthorn leaf formula granules, indicating that the thin layer identification method has good durability to humidity.

[0339] 5.8 Inspection of thin-layer boards from different manufacturers

[0340] 6 μl of the test solution of the Rosa roxburghii leaf formula granules and 1 μl of the control medicinal material solution were respectively spotted on silica gel G thin layer plates from different manufacturers (marine silica gel G plate, Sinopharm silica gel G plate, Merck silica gel G plate, high-efficiency G plate), and chloroform-ethyl acetate-formic acid (5:3:1) was used as the developing agent. The plates were developed, taken out, dried, sprayed with 5% ferric chloride ethanol solution, dried, and inspected under sunlight. The experimental results are shown in Fig.25 .

[0341] Depend on Fig.25 It can be seen that when using silica gel thin layer plates from different manufacturers (Qingdao Ocean Silica G Plate, Sinopharm Chemical Reagent Silica G Plate, Qingdao Ocean High-efficiency Silica G Plate), the main spots of the chromatogram of the test sample and the chromatogram of the reference medicinal material of the Rosa roxburghii leaf formula granules can correspond to each other without significant influence, indicating that the thin layer identification method has good durability for thin layer silica gel G plates from different manufacturers.

[0342] 5.9 Thin layer identification chromatogram of roxburghii leaf formula granules

[0343] 6 μl of the test solution of the Rosa roxburghii leaf formula granules and 1 μl of the control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and chloroform-ethyl acetate-formic acid (5:3:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 5% ferric chloride ethanol solution, dried, and inspected under sunlight. The experimental results are shown in Fig.26 .

[0344] Depend on Fig.26 It can be seen that the chromatograms of the test samples in the three batches of Rosa roxburghii leaf formula granules show spots of the same color at the corresponding positions of the chromatograms of the control medicinal materials, indicating that the thin layer identifications of the three batches of Rosa roxburghii leaf formula granules all meet the requirements.

[0345] 5.10 Summary

[0346] Judging from the spots in the chromatogram, the separation effect of the fluorescent spots of the roxburghii leaf formula granules is good, and the chromatogram of the roxburghii leaf formula granules and the chromatogram of the roxburghii leaf reference medicinal material show spots of the same color at the corresponding positions, and they can correspond one to one. This method can effectively qualitatively identify the roxburghii leaf formula granules that have lost their medicinal properties. Through the investigation of thin layer chromatography methodology, this method has good specificity and durability, and is suitable for thin layer chromatography identification of roxburghii leaf formula granules.

[0347] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for preparing a roxburghii leaf extract and a preparation thereof, characterized in that: The steps include: (1) Take Rosa roxburghii leaf slices, add water and boil twice, filter, and combine the two decoctions after the filtrate is cooled to room temperature; (2) The decoction is concentrated under reduced pressure and low temperature to obtain an extract, and the density of the obtained extract is controlled to be between 1.15 and 1.16 g / ml; the extract is obtained (3) The extract is made into a desired dosage form.

2. The method for preparing the roxburghii leaf extract and the preparation thereof according to claim 1, characterized in that: The step (1) specifically comprises taking Rosa roxburghii leaf slices, adding water and decocting twice, directly adding the slices to 18-22 times the amount of water for the first decoction, soaking for 28-32 minutes, boiling over high heat and then keeping the mixture slightly boiling over low heat for 28-30 minutes, filtering the decoction while hot, and cooling the filtrate to room temperature; adding 14-16 times the amount of water for the second decoction, heating over high heat and then boiling and then keeping the mixture slightly boiling over low heat for 18-20 minutes, filtering the decoction while hot, cooling the filtrate to room temperature, and combining the two decoctions.

3. The method for preparing the roxburghii leaf extract according to claim 1, characterized in that: In the step (2), the specific conditions for reduced pressure and low temperature concentration are: temperature: 65° C.; vacuum degree: -0.080 to -0.090 MPa.

4. The method for preparing the roxburghii leaf extract and the preparation thereof according to claim 1, characterized in that: The step (3) is to freeze-dry the obtained extract to obtain a standard decoction of roxburghii leaves; the freeze-drying parameters are: the pre-freezing temperature of the roxburghii leaf concentrate is -45°C, the pre-freezing time is 240 minutes, the sublimation drying temperature is -35°C to 0°C, the sublimation drying time is 1185 minutes, and the vacuum degree is -0.2 mbar; the analytical drying temperature is 5°C to 25°C, the analytical drying time is 445 minutes, and the vacuum degree is -0.2 mbar.

5. The method for preparing the roxburghii leaf extract and the preparation thereof according to claim 1, characterized in that: The step (3) is to add auxiliary materials to the obtained extract, dry it, add auxiliary materials again, mix them evenly, and granulate them to obtain Rosa roxburghii leaf granules.

6. The method for preparing the roxburghii leaf extract and the preparation thereof according to claim 5, characterized in that: The preparation amount of the roxburghii leaf granules is 25-30%, that is, 3333-4000g of the medicinal pieces are prepared into 1000g of granules, and the specification is that each 1g of the formula granules is equivalent to 3.3-4.0g of the medicinal pieces.

7. A method for determining the contents of multiple components in a roxburghii leaf extract and its preparation, characterized in that: The determination was carried out by high performance liquid chromatography, with octadecylsilane bonded silica gel as the filler; acetonitrile as the mobile phase A, and 0.1% phosphoric acid solution as the mobile phase B, and gradient elution was performed as specified in the following table; the flow rate was 0.2 ml per minute; the column temperature was 20°C; the detection wavelength was 278 nm for shortleaf hematoxylin and 254 nm for ellagic acid 8. The method for determining the contents of multiple components of the roxburghii leaf extract and its preparation according to claim 7, characterized in that: The chromatographic column used had a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

9. The method for determining the contents of multiple components in the roxburghii leaf extract and its preparation according to claim 7, characterized in that: The test solution is prepared by the following method: take about 0.1 g of the sample to be tested, accurately weigh it, put it in a stoppered conical flask, accurately add 30 ml of 75% ethanol, weigh it, heat and reflux it for 1 hour, cool it, weigh it again, make up the lost weight with 75% ethanol, shake it well, filter it, and take the filtrate to obtain it.

10. The method for determining the contents of multiple components in the roxburghii leaf extract and its preparation according to claim 7, characterized in that: The reference solution is a solution prepared with methanol as solvent and contains 60 μg of brevicornulinic acid and 80 μg of ellagic acid per 1 ml.

11. A method for detecting characteristic spectra of a roxburghii leaf extract and its preparation, characterized in that: The determination was carried out by high performance liquid chromatography, with octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution as specified in the following table; the flow rate was 0.2 ml per minute; the column temperature was 20°C; the detection wavelengths were 278 nm for shortleaf hematoxylin and 254 nm for ellagic acid: The characteristic spectrum standard of roxburghii leaf extract and its preparation was finally determined as follows: 4 characteristic peaks should be present in the chromatogram of the test sample, of which peak 2 should be consistent with the retention time of the ellagic acid reference peak; the peak corresponding to the ellagic acid reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.29 (peak 1), 1.44 (peak 3), 1.86 (peak 4).

12. A thin layer chromatography method for a roxburghii leaf extract and a preparation thereof, characterized in that: The specific steps are as follows: take 0.5g of the sample to be tested, grind it, add 10ml of ethyl acetate, ultrasonically treat it for 15min, filter it, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; take another 1g of the reference medicinal material of sea buckthorn leaf, add 20ml of water, heat and reflux for 30 minutes, filter it, extract the filtrate twice with ethyl acetate, 20ml each time, combine the ethyl acetate solution, evaporate it to dryness, add 1ml of methanol to dissolve the residue, and prepare the control medicinal material solution for thin layer chromatography test, respectively aspirate 6μl of the test solution and 1μl of the control medicinal material solution on the same silica gel G thin layer plate, use chloroform-ethyl acetate-formic acid (5:3:1) as the developing agent, develop it, take it out, dry it, spray it with 5% ferric chloride ethanol solution, dry it, and inspect it under sunlight. In the chromatogram of the test sample, spots of the same color should appear at the corresponding position of the chromatogram of the control medicinal material.

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