Pseudo-ginseng flower extract as well as preparation method, detection method and application thereof

Abstract

The invention relates to the field of traditional Chinese medicine analysis, in particular to a sanchi flower extract as well as a preparation method, a detection method and application thereof. The pseudo-ginseng flower extract is prepared by the following steps: decocting pseudo-ginseng flower decoction pieces twice with water, filtering to obtain decoction liquid, concentrating the decoction liquid, and drying to obtain the pseudo-ginseng flower extract. The mass content of ginsenoside Rb3 in the panax notoginseng flower extract is 29.65 mg / g to 70.13 mg / g. The method for detecting the mass content of ginsenoside Rb3 in the sanchi flower extract and the method for constructing the specific chromatogram are good in specificity, repeatability and stability, a scientific basis is provided for establishment of quality standards of the sanchi flower extract and preparations thereof, and overall quality control and effective supervision of traditional Chinese medicine formula granules are achieved.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine analysis, and in particular to a notoginseng flower extract and a preparation method, a detection method and application thereof. Background Art

[0002] Notoginseng flower is the dried inflorescence of Panax notoginseng (Burk.) FHChen, a plant of the Araliaceae family. It tastes sweet and is cool in nature. It enters the liver and kidney meridians. It has the effects of clearing heat, calming the liver, and lowering blood pressure. It is used for hypertension, dizziness, vertigo, tinnitus, acute throat paralysis, etc. The earliest readable historical data on Notoginseng flower is "Guangnan Geography Data (Part 1)", which states that "its (Notoginseng) flower can be used as tea". Notoginseng flower as a medicinal material has been included in "Yunnan Chinese Herbal Medicine Selection" and "Chinese Materia Medica Dictionary", which records that Notoginseng flower "tastes sweet and is cool in nature. It clears heat and produces body fluid, calms the liver and lowers blood pressure. It is mainly used to treat thirst caused by loss of body fluid, sore throat, hoarseness, and hypertension". It is also called "Tianqi flower". Modern pharmacological research shows that Notoginseng flower has the effects of calming the nerves, anti-inflammatory and analgesic, and lowering blood pressure. It has a wide range of pharmacological effects, few side effects, and a long-lasting and stable therapeutic effect. Panax notoginseng flower mainly contains protopanaxadiol saponins, with higher contents of ginsenosides Rb1, Rb3, Rc and notoginsenoside Fc. Ginsenosides Rb1, Rb2 and Rb3 are the pharmacologically active substances of Panax notoginseng flower, which have certain pharmacological effects on the central nervous system and cardiovascular system.

[0003] Chinese herbal medicine extracts are a traditional form of medication widely used in clinical practice. Extracts can be standardized according to the theory of traditional Chinese medicine, standardized according to the clinical decoction method, solid-liquid separation, and prepared by appropriate concentration or drying by appropriate methods. The extracts are standardized and can be used as a standard reference to measure whether Chinese herbal medicine extract preparations such as formula granules are basically consistent with clinical decoctions. Since extracts are the "bridge" connecting traditional Chinese medicine pieces and modern Chinese medicine preparations, they provide a reference for controlling the quality of Chinese medicine terminal products, for standardizing different forms of Chinese medicine, ensuring the uniformity of quality and consistency of efficacy, and for evaluating the consistency of product quality of different manufacturers. Therefore, the formulation of quality standards for Chinese herbal medicine extracts will provide a basis for the formulation of quality standards for all final products derived from decoction pieces.

[0004] The 2019 edition of the Quality Standards for Traditional Chinese and Ethnic Chinese Medicines in Guizhou Province includes Panax notoginseng flower as a national medicine in Guizhou Province. The 2020 edition of the Chinese Pharmacopoeia does not include the corresponding quality standards for Panax notoginseng flower, and its indicators cannot be effectively evaluated.

[0005] Currently, there are few reports on the quality testing of Panax notoginseng flower extract. Summary of the invention

[0006] The technical problem to be solved by the present invention is: to provide a Panax notoginseng flower extract and a preparation method and a detection method thereof, which can provide a quality control means for the quality evaluation of the Panax notoginseng flower extract and its extract preparations, and realize the overall quality control and effective supervision of the Panax notoginseng flower extract and its extract preparations.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present application provides a method for preparing a Panax notoginseng flower extract, comprising decocting Panax notoginseng flower pieces twice in water and filtering to obtain a decoction, and concentrating and drying the decoction to obtain the Panax notoginseng flower extract.

[0009] In some embodiments of the present application, the preparation method comprises the following steps:

[0010] (1) taking Panax notoginseng flower slices, adding water to decoct, and filtering to obtain a decoction; wherein, the first decoction is made with 10 to 14 times the weight of the Panax notoginseng flower slices of water, and the second decoction is made with 8 to 12 times the weight of the Panax notoginseng flower slices of water, the first decoction time is 40 to 80 minutes, and the second decoction time is 20 to 60 minutes;

[0011] (2) The decoction in step (1) is concentrated and freeze-dried to obtain a Panax notoginseng flower extract; wherein the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is -40 to -50°C; b. primary drying: the drying temperature is -30 to 0°C; c. secondary drying for analysis: the drying temperature is 5 to 25°C.

[0012] In some embodiments of the present application, the pre-freezing time is 3 to 5 hours, preferably 4 hours; and / or,

[0013] The primary drying time is 9 to 17 hours, preferably 13 hours, and more preferably the primary drying vacuum degree is 0 to 0.2 mbar; and / or,

[0014] The secondary drying time is 4 to 10 hours, preferably 7 hours.

[0015] In some embodiments of the present application, in step (2), the primary drying curve is as follows: stage 1: freeze drying at -30°C for 5-7h; stage 2: freeze drying at -20°C for 1-3h; stage 3: freeze drying at -10°C for 1-3h; stage 4: freeze drying at 0°C for 2-4h;

[0016] And / or, the secondary drying curve is as follows: stage 1: freeze drying at 5°C for 1-3 h; stage 2: freeze drying at 15°C for 1-3 h; stage 3: freeze drying at 25°C for 1-3 h; stage 4: freeze drying at 0°C for 2-4 h.

[0017] In some embodiments of the present application, in step (2), the concentration is carried out under reduced pressure, the concentration temperature is 50-65° C., and the concentration is preferably carried out to a density of 1.03-1.08 g / mL.

[0018] The present application also provides a Panax notoginseng flower extract, which is prepared by the above-mentioned preparation method; preferably, based on the dry product, the content of ginsenoside Rb3 in the Panax notoginseng flower extract is 29.65 mg / g to 70.13 mg / g.

[0019] The present application also provides a method for detecting the mass content of ginsenoside Rb3 in the above-mentioned Panax notoginseng flower extract, which comprises the following steps:

[0020] (1) Preparation of reference solution

[0021] Weigh ginsenoside Rb3 reference substance and add solvent to prepare reference substance solution;

[0022] (4) Preparation of test solution

[0023] Weigh the Panax notoginseng flower extract, add a solvent for extraction;

[0024] (5) Ultra-high performance liquid chromatography analysis

[0025] The reference solution and the test solution are injected into an ultra-high performance liquid chromatograph for analysis, wherein the chromatographic conditions for the ultra-high performance liquid chromatography analysis are as follows: octadecylsilane bonded silica gel is used as a filler, an organic solvent is used as a mobile phase A, and a 0.08-0.12 wt % phosphoric acid aqueous solution is used as a mobile phase B for gradient elution.

[0026] In some embodiments of the present application, the solvent in step (1) and step (2) is selected from 100% methanol, 75 v / v% methanol, 50 v / v% methanol, 25 v / v% methanol, 95 v / v% ethanol, 75 v / v% ethanol, 50 v / v% ethanol, 25 v / v% ethanol and water, preferably 75 v / v% methanol. Preferably, the volume amount of the solvent is 100-250 mL / g of Panax notoginseng flower extract; and / or,

[0027] The extraction in step (2) is performed by reflux or ultrasound, preferably ultrasound extraction; preferably, the extraction time in step (2) is 15-60 min, preferably 60 min; and / or,

[0028] The organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile; preferably, the conditions for the gradient elution in step (3) are: at 0-30 min, mobile phase A is 31 v / v%, mobile phase B is 69 v / v%, at 30-35 min, mobile phase A is 31-40 v / v%, mobile phase B is 69%-60 v / v%, at 35-50 min, mobile phase A is 40%-46 v / v%, mobile phase B is 60%-54 v / v%, at 50-55 min, mobile phase A is 46%-55 v / v%, mobile phase B is 54%-45 v / v%, wherein the floating range of all the above concentration values ​​is allowed to be ±1 v / v%.

[0029] The present application also provides a method for constructing the characteristic spectrum of the Panax notoginseng flower extract, which comprises the following steps:

[0030] (1) Preparation of reference solution

[0031] Weigh ginsenoside Rb3 reference substance and add solvent to prepare reference substance solution;

[0032] (2) Preparation of test solution

[0033] Weigh the Panax notoginseng flower extract, add a solvent for extraction;

[0034] (3) Ultra-high performance liquid chromatography analysis

[0035] The reference solution and the test solution are injected into an ultra-high performance liquid chromatograph for analysis, wherein the chromatographic conditions during the ultra-high performance liquid chromatography analysis are as follows: octadecylsilane bonded silica gel is used as a filler, an organic solvent is used as a mobile phase A, and an aqueous phase is used as a mobile phase B for gradient elution.

[0036] In some embodiments of the present application, the solvent in steps (1) and (2) is selected from one or more of 100% methanol, 75 v / v% methanol, 50 v / v% methanol, 25 v / v% methanol, 95 v / v% ethanol, 75 v / v% ethanol, 50 v / v% ethanol, 25 v / v% ethanol and water, preferably 75 v / v% methanol; preferably, the volume amount of the solvent is 100-250 mL / g of Panax notoginseng flower extract; and / or

[0037] The extraction in step (2) is performed by reflux extraction or ultrasonic extraction, preferably ultrasonic extraction. Preferably, the extraction time in step (2) is 15-60 min, preferably 30 min; and / or,

[0038] When ultra-high performance liquid chromatography analysis is performed in step (3), the aqueous phase is selected from water, 0.08-0.12wt% formic acid solution, 0.08-0.12wt% phosphoric acid solution or 0.08-0.12wt% acetic acid solution, preferably 0.08-0.12wt% phosphoric acid solution; preferably, the organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile; further preferably, the conditions of the gradient elution in step (3) are: at 0-30min, the mobile phase A is 31v / v%, mobile phase B is 69%, 30-35min, mobile phase A is 31%-40v / v%, mobile phase B is 69%-60v / v%, 35-50min, mobile phase A is 40%-46v / v%, mobile phase B is 60%-54v / v%, 50-55min, mobile phase A is 46%-55v / v%, mobile phase B is 54%-45v / v%, wherein the floating range of all the above concentration values ​​is allowed to be ±1v / v%.

[0039] The present application also provides an identification method for the Panax notoginseng flower extract, which comprises identifying the Panax notoginseng flower extract by thin layer chromatography; the thin layer chromatography comprises the following steps: a. preparing a Panax notoginseng flower extract test solution and a Panax notoginseng flower medicinal material reference solution; b. spotting the Panax notoginseng flower extract test solution and the Panax notoginseng flower medicinal material reference solution on the same silica gel G thin layer plate, adding a developing agent for development, taking out and drying, spraying a color developer to color the spots, and detecting to obtain;

[0040] Preferably, the Panax notoginseng flower extract test solution is prepared by the following steps: taking 0.1-2g of Panax notoginseng flower extract, adding water-saturated n-butanol to dissolve, separating to obtain a separated liquid, extracting the separated liquid with water-saturated n-butanol, taking the upper solution and evaporating to dryness, and dissolving the residue with methanol to obtain the solution; and / or, the Panax notoginseng flower medicinal material reference solution is prepared by the following steps: taking 0.1-2g of Panax notoginseng flower medicinal material, adding water-saturated n-butanol to dissolve, separating to obtain a separated liquid, extracting the separated liquid with water-saturated n-butanol, taking the upper solution and evaporating to dryness, and dissolving the residue with methanol to obtain the solution;

[0041] More preferably, the developing agent is chloroform-methanol-water, and further preferably, the volume ratio of chloroform, methanol and water is 6-8:2-4:0.1-1, preferably 7:3:0.5.

[0042] The present application also provides a method for detecting the mass content of ginsenoside Rb3 in the above-mentioned Panax notoginseng flower extract, or a method for constructing the above-mentioned characteristic spectrum of the Panax notoginseng flower extract, or the application of the above-mentioned identification method of the Panax notoginseng flower extract in Panax notoginseng flower medicinal materials or Panax notoginseng flower extract preparations.

[0043] The present application also provides the use of the Panax notoginseng flower extract in preparing health foods and Chinese herbal medicines; preferably, the Chinese herbal medicines are standard decoctions, capsules, tablets, granules or formula granules.

[0044] The present application also provides a standard decoction, which is prepared by the above-mentioned method for preparing the notoginseng flower extract.

[0045] The present application also provides a Panax notoginseng flower formula granule, which comprises the following raw materials in parts by weight: 100 parts of Panax notoginseng flower extract and 0.1-0.3 parts of pharmaceutical excipients; preferably, the pharmaceutical excipients are selected from one or two of the substance group consisting of maltodextrin, magnesium stearate, silicon dioxide, talc or microcrystalline cellulose.

[0046] The present application also provides a method for preparing Panax notoginseng flower formula granules, comprising the following steps:

[0047] (1) taking Panax notoginseng flower slices, adding water to decoct, and filtering to obtain a filtrate; wherein the water decoction is performed twice, the first water decoction is performed with 10 to 14 times the weight of the Panax notoginseng flower slices, the second water decoction is performed with 8 to 12 times the weight of the Panax notoginseng flower slices, the first water decoction time is 40 to 80 minutes, and the second water decoction time is 20 to 60 minutes;

[0048] (2) concentrating and drying the filtrate from step (1) to obtain a Panax notoginseng flower extract;

[0049] (3) Dry granulating the notoginseng flower extract and pharmaceutical excipients to obtain notoginseng flower formula granules.

[0050] Beneficial effects of the present invention:

[0051] 1. This application uses Panax notoginseng flower medicinal materials to prepare Panax notoginseng flower extract, and through a specific preparation process, the mass content of ginsenoside Rb3 in the prepared Panax notoginseng flower extract is 29.65 mg / g to 70.13 mg / g.

[0052] 2. The method described in this application provides a standard for the quality control of Panax notoginseng flower extract and its preparations such as formula granule products, thereby achieving overall quality control and effective supervision of Panax notoginseng flower extract and its preparations such as formula granule products.

[0053] 3. The characteristic spectrum method established in this application uses ultra-high phase liquid chromatography, which is simple, stable, highly precise and reproducible. In addition, the fingerprint spectrum of the obtained extract has many peaks, good peak shape, easy identification, and is accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The HPLC chromatogram is used to investigate the specificity of the determination method of ginsenoside Rb3 in Panax notoginseng flower extract;

[0055] Figure 2 This is a linear relationship investigation diagram of the determination method of the mass content of ginsenoside Rb3 in Panax notoginseng flower extract;

[0056] Figure 3 is the absorption spectrum of Panax notoginseng flower extract in the range of 190-400nm;

[0057] Figure 4 The ultra-high performance liquid chromatogram of the characteristic spectrum detection method of Panax notoginseng flower extract prepared in Example 1 with the mobile phases being acetonitrile-0.1% phosphoric acid solution and methanol-0.1% phosphoric acid solution;

[0058] Figure 5 The ultra-high performance liquid chromatograms of the characteristic spectrum detection method for preparing the Panax notoginseng flower extract in Example 1 with the mobile phases being acetonitrile-water and acetonitrile-0.1% phosphoric acid aqueous solution;

[0059] Figure 6 The results of the extraction efficiency of different extraction solvents in the characteristic spectrum detection method of the Panax notoginseng flower extract prepared in Example 1;

[0060] Figure 7 Characteristic spectra of Panax notoginseng flower extracts and control herbs prepared from different batches of medicinal pieces;

[0061] Figure 8 This is a chromatogram for investigating the specificity of the characteristic spectrum detection method of Panax notoginseng flower extract;

[0062] Fig. 9 The chromatogram is a comprehensive investigation of the characteristic spectrum detection method of Panax notoginseng flower extract;

[0063] Fig.10 Characteristic spectra of different batches of Panax notoginseng flower medicinal materials and control medicinal materials;

[0064] Fig.11 Characteristic spectra of different batches of Panax notoginseng flower granules and control medicinal materials;

[0065] Fig.12 This is the thin layer chromatogram of different sample amounts in the thin layer chromatography identification method of Panax notoginseng flower formula granules;

[0066] Fig.13 To investigate the specificity of the thin layer chromatography identification method of Panax notoginseng flower granules;

[0067] Fig.14 This is a thin layer chromatogram with a development temperature of 5°C in the thin layer chromatography identification method of Panax notoginseng flower formula granules;

[0068] Fig.15 This is a thin layer chromatogram with a development temperature of 25°C in the thin layer chromatography identification method of Panax notoginseng flower formula granules;

[0069] Fig.16 It is a thin layer chromatogram with a humidity of 33% in the thin layer chromatography identification method of Panax notoginseng flower formula granules;

[0070] Fig.17 It is a thin layer chromatogram with a humidity of 66% in the thin layer chromatography identification method of Panax notoginseng flower formula granules;

[0071] Fig.18 It is a thin layer chromatogram with a humidity of 88% in the thin layer chromatographic identification method of Panax notoginseng flower formula granules;

[0072] Fig.19 This is a thin layer chromatogram of the silica gel thin layer plate used in the thin layer chromatography identification method of Panax notoginseng flower formula granules, which is Qingdao Ocean Silica G plate;

[0073] Fig. 20 This is a thin layer chromatogram of the silica gel thin layer plate used in the thin layer chromatography identification method of Panax notoginseng flower formula granules, which is a Yantai Yinlong silica gel prefabricated silica gel G plate;

[0074] Fig.21 This is a thin layer chromatogram of the silica gel thin layer plate used in the thin layer chromatography identification method of Panax notoginseng flower formula granules, which is a German Merck plate;

[0075] Fig. 22 This is the TLC identification result of Panax notoginseng flower granules;

[0076] Fig.23 These are the thin layer chromatography identification results of different batches of Panax notoginseng flower medicinal materials. DETAILED DESCRIPTION

[0077] In order to make the purpose, technical scheme and technical effect of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention is described clearly and completely. The embodiments described below are part of the embodiments of the present invention, not all of them. In combination with the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0078] In the present application, v / v% refers to volume percentage.

[0079] In a specific embodiment of the present invention, the present invention provides a method for preparing a Panax notoginseng flower extract, wherein the preparation method comprises: decocting Panax notoginseng flower slices twice in water and filtering to obtain a decoction; concentrating and drying the decoction to obtain the Panax notoginseng flower extract.

[0080] In the present application, there is no restriction on the preparation of Panax notoginseng flower slices, which can be prepared according to conventional methods in the art, for example, the original medicinal materials can be taken, young branches and leaves can be selected, residual roots and impurities can be removed, leaves can be removed first, sieved and stored separately; the stems can be washed, watered thoroughly, cut into sections, dried, and then mixed with the leaves, or watered thoroughly, cut into sections, dried, etc.

[0081] In the present application, there is no restriction on the utensils used for decoction, and the utensils in the art can be used for decoction, for example, the provisions on decoction containers in the "Management Specifications for Traditional Chinese Medicine Decoction Rooms in Medical Institutions" National Traditional Chinese Medicine (2009) No. 3 document can be used (the decoction container should be made of ceramic, stainless steel, copper and other materials. In the present application, a 5L automatic decoction ceramic pot is used as the extract decoction equipment.

[0082] In this application, there is no restriction on the dosage of Panax notoginseng flower slices, and the dosage conventional in the art can be used. For example, according to the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", the amount of slices used for each decoction is generally not less than 100g. In this application, the dosage of Panax notoginseng flower slices is 100g.

[0083] The extract described in this application can standardize the quality control and standard research of Chinese medicine formula granules, realize the overall quality control and effective supervision of Chinese medicine formula granules, and provide a reference for the quality control of Panax notoginseng flower formula granules.

[0084] In some embodiments of the present application, the present application provides a method for preparing a Panax notoginseng flower extract, comprising the following steps:

[0085] (1) taking Panax notoginseng flower slices, adding water to decoct, and filtering to obtain a decoction; wherein the water decoction is performed twice, the first water decoction is performed with 10 to 14 times the weight of the Panax notoginseng flower slices, the second water decoction is performed with 8 to 12 times the weight of the Panax notoginseng flower slices, the first water decoction time is 40 to 80 minutes, and the second water decoction time is 20 to 60 minutes;

[0086] (2) Concentrating the filtrate of step (1) and freeze-drying to obtain a Panax notoginseng flower extract; wherein the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is -40 to -50°C; b. primary drying: the drying temperature is -30 to 0°C; c. secondary drying for analysis: the drying temperature is 5 to 25°C.

[0087] In some embodiments of the present application, in step (2), the pre-freezing time is 3 to 5 hours, preferably 4 hours; the primary drying time is 9 to 17 hours, preferably 13 hours, and the vacuum degree of the primary drying is 0 to 0.2 mbar. In some embodiments of the present application, the secondary drying time is 4 to 10 hours, preferably 7 hours; and the vacuum degree of the secondary drying is 0 mbar.

[0088] In some embodiments of the present application, in order to control the water content of the freeze-dried powder to be moderate, which is beneficial to the storage and quality stability of the freeze-dried powder, as well as easy dissolution and no need to add auxiliary materials, in step (2), the primary drying curve is as follows:

[0089] Stage 1: freeze drying at -30°C for 5-7h;

[0090] Stage 2: freeze drying at -20°C for 1-3h;

[0091] Stage 3: freeze drying at -10°C for 1-3h;

[0092] Stage 4: freeze drying at 0°C for 2-4h;

[0093] Preferably, the secondary drying curve is as follows:

[0094] Stage 1: freeze drying at 5°C for 1-3h;

[0095] Stage 2: freeze drying at 15°C for 1-3h;

[0096] Stage 3: freeze drying at 25°C for 1-3h;

[0097] Stage 4: Freeze drying at 0°C for 2-4h.

[0098] According to the present application, in step (2), the filtrate is concentrated under reduced pressure at a temperature of 50-65° C. to a density of 1.03-1.08 g / mL.

[0099] In some embodiments of the present application, in step (1), the Panax notoginseng flower slices are appropriately crushed and soaked before being decocted in water, and the soaking time is 30 minutes to 60 minutes.

[0100] According to the present application, in step (1), the filtration is performed while hot, and the filtration is performed using a 100-300 mesh sieve, preferably a 300 mesh sieve.

[0101] Preferably, the screen is a Taylor screen (Taylor Company, USA) or a filter cloth (Xinxiang Zhongxin Chemical Co., Ltd.).

[0102] In a second aspect, the present application also provides a Panax notoginseng flower extract prepared by the above-mentioned preparation method.

[0103] In some embodiments of the present application, the content of ginsenoside Rb3 in the Panax notoginseng flower extract is 29.65 mg / g to 70.13 mg / g, preferably 40-60 mg / g; preferably, the transfer rate of ginsenoside Rb3 is 15.08% to 41.20%.

[0104] It is understandable that the Panax notoginseng flower extract can be directly used as a standard decoction, or other auxiliary materials can be added to make formula granules.

[0105] In a third aspect, the present application provides a method for detecting the content of ginsenoside Rb3 in a Panax notoginseng flower medicinal material or a Panax notoginseng flower extract, comprising the following steps:

[0106] (1) Preparation of reference solution

[0107] Weigh ginsenoside Rb3 reference substance and add solvent to make a solution;

[0108] (2) Preparation of test solution

[0109] Weigh the Panax notoginseng flower medicinal material or Panax notoginseng flower extract, and add a solvent for extraction;

[0110] (3) Ultra-high performance liquid chromatography analysis

[0111] The reference solution and the test solution are respectively injected into an ultra-high performance liquid chromatograph for analysis, wherein the chromatographic conditions for the ultra-high performance liquid chromatography analysis are as follows: octadecylsilane bonded silica gel is used as a filler, an organic solvent is used as a mobile phase A, and 0.08-0.12% phosphoric acid aqueous solution is used as a mobile phase B for gradient elution.

[0112] In some embodiments of the present application, in the above-mentioned preparation method, the solvent in steps (1) and (2) is selected from one or more of 100% methanol, 75v / v% methanol, 50v / v% methanol, 25v / v% methanol, 95v / v% ethanol, 75v / v% ethanol, 50v / v% ethanol, 25v / v% ethanol and water, preferably 75v / v% methanol; preferably, the volume usage of the solvent is 100-250mL / g Panax notoginseng flower medicinal material or Panax notoginseng flower extract.

[0113] In some embodiments of the present application, in the above-mentioned preparation method, the extraction in step (2) adopts one of reflux extraction or ultrasonic extraction, preferably ultrasonic extraction. Preferably, the extraction time in step (2) is 15-60 minutes, preferably 30 minutes.

[0114] In some embodiments of the present application, after the extraction in step (2), the steps of cooling, replenishing the weight lost by the extraction with a solvent, and filtering are also included.

[0115] In some embodiments of the present application, in the above preparation method, the organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile;

[0116] Preferably, the conditions of the gradient elution in step (3) are: at 0-30 min, mobile phase A is 31 v / v%, mobile phase B is 69 v / v%, at 30-35 min, mobile phase A is 31-40 v / v%, mobile phase B is 69-60 v / v%, at 35-50 min, mobile phase A is 40-46 v / v%, mobile phase B is 60-54 v / v%, at 50-55 min, mobile phase A is 46%-55 v / v%, and mobile phase B is 54-45 v / v%.

[0117] It should be noted that the allowed floating range of all the above concentration values ​​is ±1 v / v%. For example, the allowed floating range of 31 v / v% is 31±1 v / v%, the allowed floating range of 40 v / v% is 40±1 v / v%, the allowed floating range of 46 v / v% is 46±1 v / v%, the allowed floating range of 54 v / v% is 54±1 v / v%, the allowed floating range of 60 v / v% is 60±1 v / v%, and the allowed floating range of 69 v / v% is 69±1 v / v%. Specifically for the above elution gradient, for example, when mobile phase A is 31 v / v% and mobile phase B is 69 v / v%, the floating range of mobile phase A is 30-32 v / v%, and the floating range of mobile phase B is 68-70 v / v%.

[0118] 31-40v / v% means that the concentration changes from 31±1v / v% to 40±1v / v%, 69-60v / v% means that the concentration changes from 69±1v / v% to 60±1v / v%, 40-46v / v% means that the concentration changes from 40±1v / v% to 46±1v / v%, 60-54v / v% means that the concentration changes from 60±1v / v% to 54±1v / v%, 46-55v / v% means that the concentration changes from 46±1v / v% to 55±1v / v%, and 54-45v / v% means that the concentration changes from 54±1v / v% to 45±1v / v%.

[0119] According to the present invention, the preparation method of the reference solution is as follows: taking ginsenoside R b3 The reference substance is measured accurately, and the solvent is added to make each 1mL contain ginsenoside R b3 100-200 μg of solution, preferably containing ginsenoside R per 1 mL b3 150 μg of solution.

[0120] The preparation method of the above-mentioned test solution is: take 0.1-0.15g of Panax notoginseng flower medicinal material or Panax notoginseng flower extract, accurately weigh it, add 20mL of solvent, weigh it, extract it by reflux or ultrasonic method, preferably ultrasonic extraction, weigh it again, make up the lost weight with solvent, shake well, and filter to obtain a filtrate.

[0121] According to the present invention, when performing ultra-high performance liquid chromatography analysis, the chromatographic column used can be an ACQUITY UPLCHSS T3 (2.1*150mm, 2.7μm) chromatographic column or an AmethSep C18 AQ (2.1*150mm, 1.8μm) chromatographic column; preferably an AmethSep C18 AQ (2.1*150mm, 1.8μm) chromatographic column.

[0122] According to the present invention, when ultra-high performance liquid chromatography analysis is performed, the column temperature of the chromatographic column used is 25-35°C, preferably 30°C.

[0123] According to the present invention, when performing high performance liquid chromatography analysis, the flow rate used may be 0.2-0.3 ml / min, preferably 0.25 ml / min.

[0124] In a fourth aspect, the present application provides a method for constructing a characteristic spectrum of a Panax notoginseng flower medicinal material or a Panax notoginseng flower extract, comprising the following steps:

[0125] (1) Preparation of reference solution

[0126] Weigh ginsenoside Rb3 reference substance and add solvent to prepare reference substance solution;

[0127] (2) Preparation of test solution

[0128] Take Panax notoginseng flower medicinal material or Panax notoginseng flower extract, add solvent for extraction;

[0129] (3) Ultra-high performance liquid chromatography analysis

[0130] The reference solution and the test solution are injected into an ultra-high performance liquid chromatograph for analysis, wherein the chromatographic conditions during the ultra-high performance liquid chromatography analysis are as follows: octadecylsilane bonded silica gel is used as a filler, an organic solvent is used as a mobile phase A, and an aqueous phase is used as a mobile phase B for gradient elution.

[0131] In some embodiments of the present application, in the above-mentioned preparation method, the solvent in steps (1) and (2) is selected from one or more of 100 v / v% methanol, 75 v / v% methanol, 50 v / v% methanol, 25 v / v% methanol, 95 v / v% ethanol, 75 v / v% ethanol, 50 v / v% ethanol, 25 v / v% ethanol and water, preferably 75 v / v% methanol; preferably, the volume usage of the solvent is 100-250 mL / g Panax notoginseng flower medicinal material or Panax notoginseng flower extract.

[0132] In some embodiments of the present application, in the above-mentioned preparation method, the extraction in step (2) adopts one of reflux extraction or ultrasonic extraction, preferably ultrasonic extraction. Preferably, the extraction time in step (2) is 15-60 minutes, preferably 30 minutes.

[0133] In some embodiments of the present application, after the extraction in step (2), the steps of cooling, replenishing the weight lost by the extraction with a solvent, and filtering are also included.

[0134] According to the present invention, the preparation method of the reference solution is as follows: take an appropriate amount of ginsenoside Rb3 reference substance, accurately measure, add a solvent to make a solution containing ginsenoside Rb3 per 1 mL. b3 100-200 μg of solution, preferably containing ginsenoside R per 1 mL b3 150 μg of solution.

[0135] The preparation method of the above-mentioned test solution is: take 0.1-0.15g of Panax notoginseng flower medicinal material or Panax notoginseng flower extract, accurately weigh it, add 20mL of solvent, weigh it, extract it by reflux or ultrasonic method, preferably ultrasonic extraction, weigh it again, make up the lost weight with solvent, shake well, and filter to obtain a filtrate.

[0136] In some embodiments of the present application, when ultra-high performance liquid chromatography analysis is performed in step (3), the aqueous phase may be one of water, 0.08-0.12 wt % formic acid solution, 0.08-0.12 wt % phosphoric acid solution or 0.08-0.12 wt % acetic acid solution, preferably 0.08-0.12 wt % phosphoric acid solution;

[0137] Preferably, the organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile;

[0138] Further preferably, the conditions of the gradient elution in step (3) are: at 0-30 min, mobile phase A is 31 v / v%, mobile phase B is 69 v / v%, at 30-35 min, mobile phase A is 31-40 v / v%, mobile phase B is 69%-60 v / v%, at 35-50 min, mobile phase A is 40%-46 v / v%, mobile phase B is 60%-54 v / v%, at 50-55 min, mobile phase A is 46%-55 v / v%, mobile phase B is 54%-45 v / v%, wherein the allowed floating range of all the above concentration values ​​is ±1 v / v%.

[0139] In some embodiments of the present application, when ultra-high performance liquid chromatography analysis is performed in step (3), the detection wavelength is 203 nm.

[0140] In some embodiments of the present application, when performing ultra-high performance liquid chromatography analysis, the chromatographic column used can be an ACQUITY UPLC HSS T3 (2.1*150mm, 2.7μm) chromatographic column or an AmethSep C18 AQ (2.1*150mm, 1.8μm) chromatographic column; preferably an AmethSep C18 AQ (2.1*150mm, 1.8μm) chromatographic column.

[0141] In some embodiments of the present application, when ultra-high performance liquid chromatography analysis is performed, the column temperature of the chromatographic column used is 25-35°C, preferably 30°C.

[0142] In some embodiments of the present application, when performing high performance liquid chromatography analysis, the flow rate used may be 0.2-0.3 ml / min, preferably 0.25 ml / min.

[0143] In a fifth aspect, the present invention further provides a method for identifying a Panax notoginseng flower extract, comprising identifying the Panax notoginseng flower extract by thin layer chromatography; the thin layer chromatography comprises the following steps: a. preparing a Panax notoginseng flower extract test solution and a Panax notoginseng flower medicinal material reference solution; b. spotting the Panax notoginseng flower extract test solution and the Panax notoginseng flower medicinal material reference solution on the same silica gel G thin layer plate, adding a developing agent for development, taking out and drying, spraying a color developer to color the spots, and detecting to obtain;

[0144] Preferably, the Panax notoginseng flower extract test solution is prepared by the following steps: taking 0.1-2g of Panax notoginseng flower extract, adding water-saturated n-butanol to dissolve, separating to obtain a separated liquid, extracting the separated liquid with water-saturated n-butanol, taking the upper solution and evaporating to dryness, and dissolving the residue with methanol to obtain the obtained product; Preferably, the Panax notoginseng flower medicinal material reference solution is prepared by the following steps: taking 0.1-2g of Panax notoginseng flower medicinal material, adding water-saturated n-butanol to dissolve, separating to obtain a separated liquid, extracting the separated liquid with water-saturated n-butanol, taking the upper solution and evaporating to dryness, and dissolving the residue with methanol to obtain the obtained product;

[0145] More preferably, the developing agent is chloroform-methanol-water, and further preferably, the volume ratio of chloroform, methanol and water is 6-8:2-4:0.1-1, preferably 7:3:0.5.

[0146] In the sixth aspect, the present application also provides a quality detection method for Panax notoginseng flower medicinal materials, Panax notoginseng flower extracts, or Panax notoginseng flower extract preparations, the quality detection method comprising one or more of the determination of the content of ginsenoside Rb3, the determination of the characteristic spectrum of Panax notoginseng flower medicinal materials, Panax notoginseng flower extracts, or Panax notoginseng flower extract preparations, or thin layer chromatography identification.

[0147] It is understood that the characteristic spectrum can be determined by the method for determining the characteristic spectrum of Panax notoginseng flower medicinal material or Panax notoginseng flower extract constructed above. The thin layer chromatography identification can adopt the identification method of Panax notoginseng flower extract.

[0148] The preparations of Panax notoginseng flower extract include but are not limited to standard decoctions, capsules, tablets, granules or formulated granules.

[0149] In a seventh aspect, the present application also provides the use of the Panax notoginseng flower extract in the preparation of health foods and Chinese herbal medicines; preferably, the Chinese herbal medicines are standard decoctions, capsules, tablets, granules or formula granules.

[0150] In an eighth aspect, the present application also provides a standard decoction prepared by the above-mentioned method for preparing the notoginseng flower extract.

[0151] In the ninth aspect, the present application also provides a Panax notoginseng flower formula granule, which comprises the following raw materials in parts by weight, 100 parts of Panax notoginseng flower extract and 0.1-0.3 parts of pharmaceutical excipients; preferably, the pharmaceutical excipients are selected from one or two of the substance group consisting of maltodextrin, magnesium stearate, silicon dioxide, talc or microcrystalline cellulose.

[0152] In a tenth aspect, the present application also provides a method for preparing Panax notoginseng flower formula granules, comprising the following steps:

[0153] (1) taking Panax notoginseng flower slices, adding water to decoct, and filtering to obtain a filtrate; wherein the water decoction is performed twice, the first water decoction is performed with 10 to 14 times the weight of the Panax notoginseng flower slices, the second water decoction is performed with 8 to 12 times the weight of the Panax notoginseng flower slices, the first water decoction time is 40 to 80 minutes, and the second water decoction time is 20 to 60 minutes;

[0154] (2) concentrating and drying the filtrate from step (1) to obtain a Panax notoginseng flower extract;

[0155] (3) Dry granulating the notoginseng flower extract and pharmaceutical excipients to obtain notoginseng flower formula granules.

[0156] According to the present application, in step (2), the filtrate is concentrated under reduced pressure at a temperature of 60-70° C. to a density of 1.03-1.06 g / mL.

[0157] In some embodiments of the present application, in step (1), the Panax notoginseng flower slices are appropriately crushed and soaked before being decocted in water, and the soaking time is 30 minutes to 60 minutes.

[0158] According to the present application, in step (1), the filtration is performed while hot, and the filtration is performed using a 100-300 mesh sieve, preferably a 200 mesh sieve.

[0159] The sieve described in this application is a Taylor sieve (Taylor Company, USA) or a filter cloth (Xinxiang Zhongxin Chemical Co., Ltd.).

[0160] In some embodiments of the present application, the drying in step (2) includes but is not limited to one of freeze drying, vacuum drying, spray drying, microwave drying and infrared drying, preferably spray drying. Spray drying has the advantages of high drying efficiency, fast drying speed, short drying time, less damage to effective ingredients, uniform and fine dry paste powder, low water content, good solubility, etc., and is suitable for pilot and commercial large-scale production; in addition, the control of the hot air temperature when the hot air enters and the air outlet temperature during spray drying is very important, which determines the quality of the product and the dry bulk density. The inventors have found through research that the spray drying air inlet temperature is 165-195°C, and the air outlet temperature is 85-105°C. At this temperature, the product is well formed and of high quality.

[0161] In the spray drying process, in order to prevent foreign matter mixed in the clear paste of Panax notoginseng flower extract from clogging the pipeline, the clear paste needs to be heated before drying to keep the material temperature at 60-100° C. In order to facilitate drying and reduce the problems of wall sticking and agglomeration, auxiliary materials such as maltodextrin are added to the clear paste before drying. More preferably, the amount of auxiliary materials added is 6-9% based on the weight of the Panax notoginseng flower slices added during decoction, which has a better effect.

[0162] In the present application, dry granulation mainly refers to the granules made without wetting agent or liquid adhesive, which is a method of mixing medicine and auxiliary materials, compressing them into large sheets or strips, and then crushing the required size particles. This method relies on compression force to produce binding force between ions, and its preparation method has a weight pressing method and a roller pressing method. Compared with traditional wet granulation, it saves the process of making soft materials, drying, and granulating, and the process is simple, and overcomes the disadvantage of large auxiliary material consumption, which can greatly improve the drug loading. The relevant laws and regulations of Chinese medicine formula granules require that the finished product of Chinese medicine formula granules only does not add or adds less auxiliary materials as far as possible, so it is advisable to select dry granulation as the granulation method of Chinese medicine formula granules.

[0163] The pharmaceutical excipients include but are not limited to maltodextrin, magnesium stearate, silicon dioxide, talc or microcrystalline cellulose, which can change the properties of the powder, improve the appearance and fluidity of the material, and facilitate the control of storage and transportation, solubility, porosity and specific surface area. Considering the comprehensive properties such as particle size, fluidity, one-time yield, bulk density, solubility, etc., the amount of excipients added in dry granulation should be ≤0.3% (based on the weight of the clear paste). Excessive use of materials will bring some performance defects, such as excessive use of magnesium stearate, due to its hydrophobicity, will make dissolution slow.

[0164] In the eleventh aspect, the present application also provides a method for detecting the mass content of the above-mentioned ginsenoside Rb3 or a method for constructing the above-mentioned characteristic spectrum of the Panax notoginseng flower extract or the application of the above-mentioned quality detection method in Panax notoginseng flower medicinal materials or Panax notoginseng flower extract preparations.

[0165] The beneficial effects of the present invention are further illustrated by specific examples below.

[0166] The raw materials and reagents used in the present invention are all purchased from mainstream manufacturers in the market. Those without manufacturer or concentration are all analytically pure raw materials and reagents that can be routinely obtained. As long as they can play the expected role, there is no special limitation.

[0167] If no specific technology or conditions are specified in this embodiment, the process shall be carried out according to the technology or conditions described in the literature in the art or according to the product manual.

[0168] The present invention is further described below using examples and comparative examples, but the technical scope of the present invention is not limited to these examples. It should be noted that, unless otherwise specified, all percentages, parts, and ratios used in the present invention are based on mass. The raw material information and test equipment information used in the examples and comparative examples are shown in Tables 1 and 2, respectively:

[0169] Table 1 Raw material information used in the present invention

[0170]

[0171]

[0172] Table 2 Information of experimental equipment used in the present invention

[0173]

[0174] The solid content of the Panax notoginseng flower concentrate in the following examples was determined by the following method:

[0175] 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.

[0176] Embodiment 1

[0177] 1. Preparation method of Panax notoginseng flower extract

[0178] (1) Take 100 g of Panax notoginseng flower slices (batch number P-SQH-231210-QGY-YP01), place in an electric ceramic pot, add water and boil twice. For the first decoction, add directly to water 10 times the weight of Panax notoginseng flower slices, soak for 30 minutes, boil over high heat (500W), and then keep it slightly boiling over low heat (200W) for 60 minutes, and filter the decoction through a 300-mesh sieve while hot; for the second decoction, add water 8 times the weight of Panax notoginseng flower slices, boil over high heat, and then keep it slightly boiling over low heat for 40 minutes, filter the decoction through a 300-mesh sieve while hot, and combine the two filtrates;

[0179] (2) The filtrate was transferred to a 2000 ml round-bottom flask and concentrated at low temperature under reduced pressure using a rotary evaporator (temperature: 65°C; vacuum degree: -0.085 MPa) to 100 ml of extract, the density of which was measured to be 1.078 g / mL and the extract yield was 35.74%; under magnetic stirring, the extract was dispensed into 5 ml brown vials, each with a dispensing volume of 1 ml, and half-stoppered. After dispensing, the extract was transferred to a vacuum freeze dryer for freeze drying, and pre-frozen at a temperature of -45°C for 4 hours. The extract was then dried at -30°C, -20°C, -10°C and 0°C, with a vacuum degree of 0.2 mbar, for 7 hours, 2 hours, 2 hours and 3 hours, respectively. The extract was then dried at 5°C, 15°C and 25°C, with a vacuum degree of -0.2 mbar, for 2 hours, 2 hours and 3 hours, respectively. The extract was taken out and the aluminum cover was rolled to obtain the notoginseng flower extract. The notoginseng flower extract yield and water mass content were calculated according to the following formula:

[0180] Paste yield = concentrated liquid weight * concentrated liquid solid content / decoction piece feeding amount * 100%;

[0181] Water content = water weight / sample weight*100%.

[0182] 3. Determination of mass content and transfer rate of ginsenoside Rb3 in Panax notoginseng flower extract

[0183] 2.1 Optimization of detection conditions

[0184] 2.1.1 Preparation of reference solution

[0185] Take an appropriate amount of ginsenoside Rb3 reference substance, weigh it accurately, place it in a numbered 10ml volumetric flask, add 75v / v% methanol to make a 10ml ginsenoside Rb3 reference substance. b3 150μg reference substance solution, shake well and you have it.

[0186] 2.1.2 Preparation of test solution

[0187] Take about 0.1 g of Panax notoginseng flower extract obtained in Example 1, accurately weigh it, place it in a stoppered conical flask, add 20 mL of solvent, weigh it, extract it for a period of time, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, take the filtrate, take an appropriate amount of the filtrate and pass it through a 0.22 μm microporous filter membrane, and place it in a liquid sample bottle.

[0188] 2.1.3 High performance liquid chromatography analysis

[0189] The chromatographic column was filled with octadecylsilane bonded silica gel (AmethSep C18 AQ (2.1*150mm, 1.8μm)); acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 3; the column temperature was 30°C; the flow rate was 0.25ml / min, and the detection wavelength was 203nm. The number of theoretical plates calculated based on the ginsenoside Rb3 peak should not be less than 3000. 1μL of the reference solution in 2.1.1 and 1μL of the test solution in 2.1.2 were respectively injected into the high performance liquid chromatograph for analysis.

[0190] Table 3 Elution gradient

[0191] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-30 31 69 30-35 31→40 69→60 35-50 40→46 60→54 50-55 46→55 54→45

[0192] 2.1.4 Ginsenoside R b3 The mass content and ginsenoside R b3 The transfer rate is calculated according to the following formula:

[0193] in,

[0194] w 人参皂苷Rb3 =A 供 ×C 对 ×V 供 ×10 -3 / (m 供 ×A 对 )

[0195] w 饮 =A 饮供 ×C 对 ×V 饮供 ×10 -3 / (m 饮供 ×A 对 )

[0196] Ginsenoside R b3 Transfer rate = (m 提 × 对羟基苯乙酮 ) / (m 饮 × 饮 )

[0197] Where:

[0198] w 人参皂苷Rb3 ——Ginsenoside R in Panax notoginseng flower extract b3 Mass content, mg / g;

[0199] w 饮 ——Ginsenoside R in Panax Notoginseng Flower Pieces b3 Mass content, mg / g;

[0200] A 对 ——Ginsenoside R in the HPLC spectrum of the reference solution b3 Corresponding absorption peak area;

[0201] A 供 ——Ginsenoside R in the HPLC spectrum of the test solution b3 Corresponding absorption peak area;

[0202] A 饮供 ——Ginsenoside R in the HPLC spectrum of the sample solution b3 Corresponding absorption peak area;

[0203] C 对 ——Concentration of reference substance in reference solution, μg / mL;

[0204] m 供 ——absolute dry mass of Panax notoginseng flower extract in the test solution, g;

[0205] m 饮供 ——The absolute dry mass of Panax notoginseng flower slices in the slice test solution, g;

[0206] V 供 ——Volume of test solution, ml;

[0207] V 饮供 ——Volume of the sample solution, ml;

[0208] m 提 ——The absolute dry mass of the obtained Panax notoginseng flower extract, g;

[0209] m 饮 ——The absolute dry mass of the raw material Panax notoginseng flower slices, g.

[0210] 2.1.5 Optimization of preparation conditions

[0211] Ⅰ Investigation of different extraction solvents

[0212] Take about 0.1g of Panax notoginseng flower extract prepared in Example 1, accurately weigh it, and place two parallel samples in stoppered conical bottles respectively. Accurately add 20mL 100% methanol, 75v / v% methanol, 50v / v% methanol, 25v / v% methanol, anhydrous ethanol, 75v / v% ethanol, 50v / v% ethanol, 25v / v% ethanol and water in sequence, weigh it, and ultrasonically treat it (power 500W, frequency 40kHz) for 30min, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain each test solution. Accurately take 1μL of the reference solution and the test solution respectively, inject it into the high performance liquid chromatograph, and perform chromatographic analysis according to the chromatographic conditions of 2.1.3. Ginsenoside R b3 According to the above method, the influence of different extraction solvents on its content was calculated to determine the best extraction solvent. The specific results are shown in Table 4:

[0213] Table 4 Effect of different extraction solvents on ginsenoside R in Panax notoginseng flower extract b3 The influence of content

[0214]

[0215]

[0216] It can be seen from Table 4 that different solvents have an effect on the ginsenoside R in the extract of Panax notoginseng. b3 The content of was significantly affected by the difference. Considering the content and the target peak shape comprehensively, 75v / v% methanol was selected as the extraction solvent.

[0217] Ⅱ Investigation of extraction methods

[0218] Take about 0.1g of Panax notoginseng flower extract obtained in Example 1, accurately weigh it, and make 3 portions in total. Each portion has 2 parallel samples, and place them in stoppered cones respectively. Accurately add 20mL of 75v / v% methanol in sequence, weigh the weight, ultrasonicate (power 500W, frequency 40kHz) for 30min, reflux for 30min, take it out and cool it to room temperature, add 75v / v% methanol to make up the weight, mix it, filter it, and collect the filtrate. Take an appropriate amount of the filtrate and pass it through a 0.22μm microporous filter membrane, and place it in a liquid sample bottle to obtain each test solution. Accurately aspirate 1μL of the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatograph, and perform chromatographic analysis according to the chromatographic conditions of 2.1.3 to analyze ginsenoside R b3 According to the above method, the effects of different extraction methods on its content were calculated to determine the best extraction method. The specific results are shown in Table 5:

[0219] Table 5 Effect of different extraction methods on ginsenoside R in Panax notoginseng flower extract b3 The influence of content

[0220]

[0221] It can be seen from Table 5 that the effects of different extraction methods on the content of ginsenoside Rb3 in Panax notoginseng flower extract are not significantly different. Considering the sample processing efficiency comprehensively, ultrasonic extraction was selected for extraction.

[0222] Ⅲ Investigation of different extraction times

[0223] Take about 0.1g of Panax notoginseng flower extract obtained in Example 1, accurately weigh it, and make 3 portions in total. Each portion has 2 parallel samples, and place them in stoppered cones respectively. Accurately add 20mL of 75v / v% methanol in sequence, weigh the weight, and ultrasonically treat (power 500W, frequency 40kHz) for 15min, 30min, and 60min respectively. After taking out and cooling to room temperature, add 75v / v% methanol to make up the weight, mix well, filter, and collect the filtrate. Take an appropriate amount of filtrate and pass it through a 0.22μm microporous filter membrane, place it in a liquid sample bottle, and obtain each test solution for use. Accurately aspirate 1μl of the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatograph, and perform chromatographic analysis according to the chromatographic conditions of 2.1.3, and take ginsenoside R b3 The influence of different extraction times on its content was calculated according to the above method. The specific results are shown in Table 6:

[0224] Table 6 Effect of different extraction times on ginsenoside R in Panax notoginseng flower extract b3 The influence of content

[0225]

[0226] The results in Table 6 show that different extraction times have an effect on the content of ginsenoside R in Panax notoginseng flower extract. b3 The difference in content was not significant. Considering the time cost and content difference, ultrasonic extraction for 30 minutes was selected.

[0227] The above optimized conditions were used to prepare the Panax notoginseng flower extract test solution and the Panax notoginseng flower decoction piece test solution as follows:

[0228] Take about 0.1 g of Panax notoginseng flower extract, weigh it accurately, place it in a stoppered conical flask, add 20 mL of 75 v / v% methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 min, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, take the filtrate, take an appropriate amount of the filtrate and pass it through a 0.22 μm microporous filter membrane, and place it in a liquid sample bottle to obtain the Panax notoginseng flower extract test solution.

[0229] Preparation of test solution for Panax notoginseng flower slices

[0230] Take about 1.0 g of Panax notoginseng flower slices, weigh accurately, place in a stoppered conical flask, add 25 mL of 75 v / v% methanol, weigh, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, take the filtrate, pass it through a 0.22 μm microporous filter membrane, and place it in a liquid sample bottle to obtain the Panax notoginseng flower slice test solution.

[0231] According to the above optimized method, 15 batches of Panax notoginseng flower extracts prepared from Panax notoginseng flower slices were tested, and the results are shown in Table 7.

[0232] Table 7 Test results of Panax notoginseng flower extracts prepared from different batches of Panax notoginseng flower slices

[0233]

[0234] As shown in Table 7, the mass content of ginsenoside Rb3 in the Panax notoginseng flower extract obtained by the extraction method of the present application is 40-60 mg / g, and the transfer rate of ginsenoside Rb3 in the Panax notoginseng flower extract is 21-34%.

[0235] 2.2 Methodological considerations for detection methods

[0236] 2.2.1 Specificity Investigation

[0237] Take the Panax notoginseng flower extract prepared in Example 1, and prepare the test solution of Panax notoginseng flower extract according to the optimized conditions in 2.1, and accurately pipette the test solution, ginsenoside R in 2.1.1, b3 1 μl of reference solution and 75 v / v% methanol were added and measured according to the chromatographic conditions in 2.1.3 above. The results are shown in Figure 1 .

[0238] Depend on Figure 1 It can be seen that this analytical method is specific for the determination of ginsenoside Rb3 in Panax notoginseng flower extract.

[0239] 2.2.2 Peak purity

[0240] Take the Panax notoginseng flower extract prepared in Example 1, and prepare the test solution of Panax notoginseng flower extract according to the optimized conditions in 2.1. Accurately pipette 1 μl of the above test solution and the ginsenoside Rb3 reference solution in 2.1.1 respectively, and measure according to the above chromatographic conditions in 2.1.3 to obtain the peak purity of the target peak. The results are shown in Table 8.

[0241] Table 8 Matching values ​​of peaks and peak purity

[0242] Target peak <![CDATA[Ginsenoside Rb3]]> Peak purity match value 999.95

[0243] It can be seen from Table 8 that the purity value of this index component is 999.95, which is greater than 960, indicating that its peak purity meets the analysis requirements.

[0244] 2.2.3 Investigation of linear relationship

[0245] Accurately weigh an appropriate amount of ginsenoside Rb3 reference material, place it in a numbered 10 ml volumetric flask, add 75 v / v% methanol to make a reference material solution containing 595.5168 μg of ginsenoside Rb3 per 1 ml, shake well to obtain the reference material stock solution, and place it in the refrigerator for later use.

[0246] Take the ginsenoside Rb3 reference stock solution and dilute it to 1.25, 1.56, 1.95, 2.44, 3.05, 3.81, 7.63, 15.26 times, and obtain ginsenoside Rb3 reference solution of different concentrations. Perform the test on the machine according to the chromatographic conditions of 2.1.3, and use the concentration as the horizontal axis and the peak area value as the vertical axis to examine the linear range of ginsenoside Rb3. The linearity test results are shown in Figure 2 and as shown in Table 9.

[0247] Table 9 Linearity study of ginsenoside Rb3

[0248] serial number <![CDATA[Concentration of Ginsenoside Rb3 (μg / ml)]]> Peak area (mAU) <![CDATA[Ginsenoside Rb3-1]]> 595.5168 629.017 <![CDATA[Ginsenoside Rb3-2]]> 476.4134 498.664 <![CDATA[Ginsenoside Rb3-3]]> 381.1308 386.526 <![CDATA[Ginsenoside Rb3-4]]> 304.9046 312.829 <![CDATA[Ginsenoside Rb3-6]]> 243.9237 248.063

[0249] Depend on Figure 2 As shown in Table 9, the concentration and peak area value of ginsenoside Rb3 in the range of 39.0278μg / ml-595.5168μg / ml showed a good linear relationship, the correlation coefficient r=0.9994, and the linear regression equation was: y=1.0595x-8.1662.

[0250] 2.2.4 Precision investigation

[0251] Take the Panax notoginseng flower extract prepared in Example 1, and prepare a test solution of the Panax notoginseng flower extract according to the optimized conditions in 2.1, and prepare 6 parallel samples. Each sample was injected with 2 injections, and 1 μL was injected respectively. The solution was injected into an ultra-high performance liquid chromatograph, and chromatographic analysis was performed according to the chromatographic conditions of 2.1.3. The content of ginsenoside Rb3 was calculated according to the above method, and the RSD (%) value of the target peak was calculated by the external standard one-point method based on ginsenoside Rb3. The specific results are shown in Table 10.

[0252] Table 10 Precision test results of the method for determining the content of Panax notoginseng flower extract

[0253]

[0254] As shown in Table 10, the RSD (%) value of the target peak ginsenoside Rb3 is 0.81, which is less than 1.5%, indicating that the method has good precision.

[0255] 2.2.5 Stability study

[0256] Take the Panax notoginseng flower extract prepared in Example 1, prepare a test solution of Panax notoginseng flower extract according to the optimized conditions in 2.1, perform chromatographic analysis according to the chromatographic conditions of 2.1.3, inject samples at 0, 2, 4, 6, 8, 12, and 24 hours after the preparation of the test solution, with an injection volume of 1 μl, and calculate the ginsenoside Rb3 content according to the above method. The RSD (%) value of the target peak content was calculated using the external standard one-point method, and the specific results are shown in Table 11.

[0257] Table 11 Stability Study

[0258]

[0259]

[0260] As shown in Table 11, the RSD (%) value of the target peak ginsenoside Rb3 content within 24 hours is 1.39%, which is less than 1.5%, indicating that the solution has good stability within 24 hours.

[0261] 2.2.6 Repeatability study

[0262] Take 6 portions of the Panax notoginseng flower extract prepared in Example 1, and prepare a Panax notoginseng flower extract test solution according to the optimized conditions in 2.1, with an injection volume of 1 μl. Perform chromatographic analysis according to the chromatographic conditions in 2.1.3, calculate the ginsenoside Rb3 content according to the above method, and calculate the RSD (%) value of the target peak content by using the external standard one-point method based on ginsenoside Rb3. The specific results are shown in Table 12.

[0263] Table 12 Repeatability test results

[0264]

[0265] As shown in Table 12, the RSD (%) value of the target peak ginsenoside Rb3 content is 0.67%, which is less than 1.5%, indicating that the method has good repeatability.

[0266] 2.2.7 Intermediate precision study

[0267] Six experimenters were selected to operate on two different dates and two different chromatographs. The Panax notoginseng flower extract prepared in Example 1 was used to prepare a Panax notoginseng flower extract test solution according to the optimized conditions in 2.1. The test solution was taken and chromatographic analysis was performed according to the chromatographic conditions in 2.1.3. The content of ginsenoside Rb3 was calculated according to the above method. The RSD (%) value of the target peak content was calculated based on the ginsenoside Rb3 in the test solution using the external standard one-point method. The specific results are shown in Table 13.

[0268] Table 13 Intermediate precision test results of the determination method

[0269]

[0270]

[0271] It can be seen from Table 13 that the intermediate precision RSD (%) value of the target peak ginsenoside Rb3 content is 1.33%, which is less than 1.5%, indicating that the intermediate precision of this method is good.

[0272] 2.2.8 Accuracy test

[0273] Take about 0.05g of Panax notoginseng flower extract with known content (ginsenoside Rb3 content 59.56mg / g), accurately weigh a total of 6 portions, accurately add 20ml of ginsenoside Rb3 reference solution prepared with 75v / v% methanol (concentration 0.1518555mg / ml) in sequence, weigh the weight, and prepare the test solution according to the test solution preparation method (about 0.1g, accurately weighed, placed in a stoppered conical flask, add 20mL75% methanol, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 30min, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, take the filtrate, take an appropriate amount of the filtrate to pass through a 0.22μm microporous filter membrane, and place it in a liquid sample bottle). Accurately pipette 1 μL of ginsenoside Rb3 reference solution and test solution respectively, inject into ultra-high performance liquid chromatograph, perform chromatographic analysis according to the chromatographic conditions of 2.1.3, calculate the ginsenoside Rb3 content according to the above method, calculate the content of the target peak based on ginsenoside Rb3 using the external standard one-point method, and calculate the recovery rate and RSD (%) value according to the following formula. The results are shown in Table 14.

[0274]

[0275] Table 14 Content determination method sample recovery test results

[0276]

[0277] It can be seen from Table 14 that the recovery rate of ginsenoside Rb3 in Panax notoginseng flower extract is in the range of 95%-102%, and the RSD% (0.92%) is less than 1.5%, indicating that the accuracy of the detection method is good.

[0278] 2.2.9 Durability test

[0279] 2.2.9.1 Investigation of different chromatographic columns

[0280] The effects of three chromatographic columns, namely ZORBAX Eclipse Plus C18 (2.1*150mm, 1.8μm), ACQUITYUPLC HSS T3 (2.1*150mm, 2.7μm), and AmethSep C18AQ (2.1*150mm, 1.8μm), on the peak shape and separation of ginsenoside Rb3 in Panax notoginseng flower extract were compared.

[0281] Take 6 portions of the Panax notoginseng flower extract prepared in Example 1, prepare a Panax notoginseng flower extract test solution according to the optimized conditions in 2.1, take the test solution, and measure it according to the chromatographic conditions in 2.1.3, and record the chromatographic data in terms of ginsenoside Rb3. The experimental results are shown in Table 15.

[0282] Table 15 Effects of different chromatographic columns on the chromatographic peak of ginsenoside Rb3 in Panax notoginseng flower extract

[0283]

[0284]

[0285] From the results in Table 15, it can be seen that the separation effects of the three chromatographic columns are quite different. Only the ACQUITYUPLC HSS T3 (2.1*150mm, 2.7μm) column and the AmethSep C18AQ (2.1*150mm, 1.8μm) column can meet the requirements for the determination of the content of Panax notoginseng saponin Rb3. However, the separation degree, symmetry factor and theoretical plate number of the AmethSep C18AQ (2.1*150mm, 1.8μm) column are lower than those of the ZORBAX Eclipse Plus C18 (2.1*150mm, 1.8μm) and ACQUITYUPLC HSS T3

[0286] (2.1*150mm, 2.7μm) chromatographic column is excellent, so AmethSep C18AQ (2.1*150mm, 1.8μm) was selected as the preferred chromatographic column for this experiment.

[0287] 2.2.9.2 Investigation of different chromatographs

[0288] According to the existing equipment in the laboratory, Thermo scientific and Agilent 1290 Infinity II ultra high performance liquid chromatographs were selected to compare the effects of the two chromatographs on the peak shape and separation of ginsenoside Rb3 in Panax notoginseng flower extract.

[0289] Take 6 portions of the Panax notoginseng flower extract prepared in Example 1, prepare a Panax notoginseng flower extract test solution according to the optimized conditions in 2.1, take the test solution, and measure it according to the chromatographic conditions in 2.1.3, and record the chromatographic data in terms of ginsenoside Rb3. The experimental results are shown in Table 16.

[0290] Table 16 Instrument durability investigation results

[0291] Chromatograph model Indicator ingredients Separation / R Theoretical plates Agilent, 1290InfinityII <![CDATA[Ginsenoside Rb3]]> 4.97 27572 Thermo, scientific <![CDATA[Ginsenoside Rb3]]> 9.98 22767

[0292] It can be seen from Table 16 that the durability of different chromatographs in this analysis method is good. The changes in the chromatograph can meet the system adaptability requirements.

[0293] 2.2.9.3 Investigation of different column temperatures

[0294] Take 6 portions of the Panax notoginseng flower extract prepared in Example 1, prepare a Panax notoginseng flower extract test solution according to the optimized conditions in 2.1, take the test solution, and measure according to the chromatographic conditions in 2.1.3 to compare the effects of different column temperatures of 25°C, 30°C, and 35°C on the peak shape and separation of the chromatographic peak of ginsenoside Rb3 in the Panax notoginseng flower extract. The experimental results are shown in Table 17.

[0295] Table 17 Detection results of different column temperatures for the determination of the content of Panax notoginseng flower extract

[0296]

[0297]

[0298] As shown in Table 17, the peak shape and separation effect of the chromatographic peaks are good under the three column temperatures. The baseline of the chromatogram does not drift at 30°C, and there is no significant difference in peak shape compared with the other two temperatures. Considering the tolerance of the chromatographic column and the time required for analysis, the column temperature is selected as 30°C.

[0299] 2.2.9.4 Investigation of different flow rates

[0300] Take 6 portions of the Panax notoginseng flower extract prepared in Example 1, prepare a Panax notoginseng flower extract test solution according to the optimized conditions in 2.1, take the above test solution, and measure it according to the chromatographic conditions in 2.1.3, wherein the flow rate of the mobile phase is selected as 0.20ml / min, 0.25ml / min, and 0.30ml / min, respectively, and compare the effects of different flow rates of 0.20ml / min, 0.25ml / min, and 0.30ml / min on the peak shape and separation of the chromatographic peak of ginsenoside Rb3 in the Panax notoginseng flower extract. The experimental results are shown in Table 18.

[0301] Table 18 Detection results of different flow rates for the content determination method in Panax notoginseng flower extract

[0302] Flow rate Indicator ingredients Separation / R Theoretical plates 0.20ml / min <![CDATA[Ginsenoside Rb3]]> 5.07 29900 0.25ml / min <![CDATA[Ginsenoside Rb3]]> 4.97 27572 0.30ml / min <![CDATA[Ginsenoside Rb3]]> 5.01 26767

[0303] It can be seen from Table 18 that the chromatographic peak shape and separation effect are good under the three flow rates. When the flow rate is 0.25 ml / min, the parameters of ginsenoside Rb3 are good, the baseline does not drift, and the peak shape difference is not significant, so the flow rate of 0.25 ml / min is selected in this experiment.

[0304] 3. Detection method of characteristic spectrum of Panax notoginseng flower extract

[0305] 3.1 Optimization of detection conditions

[0306] 3.1.1 Preparation of reference solution

[0307] Take an appropriate amount of ginsenoside Rb3 reference substance, weigh it accurately, add methanol to make a reference substance solution containing 150 μg of ginsenoside Rb3 per 1 ml, shake well, and you have it.

[0308] 3.1.2 Preparation of test solution

[0309] Take about 0.1 g of Panax notoginseng flower extract obtained in Example 1, accurately weigh it, place it in a stoppered conical flask, add 20 ml of solvent, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 min, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, take out the filtrate, and get an appropriate amount of the filtrate, pass it through a 0.22 μm microporous filter membrane, and place it in a liquid sample bottle.

[0310] 3.1.3 Ultra-high performance liquid chromatography analysis

[0311] 1 μL of the reference solution in 3.1.1 and the test solution in 3.1.2 were respectively injected into the ultra-high performance liquid chromatograph for analysis. The chromatographic column was filled with octadecylsilane bonded silica gel (AmethSep C18 AQ (2.1*150mm, 1.8μm)); the organic phase was used as mobile phase A and the aqueous phase was used as mobile phase B. Gradient elution was performed according to the provisions in Table 19; the determination was performed at a certain column temperature and flow rate at a certain detection wavelength.

[0312] Table 19 Gradient elution

[0313] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-30 31 69 30-35 31→40 69→60 35-50 40→46 60→54 50-55 46→55 54→45

[0314] 3.1.4 Optimization of detection conditions

[0315] 3.1.4.1 Determination of detection wavelength

[0316] Take about 0.1 g of the Panax notoginseng flower extract prepared in Example 1, accurately weigh it, place it in a stoppered conical flask, add 20 ml of 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 the corresponding solvent, shake it well, filter it, and take the filtrate to obtain an appropriate amount of the filtrate, pass it through a 0.22 μm microporous filter membrane, and place it in a liquid sample bottle to obtain a test solution of Panax notoginseng flower extract, and record the absorption spectrum in the range of 190-400 nm. The spectrum at the retention time of 28.188 min is as follows Figure 3 shown.

[0317] From the experimental results, it can be seen that at a wavelength of 203nm, the Panax notoginseng flower extract test solution can detect more chromatographic peak information and less baseline noise interference, so 203nm is selected as the detection wavelength.

[0318] 3.1.4.2 Optimization of mobile phase

[0319] ① Refer to the method in 3.1.3, the chromatographic column uses octadecylsilane bonded silica gel as the filler (AmethSep C18AQ (2.1*150mm, 1.8μm)); the column temperature is 30℃, the mobile phase flow rate is 0.25ml / min, and the Panax notoginseng flower extract test solution is prepared according to the method in 3.1.4.1. A is the organic phase (methanol or acetonitrile), and B is a 0.1% phosphoric acid aqueous solution. The results are as follows Figure 4 shown.

[0320] Depend on Figure 4 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, so acetonitrile-0.1% phosphoric acid solution was selected for condition exploration, and various acids used in the mobile phase were investigated later.

[0321] ② Referring to the method in 3.1.3, the chromatographic column was filled with octadecylsilane bonded silica gel (AmethSepC18 AQ (2.1*150mm, 1.8μm)), and the Panax notoginseng flower extract test solution was prepared according to the method in 3.1.4.1. Chromatographic analysis was performed using acetonitrile-water and acetonitrile-0.1% formic acid as the mobile phases, respectively. The results are as follows: Figure 5 shown.

[0322] Depend on Figure 5 It can be seen that the peak shape is better when adding acid than when not adding acid. The peak shape is best when the mobile phase is acetonitrile-0.1% phosphoric acid solution system, so acetonitrile-0.1% phosphoric acid solution is selected for elution.

[0323] 3.1.4.3 Investigation of different extraction solvents

[0324] Take about 0.1g of Panax notoginseng flower extract prepared in Example 1, accurately weigh it, and place two parallel samples in stoppered conical bottles, respectively, and accurately add 20ml of 100% methanol, 75v / v% methanol, 50v / v% methanol, 25v / v% methanol, anhydrous ethanol, 75v / v% ethanol, 50v / v% ethanol, 25v / v% ethanol and water, weigh it, and ultrasonically treat it (power 500W, frequency 40kHz) for 30min, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain each test solution. Inject the sample according to the method in 3.1.3, and the chromatographic conditions are: mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and the chromatographic column is AmethSep C18 AQ (2.1*150mm, 1.8μm); the column temperature is 30℃, the flow rate is 0.25ml / min, and the peak area is recorded. The results are as follows Figure 6 As shown, the solvents corresponding to the bar graph of each peak from left to right are 100% methanol, 75v / v% methanol, 50v / v% methanol, 25v / v% methanol, 95v / v% ethanol, 75v / v% ethanol, 50v / v% ethanol, 25v / v% ethanol and water.

[0325] It can be seen from Table 6 that when the extraction solvent is 75 v / v% methanol, the extraction efficiency is relatively high, so 75 v / v% methanol is selected as the extraction solvent.

[0326] The above optimized conditions were used to determine the characteristic spectrum of Panax notoginseng flower extract, and the method was as follows:

[0327] Preparation of test solution

[0328] Take about 0.1 g of Panax notoginseng flower extract, weigh it accurately, put it in a stoppered conical flask, add 20 ml of 75 v / v% methanol, weigh it, treat it ultrasonically (power 500 W, frequency 40 kHz) for 30 min, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain an appropriate amount of the filtrate, pass it through a 0.22 μm microporous filter membrane, and place it in a liquid sample bottle to obtain the Panax notoginseng flower extract test solution.

[0329] 1 μL of the reference solution in 3.1.1 and the above test solution were respectively injected into an ultra-high performance liquid chromatograph for analysis. Octadecylsilane bonded silica gel was used as filler (AmethSep C18 AQ (2.1*150mm, 1.8μm)); acetonitrile was used as mobile phase A and 0.1% phosphoric acid aqueous solution was used as mobile phase B. Gradient elution was performed according to the provisions of Table 19; the column temperature was 30°C and the flow rate was 0.25ml / min.

[0330] The characteristic spectra of Panax notoginseng flower extracts prepared from different batches of medicinal pieces (15 batches) were measured according to the above method and compared with the control medicinal materials. The results are as follows: Figure 7 As shown, S1-S15 correspond to Panax notoginseng flower extracts prepared from different batches of medicinal pieces, and R is the Panax notoginseng flower control medicinal material.

[0331] Depend on Figure 7 It can be seen that there are 6 characteristic peaks in the characteristic spectrum of Panax notoginseng flower extract, and the peak corresponding to the reference is S peak. The relative retention time of each characteristic peak and the S peak is calculated. Among them, the relative retention time of peak 1 is 0.48, the relative retention time of peak 2 is 0.51, the relative retention time of peak 3 is 0.89, the relative retention time of peak 4 (S) is 1.00, the relative retention time of peak 5 is 1.40, and the relative retention time of peak 6 is 1.47.

[0332] 3.2 Methodological considerations for detection methods

[0333] 3.2.1 Specificity inspection

[0334] Take the Panax notoginseng flower extract prepared in Example 1 and prepare a Panax notoginseng flower extract test solution according to the optimized conditions in 3.1. Take 1 μl of the test solution and 75 v / v% methanol solvent respectively, inject them into the ultra-high performance liquid chromatograph, and measure them according to the chromatographic conditions after the optimized conditions in 3.1. The results are as follows Figure 8 shown.

[0335] Depend on Figure 8 It can be seen that the solvent has no interference on the characteristic peaks in the spectrum of Panax notoginseng flower extract.

[0336] 3.2.2 Overall investigation

[0337] Take the Panax notoginseng flower extract prepared in Example 1, and prepare a Panax notoginseng flower extract test solution according to the conditions optimized in 3.1. Take 1 μl of the test solution, and measure it according to the chromatographic conditions after the optimized conditions in 3.1. Extend the elution time by one time at the mobile phase ratio at the gradient end point. The characteristic spectrum is as follows: Fig. 9 shown.

[0338] Depend on Fig. 9 It can be seen that there is no obvious chromatographic peak after doubling the elution time under this chromatographic condition, indicating that this chromatographic condition basically meets the principle of maximum information content.

[0339] 3.2.3 Precision investigation

[0340] Take the Panax notoginseng flower extract prepared in Example 1, prepare a Panax notoginseng flower extract test solution according to the optimized conditions in 3.1, take the test solution, and measure it according to the chromatographic conditions after the optimized conditions in 3.1, repeat the injection 6 times, and the injection volume is 1ul. Investigate the consistency of the relative retention time and relative peak area of ​​the characteristic peaks (calibrate 6 characteristic peaks, with peak 4 as the reference peak), and the experimental results are shown in Table 20.

[0341] Table 20 Precision results of characteristic spectrum of Panax notoginseng flower extract (relative retention time)

[0342] sample Peak 1 Peak 2 Peak 3 Peak 4(S) Peak 5 Peak 6 1 0.478 0.508 0.890 1.000 1.396 1.469 2 0.480 0.509 0.890 1.000 1.391 1.464 3 0.479 0.509 0.889 1.000 1.398 1.471 4 0.479 0.509 0.888 1.000 1.394 1.467 5 0.480 0.510 0.890 1.000 1.393 1.465 6 0.477 0.507 0.888 1.000 1.390 1.463 average value 0.48 0.51 0.89 1.00 1.39 1.47 RSD% 0.22 0.22 0.09 0.00 0.21 0.21

[0343] As can be seen from Table 20, the RSD of the relative retention time of each chromatographic peak is <3.0%, indicating that the instrument has good precision.

[0344] 3.2.4 Stability study

[0345] Take the Panax notoginseng flower extract prepared in Example 1, and prepare a test solution of Panax notoginseng flower extract according to the conditions optimized in 3.1. The test was performed according to the chromatographic conditions after the optimized conditions in 3.1. The sample was injected at 0, 2, 4, 6, 8, 12, and 24 hours after the preparation of the test solution, with an injection volume of 1 μl. The consistency of the relative retention time and relative peak area of ​​the characteristic peaks was examined (6 characteristic peaks were calibrated, with peak 4 as the reference peak). The experimental results are shown in Table 21.

[0346] Table 21 Stability results of characteristic spectrum of Panax notoginseng flower extract (relative retention time)

[0347]

[0348]

[0349] As can be seen from Table 21, the RSD of the relative retention time of the chromatographic peak is <3.0%, indicating that the sample solution is relatively stable.

[0350] 3.2.5 Repeatability study

[0351] A total of 6 portions of Panax notoginseng flower extract prepared in Example 1 were taken, and the test solution was prepared according to the conditions optimized in 3.1. The chromatographic conditions after the optimized conditions in 3.1 were measured, with an injection volume of 1 μl. The consistency of the relative retention time and relative peak area of ​​the characteristic peaks was examined (6 characteristic peaks were calibrated, with peak 4 as the reference peak). The experimental results are shown in Table 22.

[0352] Table 22 Repeatability results of characteristic spectrum of Panax notoginseng flower extract (relative retention time)

[0353] sample Peak 1 Peak 2 Peak 3 Peak 4(S) Peak 5 Peak 6 1 0.479 0.509 0.889 1.000 1.401 1.474 2 0.479 0.509 0.889 1.000 1.402 1.475 3 0.479 0.509 0.889 1.000 1.400 1.473 4 0.479 0.509 0.889 1.000 1.401 1.474 5 0.478 0.508 0.889 1.000 1.403 1.476 6 0.478 0.508 0.889 1.000 1.403 1.476 average value 0.48 0.51 0.89 1.00 1.40 1.47 RSD% 0.06 0.05 0.02 0.00 0.09 0.09

[0354] As can be seen from Table 22, the RSD of the relative retention time of each chromatographic peak is <3.0%, indicating that the method has good repeatability.

[0355] 3.2.6 Durability test

[0356] 3.2.6.1 Investigation of different chromatographic columns

[0357] Three chromatographic columns were investigated, namely: chromatographic column AmethSep C18AQ (2.1*150mm, 1.8μm); chromatographic column ZORBAX Eclipse Plus C18 (2.1*150mm, 1.8μm); chromatographic column ACQUITYUPLC HSS T3 (2.1*150mm, 2.7μm), column temperature 30°C, mobile phase flow rate 0.25ml / min, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, Panax notoginseng flower extract test solution is prepared according to the method in 3.1.4.1, and the effects of the three chromatographic columns on the peak of the characteristic spectrum of Panax notoginseng flower extract are investigated. The results are shown in Table 23.

[0358] Table 23 Results of chromatographic column investigation of characteristic spectrum of Panax notoginseng flower extract (relative retention time)

[0359] Chromatographic columns Peak 1 Peak 2 Peak 3 Peak 4(S) Peak 5 Peak 6 AmethSepC18AQ 0.478 0.508 0.889 1.000 1.395 1.468 ZORBAX Eclipse Plus C18 0.497 0.591 0.908 1.000 3.106 3.523 ACQUITY UPLC HSST3 0.490 0.520 0.894 1.000 1.475 1.650

[0360] It can be seen from Table 23 that when the ultra-high performance liquid chromatography column AmethSep C18AQ (2.1*150mm, 1.8μm) is used for elution, the chromatographic peak shape is better and the separation effect is the best. Therefore, it is recommended that this method use an ultra-high performance liquid chromatography column with model AmethSep C18AQ (2.1*150mm, 1.8μm).

[0361] 3.2.6.2 Investigation of different column temperatures

[0362] The test solution of Panax notoginseng flower extract was prepared according to the method in 3.1.4.1. The mobile phase A was acetonitrile, the mobile phase B was 0.1% phosphoric acid solution, and the chromatographic column was AmethSep C18AQ (2.1*150mm, 1.8μm); the mobile phase flow rate was 0.25ml / min, and the separation effects of different column temperatures of 25℃, 30℃, and 35℃ on the samples were compared. The results are shown in Table 24.

[0363] Table 24 Column temperature investigation results of characteristic spectrum of Panax notoginseng flower extract (relative retention time)

[0364] Column temperature Peak 1 Peak 2 Peak 3 Peak 4(S) Peak 5 Peak 6 25℃ 0.470 0.501 0.884 1.000 1.380 1.451 30℃ 0.478 0.508 0.889 1.000 1.395 1.468 35℃ 0.481 0.510 0.892 1.000 1.401 1.475

[0365] As can be seen from Table 24, the column temperature has little effect on the peak elution, but the separation effect is best and the peak shape is better when the column temperature is 30°C, so the column temperature of 30°C is selected.

[0366] 3.2.6.3 Investigation of different flow rates

[0367] The test solution of Panax notoginseng flower extract was prepared according to the method in 3.1.4.1, mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid solution, and the chromatographic column was AmethSep C18AQ (2.1*150mm, 1.8μm); the column temperature was 30°C, and the elution conditions at different flow rates (0.2ml / min, 0.25ml / min, 0.3ml / min) were investigated. The results are shown in Table 25.

[0368] Table 25 Flow rate investigation results of characteristic spectrum of Panax notoginseng flower extract (relative retention time)

[0369]

[0370] As shown in Table 25, the flow rate has little effect on the peak appearance, but the peak shape is best when the flow rate is 0.25 ml / min. Therefore, 0.25 ml / min is selected.

[0371] Embodiment 2

[0372] 1. Ginsenoside R in Panax notoginseng flower b3 Determination method of mass content

[0373] 1.1 Preparation of reference solution

[0374] Take an appropriate amount of ginsenoside Rb3 reference substance, weigh it accurately, put it into a numbered 10ml volumetric flask, add 75% methanol to make a reference solution containing 150μg of ginsenoside Rb3 per 1ml, shake well, and obtain.

[0375] 1.2 Preparation of test solution

[0376] Take about 0.1g of Panax notoginseng flower medicinal material, weigh it accurately, place it in a stoppered conical flask, add 20mL 75v / v% methanol, weigh it, heat and reflux for 2h, cool, weigh it again, make up the lost weight with 75v / v% methanol, shake well, filter, take the filtrate, take an appropriate amount of the filtrate and pass it through a 0.22μm microporous filter membrane, and place it in a liquid sample bottle to obtain the Panax notoginseng flower medicinal material test solution.

[0377] 1.3 High performance liquid chromatography analysis

[0378] The chromatographic column was filled with octadecylsilane bonded silica gel (AmethSep C18 AQ (2.1*150mm, 1.8μm)); acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 26; the column temperature was 30°C; the flow rate was 0.25ml / min, and the detection wavelength was 203nm. The theoretical plate number calculated based on the ginsenoside Rb3 peak should not be less than 3000. 1μL of the reference solution in 1.1 and the test solution in 1.2 were respectively injected into the high performance liquid chromatograph for analysis.

[0379] Table 26 Elution gradient

[0380]

[0381]

[0382] 1.4 The mass content of ginsenoside Rb3 is calculated according to the following formula:

[0383] in,

[0384] w 药材 =A 药供 ×C 对 ×V 药供 ×10 -6 / (m 药供 ×A 对 )*100%

[0385] Where:

[0386] w 药材 ——The mass content of ginsenoside Rb3 in Panax notoginseng flower medicinal material;

[0387] A 对 ——The corresponding absorption peak area of ​​ginsenoside Rb3 in the HPLC spectrum of the reference solution;

[0388] A 药供 —The corresponding absorption peak area of ​​ginsenoside Rb3 in the HPLC spectrum of the medicinal material test solution;

[0389] C 对 ——Concentration of reference substance in reference solution, μg / mL;

[0390] m 药供 ——the absolute dry mass of Panax notoginseng flower slices in the medicinal material test solution, g;

[0391] V 药供 ——Volume of the test solution of medicinal material, ml;

[0392] According to the above method, ginsenoside R in 15 batches of Panax notoginseng flower medicinal materials was determined. b3Content, as shown in Table 27,

[0393] Table 27 Test results of ginsenoside Rb3 content in Panax notoginseng flower medicinal materials

[0394]

[0395]

[0396] 2. Construction of characteristic spectrum of Panax notoginseng flower medicinal material

[0397] 2.1 Detection method of characteristic spectrum of Panax notoginseng flower medicinal material

[0398] 2.1.1 Preparation of reference solution

[0399] Take an appropriate amount of ginsenoside Rb3 reference substance, weigh it accurately, add 75v / v% methanol to make a reference substance solution containing 150μg of ginsenoside Rb3 per 1ml, shake well, and obtain.

[0400] Notoginseng flower reference medicinal material reference solution: take about 0.1g of Notoginseng flower reference medicinal material, weigh accurately, place in a stoppered conical flask, add 20ml 75v / v% methanol, weigh, heat and reflux for 2h, cool, weigh again, make up the lost weight with 75v / v% methanol, shake well, filter, take out the filtrate, and obtain, take an appropriate amount of the filtrate and pass it through a 0.22μm microporous filter membrane, and place it in a liquid sample bottle.

[0401] 2.1.2 Preparation of test solution

[0402] Take about 0.1g of Panax notoginseng flower medicinal material, weigh it accurately, put it in a stoppered conical flask, add 20ml 75v / v% methanol, weigh it, heat and reflux for 2h, cool, weigh it again, make up the lost weight with 75v / v% methanol, shake well, filter, take out the filtrate, and get it. Take an appropriate amount of the filtrate and pass it through a 0.22μm microporous filter membrane and place it in a liquid sample bottle.

[0403] 2.1.3 Ultra-high performance liquid chromatography analysis

[0404] 1 μL of the reference solution in 2.1.1 and the test solution in 2.1.2 were respectively injected into the ultra-high performance liquid chromatograph for analysis. The chromatographic column used octadecylsilane bonded silica gel as filler (AmethSep C18AQ (2.1*150mm, 1.8μm)); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B. Gradient elution was performed according to the provisions in Table 28. The theoretical plate number should be not less than 3000 calculated based on the ginsenoside Rb3 peak. The detection wavelength was 203nm, the column temperature was 30℃, and the flow rate was 0.25ml / min.

[0405] Table 28 Gradient elution

[0406] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-30 31 69 30-35 31→40 69→60 35-50 40→46 60→54 50-55 46→55 54→45

[0407] 2.2 Establishment of feature map

[0408] Take 15 batches of Panax notoginseng flower medicinal material samples, 0.1g each, and measure them according to the method in 2.1. The measurement results are as follows: Fig.10 As shown in Table 29.

[0409] Table 29 Sample measurement results (relative retention time)

[0410] Serial number batch number Peak 1 Peak 2 Peak 3 Peak 4(S) Peak 5 Peak 6 1 P-SQH-231210-QGY-YC01 0.472 0.504 0.881 1.000 1.338 1.408 2 P-SQH-231210-QGY-YC02 0.469 0.501 0.879 1.000 1.340 1.410 3 P-SQH-231210-QGY-YC03 0.472 0.504 0.881 1.000 1.345 1.415 4 P-SQH-231210-QGY-YC04 0.471 0.503 0.880 1.000 1.345 1.415 5 P-SQH-231210-QAS-YC05 0.471 0.503 0.880 1.000 1.346 1.417 6 P-SQH-231210-QGY-YC06 0.472 0.504 0.882 1.000 1.347 1.418 7 P-SQH-231210-QGY-YC07 0.472 0.504 0.882 1.000 1.347 1.417 8 P-SQH-231210-QGY-YC08 0.471 0.504 0.880 1.000 1.342 1.412 9 P-SQH-231210-QGY-YC09 0.472 0.504 0.881 1.000 1.351 1.422 10 P-SQH-231210-QGY-YC10 0.472 0.505 0.881 1.000 1.347 1.418 11 P-SQH-231210-QZY-YC11 0.471 0.504 0.882 1.000 1.347 1.417 12 P-SQH-231210-QZY-YC12 0.470 0.504 0.880 1.000 1.334 1.404 13 P-SQH-231210-QGY-YC13 0.473 0.505 0.882 1.000 1.343 1.413 14 P-SQH-231210-QGY-YC14 0.471 0.504 0.883 1.000 1.342 1.412 15 P-SQH-231210-QGY-YC15 0.472 0.504 0.882 1.000 1.351 1.421 Mean 0.471 0.504 0.881 1.000 1.344 1.415 RSD 0.18 0.17 0.10 0.00 0.35 0.35

[0411] The experimental results show that the retention time ratios of characteristic peaks in 15 batches of samples are relatively stable, and the RSDs are all less than 3.0%.

[0412] In summary, the UPLC chromatograms of different batches of Panax notoginseng flower medicinal materials and Panax notoginseng flower control medicinal materials were determined, and the results are as follows Fig.10 As shown, the “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)” recommended by the State Pharmacopoeia Committee was used to analyze the results, select the common peaks, and determine peak 4 as the reference peak (i.e., S peak).

[0413] Embodiment 3

[0414] 1. Preparation method of Panax notoginseng flower formula granules

[0415] (1) 100 kg of Panax notoginseng flower slices were taken and placed in a multifunctional extraction and concentration unit, and water was added and decocted twice. For the first decoction, the slices were directly added to water 10 times the weight of the Panax notoginseng flower slices, soaked for 30 minutes, heated to boiling and kept slightly boiling for 60 minutes, and the decoction was filtered through a 200-mesh sieve while hot; for the second decoction, water 8 times the weight of the Panax notoginseng flower slices was added, heated to boiling and kept slightly boiling for 40 minutes, and the decoction was filtered through a 200-mesh sieve while hot, and the two filtrates were combined;

[0416] (2) The filtrate was transferred to a concentrator and concentrated under reduced pressure and low temperature (temperature: 65°C; vacuum degree: -0.080 to -0.090 MPa) to a density of 1.05 g / cm 3 (45°C) to obtain a clear paste, the paste yield was measured to be 35.72%;

[0417] (3) Add the above clear paste into a batching tank, heat it to 60°C, add 3kg of maltodextrin and mix evenly, then spray dry, control the air inlet temperature to 165-195°C, and the air outlet temperature to 100-110°C, collect the dry paste powder and set it aside. There is no wall sticking phenomenon or caking phenomenon during the drying process, and it is easy to dry;

[0418] (4) Weigh 500 g of the dry paste powder obtained in step (3), crush it and pass it through an 80-mesh sieve, add 0.5 g of silicon dioxide and mix well, and dry granulate it. Adjust the feed speed to 75 r / min, the roller speed to 80 r / min, the hydraulic pressure to 6.5 MPa, the roller speed to 7.5 r / min, the crushing speed to 80 r / min, the final granulation speed to 80 r / min, the roller spacing to 0.75 mm, the mixing time to 30 min, and the mixer speed to 10 r / min to obtain Panax notoginseng flower formula granules.

[0419] Take Panax notoginseng flower slices 13 (batch number P-SQH-231210-QGY-YP13), Panax notoginseng flower slices 14 (batch number P-SQH-231210-QGY-YP14) and Panax notoginseng flower slices 15 (batch number

[0420] P-SQH-231210-QGY-YP15), and prepared Panax notoginseng flower formula granules-1, Panax notoginseng flower formula granules-2 and Panax notoginseng flower formula granules-3 according to the above method.

[0421] 2. Detection method of mass content and transfer rate of ginsenoside Rb3 in Panax notoginseng flower granules

[0422] 2.1 Preparation of reference solution

[0423] Take an appropriate amount of ginsenoside Rb3 reference substance, weigh it accurately, place it in a numbered 10ml volumetric flask, add 75% methanol to make a 10ml ginsenoside Rb3 reference substance. b3 150μg reference substance solution, shake well to obtain.

[0424] 2.2 Preparation of test solution

[0425] Take about 0.1g of Panax notoginseng flower granules, weigh accurately, place in a stoppered conical flask, add 20mL 75v / v% methanol, weigh, ultrasonically treat (power 500W, frequency 40kHz) for 30min, let cool, weigh again, make up the lost weight with 75v / v% methanol, shake well, filter, take out the filtrate, pass it through a 0.22μm microporous filter membrane, and place it in a liquid sample bottle to obtain the test solution.

[0426] 2.3 HPLC analysis

[0427] The chromatographic column was filled with octadecylsilane bonded silica gel (AmethSep C18 AQ (2.1*150mm, 1.8μm)); acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 3; the column temperature was 30°C; the flow rate was 0.25ml / min, and the detection wavelength was 203nm. The number of theoretical plates calculated based on the ginsenoside Rb3 peak should not be less than 3000. 1μL of the reference solution in 2.1 and the test solution in 2.2 were respectively injected into the high performance liquid chromatograph for analysis.

[0428] The mass content of ginsenoside Rb3 was obtained according to the calculation method of 2.1.4 in Example 1. The results are shown in Table 30. Table 30 Mass content of ginsenoside Rb3 in Panax notoginseng flower formula granules

[0429]

[0430] 3. Detection method of characteristic spectrum of Panax notoginseng flower formula granules

[0431] 3.1 Preparation of reference solution

[0432] Take an appropriate amount of ginsenoside Rb3 reference substance, weigh it accurately, and add methanol to make a ginsenoside Rb3 reference substance containing 1 ml of ginsenoside Rb3. b3 150μg of reference solution, shake well to obtain.

[0433] 3.2 Preparation of test solution

[0434] Take about 0.1g of Panax notoginseng flower granules, weigh accurately, put in a stoppered conical flask, accurately add 20ml of 75% v / v methanol, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 75v / v% methanol, shake well, filter, and take out the filtrate.

[0435] 3.3 HPLC analysis

[0436] The chromatographic column was filled with octadecylsilane bonded silica gel (AmethSep C18 AQ (2.1*150mm, 1.8μm)); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 18; the flow rate was 0.25ml / min; the column temperature was 30℃; and the detection wavelength was 203nm. 1μL of the reference solution in 3.1 and the test solution in 3.2 were respectively injected into the ultra-high performance liquid chromatograph for analysis.

[0437] The characteristic spectrum of Panax notoginseng flower formula granules measured by the above method is as follows Fig.11 As shown in Table 31,

[0438] Table 31 Characteristic spectrum test results of Panax notoginseng flower formula granules

[0439] Serial number batch number Peak 1 Peak 2 Peak 3 Peak 4(S) Peak 5 Peak 6 S1 Panax notoginseng flower formula granules-1 0.482 0.512 0.891 1.000 1.439 1.514 S2 Panax notoginseng flower formula granules-2 0.484 0.513 0.892 1.000 1.429 1.503 S3 Panax notoginseng flower formula granules-3 0.483 0.513 0.892 1.000 1.432 1.506 Mean 0.48 0.51 0.89 1.00 1.43 1.51 RSD 0.21 0.19 0.05 0.00 0.36 0.37

[0440] The experimental results show that the retention time ratios of the characteristic peaks in the three batches of samples are relatively stable, and the RSDs are all less than 3.0%.

[0441] In summary, the UPLC spectra of different batches of Panax notoginseng flower granules and control medicinal materials (R) were determined, and the results were as follows: Fig.11 As shown, the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the National Pharmacopoeia Committee was used for result analysis, and the common peaks were selected, and then the peak 4 with a relatively stable relative time was used as the reference peak (i.e., S peak), and the common peaks with relatively stable relative retention times were selected as characteristic peaks, namely, the relative retention time of peak 1 was 0.48, the relative retention time of peak 2 was 0.51, the relative retention time of peak 3 was 0.89, the relative retention time of peak 4 (S) was 1.00, the relative retention time of peak 5 was 1.43, and the relative retention time of peak 6 was 1.51.

[0442] Embodiment 4

[0443] 1. Thin layer chromatography identification method of Panax notoginseng flower granules

[0444] 1.1 Preparation of test solution

[0445] Weigh 0.5 g of Panax notoginseng flower formula granules-1, Panax notoginseng flower formula granules-2 and Panax notoginseng flower formula granules-3 prepared in Example 3 respectively, a total of 3 parts, grind them into powder, add 10 drops of water, stir well, then add 10 ml of water-saturated n-butanol, seal, shake for 10 minutes, let stand for 2 hours, centrifuge, take the supernatant, add 3 times the amount of water saturated with n-butanol, shake well, let stand to separate, take the upper solution, evaporate to dryness, add 2 ml of methanol to dissolve the residue, and use it as the test solution.

[0446] 1.2 Panax notoginseng flower medicinal material reference solution

[0447] Take 1g of Panax notoginseng flower control medicinal material (batch number 660046-202101), add 10mL of water, heat and reflux for 30min, filter, evaporate the filtrate, add 10 drops of water to the residue, stir well, add 10ml of water-saturated n-butanol, seal, shake for 10 minutes, let stand for 2 hours, centrifuge, take the supernatant, add 3 times the amount of water saturated with n-butanol, shake well, let stand to separate, take the upper solution, evaporate to dryness, add 2ml of methanol to the residue to dissolve it, as the control medicinal material solution.

[0448] 1.3 Panax notoginseng flower formula granules negative sample solution

[0449] Weigh 0.5g of maltodextrin respectively, grind it into powder, add 10 drops of water, stir well, then add 10ml of water-saturated n-butanol, seal it, shake it for 10 minutes, let it stand for 2 hours, centrifuge it, take the supernatant, add 3 times the amount of water saturated with n-butanol, shake well, let it stand to separate into layers, take the upper layer solution, evaporate it to dryness, add 2ml of methanol to dissolve the residue as the negative sample solution.

[0450] 1.4 Thin layer chromatography conditions

[0451] The thin layer plate is a silica gel G thin layer plate;

[0452] The developing solvent was chloroform-methanol-water (volume ratio 7:3:0.5);

[0453] Spotting method: spray strip spotting;

[0454] Deployment method: using double-slot deployment cylinder, deployment distance is 8cm, deployment twice;

[0455] Inspection: Spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm).

[0456] 2. Investigation of the amount of sample

[0457] Take 1.1 test solution of Panax notoginseng flower formula granules-1 and 1.2 Panax notoginseng flower medicinal material reference solution, examine the spot amount (1, 2, 4 μL) in the identification method of Panax notoginseng flower formula granules, develop according to the above thin layer chromatography conditions, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365nm). The thin layer chromatogram is as follows Fig.12 shown.

[0458] Depend on Fig.12 It can be seen that the thin layer chromatograms from left to right are: No. 1 is the sample volume of the test solution of Panax notoginseng flower formula granules, which is 1μl, No. 2 is the sample volume of the test solution of Panax notoginseng flower formula granules, which is 2μl, No. 3 is the sample volume of the test solution of Panax notoginseng flower formula granules, which is 4μl, No. 4 is the sample volume of the test solution of Panax notoginseng flower medicinal material reference solution, which is 1μl, No. 5 is the sample volume of the test solution of Panax notoginseng flower medicinal material reference solution, which is 2μl, and No. 6 is the sample volume of the test solution of Panax notoginseng flower medicinal material reference solution, which is 4μl. When the sample volume is 2-4μl, the main spots of the test sample chromatogram and the spots of the reference medicinal material chromatogram are clearly colored, the separation is good, there is no tailing phenomenon, and there is no background interference. In the test sample chromatogram, at the corresponding position of the reference medicinal material chromatogram, the fluorescent spots of the same color are displayed, so the sample volume is selected to be 2μl.

[0459] 3. Investigation on the specificity of the thin layer chromatography identification method of Panax notoginseng flower granules

[0460] 2 μl of each of the test solution of 1.1 Panax notoginseng flower formula granules, the reference solution of 1.2 Panax notoginseng flower medicinal material, and the negative sample of 1.3 Panax notoginseng flower formula granules were respectively spotted on the same silica gel G thin layer plate (Yantai Yinlong silica gel prefabricated silica gel G plate), and developed according to the above-mentioned thin layer chromatography conditions (wherein the temperature is 25°C and the humidity is 66%), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly colored, and examined under ultraviolet light (365nm). The thin layer chromatogram is as follows: Fig.13 shown.

[0461] As shown in Figure 13, the TLCs from left to right are: No. 1 is a negative test solution, No. 2 is a test solution of Panax Notoginseng Flower Formula Granule-1, No. 3 is a test solution of Panax Notoginseng Flower Formula Granule-2, No. 4 is a test solution of Panax Notoginseng Flower Formula Granule-3, and No. 5 is a test solution of Panax Notoginseng Flower Medicinal Material Reference Solution. The Panax Notoginseng Flower Formula Granule test sample chromatogram shows fluorescent spots of the same color at the corresponding position of the reference medicinal material chromatogram, and there is no interference from the negative sample. This shows that the TLC method has good specificity.

[0462] 4. Study on the durability of the thin layer chromatography identification method of Panax notoginseng flower formula granules

[0463] 4.1 Investigation at different temperatures

[0464] 2 μl of each of the test solution of Panax notoginseng flower formula granules, the reference solution of Panax notoginseng flower medicinal material and the negative sample of Panax notoginseng flower formula granules were respectively spotted on the same silica gel G thin layer plate (Yantai Yinlong silica gel prefabricated silica gel G plate), and developed under the above-mentioned thin layer chromatography conditions (humidity 66%) at different temperatures (5°C, 25°C), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The thin layer chromatograms at 5°C and 25°C were shown in Figure 2. Figure 14 to Figure 15 .

[0465] Depend on Figure 14 to Figure 15 It can be seen that the TLCs from left to right are: No. 1 is a negative test solution, No. 2 is a test solution of Panax Notoginseng Flower Formula Granule-1, No. 3 is a test solution of Panax Notoginseng Flower Formula Granule-2, No. 4 is a test solution of Panax Notoginseng Flower Formula Granule-3, and No. 5 is a test solution of Panax Notoginseng Flower Medicinal Material Reference Solution. Under different temperature conditions, the corresponding positions of the Panax Notoginseng Flower Formula Granule test sample chromatogram and the reference medicinal material chromatogram show the same color of fluorescent main spots, with clear color development, good separation, no tailing phenomenon, and no background interference. With the increase of temperature, the corresponding spot formation position has no obvious change, indicating that the decrease in temperature has no obvious effect on the thin layer identification of Panax Notoginseng Flower Formula Granule, indicating that the thin layer identification method has good durability at room temperature and low temperature.

[0466] 4.2 Investigation of different humidity

[0467] 2 μl of each of the Panax notoginseng flower formula granule test solution, Panax notoginseng flower medicinal material reference solution and Panax notoginseng flower formula granule negative sample were respectively spotted on the same silica gel G thin layer plate (Yantai Yinlong silica gel prefabricated silica gel G plate), and developed under different humidity conditions (RH: 33%, RH: 66%, RH: 88%) according to the above-mentioned thin layer chromatography conditions (temperature 25°C), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The thin layer chromatograms of RH 33%, RH 66% and RH 88%, respectively. Figures 16 to 18 .

[0468] Depend on Figures 16 to 18 It can be seen that the thin layer chromatograms from left to right are: No. 1 is the negative test solution, No. 2 is the Panax notoginseng flower formula granules-1 test solution, No. 3 is the Panax notoginseng flower formula granules-2 test solution, No. 4 is the Panax notoginseng flower formula granules-3 test solution, and No. 5 is the Panax notoginseng flower medicinal material reference solution test solution. Under different humidity conditions, the Panax notoginseng flower formula granules test sample chromatogram and the reference medicinal material chromatogram show fluorescent spots of the same color at the corresponding positions, and the main spots are clearly colored, with good separation and no tailing phenomenon, indicating that changes in humidity have no obvious effect on the thin layer identification of Panax notoginseng flower formula granules, indicating that the thin layer identification method has good durability to humidity.

[0469] 4.3 Investigation of thin-layer boards from different manufacturers

[0470] 2 μl of each of the Panax notoginseng flower formula granule test solution, Panax notoginseng flower medicinal material reference solution and Panax notoginseng flower formula granule negative sample were respectively spotted on thin layer plates from different manufacturers (Qingdao Ocean Silica G Plate, Yantai Yinlong Silica Gel Prefabricated Silica G Plate, German Merck Plate), and developed under the same temperature and humidity conditions according to the above-mentioned thin layer chromatography conditions (temperature 25°C, humidity 66%), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The thin layer chromatograms of Qingdao Ocean Silica G Plate, Yantai Yinlong Silica Gel Prefabricated Silica G Plate and German Merck Plate were Figures 19 to 21 .

[0471] Depend on Figures 19 to 21It can be seen that the thin layer chromatograms from left to right are: No. 1 is the negative test solution, No. 2 is the Panax notoginseng flower formula granules-1 test solution, No. 3 is the Panax notoginseng flower formula granules-2 test solution, No. 4 is the Panax notoginseng flower formula granules-3 test solution, and No. 5 is the Panax notoginseng flower medicinal material reference solution test solution. Silica gel thin layer plates from different manufacturers (Qingdao Ocean Silica G plate, Yantai Yinlong Silica Gel prefabricated silica gel G plate, German Merck plate) were used. The main spots of the Panax notoginseng flower formula granules test sample chromatogram and the reference sample thin layer chromatogram can correspond to each other without significant influence, indicating that the thin layer chromatography identification method has good durability for thin layer silica gel G plates from different manufacturers.

[0472] 5. Thin layer chromatography identification of Panax notoginseng flower granules

[0473] 2 μl of Panax notoginseng flower formula granule-1 test solution, Panax notoginseng flower formula granule-2 test solution, Panax notoginseng flower formula granule-3 test solution, Panax notoginseng flower medicinal material reference solution and Panax notoginseng flower formula granule negative sample were respectively spotted on the same silica gel G thin layer plate (Yantai Yinlong silica gel prefabricated silica gel G plate), and developed according to the above-mentioned thin layer chromatography conditions (temperature 25°C, humidity 66%), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The thin layer chromatogram was as shown Fig. 22 shown.

[0474] Depend on Fig. 22 It can be seen that the thin layer chromatograms from left to right are: No. 1 is the negative test solution, No. 2 is the Panax notoginseng flower formula granules-1 test solution, No. 3 is the Panax notoginseng flower formula granules-2 test solution, No. 4 is the Panax notoginseng flower formula granules-3 test solution, No. 5 is the Panax notoginseng flower medicinal material reference solution test solution, and the test sample chromatograms in the three batches of Panax notoginseng flower formula granules show spots of the same color at the corresponding positions of the Panax notoginseng flower medicinal material reference solution chromatograms, indicating that the thin layer identifications of the three batches of Panax notoginseng flower formula granules all meet the requirements.

[0475] In summary, judging from the spots in the chromatogram, the separation effect of the fluorescent spots of the Panax notoginseng flower formula granules is good, and the corresponding positions of the chromatogram of the Panax notoginseng flower formula granules and the chromatogram of the Panax notoginseng flower control medicinal material show the same color of the main fluorescent spots, and they can correspond one to one. This method can effectively qualitatively identify the Panax notoginseng flower formula granules that have lost their medicinal properties. Through the investigation of thin layer chromatography methodology, this method has good specificity and durability, and is suitable for thin layer chromatography identification of Panax notoginseng flower formula granules.

[0476] Embodiment 5

[0477] Thin layer chromatography identification method of Panax notoginseng flower medicinal material

[0478] Take 1.0g of Panax notoginseng flower powder, add 10ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate, add 10 drops of water to the residue, stir well, add 10ml of water-saturated n-butanol, plug, shake for 10 minutes, let stand for 2 hours, centrifuge, take the supernatant, add 3 times the amount of water saturated with n-butanol, shake well, let stand to separate, take the n-butanol layer, evaporate to dryness, add 2ml of methanol to the residue to dissolve it, and use it as the test solution.

[0479] Take another reference medicinal material of Panax notoginseng flower and prepare a reference medicinal material solution according to the above method.

[0480] 2 μl of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate (Yantai Yinlong silica gel prefabricated silica gel G plate), and developed with chloroform-methanol-water (7:3:0.5) as the developing agent (temperature 25°C, humidity 66%). Take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365nm).

[0481] Fifteen batches of different notoginseng flower medicinal materials (batch numbers P-SQH-231210-QGY-YC01 to P-SQH-231210-QGY-YC15) were chromatographically identified according to the above method. The results are as follows: Fig.23 As shown,

[0482] Depend on Fig.23 It can be seen that, among them, serial numbers 1-15 correspond to the medicinal material test solutions of batch numbers P-SQH-231210-QGY-YC01 to P-SQH-231210-QGY-YC15, respectively, and R is the control medicinal material test solution. In the chromatogram, at the corresponding position of the control medicinal material chromatogram, medicinal materials of different batches show fluorescent spots of the same color.

[0483] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a Panax notoginseng flower extract, characterized in that: The method comprises decocting the Panax notoginseng flower pieces twice in water and filtering to obtain a decoction liquid, and concentrating and drying the decoction liquid to obtain a Panax notoginseng flower extract.

2. The preparation method according to claim 1, characterized in that: The preparation method comprises the following steps: (1) taking Panax notoginseng flower slices, adding water to decoct, and filtering to obtain a decoction; wherein, the first decoction is made with 10 to 14 times the weight of the Panax notoginseng flower slices of water, and the second decoction is made with 8 to 12 times the weight of the Panax notoginseng flower slices of water, the first decoction time is 40 to 80 minutes, and the second decoction time is 20 to 60 minutes; (2) The decoction in step (1) is concentrated and freeze-dried to obtain a Panax notoginseng flower extract; wherein the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is -40 to -50°C; b. primary drying: the drying temperature is -30 to 0°C; c. secondary drying for analysis: the drying temperature is 5 to 25°C.

3. The preparation method according to claim 2, characterized in that: The pre-freezing time is 3 to 5 hours, preferably 4 hours; and / or, The primary drying time is 9 to 17 hours, preferably 13 hours, and more preferably the primary drying vacuum degree is 0 to 0.2 mbar; and / or, The secondary drying time is 4 to 10 hours, preferably 7 hours.

4. The preparation method according to claim 3, characterized in that: In step (2), the primary drying curve is as follows: stage 1: freeze drying at -30°C for 5-7h; stage 2: freeze drying at -20°C for 1-3h; stage 3: freeze drying at -10°C for 1-3h; stage 4: freeze drying at 0°C for 2-4h; And / or, the secondary drying curve is as follows: stage 1: freeze drying at 5°C for 1-3 h; stage 2: freeze drying at 15°C for 1-3 h; stage 3: freeze drying at 25°C for 1-3 h; stage 4: freeze drying at 0°C for 2-4 h.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step (2), the concentration is carried out under reduced pressure at a temperature of 50-65° C., preferably to a density of 1.03-1.08 g / mL.

6. A Panax notoginseng flower extract, characterized in that It is prepared by the preparation method according to any one of claims 1 to 5; preferably, based on the dry product, the content of ginsenoside Rb3 in the Panax notoginseng flower extract is 29.65 mg / g to 70.13 mg / g.

7. The method for detecting the mass content of ginsenoside Rb3 in the Panax notoginseng flower extract according to claim 6, characterized in that: The steps include: (1) Preparation of reference solution Weigh ginsenoside Rb3 reference substance and add solvent to prepare reference substance solution; (2) Preparation of test solution Weigh the Panax notoginseng flower extract, add a solvent for extraction; (3) Ultra-high performance liquid chromatography analysis The reference solution and the test solution are injected into an ultra-high performance liquid chromatograph for analysis, wherein the chromatographic conditions for the ultra-high performance liquid chromatography analysis are as follows: octadecylsilane bonded silica gel is used as a filler, an organic solvent is used as a mobile phase A, and a 0.08-0.12 wt % phosphoric acid aqueous solution is used as a mobile phase B for gradient elution.

8. The detection method according to claim 7, characterized in that: The solvent in step (1) and step (2) is selected from 100% methanol, 75 v / v% methanol, 50 v / v% methanol, 25 v / v% methanol, 95 v / v% ethanol, 75 v / v% ethanol, 50 v / v% ethanol, 25 v / v% ethanol and water, preferably 75 v / v% methanol. Preferably, the volume amount of the solvent is 100-250 mL / g of Panax notoginseng flower extract; and / or, The extraction in step (2) is performed by reflux or ultrasound, preferably ultrasound extraction; preferably, the extraction time in step (2) is 15-60 min, preferably 60 min; and / or, The organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile; preferably, the conditions for the gradient elution in step (3) are: at 0-30 min, mobile phase A is 31 v / v%, mobile phase B is 69 v / v%, at 30-35 min, mobile phase A is 31-40 v / v%, mobile phase B is 69%-60 v / v%, at 35-50 min, mobile phase A is 40%-46 v / v%, mobile phase B is 60%-54 v / v%, at 50-55 min, mobile phase A is 46%-55 v / v%, mobile phase B is 54%-45 v / v%, wherein the floating range of all the above concentration values ​​is allowed to be ±1 v / v%.

9. The method for constructing the characteristic spectrum of the Panax notoginseng flower extract according to claim 6, characterized in that: The following steps are included: (1) Preparation of reference solution Weigh ginsenoside Rb3 reference substance and add solvent to prepare reference substance solution; (2) Preparation of test solution Weigh the Panax notoginseng flower extract, add a solvent for extraction; (3) Ultra-high performance liquid chromatography analysis The reference solution and the test solution are injected into an ultra-high performance liquid chromatograph for analysis, wherein the chromatographic conditions during the ultra-high performance liquid chromatography analysis are as follows: octadecylsilane bonded silica gel is used as a filler, an organic solvent is used as a mobile phase A, and an aqueous phase is used as a mobile phase B for gradient elution.

10. The construction method according to claim 9, characterized in that: The solvent in steps (1) and (2) is selected from one or more of 100% methanol, 75 v / v% methanol, 50 v / v% methanol, 25 v / v% methanol, 95 v / v% ethanol, 75 v / v% ethanol, 50 v / v% ethanol, 25 v / v% ethanol and water, preferably 75 v / v% methanol; preferably, the volume amount of the solvent is 100-250 mL / g of Panax notoginseng flower extract; and / or The extraction in step (2) is performed by reflux extraction or ultrasonic extraction, preferably ultrasonic extraction. Preferably, the extraction time in step (2) is 15-60 min, preferably 30 min; and / or, When ultra-high performance liquid chromatography analysis is performed in step (3), the aqueous phase is selected from water, 0.08-0.12wt% formic acid solution, 0.08-0.12wt% phosphoric acid solution or 0.08-0.12wt% acetic acid solution, preferably 0.08-0.12wt% phosphoric acid solution; preferably, the organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile; further preferably, the conditions of the gradient elution in step (3) are: at 0-30min, the mobile phase A is 31v / v%, mobile phase B is 69%, 30-35min, mobile phase A is 31%-40v / v%, mobile phase B is 69%-60v / v%, 35-50min, mobile phase A is 40%-46v / v%, mobile phase B is 60%-54v / v%, 50-55min, mobile phase A is 46%-55v / v%, mobile phase B is 54%-45v / v%, wherein the floating range of all the above concentration values ​​is allowed to be ±1v / v%.

11. The method for identifying the Panax notoginseng flower extract according to claim 6, characterized in that: The method comprises identifying the Panax notoginseng flower extract by thin layer chromatography; The thin layer chromatography method comprises the following steps: a. preparing a test solution of Panax notoginseng flower extract and a reference solution of Panax notoginseng flower medicinal material; b. spotting the test solution of Panax notoginseng flower extract and the reference solution of Panax notoginseng flower medicinal material on the same silica gel G thin layer plate, adding a developing agent to develop, taking out and drying, spraying a color developer to color the spots, and detecting, thereby obtaining; Preferably, the Panax notoginseng flower extract test solution is prepared by the following steps: taking 0.1-2g of Panax notoginseng flower extract, adding water-saturated n-butanol to dissolve, separating to obtain a separated liquid, extracting the separated liquid with water-saturated n-butanol, taking the upper solution and evaporating to dryness, and dissolving the residue with methanol to obtain the solution; and / or, the Panax notoginseng flower medicinal material reference solution is prepared by the following steps: taking 0.1-2g of Panax notoginseng flower medicinal material, adding water-saturated n-butanol to dissolve, separating to obtain a separated liquid, extracting the separated liquid with water-saturated n-butanol, taking the upper solution and evaporating to dryness, and dissolving the residue with methanol to obtain the solution; More preferably, the developing agent is chloroform-methanol-water, and further preferably, the volume ratio of chloroform, methanol and water is 6-8:2-4:0.1-1, preferably 7:3:0.

5.

12. Use of the method for detecting the mass content of ginsenoside Rb3 in the Panax notoginseng flower extract according to claim 7 or 8, or the method for constructing the characteristic spectrum of the Panax notoginseng flower extract according to claim 9 or 10, or the method for identifying the Panax notoginseng flower extract according to claim 11 in Panax notoginseng flower medicinal materials or Panax notoginseng flower extract preparations.

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