Preparation method of Bupleurum marginatum Wall. ex DC. var. stenophyllum (Wolff) Shan & Y. Li extract, detection method and construction method of characteristic chromatogram

Abstract

This application relates to the technical field of traditional Chinese medicine, and specifically relates to a preparation method of Bupleurum marginatum Wall. ex DC. extract, a construction method of characteristic fingerprint, and a detection method of rutin in Bupleurum marginatum Wall. ex DC. extract. The preparation method includes: decocting Bupleurum marginatum Wall. ex DC. slices twice to obtain a decoction, and filtering; concentrating the decoction to obtain a concentrated solution, and freeze-drying the concentrated solution to obtain Bupleurum marginatum Wall. ex DC. extract. This application uses Bupleurum marginatum Wall. ex DC. medicinal materials to prepare Bupleurum marginatum Wall. ex DC. extract. The extract yield of the Bupleurum marginatum Wall. ex DC. extract is 6-25%, and the extract yield is relatively high. Through the accelerated stability test, the rutin content and moisture content are relatively stable, and the characteristic fingerprint and content detection method have good specificity, good repeatability, and good stability.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine, and specifically relates to a preparation method of Bupleuri Radix Extract, a construction method of characteristic chromatogram, and a detection method of rutin and / or total transfer rate in Bupleuri Radix Extract. Background Art

[0002] The Chinese medicinal material Bupleuri Radix is a plant of the Umbelliferae family, which relieves exterior and interior syndromes, and promotes yang and relieves depression. It is used for alternating chills and fever, fullness in the chest, pain in the hypochondrium, bitter taste in the mouth, deafness, dizziness, malaria, qi deficiency in the middle-jiao, rectal prolapse, irregular menstruation, and uterine prolapse. The extract, also known as the standard decoction, is a traditional dosage form widely used clinically. The extract is prepared by standard decocting according to the theory of traditional Chinese medicine, following the clinical decoction method, separating the solid and liquid, and appropriately concentrating or drying by a suitable method. It serves as a standard reference for measuring whether the traditional Chinese medicine formula granules are basically consistent with the clinical decoction.

[0003] With the development of new dosage forms of traditional Chinese medicine, the quality control of Chinese medicinal material extracts and their preparations has become an urgent problem to be solved. At present, standards for Bupleuri Radix have been issued in Sichuan Province, Gansu Province, etc., but there is no standard reference for the extracts and their preparations of the medicinal material. It cannot provide a reference for controlling the quality of the terminal products of traditional Chinese medicine extracts. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present application provides a preparation method of Bupleuri Radix Extract, a construction method of characteristic chromatogram, and a detection method of rutin in Bupleuri Radix Extract.

[0005] Specifically, the present application proposes the following technical solutions.

[0006] The present application provides a method for preparing Bupleuri Radix Extract, wherein the method includes:

[0007] Performing two decoctions on the Bupleuri Radix slices to obtain a decoction, and filtering;

[0008] Concentrating the decoction to obtain a concentrated solution, and subjecting the concentrated solution to freeze-drying to obtain Bupleuri Radix Extract.

[0009] Preferably, for the above-mentioned method, 11 - 13 times the amount of water is added for the first decoction, and preferably, 9 - 11 times the amount of water is added for the second decoction.

[0010] Preferably, for the above-mentioned method, the freeze-drying includes pre-freezing, primary drying, and secondary drying. Preferably, the pre-freezing is carried out at -60 to -48 °C for 150 - 200 minutes, preferably at -50 °C for 180 minutes;

[0011] Preferably, the primary drying is carried out at -45 to 0 °C for 120 - 2340 minutes;

[0012] Preferably, the secondary drying is carried out at 10 - 30°C for 60 - 360 minutes.

[0013] Preferably, for the method described above, filtration is carried out through a sieve with 100 - 300 meshes.

[0014] Preferably, for the method described above, the paste yield of the concentrated solution is 6 - 25%, preferably 6.1 - 20.7%.

[0015] This application provides the Bupleurum marginatum Wall. ex DC. extract prepared by the method described above.

[0016] This application provides a method for constructing a characteristic chromatogram of Bupleurum marginatum Wall. ex DC. extract, which includes:

[0017] Preparation of the reference substance solution: Add Bupleurum marginatum Wall. ex DC. to the first solvent for extraction to obtain the reference substance solution;

[0018] Preparation of the reference solution: Add rutin and quercetin to the first solvent respectively to obtain the reference solution;

[0019] Preparation of the test solution: Add the Bupleurum marginatum Wall. ex DC. extract prepared by the method for preparing Bupleurum marginatum Wall. ex DC. extract described above to the first solvent for extraction to obtain the test solution;

[0020] Perform high - performance liquid analysis on the reference substance solution, reference solution, and test solution to obtain the characteristic chromatogram of Bupleurum marginatum Wall. ex DC. extract.

[0021] Preferably, for the construction method described above, the first solvent is water, 95% (v / v) ethanol, 75% (v / v) ethanol, dilute ethanol, methanol, 75% (v / v) methanol, or 50% (v / v) methanol, preferably 75% methanol.

[0022] Preferably, for the construction method described above, mobile phase A is acetonitrile or methanol, preferably acetonitrile;

[0023] Preferably, mobile phase B is an aqueous solution containing 0.1 wt% phosphoric acid, water, an aqueous solution containing 0.1 wt% formic acid, or an aqueous solution containing 0.1 wt% acetic acid, preferably an aqueous solution containing 0.1 wt% acetic acid.

[0024] Preferably, for the construction method described above, gradient elution is adopted. Preferably, the gradient mode is as follows:

[0025] 0 - 25 min, mobile phase A increases from 5% to 25%, and mobile phase B decreases from 95% to 75%;

[0026] 25 - 30 min, mobile phase A increases from 25% by volume to 45% by volume, and mobile phase B decreases from 75% by volume to 55% by volume;

[0027] 30 - 33 min, mobile phase A increases from 45% by volume to 95% by volume, and mobile phase B decreases from 55% by volume to 5% by volume;

[0028] 33 - 33.2 min, mobile phase A decreases from 95% by volume to 5% by volume, and mobile phase B increases from 5% by volume to 95% by volume;

[0029] 33.2 - 35 min, 5% by volume of mobile phase A: 95% by volume of mobile phase B.

[0030] Preferably, for the construction method described above, the column temperature is 30 - 40 °C;

[0031] Preferably, the flow rate is 0.25 - 0.35 ml / min;

[0032] Preferably, the detection wavelength is 255 nm;

[0033] Preferably, the injection volume is 1 - 5 μL.

[0034] Preferably, for the construction method described above, the characteristic chromatogram of the Bupleurum chinense DC. var. glabratum Hsiao et K. Hsuan extract contains 6 characteristic peaks. Preferably, the characteristic peaks are the characteristic peaks at a relative retention time of 0.30 ± 10%, 0.48 ± 10%, 1.04 ± 10% and 1.20 ± 10%, as well as the characteristic peaks of rutin and quercetin.

[0035] The present application provides a method for determining the content and / or total transfer rate of rutin in Bupleurum chinense DC. var. glabratum Hsiao et K. Hsuan extract, which includes:

[0036] Preparation of the reference solution: Rutin is added to a second solvent for extraction to obtain the reference solution;

[0037] Preparation of the test solution: The Bupleurum chinense DC. var. glabratum Hsiao et K. Hsuan extract prepared by the method for preparing Bupleurum chinense DC. var. glabratum Hsiao et K. Hsuan extract described above is added to a second solvent for extraction to obtain the test solution;

[0038] The reference solution and the test solution are subjected to high - performance liquid analysis to obtain the content and / or total transfer rate of rutin in the Bupleurum chinense DC. var. glabratum Hsiao et K. Hsuan extract.

[0039] Preferably, for the determination method described above, the second solvent is water, dilute ethanol, 75% by volume of ethanol, ethanol, 50% by volume of methanol, 75% by volume of methanol or methanol, preferably 75% by volume of methanol.

[0040] Preferably, for the above-mentioned determination method, the extraction method is ultrasonic extraction, shaking extraction or reflux extraction, preferably ultrasonic extraction.

[0041] Preferably, for the above-mentioned determination method, the extraction time is 15 - 60 min, preferably 60 min.

[0042] Preferably, for the above-mentioned determination method, mobile phase A is acetonitrile and mobile phase B is an aqueous solution containing 0.1 wt% formic acid.

[0043] Preferably, for the above-mentioned determination method, gradient elution is performed using mobile phase A and mobile phase B, preferably according to the following method:

[0044] From 0 - 6 min, mobile phase A decreases from 90% by volume to 80% by volume, and mobile phase B increases from 10% by volume to 20% by volume;

[0045] From 6 - 12 min, mobile phase A decreases from 80% by volume to 79% by volume, and mobile phase B increases from 20% by volume to 21% by volume;

[0046] From 12 - 22 min, mobile phase A decreases from 79% by volume to 52% by volume, and mobile phase B increases from 21% by volume to 48% by volume; and

[0047] From 22 - 25 min, 90% by volume of mobile phase A: 10% by volume of mobile phase B.

[0048] Preferably, for the above-mentioned determination method, the flow rate of the mobile phase is 0.20 - 0.30 ml / min;

[0049] Preferably, the column temperature is 25 - 35 °C;

[0050] Preferably, the detection wavelength is 350 - 360 nm;

[0051] Preferably, the injection volume is 1 - 5 μL.

[0052] Preferably, for the above-mentioned determination method, the content of rutin in the extract is 0.5 - 2%, preferably 0.51 - 1.44%; and / or

[0053] The total transfer rate of rutin in the extract is 6 - 25%, preferably 6.96 - 21.95%.

[0054] This application provides the use of the characteristic spectrum constructed by the above-mentioned method in Bupleurum marginatum Wall. ex DC. extract and its preparations.

[0055] The present application provides the use of the method for determining the content and / or total transfer rate as described above in Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract and its preparations.

[0056] Beneficial effects achieved by the present application:

[0057] 1. The preparation method described in the present application can provide a standard for the quality control of Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract and its preparations such as formula granule products, realizing the overall quality control and effective supervision of Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract and its preparations such as formula granule products.

[0058] 2. The present application uses Bupleurum chinense DC. var. marginatum Wulf. ex DC. medicinal materials to prepare Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract. The extract yield of the Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract is 6 - 25%, and the extract yield is relatively high. After the accelerated stability test, the rutin content and moisture content are relatively stable, the relative retention time and relative peak area of the characteristic chromatogram are relatively stable, it is moisture-absorption resistant, and no impurity components are generated. The prepared Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract is analyzed by high performance liquid chromatography and ultra-high performance liquid chromatography. The method has good specificity, good repeatability, and good stability.

[0059] 3. The characteristic chromatogram method established in the present application uses ultra-high performance liquid chromatography, and has the characteristics of simplicity, stability, high precision, and good reproducibility. Moreover, the fingerprint chromatogram of the obtained extract has many peaks, good peak shapes, is easy to identify, and is accurate and reliable.

[0060] 4. The characteristic chromatogram method established in the present application is simple, time-saving, environmentally friendly. The analysis of a batch of preparations only takes 30 minutes, which can greatly shorten the detection and analysis time, improve production efficiency, and enable the quality control purpose to be quickly achieved in large-scale production sampling detection. Description of the drawings

[0061] Figure 1 It is the DAD diagram of the Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract in Example 2.

[0062] Figure 2 It is the schematic diagram of the influence of different mobile phase A on the characteristic chromatogram of the Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract in Example 2.

[0063] Figure 3 It is the schematic diagram of the influence of different mobile phase B on the characteristic chromatogram of the Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract in Example 2.

[0064] Figure 4 It is the schematic diagram of the common pattern of the characteristic chromatogram of the Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract in Example 2.

[0065] Figure 5 It is the schematic diagram of the reference chromatogram of the Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract in Example 2.

[0066] Figure 6 is the schematic diagram of the influence of different extraction methods on the characteristic chromatogram of the Bupleurum chinense DC. var. marginatum Wulf. ex DC. extract in Example 2.

[0067] Figure 7 It is a comparison chart of the extraction efficiency of different extraction solvents for the characteristic fingerprint of Bupleurum chinense DC. var. marginatum W. W. Smith extract in Example 2.

[0068] Figure 8 is a schematic diagram of the comparison chromatogram of the reference substance, reference medicinal material and extract in Example 2.

[0069] Figure 9 It is a schematic diagram for investigating the specificity of the characteristic fingerprint of Bupleurum chinense DC. var. marginatum W. W. Smith extract in Example 2.

[0070] Figure 10 It is a schematic diagram for investigating the integrity of the characteristic fingerprint of Bupleurum chinense DC. var. marginatum W. W. Smith extract in Example 2.

[0071] Figure 11 is a schematic diagram of the influence of different chromatographic columns on the characteristic fingerprint of Bupleurum chinense DC. var. marginatum W. W. Smith extract in Example 2.

[0072] Figure 12 It is a schematic diagram of the influence of different column temperatures on the characteristic fingerprint of Bupleurum chinense DC. var. marginatum W. W. Smith extract in Example 2.

[0073] Figure 13 It is the influence of different flow rates on the durability of the characteristic fingerprint of Bupleurum chinense DC. var. marginatum W. W. Smith extract in Example 2.

[0074] Figure 14 is a schematic diagram of the specificity of different substances in Example 3.

[0075] Figure 15 It is a schematic diagram of the peak purity of the target peak of different substances in Example 3.

[0076] Figure 16 It is a schematic diagram of the linear regression equation of rutin in Example 3.

[0077] Figure 17 It is a chromatogram of the rutin content in Bupleurum chinense DC. var. marginatum W. W. Smith extract with different chromatographic columns in Example 3.

[0078] Figure 18 It is a chromatogram of the rutin content in Bupleurum chinense DC. var. marginatum W. W. Smith extract with different chromatographic instruments in Example 3.

[0079] Figure 19 is a chromatogram of the rutin content in Bupleurum chinense DC. var. marginatum W. W. Smith extract at different column temperatures in Example 3.

[0080] Figure 20 It is a chromatogram of the rutin content in Bupleurum chinense DC. var. marginatum W. W. Smith extract at different flow rates in Example 3. Detailed implementation manners

[0081] This application provides a method for preparing Bupleurum chinense DC. var. marginatum W. W. Smith extract, wherein the method includes:

[0082] Performing two decoctions on the Bupleurum chinense DC. var. marginatum W. W. Smith decoction pieces to obtain a decoction solution, and filtering;

[0083] The decoction is concentrated to obtain a concentrated solution, and the concentrated solution is freeze-dried to obtain the Bupleurum marginatum Wall. ex DC. extract.

[0084] In this application, there is no restriction on the processing of the Bupleurum marginatum Wall. ex DC. slices. It can be prepared according to the conventional methods in the art. For example, the original medicinal materials can be taken, impurities removed, cut into sections, and dried, etc.

[0085] In this application, there is no restriction on the utensils used for decocting. The common utensils in the art can be used for decocting. For example, according to the regulations on decocting containers in the "Management Specification for Traditional Chinese Medicine Decocting Rooms in Medical Institutions" issued by the Ministry of Health and the State Administration of Traditional Chinese Medicine in 2009 (decocting containers should preferably be utensils made of ceramics, stainless steel, copper, etc.), in this application, a 6L automatic decocting ceramic pot is selected as the extract decocting equipment.

[0086] In this application, there is no restriction on the dosage of the Bupleurum marginatum Wall. ex DC. slices. The conventional dosage in the art can be used. For example, according to the suggestions in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules" under "Extract Preparation", the dosage of slices used for each decoction is generally not less than 100 g, and the dosage of slices such as flowers and leaves can be appropriately reduced. 100 g of Bupleurum marginatum Wall. ex DC. (Bupleurum marginatum Wall. ex DC.) slices are convenient for operation, and the dry extract obtained from the decoction is sufficient for experimental research on the extract. Therefore, the dosage of the slices in the Bupleurum marginatum Wall. ex DC. (Bupleurum marginatum Wall. ex DC.) extract is set at 100 g.

[0087] The extract described in this application can standardize the quality control and standard research of traditional Chinese medicine formula granules, achieve the overall quality control and effective supervision of traditional Chinese medicine formula granules, and provide a reference for the quality control of Bupleurum marginatum Wall. ex DC. formula granules.

[0088] In some embodiments, 11 - 13 times the amount of water is added for the first decoction, preferably 12 times the amount of water. Preferably, 9 - 11 times the amount of water is added for the second decoction, preferably 10 times the amount of water.

[0089] In the present application, 11 - 13 times the amount of water is added for the first decoction, preferably 12 times the amount of water. Preferably, 9 - 11 times the amount of water is added for the second decoction, preferably 10 times the amount of water. This is in accordance with the suggestion in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules" under "Preparation of Extracts". Generally, the amount of water added should preferably soak the medicinal materials by 2 - 5 cm. For Chinese herbal medicine pieces of flowers and herbs or those with a longer decoction time, the amount of water can be appropriately increased. In the present application, when 11 - 13 times the amount of water is added for the first decoction, preferably 12 times the amount of water, which meets the requirement that the solvent amount soaks the medicinal materials by 2 - 5 cm, and 9 - 11 times the amount of water is added for the second decoction, preferably 10 times the amount of water, the water level has exceeded 2 cm. Therefore, in the present application, 11 - 13 times the amount of water is added for the first decoction, preferably 12 times the amount of water, and preferably, 9 - 11 times the amount of water is added for the second decoction, preferably 10 times the amount of water.

[0090] In the present application, after adding water, soaking is required before decoction. In the present application, no limitation is imposed on the soaking time, and it can be designed according to the conventional methods in the art. For example, it can be based on the suggestion in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules" under "Preparation of Extracts" that the medicinal materials 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 of Bupleurum chinense DC. var. glabrum Kitag. extract is determined to be 30 minutes.

[0091] In the present application, no limitation is imposed on the decoction time, and it can be designed according to the conventional methods in the art. For example, it can be based on the suggestion in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules" under "Preparation of Extracts" that generally each dose of medicine is decocted twice. Generally, after boiling, decoct for another 30 minutes; medicines for relieving exterior syndrome, clearing heat, and aromatic medicines should not be decocted for a long time, and it is appropriate to decoct for another 20 minutes after boiling. For Chinese herbal medicine pieces with a harder texture, the decoction time can be appropriately extended; for tonic Chinese herbal medicine pieces, first bring to a boil with strong fire and then change to slow fire and simmer for about 30 minutes. The decoction time for the second decoction can be appropriately shortened. Therefore, according to the "Technical Requirements", the number of decoctions of Bupleurum chinense DC. var. glabrum Kitag. extract is determined to be two times. Decoct for another 30 minutes after the first boil, and decoct for another 20 minutes after the second boil.

[0092] In some embodiments, filtration is carried out through a 100 - 300 - mesh sieve.

[0093] For example, the filtration can be carried out through a 100-mesh sieve, a 200-mesh sieve or a 300-mesh sieve. It is designed according to the requirement in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules" that the mesh number of the solid-liquid separation filter material under "Extract Preparation" should be above 100 meshes. In order to obtain a decoction consistent with the traditional extract through modern solid-liquid separation methods, the filtration effects of 100-mesh sieve, 200-mesh sieve and 300-mesh sieve on the Bupleuri Radix Decoction were mainly investigated. The results showed that when solid-liquid separation was carried out, the 100-mesh, 200-mesh and 300-mesh sieves were all relatively easy 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 300-mesh sieve was good, and the amount of sediment after standing was small. Therefore, a 300-mesh sieve was used for the solid-liquid separation of Bupleuri Radix extract.

[0094] In this application, for the concentration method, this application does not make any restrictions. It can be concentrated according to the conventional concentration methods in the art. For example, according to the suggestion in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules" under "Extract Preparation", a vacuum concentration method can be used for low-temperature concentration to a specified amount of extract. Now, by comparing different concentration temperatures, a suitable concentration temperature is determined. Temporarily, the concentration ratio is determined to be 1:1, and its concentration state is observed. By comparing and measuring the density, extract yield of Bupleuri Radix extract at different concentration temperatures and observing the state of the concentrated solution. The results show that: the extract yields at different concentration temperatures are basically the same, and there is not much difference between the concentration temperatures of 50°C and 65°C. Considering comprehensively, 65°C is selected as the concentration temperature. When the concentration ratio is 1:1, the viscosity of the extract is moderate, the fluidity is good, it is convenient to transfer, and the density is between 1.06 and 1.09 g / ml. Therefore, the concentration ratio is determined to be 1:1, and other concentration ratios are not considered for the time being.

[0095] Therefore, for concentration, a rotary evaporator can be used for vacuum concentration (temperature: 65°C; vacuum degree: -0.080 to -0.090 MPa) to 100 ml.

[0096] In some embodiments, the freeze-drying includes pre-freezing, primary drying and secondary drying. Preferably, the pre-freezing is maintained at -60 to -48°C for 150 - 200 minutes, preferably at -50°C for 180 minutes;

[0097] Preferably, the primary drying is carried out at -45 to 0°C for 120 - 2340 minutes;

[0098] Preferably, the secondary drying is carried out at 10 - 30°C for 60 - 360 minutes.

[0099] In this application, the freeze-drying is carried out according to the suggestion in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules" that the drying of the extract is generally preferably carried out by freeze-drying, which can ensure the stability of its quality, easy dissolution and the avoidance of adding excipients. Therefore, freeze-drying is first selected as the drying method for the Bupleuri Radix extract.

[0100] In this application, since the eutectic point test result of the vacuum freeze-dryer for Bupleuri Radix is -43.6 °C, the pre-freezing temperature is -50 °C, the primary drying temperature is -45 °C to 0 °C, the secondary drying temperature is 10 °C to 30 °C, and the total drying time is 48 hours. Therefore, under these freeze-drying conditions, the water content of the Bupleuri Radix extract is relatively low and it is easy to preserve.

[0101] In this application, the primary drying refers to sublimation drying, and the secondary drying refers to desorption drying.

[0102] In this application, for the primary drying, for example, it can be maintained at -45 °C for 120 min, for example, it can be maintained at -35 °C for 360 min; for example, it can be maintained at -30 °C for 1080 min; for example, it can be maintained at -25 °C for 540 min; for example, it can be maintained at -20 °C for 120 min; for example, it can be maintained at -10 °C for 60 min; for example, it can be maintained at 0 °C for 60 min.

[0103] In this application, for the secondary drying, for example, it can be maintained at 10 °C for 60 min; for example, it can be maintained at 20 °C for 60 min; for example, it can be maintained at 30 °C for 240 min.

[0104] In some embodiments, the extract yield of the concentrated liquid is 6 - 25%.

[0105] The extract yield of the concentrated liquid can be 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, etc.

[0106] In this application, for the determination method of the solid content (9.09 - 17.41%) of the decoction liquid, this application does not make any restrictions, and it can be determined according to the conventional methods in the art. For example, it can be determined by referring to the "Hot Infusion Method" in the General Rules 2201 for the Determination of Extracts in Part IV of the Chinese Pharmacopoeia 2020 Edition. Precisely pipette 25 ml of the extract solution, place it in a pre-weighed evaporating dish, evaporate to dryness on a water bath, dry at 105 °C for 3 hours, cool in a desiccator for 30 min, and quickly and precisely weigh to obtain the solid content.

[0107] Calculation formula for the extract yield of the extract solution: Extract yield % = Extract solution volume * Extract solution solid content / Decoction piece amount * 100%.

[0108] In this application, there is no limitation on the method for measuring the extract yield of the concentrated liquid. It can be measured according to the conventional methods in the art. For example, it can refer to the determination of the extract content by the "hot extraction method" in the general rules 2201 of the fourth part of the Chinese Pharmacopoeia (2020 edition): Accurately weigh 10 g of the concentrated liquid, place it in a pre-weighed evaporating dish, evaporate it to dryness on a water bath, dry it at 105 °C for 3 hours, cool it in a desiccator for 30 min, quickly and accurately weigh it, calculate the solid content rate of the concentrated liquid, and then calculate the extract yield according to the following calculation formula: Extract yield % = weight of concentrated liquid * solid content rate of concentrated liquid / amount of Chinese herbal pieces * 100%.

[0109] This application provides a Bupleurum marginatum Wall. ex DC. extract prepared by the method described above.

[0110] This application provides a method for constructing a characteristic fingerprint of Bupleurum marginatum Wall. ex DC. extract, which includes:

[0111] Preparation of the reference substance solution: Add Bupleurum marginatum Wall. ex DC. to the first solvent for extraction to obtain the reference substance solution;

[0112] Preparation of the reference solution: Add rutin and quercetin to the first solvent respectively to obtain the reference solution;

[0113] Preparation of the test solution: Add the Bupleurum marginatum Wall. ex DC. extract prepared by the method described above to the first solvent for extraction to obtain the test solution;

[0114] The reference solution, reference substance solution and test solution are subjected to high performance liquid analysis to obtain the characteristic chromatogram of the Bupleurum falcatum L. extract. In some embodiments, the first solvent is water, 95% by volume ethanol, 75% by volume ethanol, dilute ethanol, methanol, 75% by volume methanol or 50% by volume methanol, preferably 75% methanol. In some embodiments, mobile phase A is acetonitrile or methanol, preferably acetonitrile; preferably, mobile phase B is an aqueous solution containing 0.1 wt% phosphoric acid, water, an aqueous solution containing 0.1 wt% formic acid or an aqueous solution containing 0.1 wt% acetic acid, preferably an aqueous solution containing 0.1 wt% acetic acid. In some embodiments, gradient elution is employed, preferably, the gradient is as follows: 0 - 25 min, mobile phase A increases from 5% to 25%, and mobile phase B decreases from 95% to 75%; 25 - 30 min, mobile phase A increases from 25% to 45%, and mobile phase B decreases from 75% to 55%; 30 - 33 min, mobile phase A increases from 45% to 95%, and mobile phase B decreases from 55% to 5%; 33 - 33.2 min, mobile phase A decreases from 95% to 5%, and mobile phase B increases from 5% to 95%; 33.2 - 35 min, 5% mobile phase A: 95% mobile phase B. In some embodiments, the column temperature is 30 - 40 °C;

[0115] Preferably, the flow rate is 0.25 - 0.35 ml / min;

[0116] Preferably, the detection wavelength is 255 nm;

[0117] Preferably, the injection volume is 1 - 5 μL.

[0118] For example, the column temperature can be 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, etc.

[0119] For example, the flow rate can be 0.25 ml / min, 0.26 ml / min, 0.27 ml / min, 0.28 ml / min, 0.29 ml / min, 0.30 ml / min, 0.31 ml / min, 0.32 ml / min, 0.33 ml / min, 0.34 ml / min, 0.35 ml / min, etc.

[0120] In the present application, the dilute ethanol is prepared according to the provisions of the Chinese Pharmacopoeia, that is, 529 ml of ethanol is taken and diluted with water to 1000 ml to obtain it. This solution contains C 2 H 5 OH and should be 49.5 - 50.5% (ml / ml) at 20 °C.

[0121] In the present application, the chromatographic column is a chromatographic column filled with octadecylsilyl-bonded silica gel (column length is 150 mm, inner diameter is 2.1 mm, and particle size is 1.9 μm).

[0122] In some embodiments, the characteristic chromatogram of the Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract contains 6 characteristic peaks. Preferably, the characteristic peaks are the characteristic peaks at a relative retention time of 0.30 ± 10%, 0.48 ± 10%, 1.04 ± 10%, and 1.20 ± 10%, as well as the characteristic peaks of rutin and quercetin.

[0123] The characteristic chromatogram described in the present application is determined by the research and establishment of the analysis method for the characteristic chromatogram of the Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract and the determination of the characteristic chromatograms of 15 batches of Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extracts, and finally the reference characteristic chromatogram of the Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract is determined.

[0124] The present application provides a method for determining the content and / or total transfer rate of rutin in the Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract, which includes:

[0125] Preparation of the reference solution: Rutin is added to a second solvent for extraction to obtain the reference solution;

[0126] Preparation of the test solution: The Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract prepared by the above-mentioned preparation method is added to a second solvent for extraction to obtain the test solution;

[0127] The reference solution and the test solution are subjected to high-performance liquid chromatography analysis to obtain the content and / or total transfer rate of rutin in the Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract. In some embodiments, the second solvent is water, dilute ethanol, ethanol with a volume fraction of 75%, ethanol, methanol with a volume fraction of 50%, methanol with a volume fraction of 75%, or methanol, preferably methanol with a volume fraction of 75%. In some embodiments, the extraction method is ultrasonic extraction, shaking extraction, or reflux extraction, preferably ultrasonic extraction. In some embodiments, the extraction time is 15 - 60 min, preferably 60 min. For example, the extraction time can be 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.

[0128] In some embodiments, mobile phase A is acetonitrile, and mobile phase B is an aqueous solution containing 0.1 wt% formic acid. In some embodiments, gradient elution is performed using mobile phase A and mobile phase B, preferably according to the following method for gradient elution:

[0129] 0 - 6 min, mobile phase A decreases from 90% by volume to 80% by volume, and mobile phase B increases from 10% by volume to 20% by volume;

[0130] From 6 - 12 min, mobile phase A is reduced from 80% by volume to 79% by volume, and mobile phase B is increased from 20% by volume to 21% by volume;

[0131] From 12 - 22 min, mobile phase A is reduced from 79% by volume to 52% by volume, and mobile phase B is increased from 21% by volume to 48% by volume; and

[0132] From 22 - 25 min, 90% by volume of mobile phase A: 10% by volume of mobile phase B.

[0133] In some embodiments, the flow rate of the mobile phase is 0.20 - 0.30 ml / min;

[0134] Preferably, the column temperature is 25 - 35 °C;

[0135] Preferably, the detection wavelength is 350 - 360 nm;

[0136] Preferably, the injection volume is 1 - 5 μL.

[0137] For example, the flow rate of the mobile phase can be 0.20 ml / min, 0.21 ml / min, 0.22 ml / min, 0.23 ml / min, 0.24 ml / min, 0.25 ml / min, 0.26 ml / min, 0.27 ml / min, 0.28 ml / min, 0.29 ml / min, 0.30 ml / min, etc.;

[0138] For example, the column temperature can be 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, etc.

[0139] In some embodiments, the content of rutin in the extract is 0.5 - 2%, preferably 0.51 - 1.44%; and / or

[0140] The total transfer rate of rutin in the extract is 6 - 25%, preferably 6.96 - 21.95%.

[0141] For example, the content of rutin in the extract can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, etc.

[0142] The total transfer rate of rutin in the extract can be 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, etc.

[0143] In the present application, the chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as a filler (2.1×150 mm, inner diameter 1.8 μm).

[0144] The present application conducts high performance liquid chromatography determination of the bamboo leaf bupleurum extract, takes rutin content as an indicator, formulates a quantitative standard, and provides a scientific basis for the establishment of the quality standard of the bamboo leaf bupleurum extract.

[0145] The present application provides the application of the characteristic spectrum obtained by the above-mentioned construction method in the extract of Bupleurum chinense and its preparation.

[0146] The present application provides the application of the above-mentioned method for determining the content and / or total transfer rate in the extract of Bupleurum chinense and its preparation.

[0147] Example

[0148] The manufacturers of the raw materials and equipment used in this example, as well as the equipment and analysis methods used in the product analysis are described below. The chemical substances described without being marked are of the chemical purity level of conventional reagents.

[0149] Example 1 Preparation of extract

[0150] Optimization of preparation process: including optimization of extraction process, optimization of concentration process and optimization of drying process.

[0151] A: Optimization of extraction process

[0152] (1) Preparation of medicinal pieces: Take the medicinal materials, remove impurities, wash, cut into sections, and dry;

[0153] (2) Inspection of cooking utensils:

[0154] According to the provisions on decoction containers in the "Management Standards for Chinese Medicine Decoction Rooms in Medical Institutions" issued by the Ministry of Health and the State Administration of Traditional Chinese Medicine in 2009 (decoction containers should be made of ceramic, stainless steel, copper and other materials, and iron and other corrosive containers are prohibited) and the habit of decocting soups at home, a 6L automatic decoction ceramic pot was selected as the extract decoction equipment.

[0155] (3) Investigation of dosage of medicinal pieces

[0156] According to the recommendations under "Extract 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 bamboo leaf bupleurum slices is easy to operate, and the dry paste is sufficient for experimental research on the extract, so the amount of slices used in the bamboo leaf bupleurum extract is set at 100g.

[0157] (4) Investigation of water addition amount

[0158] According to the suggestions under "Extract Preparation" in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules", the amount of water added is generally appropriate when it submerges the medicinal materials by 2 cm to 5 cm. For Chinese herbal medicine pieces of flowers and herbs or those with a longer decocting time, the amount of water added can be appropriately increased. When 12 times the amount of water is added for the first decoction to meet the solvent usage of "submerging the medicinal materials by 2 - 5 cm" and 10 times the amount of water is added for the second decoction, the water level already exceeds 2 cm. The final amount of water added is determined as 12 times the amount of water for the first decoction and 10 times the amount of water for the second decoction.

[0159] (5) Investigation of soaking time

[0160] According to the suggestions under "Extract Preparation" in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules", the medicinal materials 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 of Bupleuri Radix Extractum is determined to be 30 minutes.

[0161] (6) Investigation of decocting time

[0162] According to the suggestions under "Extract Preparation" in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules", generally, each dose of medicine is decocted twice. Generally, it is decocted for another 30 minutes after boiling; medicines for relieving exterior syndrome, clearing heat, and aromatic medicines should not be decocted for a long time, and it is appropriate to decoct for another 20 minutes after boiling. For Chinese herbal medicine pieces with a harder texture, the decocting time can be appropriately extended; for tonic Chinese herbal medicine pieces, first bring to a boil over high heat and then decoct slowly over low heat for about 60 minutes. The time for the second decoction can be appropriately shortened. Therefore, according to the "Technical Requirements", the number of decoctions of Bupleuri Radix Extractum is determined to be twice. For the first decoction, it is decocted for another 30 minutes after boiling, and for the second decoction, it is decocted for another 20 minutes after boiling.

[0163] B. Optimization of concentration process

[0164] (1) Investigation of solid - liquid separation conditions

[0165] According to the suggestions under "Extract Preparation" in the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules", the mesh number of the solid - liquid separation filter material should be above 100 meshes. In order to obtain a decoction consistent with the traditional decoction by modern solid - liquid separation methods, the filtration effects of 100 - mesh sieve, 200 - mesh sieve, and 300 - mesh sieve on the Bupleuri Radix decoction were mainly investigated. The results showed that when performing solid - liquid separation, 100 - mesh, 200 - mesh, and 300 - mesh were all relatively easy to filter. However, 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 300 - mesh sieve was better, and the amount of sediment after standing was small. Therefore, 300 - mesh sieve is used for solid - liquid separation of Bupleuri Radix Extractum.

[0166] The extraction process parameters are determined as follows: Take 100 g of Bupleuri Radix Thunb. var. lanceolatus Hsiao et K. Hsuan slices, place them in an electric ceramic pot, and decoct with water twice. For the first decoction, add 12 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500 W), and then keep it simmering gently over low heat (200 W) for 30 minutes. Filter the decoction while it is hot through a 300-mesh sieve, and record the mass of the decoction after it cools to room temperature. For the second decoction, add 10 times the amount of water, bring to a boil over high heat, and then keep it simmering gently over low heat for 20 minutes. Filter the decoction while it is hot through a 300-mesh sieve, record the mass of the decoction after it cools to room temperature, and combine the two decoctions.

[0167] (2) Study on concentration process parameters

[0168] According to the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules", it is recommended to use vacuum concentration method for low-temperature concentration to the specified amount of extract under the item of "Extract Preparation". Now, by comparing different concentration temperatures, the appropriate concentration temperature is determined. The concentration ratio is temporarily determined as 1:1, and its concentration state is observed.

[0169] By comparing and measuring the density, extract yield of Bupleuri Radix Thunb. var. lanceolatus Hsiao et K. Hsuan extract at different concentration temperatures and observing the state of the concentrated solution. The results show that the extract yields at different concentration temperatures are basically the same, and there is not much difference between the concentration temperatures of 50 °C and 65 °C. Considering comprehensively, 65 °C is selected as the concentration temperature. When the concentration ratio is 1:1, the viscosity of the extract is moderate, the fluidity is good, which is convenient for transfer, and the density is between 1.06 and 1.09 g / ml. Therefore, the concentration ratio is determined as 1:1, and other concentration ratios are not considered for the time being. The final preparation method of the concentrated solution is determined as follows: Transfer the decoction to a 2000-ml round-bottom flask, and concentrate it under reduced pressure and low temperature (temperature: 65 °C; vacuum degree: -0.080 to -0.090 MPa) using a rotary evaporator to 100 ml of extract.

[0170] C. Optimization of drying process

[0171] Study on freeze-drying process parameters

[0172] According to the "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules", it is recommended that the drying of the extract generally uses freeze-drying for preparation, which can ensure the stability of its quality, easy dissolution and no need to add excipients. Therefore, freeze-drying is first selected as the drying method for Bupleuri Radix Thunb. var. lanceolatus Hsiao et K. Hsuan extract.

[0173] Under magnetic stirring, the extract was dispensed into 10-ml brown vials, with a volume of 1 ml per vial. It was half-capped, transferred to a vacuum freeze dryer after dispensing, freeze-dried, taken out, and sealed with an aluminum cap to obtain the product. The eutectic point test result of the Bupleuri Radix extract in the vacuum freeze dryer was -43.6 °C, the pre-freezing temperature was -50 °C, the sublimation drying temperature was -45 °C to 0 °C, the analytical drying temperature was 10 °C to 30 °C, and the total drying time was 48 hours. The results showed that under these freeze-drying conditions, the water content of the Bupleuri Radix extract was low and it was easy to preserve. The final freeze-drying process parameters were confirmed as follows:

[0174] Under magnetic stirring, it was dispensed into 10-ml brown vials, with a volume of 2 ml per vial. It was half-capped, transferred to a vacuum freeze dryer after dispensing, and freeze-dried. The freeze-drying parameters were as follows: the pre-freezing temperature of the Bupleuri Radix concentrated solution was -50 °C, the pre-freezing time was 180 minutes, the sublimation drying temperature was -45 °C to 0 °C, the sublimation drying time was 2340 minutes, and the vacuum degree was -0.2 mbar; the analytical drying temperature was 10 °C to 30 °C, the analytical drying time was 360 minutes, and the vacuum degree was 0 mbar.

[0175] The optimized preparation method of the extract was as follows:

[0176] Take 100 g of Bupleuri Radix slices, put them in an electric ceramic pot, and decoct them twice with water. For the first decoction, directly add 12 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500 W), and then keep it simmering gently over low heat (200 W) for 30 minutes. The decoction was filtered through a 300-mesh sieve while it was still hot, and the mass of the decoction was recorded after it cooled to room temperature. For the second decoction, add 10 times the amount of water, bring to a boil over high heat, and then keep it simmering gently over low heat for 20 minutes. The decoction was filtered through a 300-mesh sieve while it was still hot, and the mass of the decoction was recorded after it cooled to room temperature. The two decoctions were combined. Transfer the decoction to a 2000-ml round-bottom flask, and concentrate it under reduced pressure and low temperature (temperature: 65 °C; vacuum degree: -0.080 to -0.090 MPa) using a rotary evaporator to obtain an extract of 100 ml. Measure its density and extract yield; under magnetic stirring, dispense it into 10-ml brown vials, with a volume of 1 ml per vial. It was half-capped, transferred to a vacuum freeze dryer after dispensing, and freeze-dried. The freeze-drying parameters were as follows: the pre-freezing temperature of the Bupleuri Radix concentrated solution was -50 °C, the pre-freezing time was 180 minutes, the sublimation drying temperature was -45 °C to 0 °C, the sublimation drying time was 2340 minutes, and the vacuum degree was -0.2 mbar; the analytical drying temperature was 10 °C to 30 °C, the analytical drying time was 360 minutes, and the vacuum degree was 0 mbar. Take it out and seal it with an aluminum cap to obtain the product.

[0177] And verify the preparation method:

[0178] Take 3 batches of bamboo leaf bupleurum slices (batch number YP2105-1, purchased from Maochang Town, Dafang County, Bijie City, Guizhou Province; batch number YP2105-2, purchased from Yangchang Township, Dafang County, Bijie City, Guizhou Province; batch number YP2105-3, purchased from Longjinggou, Dafang County, Bijie City, Guizhou Province), each batch is parallel to three parts, each part is 100g, according to the bamboo leaf bupleurum (bamboo leaf bupleurum) slice extract preparation process regulations, prepare bamboo leaf bupleurum (bamboo leaf bupleurum) extract; determine the specific gravity, solid content and paste yield of the bamboo leaf bupleurum (bamboo leaf bupleurum) extract concentrate, evaluate the stability and rationality of the extract preparation process, the results are shown in Table 1, wherein, the method for determining the solid content of the bamboo leaf bupleurum extract is as follows:

[0179] Refer to the "hot leaching method" in 2201 Extract Determination Method of Part IV of the 2020 Edition of the Chinese Pharmacopoeia. Accurately pipette 25 ml of the extract into an evaporating dish of constant weight. Evaporate it to dryness on a water bath, dry it at 105°C for 3 hours, cool it in a desiccator for 30 minutes, and quickly and accurately weigh it.

[0180] The calculation formula for the extract paste yield is: paste yield (%) = extract volume * extract solid content / slice volume * 100%, extract solid content (%) = dry constant weight / original material weight × 100%.

[0181] The method for determining the solid content of the concentrated solution of Bupleurum chinense is as follows:

[0182] 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.

[0183] 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.

[0184] The calculation formula for the paste yield of the concentrate is: paste yield (%) = concentrate weight * concentrate solid content / amount of decoction pieces * 100%.

[0185] The extract concentrate yield value is the extract yield.

[0186] The specific gravity of the concentrated solution of the Bupleurum chinense extract is determined using a densitometer or a specific gravity bottle.

[0187] Table 1 Verification of the preparation method of Bupleurum chinense extract

[0188]

[0189] As can be seen from the above table, the specific gravity, solid content and extract yield of three parallel concentrated extracts of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan are basically the same, indicating that this process is stable and repeatable and can be used as the preparation process of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan extract.

[0190] In addition, 15 batches of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan extracts were prepared by the method described in Example 1, and the detailed parameters of the preparation process are shown in the following table.

[0191] Table 2 Preparation process table of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan extract

[0192]

[0193]

[0194] Among them, the information of the cut crude drugs is shown in the following table:

[0195] Table 3 Information of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan cut crude drugs

[0196] Serial number Batch number Place of origin 1 YP2105-1 Maochang Town, Dafang County, Bijie City, Guizhou Province 2 YP2105-2 Yangchang Township, Dafang County, Bijie City, Guizhou Province 3 YP2105-3 Longjinggou, Dafang County, Bijie City, Guizhou Province 4 YP2105-4 Baiwachang, Dafang County, Bijie City, Guizhou Province 5 YP2105-5 Luoshuidong, Dafang County, Bijie City, Guizhou Province 6 YP2105-6 Jinlong Township, Dafang County, Bijie City, Guizhou Province 7 YP2105-7 Rena Town, Dafang County, Bijie City, Guizhou Province 8 YP2105-8 Shuangshan Town, Dafang County, Bijie City, Guizhou Province 9 YP2105-9 Xiaotun Township, Dafang County, Bijie City, Guizhou Province 10 YP2105-10 Xijiang Town, Qiandongnan Prefecture, Guizhou Province 11 YP2105-11 Shabao Town, Dafang County, Bijie City, Guizhou Province 12 YP2105-12 Lvtang Township, Dafang County, Bijie City, Guizhou Province 13 YP2105-13 Machang Town, Dafang County, Bijie City, Guizhou Province 14 YP2105-14 Xiaopo, Dafang County, Bijie City, Guizhou Province 15 YP2105-15 Dayandong, Dafang County, Bijie City, Guizhou Province

[0197] Prepare the extract according to the process in Table 2. The extract yield is calculated based on the amount of the extract. Take 10 g of the concentrated solution and place it in a pre-weighed evaporating dish. Dry it on a water bath and then dry it in a drying oven at 105 °C for 3 hours. Cool it in a desiccator for half an hour and quickly weigh it. Calculate the extract yield according to the following formula. The extract yield results of 15 batches of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan extracts are shown in Table 4.

[0198]

[0199] Table 4 Extract yields of 15 batches of extracts

[0200]

[0201]

[0202] Based on the extract yields of 15 batches of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan extracts in the above table, the fluctuation range is 9.15% - 17.45%, the average value is 13.39%, and the SD is 2.43%; the range of 70% - 130% of the average extract yield is 9.37% - 17.41%, and the range of ±3SD of the average extract yield is 6.10% - 20.69%. Temporarily determine the extract yield range of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan extract as the range of ±3SD of the average extract yield, which is 6.10% - 20.69%. Approximately round off to 6.1% - 20.7%.

[0203] Determination method for characteristic chromatogram of Bupleurum marginatum Wall. ex DC. var. stenophyllum Hsiao et K. Hsuan extract in Example 2

[0204] 1. Preparation of reference solution: Take about 1 g of the reference medicinal material of Bupleurum marginatum Wall. ex DC., accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 75% methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 75% methanol, shake well, filter, and take the subsequent filtrate, that is, obtain it.

[0205] Take rutin reference substance and make a solution containing 0.1 mg per 1 ml with methanol, shake well, and use it as the reference substance solution. Take quercetin reference substance and make a solution containing 0.1 mg per 1 ml with methanol, shake well, and use it as the reference substance solution. This reference substance solution can be used to determine the corresponding technical parameters such as the peak area and retention time of rutin and quercetin in the extract of Bupleurum marginatum Wall. ex DC..

[0206] 2. Confirmation of chromatographic conditions

[0207] (1) Confirmation of detection wavelength

[0208] Inject the test solution of the extract of Bupleurum marginatum Wall. ex DC. for analysis, record the absorption spectrum in the range of 190 - 400 nm, as Figure 1 shown.

[0209] From Figure 1 it can be seen that at the wavelength of 255 nm, more chromatographic peaks can be detected in the test solution of the extract of Bupleurum marginatum Wall. ex DC., the baseline is stable, and the response of each chromatographic peak is also good. Therefore, 225 nm is selected as the detection wavelength.

[0210] (2) Optimization of mobile phase

[0211] ① Examine the organic phase. A is the organic phase, B is the aqueous phase (0.1% phosphoric acid solution), and the elution gradient is shown in Table 5. The obtained chromatogram is as Figure 2 shown.

[0212] Table 5 Elution gradient table

[0213] Time min A B 0-20 5→100 95→0 20-20.2 100→5 0→95 20.2-20 5 95

[0214] From Figure 2 it can be seen that the elution ability of acetonitrile - 0.1% phosphoric acid solution is stronger than that of methanol - 0.1% phosphoric acid solution. Therefore, acetonitrile - 0.1% phosphoric acid solution is selected to explore the conditions, and various acids used in the mobile phase will be examined later.

[0215] ② Adjust the gradient and examine the cases of adding acid and not adding acid to the aqueous phase. The results are as Figure 3 shown.

[0216] From Figure 3 it can be seen that adding acid has a better effect, and the peak shape is the best when the mobile phase is the acetonitrile - 0.1% acetic acid solution system. Therefore, acetonitrile - 0.1% acetic acid solution is selected for elution.

[0217] (3) Determination of Chromatographic Conditions

[0218] After a series of condition explorations, the chromatographic conditions were finally determined as follows: using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); using acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, and performing gradient elution according to the regulations in the following table; flow rate 0.30 ml per minute; column temperature 35 °C; detection wavelength 255 nm.

[0219] Table 6 Gradient Elution Table

[0220] Time (min) Mobile phase A (%) Mobile phase B (%) 0-25 5→25 95→75 25-30 25→45 75→55 30-33 45→95 55→5 33-33.2 95→5 5→95 33.2-35 5 95

[0221] 3. Optimization of the Conditions for the Preparation Process of the Test Solution:

[0222] First, the UPLC chromatograms of Bupleurum chinense DC. var. officinale Shan et Y. Li extracts from different batches were determined according to the above chromatographic conditions, and their superimposed chromatogram is as Figure 4 shown, where S16 is the reference medicinal material, and the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)" recommended by the Pharmacopoeia Commission of the People's Republic of China was used for result analysis. A total of 6 common peaks were determined, and the relative retention times of the 6 common peaks are shown in Table 7.

[0223] Table 7 Relative Retention Times of 6 Common Peaks

[0224] Serial number Batch number Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 Peak 6 S1 BT2105-1 0.298 0.476 1.000 1.043 1.198 1.577 S2 BT2105-2 0.297 0.475 1.000 1.043 1.198 1.575 S3 BT2105-3 0.297 0.475 1.000 1.043 1.198 1.576 S4 BT2105-4 0.297 0.475 1.000 1.043 1.197 1.575 S5 BT2105-5 0.297 0.475 1.000 1.043 1.198 1.577 S6 BT2105-6 0.297 0.476 1.000 1.043 1.198 1.578 S7 BT2105-7 0.297 0.475 1.000 1.043 1.198 1.579 S8 BT2105-8 0.297 0.476 1.000 1.043 1.198 1.579 S9 BT2105-9 0.298 0.476 1.000 1.043 1.198 1.579 S10 BT2105-10 0.297 0.476 1.000 1.043 1.198 1.579 S11 BT2105-11 0.298 0.476 1.000 1.043 1.198 1.578 S12 BT2105-12 0.298 0.476 1.000 1.043 1.198 1.578 S13 BT2105-13 0.297 0.475 1.000 1.043 1.198 1.577 S14 BT2105-14 0.298 0.476 1.000 1.043 1.198 1.578 S15 BT2105-15 0.298 0.476 1.000 1.043 1.198 1.578 Average value 0.297 0.476 1.000 1.043 1.198 1.578 RSD % 0.10 0.06 0.00 0.01 0.02 0.08

[0225] As can be seen from Table 7, taking peak 3 (rutin) as the reference peak, the RSD values of the relative retention times of the remaining 5 characteristic peaks in the characteristic chromatograms of 15 batches of Bupleurum chinense DC. var. officinale Shan et Y. Li extracts are between 0.02% and 0.10%, all less than 3.0%, meeting the standard requirements of the characteristic chromatogram of Bupleurum chinense DC. var. officinale Shan et Y. Li extracts. According to the above analysis, taking peak 3 (rutin) as the reference peak, a control characteristic chromatogram of Bupleurum chinense DC. var. officinale Shan et Y. Li extracts was tentatively established, and it is as Figure 5 shown (the chromatographic column used is Waters ACQUITY HSS T3 (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm)).

[0226] From Figure 5It can be seen that 6 characteristic peaks should be presented in the characteristic chromatogram of the test sample, and correspond to the retention times of the 6 characteristic peaks in the reference chromatogram of the control crude drug; among them, peak 3 (S) corresponds to the peak of the rutin reference substance, and peak 6 corresponds to the peak of the quercetin reference substance. The peak 3 (rutin) corresponding to the reference substance is the S peak. Calculate the relative retention times of each characteristic peak and the S peak, and the relative retention times should be within ±10% of the specified values, and the specified values are 0.30 (peak 1), 0.48 (peak 2), 1.04 (peak 4), 1.20 (peak 5).

[0227] Then, the effects of different extraction solvents on the characteristic chromatogram of Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract were investigated respectively. Water, 95% ethanol, 75% ethanol, dilute ethanol, methanol, 75% methanol, and 50% methanol were selected as extraction solvents. The results of the characteristic chromatograms of different extraction solvents were compared through the total peak area / sample weight of the temporarily determined 6 chromatographic peaks and the chromatograms. The chromatograms are shown in Figure 6, and the extraction efficiencies are shown respectively as Figure 7 and Table 8.

[0228] Table 8 Extraction efficiencies of different extraction methods (peak area / sample weight)

[0229]

[0230]

[0231] As can be seen from Figures 6 - 7 and Table 8, the extraction efficiency is relatively high when the extraction solvent is 75% methanol. Therefore, 75% methanol is selected as the extraction solvent.

[0232] According to the above experimental results, the sample pretreatment method for the characteristic chromatogram of Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract can be determined as follows: Take about 1 g of Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 75% methanol, weigh it, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 75% methanol, shake well, filter, and take the continuous filtrate, that is obtained.

[0233] 4. Determination and identification of common peaks

[0234] As mentioned above, the UPLC chromatograms of Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extracts from different batches have been determined, as shown in Figure 4 and the common peaks have been identified based on the chromatograms of the reference substance, control crude drug and extract, and finally the attribution of the common peaks of the characteristic chromatogram of Bupleurum marginatum Wall. ex DC. var. wilsonii (Franch.) Shan et Y. Li extract has been confirmed. Among them, the chromatograms of the blank solvent, reference substance (rutin, quercetin), control crude drug and extract are shown in Figure 8, and the characteristic chromatogram is shown in Figure 5 as shown.

[0235] As can be seen from Figure 8, in the characteristic chromatogram of Bupleurum chinense DC. var. marginatum Wight var. sutchuenense Shan et Y. Li extract, characteristic peaks with the same retention time as peak 3 (S) of the rutin reference substance and characteristic peaks with the same retention time as peak 6 of the quercetin reference substance can be found.

[0236] 5. Methodological investigation

[0237] (1) Specificity investigation

[0238] Precisely pipette 1 μl each of the test solution of Bupleurum chinense DC. var. marginatum Wight var. sutchuenense Shan et Y. Li extract and the blank solvent, inject them into the liquid chromatograph, and determine according to the chromatographic conditions under the above items. The results are as Figure 9 shown.

[0239] From Figure 9 it can be seen that the solvent has no interference on the characteristic peaks in the chromatogram of Bupleurum chinense DC. var. marginatum Wight var. sutchuenense Shan et Y. Li extract.

[0240] (2) Integrity investigation

[0241] Take the test solution of Bupleurum chinense DC. var. marginatum Wight var. sutchuenense Shan et Y. Li extract, 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 chromatogram. The results are as Figure 10 shown.

[0242] From Figure 10 it can be seen that there are no obvious chromatographic peaks after extending the elution time by one time under this chromatographic condition, indicating that this chromatographic condition basically meets the principle of the largest amount of information.

[0243] (3) Precision investigation

[0244] Take the test solution of Bupleurum chinense DC. var. marginatum Wight var. sutchuenense Shan et Y. Li extract, inject it repeatedly 6 times with an injection volume of 1 μl. Tentatively calibrate 6 characteristic peaks, take rutin (peak 3) as the reference peak, and calculate the relative retention time. The experimental results are shown in Table 9.

[0245] Table 9 Precision results of the characteristic chromatogram of Bupleurum chinense DC. var. marginatum Wight var. sutchuenense Shan et Y. Li extract (relative retention time)

[0246] Number 1 2 3 4 5 6 Average value RSD % Peak 1 0.299 0.299 0.299 0.299 0.299 0.299 0.299 0.03 Peak 2 0.477 0.476 0.477 0.477 0.477 0.477 0.477 0.05 Peak 3 (S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00 Peak 4 1.043 1.043 1.044 1.043 1.044 1.044 1.043 0.01 Peak 5 1.198 1.197 1.198 1.198 1.198 1.197 1.198 0.02 Peak 6 1.578 1.578 1.578 1.578 1.578 1.577 1.578 0.02

[0247] As can be seen from Table 9, the RSD of the relative retention time of each chromatographic peak is < 3.0%, and the precision of this instrument is good.

[0248] (4) Stability investigation

[0249] Take the test solution of Bupleurum chinense DC. var. marginatum Wight var. sutchuenense Shan et Y. Li extract, inject it at 0, 2, 4, 8, 12, and 24 hours respectively with an injection volume of 1 μl. Tentatively calibrate 6 characteristic peaks, take (peak 3) as the reference peak, and calculate the relative retention time. The results are shown in Table 10.

[0250] Table 10 Stability results of the characteristic chromatogram of Bupleurum chinense DC. var. marginatum Wight var. sutchuenense Shan et Y. Li extract (relative retention time)

[0251] Number 0h 2h 4h 8h 12h 24h Average value RSD % Peak 1 0.297 0.299 0.299 0.299 0.299 0.299 0.299 0.23 Peak 2 0.474 0.476 0.476 0.476 0.476 0.476 0.476 0.18 Peak 3 (S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00 Peak 4 1.044 1.043 1.043 1.043 1.044 1.043 1.043 0.00 Peak 5 1.197 1.198 1.198 1.197 1.197 1.198 1.198 0.02 Peak 6 1.578 1.579 1.578 1.578 1.578 1.579 1.578 0.02

[0252] As can be seen from Table 10, the RSD of the relative retention time of the chromatographic peaks is < 3.0%, indicating that the sample solution is relatively stable.

[0253] (5) Repeatability investigation

[0254] Take about 1 g of the same batch of Bupleuri chinensis DC. var. officinalis (Boiss.) Shan et Y. Li extract, accurately weigh it, make 6 parallel portions, prepare the test solution according to the preparation method of the test solution, and inject 1 μl respectively. Tentatively calibrate 6 characteristic peaks, take rutin as the reference peak, calculate the relative retention time, and the results are shown in Table 11.

[0255] Table 11 Repeatability results of the characteristic chromatogram of Bupleuri chinensis DC. var. officinalis (Boiss.) Shan et Y. Li extract (relative retention time)

[0256]

[0257]

[0258] The experimental results show that the RSD of the relative retention time of each chromatographic peak is < 3.0%, indicating that the method has good repeatability.

[0259] (6) Robustness investigation

[0260] 1) Investigation of chromatographic columns

[0261] Three chromatographic columns were investigated, namely: Chromatographic column 1 - Waters ACOUITY HSS T3 (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); 2 - Dikma C18 (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm), 3 - Shim - pack GISS - HP C18 - AQ (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.9 μm); The influence of the three chromatographic columns on the peak emergence of the characteristic chromatogram of Bupleuri chinensis DC. var. officinalis (Boiss.) Shan et Y. Li extract was investigated, and the chromatograms and the relative retention times of each peak are shown in Figure 11 and Table 12 respectively.

[0262] Table 12 Investigation results of different chromatographic columns (relative retention time)

[0263] Chromatographic column Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 Peak 6 1 0.298 0.476 1.000 1.043 1.198 1.578 2 0.282 0.507 1.000 1.042 1.205 1.624 3 0.330 0.512 1.000 1.051 1.200 1.497

[0264] As can be seen from Figure 11 and Table 12, the chromatographic column has a greater influence on the characteristic chromatogram. Using 1 - Waters ACOUITY Elution was carried out using a Waters ACOUITY HSS T3 ultra-high performance liquid chromatography column (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm), and the chromatographic peak shape was good and the separation effect was the best. Therefore, this method uses 1-Waters ACOUITY HSS T3 liquid chromatography column (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm).

[0265] 2) Column temperature investigation

[0266] The elution conditions at different column temperatures (30 °C, 35 °C, 40 °C) were investigated, and the chromatograms and the relative retention times of each peak are shown respectively as Figure 12 and Table 13.

[0267] Table 13 Results of column temperature investigation (relative retention time)

[0268] Column temperature Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 Peak 6 30℃ 0.308 0.486 1.000 1.045 1.195 1.561 35℃ 0.298 0.476 1.000 1.043 1.198 1.579 40℃ 0.289 0.466 1.000 1.042 1.202 1.592

[0269] From Figure 12 and Table 13, it can be seen that the column temperature has a certain influence on the peak emergence situation. The separation effect is the best and the peak shape is good at a column temperature of 35 °C. Therefore, it is recommended to use a column temperature of 35 °C for determination.

[0270] 3) Investigation of different flow rates

[0271] The effects of different flow rates, namely 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min, on the durability of the characteristic chromatogram of Bupleurum chinense DC. var. glabrum Hsiao et Keng f. extract were compared. The chromatograms and the relative retention times of each peak are shown respectively as Figure 13 and Table 14.

[0272] Table 14 Results of flow rate investigation (relative retention time)

[0273] Flow rate Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 Peak 6 0.25 ml / min 0.330 0.505 1.000 1.046 1.194 1.542 0.30 ml / min 0.290 0.469 1.000 1.043 1.200 1.586 0.35 ml / min 0.273 0.452 1.000 1.041 1.201 1.599

[0274] From Figure 13 and Table 14, it can be seen that the flow rate has a greater influence on the peak emergence situation, and the peak shape is the best at a flow rate of 0.30 ml per minute. Therefore, it is recommended to use 0.30 ml / min as the determination flow rate.

[0275] Example 3 Determination method for rutin content and total transfer rate in Bupleurum chinense DC. var. glabrum Hsiao et Keng f. extract

[0276] Determination of content:

[0277] Determination of chromatographic conditions

[0278] Using octadecylsilane-bonded silica gel as the filler (2.1×150 mm, inner diameter 1.8 μm); using acetonitrile-0.1% formic acid water as the mobile phase, gradient elution was carried out according to Table 15, the column temperature was 30 °C; the flow rate was 0.20 ml / min, and the detection wavelength was 354 nm. The number of theoretical plates calculated based on the rutin peak should be not less than 8000.

[0279] Table 15 Gradient elution table

[0280] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 6 80 20 12 79 21 22 52 48 22.5 90 10 25 90 10

[0281] Preparation of reference substance solution: Take an appropriate amount of rutin reference substance (batch number 100080-202012, its purity is calculated as 91.6%, purchased from the National Institutes for Food and Drug Control. For content determination, no treatment is required before use), accurately weigh it, add 50% methanol to make a solution containing 50 μg of rutin reference substance per 1 ml, mix well to obtain.

[0282] 1. Optimization of the preparation conditions of the test solution:

[0283] (1) Investigation of different extraction solvents

[0284] Take an appropriate amount of Bupleurum chinense DC. var. marginatum Wight et Arn. extract BT2105-1, a total of 7 portions, with 2 parallel samples for each portion. Respectively place them in stoppered conical flasks, accurately add 25 ml of water, dilute ethanol, 75% ethanol, ethanol, 50% methanol, 75% methanol, and methanol in sequence, weigh, ultrasonically treat for 30 min, cool, make up the weight with the corresponding solvent, mix well, filter, and collect the subsequent filtrate to obtain each test solution. Detect on the machine according to the above chromatographic conditions, the injection volume is 1 μl, calculate the influence of different extraction solvents on its content based on rutin, determine the best extraction solvent, and the experimental results are calculated by the external standard single-point method. The results are shown in Table 16.

[0285] Table 16 Influence of different extraction solvents on the rutin content in Bupleurum chinense DC. var. marginatum Wight et Arn. extract

[0286]

[0287] It can be seen from Table 16 that: the influence of different solvents on the rutin content in Bupleurum chinense DC. var. marginatum Wight et Arn. extract is significantly different. Considering its content comprehensively, 75% methanol was selected as the extraction solvent for subsequent research.

[0288] (2) Investigation of extraction methods

[0289] Take an appropriate amount of Bupleurum falcatum L. extract BT2105-1, a total of 3 portions, with 2 parallel samples for each portion. Respectively place them in stoppered conical flasks, precisely add 25 ml of 75% methanol in sequence, weigh, and respectively perform ultrasonic treatment, reflux treatment, and shaking treatment for 30 min. After taking out and cooling to room temperature, make up the weight with 75% methanol, mix well, filter, and collect the subsequent filtrate. Take an appropriate amount of the subsequent filtrate and pass it through a 0.22 μm microporous filter membrane, place it in a high-performance liquid chromatography sample bottle to obtain each test solution. According to the chromatographic conditions under the above items, perform on-machine detection, with an injection volume of 1 μl. Calculate the influence of different extraction methods on its content using rutin as the reference, and determine the optimal extraction method. The experimental results are calculated by the external standard single-point method, and the results are shown in Table 17.

[0290] Table 17 Influence of Different Extraction Methods on the Rutin Content in Bupleurum falcatum L. Extract

[0291]

[0292]

[0293] As can be seen from Table 17, the differences in the influence of different extraction methods on the rutin content in Bupleurum falcatum L. extract are not significant. Considering the simplicity of operation and the content differences comprehensively, ultrasonic extraction is selected as the extraction method for subsequent research.

[0294] (3) Investigation of Different Extraction Times

[0295] Take an appropriate amount of Bupleurum falcatum L. extract BT2105-1, a total of 3 portions, with 2 parallel samples for each portion. Respectively place them in stoppered conical flasks, precisely add 25 ml of 75% methanol in sequence, weigh, and reflux for 15 min, 30 min, and 60 min respectively. After taking out and cooling to room temperature, make up the weight with 75% methanol, mix well, filter, and collect the subsequent filtrate. Take an appropriate amount of the subsequent filtrate and pass it through a 0.22 μm microporous filter membrane, place it in a high-performance liquid chromatography sample bottle to obtain each test solution for standby. According to the chromatographic conditions under the above items, perform on-machine detection, with an injection volume of 1 μl. Calculate the influence of different extraction times on its content using rutin as the reference, and determine the optimal extraction time. The experimental results are calculated by the external standard single-point method, and the results are shown in Table 18.

[0296] Table 18 Influence of Different Extraction Times on the Rutin Content in Bupleurum falcatum L. Extract

[0297]

[0298] As can be seen from Table 18, the differences in the influence of different extraction times on the rutin content in Bupleurum falcatum L. extract are not significant. Considering the time cost and the content differences comprehensively, ultrasonic extraction for 60 min is selected for subsequent research.

[0299] (4) Determination of the Preparation Method of the Test Solution

[0300] According to the experimental results of sample pretreatment, the preparation method of the test sample can be determined as follows:

[0301] Take about 0.14 g of Bupleuri Chinensis Extractum Folium, weigh it precisely, place it in a stoppered conical flask, precisely add 25 ml of 75% methanol, weigh it, ultrasonically treat it for 60 min, let it cool, replenish the weight with 75% methanol, filter and collect the subsequent filtrate, pass it through a 0.22 μm microporous filter membrane, and place it in a high-performance liquid sample bottle to obtain the test sample solution.

[0302] 2. Methodological verification

[0303] (1) Specificity investigation

[0304] Precisely pipette 1 μl of the test sample solution of Bupleuri Chinensis Extractum Folium (BT2105-1), rutin reference substance solution and blank solvent respectively, perform on-machine detection according to the above chromatographic conditions, and record the chromatographic elution pattern. The results are shown in the following figure, as shown in Figure 14.

[0305] As can be seen from Figure 14, the analytical method has good specificity for rutin in Bupleuri Chinensis Extractum Folium.

[0306] (2) Peak purity

[0307] Precisely pipette the test sample solution of Bupleuri Chinensis Extractum Folium (BT2105-1), perform on-machine detection according to the above chromatographic conditions, and detect the peak purity of the target peak. The results are as follows Figure 15 and shown in Table 19.

[0308] Table 19 Matching values of the target peak and peak purity

[0309] Target peak Rutin Peak purity matching value 999

[0310] From Figure 15 and Table 19, it can be seen that the peak purity matching value of the target component rutin is 999, which is greater than 960, indicating that its peak purity meets the analysis requirements.

[0311] (3) Linear relationship investigation

[0312] Precisely weigh rutin and place it in a numbered 20 ml volumetric flask, dissolve it with 75% methanol and make up to the mark to obtain the stock solution of each reference substance solution (concentration: 0.2001918 mg / ml), and store it in the refrigerator for later use.

[0313] Dilute the rutin reference substance stock solution by 20, 10, 5, 4, 2, and 1 times to obtain reference substance solutions of different concentrations of rutin. Perform on-machine detection according to the above chromatographic conditions. Take the concentration as the abscissa and the peak area value as the ordinate to investigate the linear range of rutin. The results of the linear investigation are shown in Table 20 and Figure 16 .

[0314] Table 20 Rutin linearity investigation table

[0315] Reference substance number Rutin concentration (mg / ml) Peak area value (mAU) Rutin-1 0.01 1.38 Rutin-2 0.02 2.66 Rutin-3 0.04 5.26 Rutin-4 0.05 6.34 Rutin-5 0.10 13.48 Rutin-6 0.20 27.02

[0316] From Table 20 and Figure 16 it can be seen that rutin has a good linear relationship between its concentration and peak area value in the range of 0.01 mg / ml to 0.20 mg / ml, and the correlation coefficient r = 0.9998.

[0317] (4) Precision investigation

[0318] 1) Instrument precision test

[0319] Precisely pipette the test solution of Bupleurum chinense DC. var. officinale (Wolff) Shan & Y. Li extract (BT2105-1) and the rutin reference substance solution, and perform on-machine detection according to the above chromatographic conditions with an injection volume of 1 μl. Using rutin as the reference, the RSD (%) value of the target peak is calculated by the external standard single-point method, and the results are shown in Table 21.

[0320] Table 21 Precision test results of the determination method for rutin content in Bupleurum chinense DC. var. officinale (Wolff) Shan & Y. Li extract

[0321]

[0322] From the results in Table 21, it can be seen that the RSD (%) value of the target peak rutin is 0.24% < 2.0%, indicating that the precision of this method is good.

[0323] 2) Repeatability test

[0324] Take about 0.14 g of the same batch of Bupleurum chinense DC. var. officinale (Wolff) Shan & Y. Li extract (BT2105-1), accurately weigh it, and prepare 6 parallel portions to prepare 6 test solutions for standby. Perform on-machine detection according to the above chromatographic conditions. Using rutin in the test solution as the reference, the RSD (%) value of the target peak content is calculated by the external standard single-point method, and the results are shown in Table 22.

[0325] Table 22 Repeatability test results of the determination method for rutin content in Bupleurum chinense DC. var. officinale (Wolff) Shan & Y. Li extract

[0326]

[0327] From the results in Table 22, it can be seen that the RSD (%) value of the target peak rutin content is 1.67% < 2.0%, indicating that the precision of this method is good.

[0328] 3) Intermediate precision

[0329] Operated by other analysts in the project team on different dates and different chromatographs, approximately 0.14 g of the same batch of Bupleuri Radix Extractum (BT2105-1) was accurately weighed, in parallel for 6 portions. According to the preparation method of the test solution, the test solution was prepared, and detected on the machine under the above chromatographic conditions. Calculated by the external standard single-point method based on rutin in the test solution, the RSD (%) value of the target peak content was calculated, and the results are shown in Table 23 in detail.

[0330] Table 23 Intermediate Precision Test Results of Rutin Content Determination Method in Bupleuri Radix Extractum

[0331]

[0332] It can be seen from the experimental results in Table 23 that the RSD (%) value of the intermediate precision of the rutin content in the target peak is 1.38% < 2.0%, indicating that the intermediate precision of this method is good.

[0333] (5) Accuracy Test

[0334] 0.07 g of Bupleuri Radix Extractum with known content (batch number BT2105-1, rutin content 1.06%) was accurately weighed in 6 portions in sequence. 1 ml of rutin reference substance solution (concentration 0.7420 mg / ml) prepared with 75% methanol was accurately added to each portion respectively, and another 24 ml of 75% methanol was accurately added; weighed, and prepared into a test solution according to the preparation method of the test solution. Detected on the machine under the above chromatographic conditions, injected 1 μl respectively. Calculated by the external standard single-point method based on rutin, the content of the target peak was calculated, and the recovery rate and RSD were calculated according to the following formula, and the results are shown in Table 24.

[0335]

[0336] Table 24 Results of the Spiked Recovery Experiment of the Content Determination Method of Bupleuri Radix Extractum

[0337]

[0338] It can be seen from the experimental results that the recovery rate range of rutin in the extract is within 92% - 105%, and the RSD% (1.74%) is less than 2.0%, indicating that the accuracy of this content determination method is good.

[0339] (6) Stability Investigation

[0340] The test solution of Bupleuri Radix Extractum (BT2105-1) was prepared according to the preparation method of the test solution. According to the above chromatographic conditions, injected at 0, 2, 4, 6, 8, 10, 12, and 24 hours respectively, with an injection volume of 1 μl. Calculated by the external standard single-point method based on rutin, the RSD (%) value of the target peak content was calculated, and the results are shown in Table 25.

[0341] Table 25 Results of the stability experiment on the content determination method of Bupleurum chinense DC. var. marginatum W. W. Smith extract

[0342]

[0343] The experiment shows that the RSD(%) value of the content of the target peak rutin within 24 hours is 0.96% < 2.0%, indicating that the solution has good stability within 24 hours.

[0344] (7) Robustness investigation

[0345] 1) Investigation with different chromatographic columns

[0346] The effects of three chromatographic columns of Shimadzu, Dima, and Thermo Fisher on the peak shape and resolution of the rutin chromatographic peak in the Bupleurum chinense DC. var. marginatum W. W. Smith extract were compared. The test solution of the Bupleurum chinense DC. var. marginatum W. W. Smith extract (BT2105-1) was taken and determined under the above chromatographic conditions. Taking rutin as the reference, the chromatographic data were recorded. The results are shown in Table 26 and Figure 17 as follows.

[0347] Table 26 Effects of different chromatographic columns on the results of content determination in the Bupleurum chinense DC. var. marginatum W. W. Smith extract

[0348] Chromatographic column model Index component Resolution / R Theoretical plate number Peak purity Dikma Endeavorsil C18 Rutin 8.28 163220 999 Inerstil ODS-3 C18 Rutin 6.64 56744 998 Acclaim Vanquish C18 Rutin 7.47 110592 999

[0349] As can be seen from Table 26 and Figure 17 it can be seen that the durability of different chromatographic columns is good. Considering the peak shape and resolution of the target chromatographic peak comprehensively, the chromatographic column was selected as Dikma Endeavorsil C18.

[0350] 2) Investigation with different chromatographs

[0351] According to the existing equipment in the laboratory, the Thermo ultra-high performance liquid chromatograph and the Agilent ultra-high performance liquid chromatograph were selected to compare the effects of the two chromatographs on the peak shape and resolution of the rutin chromatographic peak in the Bupleurum chinense DC. var. marginatum W. W. Smith extract. The test solution of the Bupleurum chinense DC. var. marginatum W. W. Smith extract (BT2105-1) was taken and determined under the above chromatographic conditions. Taking rutin as the reference, the chromatographic data were recorded. The experimental results are shown in Table 27 and Figure 18 as follows.

[0352] Table 27 Results of the investigation on the durability of the instrument

[0353] Chromatograph brand Index component Resolution / (R / min) Theoretical plate number Peak purity Agilent UPLC1290 Rutin 15.44 48394 999 Thermo VanquishF1 Rutin 8.27 165212 999

[0354] As can be seen from Table 27 and Figure 18 it can be seen that the durability of the analysis method with different chromatographs is good. The change of the chromatograph can meet the requirements of system suitability.

[0355] 3) Investigation with different column temperatures

[0356] Compare the effects of different column temperatures (25°C, 30°C, and 35°C) on the peak shape of rutin chromatographic peaks in the extract of Bupleurum chinense DC. var. marginatum Wight. Take the test solution of the extract of Bupleurum chinense DC. var. marginatum Wight. (BT2105-1), and determine it according to the above chromatographic conditions. Record the chromatographic data based on rutin. The experimental results are shown in Table 28 and Figure 19.

[0357] Table 28 Chromatogram results of the target components for the content determination of the extract of Bupleurum chinense DC. var. marginatum Wight. at different column temperatures

[0358] Column temperature / °C Index component Resolution / R Theoretical plate number Peak purity 25 Rutin 8.80 156644 999 30 Rutin 8.26 162428 999 35 Rutin 7.73 168251 1000

[0359] As can be seen from Table 28 and Figure 19, the peak shapes and separation effects of the chromatographic peaks are good at the three column temperatures. The baseline of the chromatogram has no drift at 30°C, and there is no significant difference in the retention time compared with the other two temperatures. Considering the tolerance of the chromatographic column and the analysis time required, the column temperature of 30°C is selected.

[0360] 4) Investigation of different flow rates

[0361] Compare the effects of different flow rates (0.20 ml / min, 0.25 ml / min, and 0.30 ml / min) on the peak shape of rutin chromatographic peaks in the extract of Bupleurum chinense DC. var. marginatum Wight. Take the test solution of the extract of Bupleurum chinense DC. var. marginatum Wight. (BT2105-1), and determine it according to the above chromatographic conditions. Record the chromatographic data based on rutin. The experimental results are shown in Table 29 and Figure 20 as follows.

[0362] Table 29 Detection results of the content determination method of the standard decoction of Bupleurum chinense DC. var. marginatum Wight. at different flow rates

[0363] Flow rate / (ml / min) Index component Resolution / R Theoretical plate number Peak purity 0.20 Rutin 8.26 162486 999 0.25 Rutin 7.82 177489 998 0.30 Rutin 7.34 191345 1000

[0364] As can be seen from Table 29 and Figure 20 it can be seen that the peak shapes and separation effects of the chromatographic peaks are good at the three flow rates. The resolution of each component is good when the flow rate is 0.20 ml / min, and the baseline has no drift. The flow rate of 0.20 ml / min is selected in this experiment.

[0365] Determination of the transfer rate:

[0366] The determination method of the transfer rate of rutin is the same as that of the content determination method. Use the content determination method to detect the content of rutin in the medicinal material of Bupleurum chinense DC. var. marginatum Wight. and the corresponding extract of Bupleurum chinense DC. var. marginatum Wight., and calculate the transfer rate of rutin in the extract of Bupleurum chinense DC. var. marginatum Wight. The formula is: Transfer rate of rutin in the extract of Bupleurum chinense DC. var. marginatum Wight. (%) = (Mass of the extract of Bupleurum chinense DC. var. marginatum Wight. × Content of rutin in the extract) / (Mass of the medicinal material of Bupleurum chinense DC. var. marginatum Wight. × Content of rutin in the medicinal material) × 100%. The average value of the total transfer rate of rutin in the standard decoction of Bupleurum chinense DC. var. marginatum Wight. is 14.45%, the SD is 2.50%, and the fluctuation range is 6.96% - 21.95%.

[0367] As described above, it is only a preferred embodiment of the implementation of the present invention, and does not impose any formal restrictions on the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing Bupleuri Chinensis Extractum Folium Bambusae, wherein, the method comprises: performing double decoctions on the cut pieces of Bupleuri Chinensis Extractum Folium Bambusae to obtain a decoction, and filtering; concentrating the decoction to obtain a concentrated solution, and subjecting the concentrated solution to freeze-drying to obtain Bupleuri Chinensis Extractum Folium Bambusae; wherein, 11 - 13 times the amount of water is added in the first decoction, and 9 - 11 times the amount of water is added in the second decoction; the freeze-drying includes pre-freezing, primary drying, and secondary drying, and the pre-freezing is to maintain at -60 to -48 °C for 150 - 200 minutes; the primary drying is to maintain at -45 to 0 °C for 120 - 2340 minutes; the secondary drying is to maintain at 10 - 30 °C for 60 - 360 minutes.

2. The method according to claim 1, wherein, the pre-freezing is to maintain at -50 °C for 180 minutes.

3. The method according to claim 1, wherein, the filtering is performed through a sieve with 100 - 300 meshes.

4. The method according to claim 1, wherein, the extract yield of the concentrated solution is 6 - 25%.

5. The method according to claim 4, wherein, the extract yield of the concentrated solution is 6.1 - 20.7%.

6. Bupleuri Chinensis Extractum Folium Bambusae prepared by the method according to any one of claims 1 - 5.

7. A method for constructing a characteristic chromatogram of Bupleuri Chinensis Extractum Folium Bambusae, which comprises: preparation of a reference substance solution: adding Bupleuri Chinensis Extractum Folium Bambusae into a first solvent for extraction to obtain a reference substance solution; preparation of a reference standard solution: adding rutin and quercetin respectively into the first solvent to obtain a reference standard solution; preparation of a test solution: adding the Bupleuri Chinensis Extractum Folium Bambusae according to claim 6 into the first solvent for extraction to obtain a test solution; performing high performance liquid analysis on the reference substance solution, the reference standard solution, and the test solution to obtain a characteristic chromatogram of Bupleuri Chinensis Extractum Folium Bambusae; wherein, the first solvent is water, 95% (v / v) ethanol, 75% (v / v) ethanol, dilute ethanol, methanol, 75% (v / v) methanol, or 50% (v / v) methanol; the chromatographic conditions for high performance liquid analysis are: using octadecylsilane chemically bonded silica as the filler, using acetonitrile as mobile phase A, and an aqueous solution containing 0.1 wt% acetic acid as mobile phase B, and performing gradient elution in the following gradient manner: 0 - 25 min, mobile phase A increases from 5% (v / v) to 25% (v / v), and mobile phase B decreases from 95% (v / v) to 75% (v / v); 25 - 30 min, mobile phase A increases from 25% (v / v) to 45% (v / v), and mobile phase B decreases from 75% (v / v) to 55% (v / v); 30 - 33 min, mobile phase A increases from 45% (v / v) to 95% (v / v), and mobile phase B decreases from 55% (v / v) to 5% (v / v); 33 - 33.2 min, mobile phase A decreases from 95% (v / v) to 5% (v / v), and mobile phase B increases from 5% (v / v) to 95% (v / v); 33.2 - 35 min, 5% (v / v) mobile phase A: 95% (v / v) mobile phase B.

8. The construction method according to claim 7, wherein, the first solvent is 75% methanol.

9. The construction method according to claim 7, wherein, the column temperature is 30 - 40 °C.

10. The construction method according to claim 9, Among them, The flow rate is 0.25 - 0.35 ml / min.

11. The construction method according to claim 9, Among them, The detection wavelength is 255 nm.

12. The construction method according to claim 9, Among them, The injection volume is 1 - 5 μL.

13. The construction method according to any one of claims 7 - 12, Among them, The characteristic chromatogram of the Bupleurum chinense DC. var. officinale Shan et Y. Li extract contains 6 characteristic peaks.

14. The construction method according to claim 13, Among them, The characteristic peaks are the characteristic peaks at relative retention times of 0.30 ± 10%, 0.48 ± 10%, 1.04 ± 10% and 1.20 ± 10%, and the characteristic peaks of rutin and quercetin.

15. A method for determining the content and / or total transfer rate of rutin in Bupleurum chinense DC. var. officinale Shan et Y. Li extract, which includes: Preparation of reference solution: Rutin is added to the second solvent for extraction to obtain the reference solution; Preparation of test solution: The Bupleurum chinense DC. var. officinale Shan et Y. Li extract described in claim 6 is added to the second solvent for extraction to obtain the test solution; Performing high - performance liquid analysis on the reference solution and the test solution to obtain the content and / or total transfer rate of rutin in the Bupleurum chinense DC. var. officinale Shan et Y. Li extract; The second solvent is water, dilute ethanol, 75% (v / v) ethanol, ethanol, 50% (v / v) methanol, 75% (v / v) methanol or methanol; The chromatographic conditions for high - performance liquid analysis are: Using octadecylsilane - bonded silica gel as the filler, acetonitrile as mobile phase A, and an aqueous solution containing 0.1 wt% formic acid as mobile phase B, and performing gradient elution in the following gradient manner: 0 - 6 min, mobile phase A decreases from 90% (v / v) to 80% (v / v), and mobile phase B increases from 10% (v / v) to 20% (v / v); 6 - 12 min, mobile phase A decreases from 80% (v / v) to 79% (v / v), and mobile phase B increases from 20% (v / v) to 21% (v / v); 12 - 22 min, mobile phase A decreases from 79% (v / v) to 52% (v / v), and mobile phase B increases from 21% (v / v) to 48% (v / v); and 22 - 25 min, 90% (v / v) mobile phase A: 10% (v / v) mobile phase B.

16. The method according to claim 15, Among them, The second solvent is 75% (v / v) methanol; and / or, The extraction method is ultrasonic extraction, shaking extraction or reflux extraction; and / or, The extraction time is 15 - 60 min.

17. The method according to claim 16, Among them, The extraction method is ultrasonic extraction, and / or the extraction time is 60 min.

18. The determination method according to claim 15, Among them, The flow rate of the mobile phase is 0.20 - 0.30 ml / min.

19. The determination method according to claim 18, Among them, The column temperature is 25 - 35 °C.

20. The determination method according to claim 18, Among them, The detection wavelength is 350 - 360 nm.

21. The determination method according to claim 18, Among them, The injection volume is 1 - 5 μL.

22. The determination method according to any one of claims 15 - 21, Among them, The content of rutin in the extract is 0.5 - 2%; and / or, The total transfer rate of rutin in the extract is 6-25%.

23. The assay method according to claim 22, in, The content of rutin in the extract is 0.51-1.44%; and / or, The total transfer rate of rutin in the extract is 6.96-21.95%.

24. Application of the characteristic spectrum obtained by the construction method according to any one of claims 7 to 14 in an extract of Bupleurum chinense and its preparation.

25. Use of the method for determining the content and / or total transfer rate according to any one of claims 15 to 23 in an extract of Bupleurum chinense and a preparation thereof.

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