A method for constructing a characteristic spectrum of nard and its preparations and a method for quality detection

Abstract

The invention relates to the field of quality detection of traditional Chinese medicine preparations and specifically provides a method for constructing a characteristic spectrum of nardostachys radix and a preparation thereof and a quality detection method. In the characteristic spectrum construction method, chromatographic conditions include: using acetonitrile and an aqueous solution containing formic acid as mobile phases for gradient elution, the gradient elution program includes: 0-5 min-40 min-50 min, and the volume percentage of acetonitrile in the mobile phase is: 10%-10%-35%-50%. Under the specific elution program, 9 common characteristic peaks are obtained, and good separation of the common characteristic peaks is achieved. The obtained characteristic spectrum has a stable baseline, good characteristic peak shape, good separation effect, and strong characteristic component specificity. The method provides a basis for quality detection and control of nardostachys radix and a preparation thereof, achieves characterization of the overall components of nardostachys radix and a preparation thereof, and has stable reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic spectrum of nard and its preparations and a quality detection method. Background Art

[0002] Nardostachys jatamansi DC., a plant of the Patrinaceae family, is a plant derived from dried roots and rhizomes. The main active ingredients of Nardostachys jatamansi, including volatile oils, terpenes, and phenolic acids, were evaluated. Nardostachys jatamansi has the following benefits: regulating qi and relieving pain, relieving depression and invigorating the spleen; and, when applied externally, dispelling dampness and reducing swelling. It is used for abdominal distension, loss of appetite, and vomiting; it can also be used externally to treat toothache and beriberi swelling.

[0003] The 2020 edition of the Chinese Pharmacopoeia only uses medicinal material properties, microscopic identification, thin layer identification, total amount of volatile oil, and naringenone as characteristic components to control its quality. The literature also describes the chemical components of naringensis. However, on the one hand, through the above-mentioned existing standards or related research, there is no specific quality control method for naringensis pharmaceutical preparations, let alone its overall quality control; on the other hand, it is time-consuming and labor-intensive to determine the content of a single component combined with the identification of other components in naringensis pharmaceutical preparations, making it difficult to be widely used in production practice.

[0004] In the existing technology, the quality control of ampoules is aimed at ampoules medicinal materials and decoction pieces, not at ampoules pharmaceutical preparations, and there are defects such as little common information and insufficient characteristic. The detection methods for ampoules medicinal materials and decoction pieces in the existing technology cannot be applied to ampoules preparations.

[0005] Common types of naringa preparations include decoctions, powders, and formulated granules. For example, a standard lyophilized decoction powder is obtained from naringa slices through extraction, concentration, and drying. However, because the lyophilized decoction powder of traditional Chinese medicine no longer possesses the characteristics required for identifying medicinal properties, existing methods for identifying naringa are unsuitable for quality testing of preparations such as standard lyophilized decoction powder made from naringa water extracts. These methods often lack characteristic peaks and result in poor separation. Summary of the Invention

[0006] Therefore, the object of the present invention is to provide a method for constructing a characteristic spectrum of nard and its preparations and a quality detection method. The method establishes a characteristic spectrum of this variety based on the characteristics of nard and its preparations, realizes the effective separation of multiple characteristic peaks, saves detection time, and improves the characteristic, providing a scientific basis for comprehensively and rapidly establishing the quality control standards of nard and its preparations.

[0007] To this end, the present invention provides a method for constructing a characteristic spectrum of nard and its preparation, comprising the following steps:

[0008] (1) Preparation of test solution;

[0009] (2) The sample solution was tested by high performance liquid chromatography to obtain a characteristic spectrum of the sample. The chromatographic conditions included: octadecylsilane bonded silica gel as a filler, acetonitrile and an aqueous solution containing formic acid as a mobile phase, and gradient elution. The gradient elution program included: 0→5 min→40 min→50 min, and the volume percentage of acetonitrile in the mobile phase was: 10%→10%→35%→50%.

[0010] Furthermore, in step (2), the detection wavelength is 234-344 nm; and / or the column temperature is 30-40° C.; and / or the flow rate is 0.8-1.2 mL / min; and / or the gradient elution program further includes: 50→56-60 min, the volume percentage of acetonitrile in the mobile phase is: 50%→90%; and / or the volume percentage of formic acid in the formic acid-containing aqueous solution is 0.05-0.2%; and / or the injection volume is 5 μL-20 μL.

[0011] Furthermore, step (1) includes the following steps:

[0012] 1) Take the test sample and add the extraction solvent to obtain an extract;

[0013] 2) performing solid-liquid separation on the extract, taking the liquid to obtain the test solution; preferably, step (1) satisfies any one or more of the following AD:

[0014] A. In step 1), the mass-to-volume ratio of the test sample to the extraction solvent is 0.1-0.4:10-50; the mass-to-volume ratio is g / mL;

[0015] B. The extraction solvent comprises methanol or a methanol aqueous solution, preferably a 50% methanol aqueous solution;

[0016] C. In step 1), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 15min-45min;

[0017] D. In step 2), the solid-liquid separation is centrifugation or filtration.

[0018] Furthermore, the naringa and its preparation are selected from naringa medicinal materials, naringa decoction pieces, naringa water extract or naringa preparations.

[0019] Optionally, the pharmaceutical preparation is a powder (such as a standard decoction freeze-dried powder), tablet, capsule, pill, granule, honey-refined pill, sustained-release preparation, rapid-release preparation, controlled-release preparation, oral liquid preparation or injection preparation.

[0020] Optionally, the preparation method of the pharmaceutical preparation of nardus truncatula comprises the following steps:

[0021] Take nardus truncatus, heat and reflux extract at least once, add at least 8 times the weight of water each time and extract for at least 20 minutes, collect an appropriate amount of volatile oil at the same time (no less than 0.21% (ml / g) volatile oil equivalent to the amount of the decoction piece), encapsulate and prepare for use (volatile oil: β-cyclodextrin: water = 1:8:21, colloid mill grinding for 30 minutes), filter, concentrate the filtrate to a relative density of 1.0-1.2 at 60°C, add conventional excipients, and prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, quick-release preparations, controlled-release preparations, oral liquid preparations or injection preparations according to conventional processes; the excipients are pharmaceutically acceptable excipients: fillers, disintegrants, lubricants, suspending agents, adhesives, sweeteners, flavorings, preservatives, matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include: polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavorings include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include: PEG6000, PEG4000, insect wax, etc.

[0022] Optionally, the preparation method of the pharmaceutical preparation of nardus truncatula comprises the following steps:

[0023] Take nardus truncatus, heat and reflux extract 1 to 5 times, add 8 to 25 times the weight of water each time, extract for 20 minutes to 1 hour, and collect an appropriate amount of volatile oil (no less than 0.21% (ml / g) volatile oil equivalent to the amount of the decoction piece), include it for later use (volatile oil: β-cyclodextrin: water = 1:8:21, colloid mill grinding for 30 minutes), filter the water extract, concentrate the filtrate to a relative density of 1.05 to 1.15 / mL at 70°C, add the volatile oil inclusion compound, mix well, add conventional excipients, and prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations or injection preparations according to conventional processes;

[0024] More optionally, take nard, heat and reflux extract once, add 18 times the weight of water to extract for 45 minutes, and at the same time collect an appropriate amount of volatile oil (not less than 0.21% (ml / g) volatile oil equivalent to the amount of the decoction piece), include for use (volatile oil: β-cyclodextrin: water = 1:8:21, colloid mill grinding for 30 minutes), filter the aqueous extract, concentrate the filtrate to a relative density of 1.06-1.14 g / mL at 60°C, add the volatile oil inclusion compound, mix well, add conventional excipients, and prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, rapid-release preparations, controlled-release preparations, oral liquid preparations or injection preparations according to conventional processes. Conventional processes may include spray drying (for example, inlet air temperature: 150-210°C, outlet air temperature: 70-115°C, wind speed 30-50 Hz).

[0025] Furthermore, the construction method further includes the step of using one or more of chlorogenic acid, cryptochlorogenic acid, deoxygalactone A, galactone diol and galactone A as a reference substance and a solubilizing agent to prepare a reference substance solution, and the step of detecting the reference substance solution by high performance liquid chromatography according to any of the above construction methods to obtain a reference spectrum of the reference substance; preferably, the solvent is selected from methanol or a methanol aqueous solution with a volume percentage of more than 70%; preferably, each 1 mL of the reference substance solution contains 0.04-0.2 mg of each reference substance.

[0026] Furthermore, the construction method also includes extracting the solid obtained by adding water to the control medicinal material of Nardostachys rapa, filtering, and drying, extracting it with an extraction solvent, separating the solid and the liquid, and taking the liquid to obtain a control medicinal material reference solution, and detecting the control medicinal material reference solution by high performance liquid chromatography according to any of the construction methods described above to obtain a control medicinal material reference atlas.

[0027] Wherein, the extraction solvent is as defined above. The solid-liquid separation is centrifugation or filtration.

[0028] Furthermore, the characteristic spectrum of the described cassia bark and its preparation has 9 common characteristic peaks, peak 2 and peak 7 should correspond to the retention time of the chlorogenic acid reference substance and the deoxycandrapine A reference substance reference substance peak, respectively. The peak corresponding to the chlorogenic acid reference substance is the S1 peak, and the peak corresponding to the deoxycandrapine A reference substance is the S2 peak. The relative retention time of peak 1, peaks 3 to 5 and the S1 peak is within ±10% of the specified value, and the specified values ​​of peak 1, peak 3 to 5 and the S1 peak are: 0.47, 1.06, 1.31, and 1.63, respectively; the relative retention time of peak 6, peak 8 to 9 and the S2 peak is within ±10% of the specified value, and the specified values ​​of peak 6, peak 8 to 9 and the S2 peak are: 0.92, 1.14, and 1.30, respectively; preferably, the relative peak area of ​​peak 5 and peak 2 is not less than 0.13; preferably, the relative peak area of ​​peak 7 and peak 2 is not less than 1.5.

[0029] Furthermore, the characteristic spectrum of the described spikenard and its preparation has 9 common characteristic peaks, wherein peak 2 is chlorogenic acid; peak 3 is cryptochlorogenic acid; peak 7 is deoxysapranol A; peak 8 is spikenardone diol; and peak 9 is spikenardone A.

[0030] The present invention also provides a quality detection method for nard and its preparation products, comprising the step of obtaining a characteristic spectrum of the product to be tested according to any of the construction methods described above; preferably, it also includes the step of determining the content of deoxynapthol A and / or chlorogenic acid in the nard and its preparation products.

[0031] Furthermore, the step of determining the content of deoxynarconol A and / or chlorogenic acid in naringa and its preparation products includes:

[0032] (1) Preparation of test solution and deoxycandol A and / or chlorogenic acid reference solution;

[0033] (2) The test solution and the reference solution were tested by high performance liquid chromatography, and the content of the reference substance in the test solution was calculated by the external standard method.

[0034] Furthermore, the chromatographic conditions for the determination of deoxycandol A include: using octadecylsilane bonded silica gel as a filler and acetonitrile-water (30:70) as a mobile phase.

[0035] The detection wavelength is 234-344 nm; and / or the column temperature is 20-40° C.; and / or the flow rate is 0.8-1.2 mL / min.

[0036] Furthermore, the chromatographic conditions for the determination of chlorogenic acid include: using octadecylsilane bonded silica gel as a filler and acetonitrile-0.1% phosphoric acid solution (10:90) as a mobile phase.

[0037] The detection wavelength is 234-344 nm; and / or the column temperature is 20-40° C.; and / or the flow rate is 0.8-1.2 mL / min.

[0038] Furthermore, the preparation method of the test solution comprises the following steps:

[0039] 1) Take the test sample and add the extraction solvent to obtain an extract;

[0040] 2) performing solid-liquid separation on the extract, taking the liquid, and drying it to obtain the test solution; preferably, step (1) satisfies any one or more of the following AD:

[0041] A. In step 1), the mass-to-volume ratio of the test sample to the extraction solvent is 0.05-0.4:10-50; the mass-to-volume ratio is g / mL;

[0042] B. The extraction solvent comprises one or more of water, methanol and ethanol, preferably a 50%-70% methanol aqueous solution;

[0043] C. In step 1), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 15min-45min;

[0044] D. In step 2), the solid-liquid separation is centrifugation or filtration.

[0045] Furthermore, the naringa and its preparation are selected from naringa medicinal materials, naringa decoction pieces, naringa water extract or naringa preparations.

[0046] Optionally, the pharmaceutical preparation is a powder (such as a standard decoction freeze-dried powder), tablet, capsule, pill, granule, honey-refined pill, sustained-release preparation, rapid-release preparation, controlled-release preparation, oral liquid preparation or injection preparation.

[0047] Furthermore, the solvent of the reference solution is selected from methanol or a methanol aqueous solution with a volume percentage of more than 70%.

[0048] Furthermore, each reference substance is contained in 40-80 μg per 1 mL of reference substance solution.

[0049] Specifically, determining the content of deoxynarconol A in naringa and its preparations includes:

[0050] The chromatographic conditions and system suitability test used octadecylsilane bonded silica as the filler; acetonitrile-water (30:70) as the mobile phase; a flow rate of 1.0 ml / min, a column temperature of 25°C, and a detection wavelength of 236 nm. The theoretical plate number, calculated based on the deoxygalactopyranol A peak, should be no less than 3000.

[0051] Preparation of reference solution: Take an appropriate amount of deoxycandol A reference substance, accurately weigh it, place it in a brown volumetric flask, and add methanol to make a solution containing 0.1 mg per 1 ml.

[0052] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take 0.1 g, accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 70% methanol, stopper it tightly, weigh it, and treat it ultrasonically (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0053] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0054] Specifically, the determination of the content of chlorogenic acid in nard and its preparations includes:

[0055] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile-0.1% phosphoric acid solution (10:90) as the mobile phase; a flow rate of 1.0 ml / min; a column temperature of 30°C; and a detection wavelength of 327 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 3000.

[0056] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, and add 50% methanol to make a solution containing 40 μg per 1 ml.

[0057] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.1 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, and treat it ultrasonically (power 250 W, frequency 30 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate.

[0058] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0059] The present invention also provides a method for identifying nardus serrata with different origins and its preparation products, comprising the step of obtaining a characteristic spectrum of the product to be identified according to any of the construction methods described above; preferably, if the relative peak area of ​​peak 5 and peak 2 is not greater than 0.13, the origin is nardus serrata origin; otherwise, the origin is nardus spathulata origin.

[0060] The present invention also provides a method for identifying nardus truncatus and its preparation products from different parts, comprising the step of obtaining a characteristic spectrum of the product to be identified according to any of the construction methods described above; preferably, if the relative peak area of ​​peak 7 and peak 2 is not less than 1.5, the part source is the root and rhizome; otherwise, the part source is the nardus truncatus leaves.

[0061] The technical solution of the present invention has the following advantages:

[0062] 1. The method for constructing the characteristic spectrum of jasmine and its preparation provided by the present invention is to take the test sample solution and adopt high performance liquid chromatography to detect it to obtain the characteristic spectrum of the test sample. The chromatographic conditions include: using octadecylsilane bonded silica gel as a filler, acetonitrile and an aqueous solution containing formic acid as the mobile phase, and obtaining 9 common characteristic peaks under a specific elution program. And a good separation of these common characteristic peaks is achieved. The obtained characteristic spectrum has a stable baseline, good characteristic peak shape, good separation effect, and is simple. It provides a basis for the quality detection and control of jasmine and its preparations, and realizes the characterization of the overall components of jasmine and its preparations with stable reproducibility. More importantly, the simultaneous identification of specific components such as chlorogenic acid, cryptochlorogenic acid, deoxyjasminol A, jasminone diol and jasminone A is achieved through the screening of the mobile phase gradient, and the characteristic is significant.

[0063] 2. The method for constructing the characteristic spectrum of naringa and its preparations provided by the present invention takes medicinal materials, decoction pieces and related preparations as research objects, confirms 9 common characteristic peaks, identifies key characteristic components such as chlorogenic acid, cryptochlorogenic acid, deoxynaringaol A, naringaone diol and naringaone A, and comprehensively analyzes the quality of naringa with 9 key information including terpenes and phenolic acid characteristic fingerprints, and guides the development and utilization of naringa pharmaceutical preparations. The formulated technical method has strong specificity, low requirements for detection equipment, simple and convenient operation, and good reproducibility. The quality of naringa-related pharmaceutical preparations is objectively and reasonably evaluated. It can comprehensively and quickly detect the quality of naringa and its pharmaceutical preparations, which is beneficial to its comprehensive quality detection and overall quality control, thereby helping to improve the safety and stability of the use of the drug. At the same time, the method has the advantages of high stability, high precision and good repeatability.

[0064] 3. The identification method of different origins of nardus and its preparations provided by the present invention can conveniently and quickly identify the origin of nardus spathulatus or the origin of nardus spathulatus and its preparations, which is beneficial to its comprehensive quality inspection and overall quality control, thereby helping to improve the safety and stability of the use of the drug.

[0065] 4. The method for identifying nardus odoratus and its preparation products from different parts provided by the present invention can conveniently and quickly identify nardus odoratus and its preparations from roots and rhizomes or leaves, which is beneficial to its comprehensive quality inspection and overall quality control, thereby helping to improve the safety and stability of the use of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0067] Figure 1 This is the chromatogram of the chlorogenic acid reference substance in Example 1;

[0068] Figure 2 This is the chromatogram of the deoxycandol A reference substance in Example 1;

[0069] Figure 3 The chromatogram is a sample of the standard decoction of Nardostachys rapa slices (lyophilized powder) in Example 1;

[0070] Figure 4 This is the chromatogram of gradient condition 1 in Example 2;

[0071] Figure 5This is the chromatogram of gradient condition 2 in Example 2;

[0072] Figure 6 This is the chromatogram of gradient condition 3 in Example 2;

[0073] Figure 7 This is the chromatogram of gradient condition 4 in Example 2;

[0074] Figure 8 This is the chromatogram of gradient condition 5 in Example 2;

[0075] Figure 9 This is the chromatogram at a wavelength of 214 nm in Example 2;

[0076] Figure 10 This is the chromatogram at a wavelength of 234 nm in Example 2;

[0077] Figure 11 This is the chromatogram at a wavelength of 254 nm in Example 2;

[0078] Figure 12 This is the chromatogram at a wavelength of 264 nm in Example 2;

[0079] Figure 13 This is the chromatogram at a wavelength of 274 nm in Example 2;

[0080] Figure 14 This is the chromatogram at a wavelength of 284 nm in Example 2;

[0081] Figure 15 This is the chromatogram at a wavelength of 304 nm in Example 2;

[0082] Figure 16 This is the chromatogram at a wavelength of 324 nm in Example 2;

[0083] Figure 17 This is the chromatogram at a wavelength of 344 nm in Example 2;

[0084] Figure 18 This is the chromatogram at a wavelength of 364 nm in Example 2;

[0085] Figure 19 This is the 0.05% formic acid chromatogram in Example 2;

[0086] Figure 20 This is the 0.1% formic acid chromatogram in Example 2;

[0087] Figure 21 This is the 0.2% formic acid chromatogram in Example 2;

[0088] Figure 22 This is the chromatogram at a flow rate of 0.8 mL / min in Example 2;

[0089] Figure 23This is the chromatogram at a flow rate of 1.0 mL / min in Example 2;

[0090] Figure 24 This is the chromatogram at a flow rate of 1.2 mL / min in Example 2;

[0091] Figure 25 This is a comparison chart under the column temperature of 30°C in Example 2;

[0092] Figure 26 This is a comparison chart under the column temperature of 35°C in Example 2;

[0093] Figure 27 This is a comparison chart under the column temperature of 40°C in Example 2;

[0094] Figure 28 The chromatogram of the Dikma Platisil ODS column in Example 2 is shown;

[0095] Figure 29 The chromatogram of the Agilent 5TC-C18(2) column in Example 2 is shown;

[0096] Figure 30 This is the chromatogram of the Shimadzu-GL Wondacract ODS-2 column in Example 2;

[0097] Figure 31 This is the Waters liquid phase chromatogram in Example 2;

[0098] Figure 32 This is the chromatogram of the Dionex liquid phase investigation in Example 2;

[0099] Figure 33 This is the Shimadzu liquid phase investigation chromatogram in Example 2;

[0100] Figure 34 This is the chromatogram of the reference medicinal material Nardostachys grandiflora in Example 3;

[0101] Figure 35 Characteristic spectra of multiple batches of freeze-dried powder of standard decoction of Gansong slices in Example 3; (S1 to S18 are: A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18; R is the control characteristic spectrum);

[0102] Figure 36 This is the reference characteristic spectrum of the freeze-dried powder of the standard decoction of Nardostachys rapa in Example 3; Peak 2: chlorogenic acid; Peak 3: cryptochlorogenic acid; Peak 7: deoxynapthol A; Peak 8: narostane diol; Peak 9: narostane A;

[0103] Figure 37Positioning chromatograms of different reference substances in Example 3; S1: reference chromatogram of naringa; S2: chlorogenic acid; S3: cryptochlorogenic acid; S4: naringapine diol; S5: naringapine ketone A; S6: deoxynaringapine A;

[0104] Figure 38 Comparison of the herbal atlas of Nardostachys chinensis and Nardostachys spathiphyllum in Example 5; S1: Nardostachys chinensis sample; S2: Nardostachys spathiphyllum sample;

[0105] Figure 39 Comparison of the atlases of different parts of Nardostachys rapa in Example 5; S1: roots and rhizomes of Nardostachys rapa; S2: leaves of Nardostachys rapa. DETAILED DESCRIPTION

[0106] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0107] If the specific experimental steps or conditions are not specified in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be carried out. If the manufacturer of the reagents or instruments used is not specified, they are all conventional reagent products that can be obtained commercially. Unless otherwise specified, the base of the Nardostachys jatamansi decoction pieces in the freeze-dried powder of the standard decoction of Nardostachys jatamansi in the present invention is the base of the plant Nardostachys jatamansi DC. of the Patrinaceae family, and the part source is the dried root and rhizome.

[0108] In the present invention, the freeze-dried powder of the standard decoction of Nardostachys galanga, the formula granule extract and the formula granule are all prepared using Nardostachys galanga slices as raw materials according to conventional methods in the art. For example, in the present invention, the sample is prepared as follows: taking Nardostachys galanga medicinal materials, and processing them according to the relevant provisions of the 2020 edition of the Chinese Pharmacopoeia under the Nardostachys galanga medicinal materials (processing process: taking an appropriate amount of Nardostachys galanga medicinal materials, removing impurities and sediment, washing, cutting into long segments, and drying to prepare Nardostachys galanga slices.

[0109] 1. Freeze-dried powder of standard decoction of Gansong slices: Take about 600g of Gansong slices, put them in a casserole, extract twice, add 10 times (mass times) of water and soak for 30 minutes, first boil over high heat (5 gears), then simmer over low heat (3 gears) for 30 minutes, filter with 200 mesh gauze (nylon) while hot; add 8 times (mass times) of water to the residue, boil over high heat (5 gears), then simmer over low heat (3 gears) for 25 minutes, filter with 200 mesh gauze (nylon) while hot, and combine the filtrate; the filtrate is the standard decoction of Gansong slices. Take the standard decoction of Gansong slices corresponding to 500g of the slices, and extract the volatile oil according to the volatile oil determination method (General Rules 2204 of the 2020 edition of the Chinese Pharmacopoeia). The remaining 100 g of the pine decoction corresponding to the decoction piece is concentrated under reduced pressure (temperature 60°C, vacuum degree 50 mbar) to a concentrated extract with a relative density of 1.02 g / ml to 1.08 g / ml; placed in a freeze dryer, freeze-dried (-80°C, 0 MPa) to a dry state, taken out, weighed, ground into powder, and packaged in vials to obtain the freeze-dried powder of the standard decoction of the pine decoction piece.

[0110] 2. Formula granule extract: Take 6700g of sweet osmanthus slices, decoct once with water, add 18 times (mass times) of water to soak for 30 minutes, decoct for 45 minutes to obtain an aqueous extract and collect an appropriate amount of volatile oil (not less than 0.21% (ml / g) of volatile oil equivalent to the amount of slices), enclose for later use (the mass ratio of volatile oil: β-cyclodextrin: water is 1:8:21, and the colloid mill is ground for 30 minutes to obtain a volatile oil inclusion compound), the aqueous extract is filtered through 200 mesh (the dry extract yield is 8.0%-13.0%), and concentrated under reduced pressure at 60°C to a flow paste with a relative density of 1.06-1.14g / mL (60°C ± 5°C). The flow paste is kept warm to 65°C, the volatile oil inclusion compound is added, mixed, and spray dried (inlet air temperature: 180°C, outlet air temperature: 100°C, wind speed 40Hz), an appropriate amount of maltodextrin is added, mixed, and obtained.

[0111] 3. Formula granules: Take 6700g of Nardostachys rapa slices, add water and boil once, add 18 times (mass times) of water to soak for 30 minutes, boil for 45 minutes, get water extract and collect appropriate amount of volatile oil (not less than 0.21% (ml / g) of volatile oil equivalent to the slice amount), encapsulate for standby use (mass ratio of volatile oil: β-cyclodextrin: water is 1:8:21, grind in colloid mill for 30 minutes to get volatile oil inclusion compound), water extract 2 The mixture was filtered through 00 mesh (the dry extract yield was 8.0%-13.0%), concentrated under reduced pressure at 60°C to a fluid paste with a relative density of 1.06-1.14 g / mL (60°C±5°C), and the fluid paste was kept warm at 65°C. The volatile oil inclusion compound was added and mixed, and spray-dried (inlet air temperature: 180°C, outlet air temperature: 100°C, wind speed 40 Hz). An appropriate amount of maltodextrin was added, mixed, and granulated to obtain 1000 g.

[0112] Example 1

[0113] This embodiment provides a method for constructing a characteristic spectrum of a freeze-dried powder of standard nard decoction, comprising the following steps:

[0114] (1) Preparation of test solution: Take the freeze-dried powder of standard decoction of nard as the test sample, take an appropriate amount of the test sample, grind it into powder, take 0.2 g, accurately weigh it, place it in a stoppered conical flask, add 20 ml of 50% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate to obtain the solution.

[0115] (2) Preparation of reference solution: Take an appropriate amount of deoxygalactanol A reference substance, accurately weigh it, place it in a brown volumetric flask, and add methanol to make a solution containing 0.1 mg per 1 ml, thus obtaining reference solution 1; take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, and add 50% methanol to make a solution containing 40 μg per 1 ml, thus obtaining reference solution 2; take an appropriate amount of galactanone diol, accurately weigh it, and add 50% methanol to make a solution containing 40 μg per 1 ml, thus obtaining reference solution 3; take an appropriate amount of galactanone A, accurately weigh it, and add 50% methanol to make a solution containing 40 μg per 1 ml, thus obtaining reference solution 4.

[0116] (3) Detection by high performance liquid chromatography: 10 μl of each reference solution and test solution were injected into a high performance liquid chromatograph. The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler (Dikma Platisil ODS, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid water was used as mobile phase B. Elution was performed according to the conditions specified in the table below; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 254 nm; and the number of theoretical plates calculated based on the chlorogenic acid peak should be no less than 3000.

[0117]

[0118] The results are shown in the table below and Figure 1-3 As shown, the chromatographic method has good system adaptability. The characteristic chromatogram of the freeze-dried powder of standard decoction of nard has a stable baseline, good characteristic peak shape, good separation effect, and uniform distribution of characteristic peaks. It has 9 common characteristic peaks. Peak 2 and peak 7 should correspond to the retention times of the chlorogenic acid reference substance and the deoxynardinol A reference substance, respectively. The peak corresponding to the chlorogenic acid reference substance is the S1 peak, and the peak corresponding to the deoxynardinol A reference substance is the S2 peak. The relative retention times of peak 1, peaks 3-5 and S1 peak are 0.47, 1.06, 1.31, and 1.63, respectively; the relative retention times of peak 6, peak 8-9 and S2 peak are 0.92, 1.14, and 1.30, respectively; the relative peak area of ​​peak 5 and S1 peak (peak 2) is 0.28 (not less than 0.13), and the relative peak area of ​​peak S2 (peak 7) and S1 peak (peak 2) is 3.3 (not less than 1.5).

[0119] Table 1 Characteristic spectrum system adaptability parameters of lyophilized powder of standard decoction of nard

[0120]

[0121] Example 2

[0122] 1. Instruments and test drugs

[0123] 1.1 Instrument

[0124] Chromatograph 1: Waters E2695 chromatography system, including a quaternary gradient infusion pump (Alliance 2695), a 120-position high-performance autosampler, an imported column oven, a Waters 2489 UV detector, and an Empower chromatography workstation. Chromatograph 2: Agilent 1260 Infinity II, including a G711B quaternary pump, a G7129A autosampler, a G7114A VWD detector, a G7116A column oven, and an OpenLab CDS chromatography workstation. Chromatograph 3: Shimadzu LC-2030C 3Dplus chromatography system, including an LC-40B XR quaternary pump, a SIL-40C automatic temperature-controlled autosampler, an SPD-40V PAD detector, a CTO-40C column oven, and a Lab Solution chromatography workstation. Chromatographic columns: (1) Dikma Platisil ODS (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); (2) Agilent 5TC-C18(2) (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); (3) Shimadzu-GL Wondacract ODS-2 (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm).

[0125] 1.2 Drug testing

[0126] Chlorogenic acid reference substance: purchased from the National Institutes for Food and Drug Control, batch number 110753-202018, content calculated as 96.1%; deoxygalactopyranol A reference substance: purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd., batch number 001810-202301, purity HPLC ≥ 98%; galactopyranone diol reference substance: purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 070100-202111, purity HPLC ≥ 98%; galactopyranone A reference substance: purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 070102-202111, purity HPLC ≥ 98%. Galactopyranthes rhizome reference medicinal material: purchased from the National Institutes for Food and Drug Control, batch number 121402-201805.

[0127] Batches of freeze-dried powder of Gansong decoction slices: A1-A18, the places of origin are: Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, Banma County, Golog Tibetan Autonomous Prefecture, Qinghai, Banma County, Golog Tibetan Autonomous Prefecture, Qinghai, Banma County, Golog Tibetan Autonomous Prefecture, Qinghai, Aba County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan, Aba County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan, Aba County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan, Maqu County, Gannan Tibetan Autonomous Prefecture, Gansu, Maqu County, Gannan Tibetan Autonomous Prefecture, Gansu, Maqu County, Gannan Tibetan Autonomous Prefecture, Gansu, Songpan County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan, Songpan County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan.

[0128] 2. Investigation of chromatographic conditions and extraction solvents

[0129] (1) Investigation of gradient elution procedure

[0130] The sample solution prepared in Example 1 was injected into a high performance liquid chromatograph using the following chromatographic conditions: octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); Dikma Platisil ODS column; acetonitrile as mobile phase A, 0.1% formic acid in water as mobile phase B; elution was performed according to the conditions specified in the table below; flow rate: 1.0 ml / min; column temperature: 30°C; detection wavelength: 254 nm; injection volume: 10 μl.

[0131] Table 2 Gradient conditions 1 to 3

[0132]

[0133] Table 3 Elution optimization conditions 4 and 5

[0134]

[0135] Table 4 System suitability parameters

[0136]

[0137]

[0138]

[0139] In the present invention, na in asymmetry means that the system does not display data, and na in separation means that the system does not display data.

[0140] The results are shown in the table above and Figure 4-8As shown, the chromatographic peaks of gradient condition 3 showed better separation and richer chromatographic information than those of the previous gradients. Gradient conditions 4 and 5 significantly improved separation compared to gradient conditions 1-3. The chromatogram presented after elution with gradient condition 5 showed richer chromatographic information, more major chromatographic peaks, better resolution, a smoother baseline, and shorter detection time. Therefore, gradient condition 5 was the preferred choice for subsequent condition screening.

[0141] (2) Selection of detection wavelength

[0142] The sample solution prepared in Example 1 was injected into a high performance liquid chromatograph, and chromatograms were compared at 10 different absorption wavelengths (214 nm, 234 nm, 254 nm, 264 nm, 274 nm, 284 nm, 304 nm, 324 nm, 344 nm, and 364 nm). The remaining chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); the chromatographic column was Dikma Platisil ODS; acetonitrile was used as mobile phase A, and 0.1% formic acid in water was used as mobile phase B, with elution using the gradient conditions described above in step 5. The flow rate was 1.0 ml / min; the column temperature was 30°C; and the injection volume was 10 μl.

[0143] Table 5 System adaptability parameters of chromatographic peaks at different absorption wavelengths

[0144]

[0145]

[0146]

[0147] The active ingredients in Nardostachys rapa are mainly volatile oils and terpenoids, and their effective detection wavelength range is 234-364nm. Figure 9-18 It can be seen that the chromatogram under the wavelength of 234-344nm has more chromatographic peaks, a larger amount of information, a smoother baseline, and better separation. Combined with the peak area, the peak area uniformity at a wavelength of 254nm is the best.

[0148] (3) Investigation of formic acid concentration

[0149] The sample solution prepared in Example 1 was injected into a high-performance liquid chromatograph, and the chromatograms obtained with 0.05% formic acid solution, 0.1% formic acid, and 0.2% formic acid as mobile phase B were compared. The remaining chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); the column was Dikma Platisil ODS; acetonitrile was used as mobile phase A, and the above-mentioned acid solutions were used as mobile phase B, with the gradient elution conditions described above as 5. The flow rate was 1.0 ml / min; the column temperature was 30°C; the detection wavelength was 254 nm; and the injection volume was 10 μl.

[0150] Table 6 System suitability parameters for different formic acid concentrations

[0151]

[0152]

[0153] The results showed that different formic acid concentrations had a certain effect on the chromatographic peaks. The peak shape and separation of the main chromatographic peaks in the 0.05-0.2% formic acid solution elution were better (see the table above and Figure 19-21 ), combined with the peak area and baseline, the peak areas of the characteristic peaks of the chromatogram obtained by 0.1%-0.2% formic acid solution are higher and more uniform, and the baseline is more stable, so 0.1%-0.2% formic acid solution is preferred.

[0154] (4) Investigation of flow rate

[0155] The effects of different flow rates of 0.8, 1.0, and 1.2 mL / min on the separation of each characteristic peak were investigated. The test solution prepared in item (1) of this example was subjected to gradient analysis at flow rates of 0.8, 1.0, and 1.2 mL / min, respectively, according to gradient condition 5. The remaining chromatographic conditions were the same as those in item (1).

[0156] Table 7 System suitability parameters for different flow rates

[0157]

[0158]

[0159] The results are shown in the table above and Figure 22-24 , 0.8, 1.0, and 1.2 mL / min can all achieve separation of multiple characteristic peaks, with 1.0 mL / min being the best. Considering the peak area and elution time, the chromatogram obtained at 1.0 mL / min has higher and more uniform peak areas for each characteristic peak, and a shorter elution time, so 1.0 mL / min is preferred.

[0160] (5) Investigation of column temperature

[0161] The effects of different column temperatures of 30, 35, and 40°C on the separation of each characteristic peak were investigated. The test solution prepared in item (1) of this example was subjected to gradient analysis at column temperatures of 30, 35, and 40°C, respectively, according to gradient condition 5. The remaining chromatographic conditions were the same as those in item (1).

[0162] Table 8 System suitability parameters for different column temperatures

[0163]

[0164]

[0165]

[0166] The results are shown in the table above and Figures 25-27 , using 20, 25, and 30 °C can achieve the separation of multiple characteristic peaks, especially 35 °C has the best separation effect.

[0167] (6) Inspection of chromatographic columns

[0168] The influence of the chromatographic column on the separation and peak shape of the chromatographic peaks was investigated. The test solution prepared in item (1) of this embodiment was taken and gradient analysis was performed according to gradient condition 5. The following three chromatographic columns were used for detection: Dikma Platisil ODS (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm) chromatographic column; Agilent 5TC-C18 (2) (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm) chromatographic column; Shimadzu-GL Wondacract ODS-2 (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm) chromatographic column, column temperature 35°C, and the other chromatographic conditions were the same as those in item (1).

[0169] Table 9 Dikma Platisil ODS column system suitability parameters

[0170]

[0171] Table 10 System suitability parameters for the Agilent 5TC-C18(2) column

[0172]

[0173]

[0174] Table 11 System suitability parameters for Shimadzu-GL Wondacract ODS-2 chromatographic column

[0175]

[0176] See the results Figures 28-30 The above three C18 columns can all achieve the separation of multiple characteristic peaks, especially when the Dikma Platisil ODS column is used, the column has a better separation effect on peak 1. Therefore, the Dikma Platisil ODS column was selected as the chromatographic column for the determination of the characteristic spectrum of the freeze-dried powder of the standard decoction of nard.

[0177] (7) Inspection of chromatograph

[0178] The effects of three types of chromatographs, Waters, Dionex, and Shimadzu, on chromatographic peak separation and peak shape were investigated. The test solution prepared in item (1) of this example was subjected to gradient analysis according to gradient condition 5, with a column temperature of 35°C and the same chromatographic conditions as in item (1).

[0179] Comparing the characteristic spectra obtained by different brands of HPLC, it can be found that the chromatographic information presented by the HPLC methods of Waters, Dionex and Shimadzu is relatively complete, and none of the characteristic peaks are missing (see Figures 31-33 ), therefore, there is no need to fix the liquid chromatography model to determine the characteristic spectrum of the freeze-dried powder of Gansong decoction pieces (standard decoction).

[0180] 3. Preparation of test solution

[0181] (1) Investigation of extraction solvent

[0182] The effects of different extraction solvents (20% methanol, 50% methanol, methanol) on the characteristic spectrum of the standard decoction of Nardostachys chinensis (lyophilized powder) were investigated. About 0.1 g of the lyophilized powder of the standard decoction of Nardostachys chinensis was taken respectively, accurately weighed, placed in a stoppered conical flask, and 50 mL of different extraction solvents were accurately added respectively, plugged, and ultrasonically treated (power 250 W, frequency 40 kHz) for 30 minutes, cooled, shaken, filtered, and the filtrate was obtained. HPLC detection, chromatographic conditions were the same as in Example 1, and the results of the investigation are shown in the following table and Figure 23-28 shown.

[0183] Table 12 Extraction solvent methanol concentration to investigate the chromatographic peak system adaptability parameters

[0184]

[0185]

[0186] Conclusion: 20% methanol, 50% methanol and methanol can all achieve effective separation of each peak. The chromatographic peak area obtained by 50% methanol extraction detection is larger and the peak shape of each peak is better. Therefore, 50% methanol is preferred as the extraction solvent for the characteristic spectrum of the freeze-dried powder of standard decoction of nard.

[0187] (2) Investigation of extraction time

[0188] The extraction effect of different extraction times (15, 30, and 45 minutes) on the main characteristic peaks of the characteristic spectrum of the standard decoction of Nardostachys chinensis slices was investigated. Approximately 0.1 g of freeze-dried powder of the standard decoction of Nardostachys chinensis was taken, accurately weighed, placed in a stoppered conical flask, and 50 mL of 50% methanol was accurately added. The flask was sealed and ultrasonicated (power 250 W, frequency 40 kHz) for 15, 30, and 45 minutes, respectively. The filtrate was cooled, shaken, filtered, and the filtrate was obtained. HPLC detection was carried out under the same chromatographic conditions as in Example 1.

[0189] The experimental results show that, a comprehensive comparison of the results of the above three different extraction times shows that different extraction times have no effect on the retention time of each peak, and all peaks can be completely extracted; there are certain differences in peak area when ultrasonic treatment is performed for 15, 30, and 45 minutes, and the extraction is relatively complete when ultrasonic treatment is performed for 30 minutes, and the separation, asymmetry, theoretical plate number, etc. of each peak are better. The optimal extraction time is 30 minutes.

[0190] (3) Selection of sampling volume

[0191] 0.1 g, 0.2 g, and 0.4 g of lyophilized powder of standard decoction of nard were accurately weighed and placed in a stoppered conical flask. 20 mL of 50% methanol was accurately added, the flask was sealed, and ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 30 minutes. The flask was cooled, shaken, and filtered. The filtrate was collected to obtain the product. HPLC analysis was performed under the same chromatographic conditions as in Example 1.

[0192] The test results show that when the sampling amount is between 0.1g and 0.4g, the peak area increases proportionally, indicating that complete extraction can be achieved when the sampling amount is between 0.1g and 0.4g. The peak area of ​​the characteristic peak when the sampling amount is 0.2g is relatively moderate, so 0.2g is preferably used as the sampling amount of the freeze-dried powder of the standard decoction of nard.

[0193] (4) Investigation of solvent usage

[0194] About 0.2 g of lyophilized powder of standard decoction of nard was accurately weighed and placed in a stoppered conical flask. 10 mL, 20 mL, and 50 mL of 50% methanol were accurately added, respectively. The flask was sealed tightly and ultrasonicated (power 250 W, frequency 40 kHz) for 30 minutes. The flask was cooled, shaken, filtered, and the filtrate was collected to obtain the product. HPLC analysis was performed under the same chromatographic conditions as in Example 1.

[0195] The test results show that complete extraction can be achieved when the solvent volume is 10-50 ml. Taking the peak area response into comprehensive consideration, the peak area of ​​the characteristic peak when the solvent volume is 20 mL is relatively moderate, so the extraction solvent volume is preferably 20 ml.

[0196] Example 3

[0197] (1) Characteristic spectrum determination of multiple batches of freeze-dried powder of standard decoction of nardus

[0198] Take 1 g of Nardostachys grandiflora as a control medicinal material, accurately weigh it, place it in a stoppered conical flask, add 20 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the control medicinal material reference solution.

[0199] 18 batches of lyophilized powder of standard decoction of nard were prepared according to the method of Example 1 to obtain test solution. 18 batches of test solution and control medicinal material reference solution were tested according to the method of Example 1 (3) to obtain characteristic spectra of the control medicinal material of nard and characteristic spectra of 18 batches of lyophilized powder of standard decoction of nard. Figure 34 and 35 As shown in Figure 1, the characteristic spectrum detection AIA data of 18 batches of lyophilized powder of standard decoction of nardus root were imported into the calculation software of "Chinese medicine chromatographic fingerprint similarity evaluation system", and a total of 8 common peaks were calibrated to form a common pattern map, and a reference map was established, as shown in Figure 1. Figure 36 shown.

[0200] Table 13 Relative retention time of characteristic spectra of 18 batches of standard decoctions of Gansong slices

[0201]

[0202]

[0203] Table 14 Relative peak areas of characteristic spectra of 18 batches of standard decoctions of Gansong slices

[0204]

[0205] Based on the research results, the characteristic spectrum of the freeze-dried powder of standard naringa decoction should show nine common characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the nine common characteristic peaks in the chromatogram of the reference medicinal material. Peak 2 and Peak 7 should correspond to the retention times of the chlorogenic acid reference and the deoxynaringaol A reference, respectively. The peak corresponding to the chlorogenic acid reference is the S1 peak. The relative retention times of Peak 1 and Peaks 3-5 to the S1 peak are calculated. The peak corresponding to the deoxynaringaol A reference is the S2 peak. The relative retention times of Peaks 6, Peaks 8-9 to the S2 peak are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.47 (Peak 1), 1.06 (Peak 3), 1.31 (Peak 4), 1.63 (Peak 5), 0.92 (Peak 6), 2.67 (Peak 7), 1.14 (Peak 8), and 1.30 (Peak 9). Calculate the relative peak areas of Peak 5 and Peak 7 (S2) relative to Peak S1. These relative peak areas should be within the specified range. The specified values ​​are: no less than 0.13 (Peak 5) and no less than 1.5 (Peak 7). The measurement results indicate that the relative retention times of each characteristic peak in 18 batches of standard lyophilized powder of Nardostachys raphanus decoction were within ±10% of the specified values.

[0206] The reference spectrum was fitted using Mark peak fitting.

[0207] Table 15 Relative retention time of the comparison chart of standard decoction of Gansong slices (lyophilized powder)

[0208]

[0209] Table 16 Relative peak areas of the comparison chart of standard decoction of Gansong slices (lyophilized powder)

[0210]

[0211] Table 17 Relative retention time of characteristic spectrum of Nardostachys grandiflora reference medicinal materials

[0212]

[0213] Table 18 Relative peak areas of characteristic spectra of Nardostachys grandiflora control medicinal materials

[0214]

[0215] (2) Feature Spectrum Transfer

[0216] 18 batches of Nardostachys radix slices were used as test samples to construct characteristic maps according to the method of Example 1, and a control characteristic map of Nardostachys radix slices was established and compared with the control characteristic map of Nardostachys radix standard decoction freeze-dried powder. The results are shown in the table below.

[0217] Table 19 Relative retention time of the comparison chart of Nardostachys glutinosa slices and standard decoction of Nardostachys glutinosa slices

[0218]

[0219] Table 20 Relative peak areas of comparison charts of Nardostachys grandiflora slices and Nardostachys grandiflora slices standard decoction

[0220]

[0221] The results showed that the 18 batches of Nardostachys galangal slices and the corresponding 18 batches of Nardostachys galangal slice standard decoctions selected for the study were measured according to the method under Example 1, and the number of characteristic peaks obtained was consistent, and the relative retention times were also basically consistent. This indicates that the chemical composition of the freeze-dried powder of the standard decoction prepared by the traditional preparation method of Nardostachys galangal slices is consistent with that of the raw material, indicating that the material basis of the Nardostachys galangal slices has not changed after being boiled in water, and the quantity transfer relationship is good.

[0222] (3) Peak identification

[0223] Preparation of reference solution: Take appropriate amounts of chlorogenic acid, cryptochlorogenic acid, deoxygalbanol A, galbanolone diol and galbanolone A, accurately weigh them, and add methanol to each to make a solution containing 40 μg chlorogenic acid, 40 μg cryptochlorogenic acid, 100 μg deoxygalbanol A, 40 μg galbanolone diol and 40 μg galbanolone A per 1 mL of reference solution.

[0224] Take the test solution of the freeze-dried powder of the standard decoction of nard prepared in Example 1 above, and compare the reference solution and the test solution by high performance liquid chromatography according to the method of Example 1. Figure 37 .

[0225] Summary: In the chromatogram of the freeze-dried powder of standard decoction of naringa, peak 2 is chlorogenic acid; peak 3 is cryptochlorogenic acid; peak 7 is deoxynaringapolone A; peak 8 is naringapolone diol; and peak 9 is naringapolone A.

[0226] LC / MS / MS was also used to analyze the molecular weight and molecular structure of Peaks 1 and 4-5, providing a reference for the subsequent qualitative analysis of the characteristic component peaks. The LC / MS / MS analysis structure is shown in the report.

[0227] Table 21 LC / MS / MS analysis results of standard decoction of Gansong slices

[0228]

[0229] Example 4 Methodology Verification

[0230] 1. Exclusivity

[0231] Accurately pipette 10 μL of the test solution and negative blank solution (50% methanol) prepared in Example 1, respectively, and inject them into the liquid chromatograph. The results were detected according to the method of Example 1, and the results were negative without interference.

[0232] 2. Instrument precision test

[0233] A test solution was prepared using the freeze-dried powder of standard decoction of nardostachys as the test sample according to the method of Example 1. The same test solution of the freeze-dried powder of standard decoction of nardostachys was taken and injected 6 times according to the chromatographic conditions under Example 1. The relative retention times of the 9 common characteristic peaks were measured and analyzed.

[0234] The results showed that the RSDs of the relative retention times of each characteristic peak and the reference S peak were all less than 2%, indicating that the instrument had good precision.

[0235] 3. Method repeatability test

[0236] Six portions of the same batch of freeze-dried powder of standard nardostachys rapa decoction were taken, and the relative retention times of the nine common characteristic peaks were measured and analyzed according to the following method of Example 1.

[0237] The results showed that the RSD of the relative retention time between each characteristic peak and the reference S peak was less than 2.0%, indicating that the method had good repeatability.

[0238] 4. Intermediate precision (different operators)

[0239] Three inspectors, at different times, used the same equipment to measure the same batch of freeze-dried powder of standard decoction of nardus galanga under the chromatographic conditions of Example 1, and measured the relative retention times of the nine common characteristic peaks and performed analysis.

[0240] The results showed that the RSDs of the relative retention times of the characteristic peaks and the reference S peak were all less than 2.0%, indicating that the intermediate precision (different personnel) of the method was good.

[0241] 5. Stability inspection

[0242] The same lyophilized powder of standard nardostachys odorata decoction was used as the test sample, and the test solution was prepared and tested by high performance liquid chromatography according to Example 1. The samples were injected at 0 h, 2 h, 4 h, 6 h, 10 h, 12 h, and 24 h after preparation, and the relative retention times of the 9 common characteristic peaks were measured and analyzed to determine the stability of the test solution.

[0243] The results showed that the RSDs of the relative retention times of the characteristic peaks and the reference S peak were all less than 2%, indicating that the test solution was stable within 24 hours and met the determination requirements.

[0244] Example 5

[0245] This embodiment provides a method for identifying freeze-dried powders of standard naringa decoction of different origins, comprising the following steps:

[0246] (1) Preparation of test solution: Three batches of freeze-dried powder of standard decoction of Nardostachys lanceolata were used as test samples to prepare test solutions according to the method of Example 1.

[0247] (2) HPLC detection: Same as Example 1. Three batches of freeze-dried powder of standard decoction of Nardostachys lanceolata were prepared.

[0248] The results are shown in the table below and Figure 38 As shown in the figure, by comparing the characteristic spectra of freeze-dried powder of standard decoction of 18 batches of naringa base, it can be seen that there are 9 peaks in different varieties of naringa samples. The response of peak 5 in the spathiphyllum naringa sample is lower than that of the naringa sample. Therefore, the relative peak area range of peak 5 and peak 2 (S1, chlorogenic acid) can be specified to distinguish different varieties of naringa and spathiphyllum naringa samples. The relative peak area of ​​peak 5 and peak 2 (S1, chlorogenic acid) of the characteristic spectra of standard decoction of 3 batches of spathiphyllum naringa slices was measured and analyzed, ranging from 0.012 to 0.058, with an average of 0.039. The relative peak area range of peak 5 and peak 2 (S1, chlorogenic acid) of standard decoction of 18 batches of naringa slices is 0.19-0.76, with an average of 0.36, SD of 0.12, and a lower limit of -30% of 0.13. Therefore, the relative peak area value of peak 5 and peak S1 is specified to be no more than 0.13. That is, it is stipulated that the relative peak area between Peak 5 and Peak 2 (S1, chlorogenic acid) of the agave formula granules should not be greater than 0.13, which can be used to distinguish and identify different varieties of agave.

[0249] This embodiment also provides a method for identifying freeze-dried powders of standard nardus root decoction from different parts of the body, comprising the following steps:

[0250] (1) Preparation of test solution: 5 batches of freeze-dried powder of standard decoction of nardus truncatus prepared from nardus truncatus leaves were used as test samples, and test solutions were prepared according to the method of Example 1.

[0251] (2) HPLC detection: Same as Example 1. Five batches of freeze-dried powder of standard decoction of Nardostachys pinnatifida, the source of which was Nardostachys pinnatifida leaves, were prepared.

[0252] The results are shown in the table below and Figure 39As shown in the figure, by comparing the characteristic spectra of 18 batches of lyophilized powder of standard decoction of nardus truncatus from roots and rhizomes, it can be seen that the response of the chromatographic peak of chlorogenic acid in the characteristic spectra of nardus truncatus roots and rhizomes is small, while the response of the chromatographic peak of deoxynardol A is large. In the characteristic spectra of nardus truncatus leaves, the response of the chromatographic peak of chlorogenic acid is large, while the response of the chromatographic peak of deoxynardol A is very small. Therefore, the quality of nardus truncatus can be controlled by specifying the relative peak area of ​​peak 7 (S2, deoxynardol A) and peak 2 (S1, chlorogenic acid). The relative peak area of ​​peak 7 (S2, deoxynardol A) and peak 2 (S1, chlorogenic acid) in the characteristic spectra of 18 batches of nardus truncatus medicinal materials ranges from 2.21 to 10.41, with an average of 4.04. Therefore, it is recommended that the relative peak area of ​​peak 7 (S2, deoxynardol A) and peak 2 (S1, chlorogenic acid) in the characteristic spectra of nardus truncatus medicinal materials should not be less than 1.5, which can control the quality of nardus truncatus medicinal materials.

[0253] Table 22 Relative peak area ratios of 18 batches of standard decoctions of Gansong slices

[0254]

[0255]

[0256] Table 23 Peak 5 / Peak S1 relative peak area ratios of three batches of standard decoctions of Nardostachys chinensis slices

[0257]

[0258] Table 24 Peak area ratios of different parts of 5 batches of whole herb Nardostachys rapa

[0259]

[0260] Example 6

[0261] This embodiment provides a method for determining the content of chlorogenic acid in freeze-dried powder of standard decoction of Nardostachys grandiflora, comprising:

[0262] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile-0.1% phosphoric acid solution (10:90) as the mobile phase; a flow rate of 1.0 ml / min; a column temperature of 30°C; and a detection wavelength of 327 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 3000.

[0263] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, and add 50% methanol to make a solution containing 40 μl per 1 ml.

[0264] Preparation of test solution: Take 0.1 g of the powder of this product, weigh it accurately, place it in a stoppered conical flask, add 25 ml of 50% methanol accurately, weigh the weight, ultrasonically treat (power 250 W, frequency 30 kHz) for 30 minutes, let cool, weigh the weight again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate.

[0265] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0266] Example 7

[0267] This embodiment provides a method for determining the content of deoxynarconol A in lyophilized powder of standard naringa decoction, comprising:

[0268] The chromatographic conditions and system suitability test used octadecylsilane bonded silica as the filler; acetonitrile-water (30:70) as the mobile phase; a flow rate of 1.0 ml / min, a column temperature of 25°C, and a detection wavelength of 236 nm. The theoretical plate number, calculated based on the deoxygalactopyranol A peak, should be no less than 3000.

[0269] Preparation of reference solution: Take an appropriate amount of deoxycandol A reference substance, accurately weigh it, and add methanol to make a solution containing 0.1 mg per 1 ml.

[0270] Preparation of test solution: Take 0.1 g of the powder of this product, weigh it accurately, place it in a stoppered conical flask, add 20 ml of 70% methanol accurately, stopper it tightly, weigh it, and treat it ultrasonically (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0271] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0272] Example 8 Investigation of the method for determining the content of chlorogenic acid

[0273] (1) Investigation of mobile phase composition

[0274] The test solution prepared in Example 6 was subjected to HPLC testing, using the following mobile phases: ①: 0.1% phosphoric acid-acetonitrile (volume ratio 90:10), ②: 0.05% phosphoric acid-acetonitrile (volume ratio 90:10), and ③: 0.2% phosphoric acid-acetonitrile (volume ratio 90:10), respectively. The effects of different mobile phases on the separation of the index components were investigated, and the remaining chromatographic conditions were the same as in Example 6. The results showed that different concentrations of phosphoric acid had little effect on the separation of the index components, so the mobile phase system selected for this method was 0.1% phosphoric acid-acetonitrile.

[0275]

[0276] (2) Selection of flow rate

[0277] The test solution prepared in Example 6 was subjected to HPLC testing at flow rates of 0.9, 1.0, and 1.1 ml / min, respectively, to investigate the effect of different flow rates on the separation of the index components. The other chromatographic conditions were the same as in Example 6. The results showed that the separation effect of the index components and the system suitability parameters were relatively good when the flow rate was 1.0 ml / min, so the flow rate of 1.0 ml / min was selected.

[0278]

[0279] (3) Selection of column temperature

[0280] The test solution prepared in Example 6 was subjected to HPLC testing, and column temperatures of 20°C, 25°C, and 30°C were used to investigate the effect of different column temperatures on the separation of the index components. The other chromatographic conditions were the same as in Example 6. The results showed that when the column temperature was 25°C, the separation effect of the index components and the system adaptability parameters were relatively good, so the column temperature was determined to be 25°C.

[0281]

[0282] (4) Selection of chromatographic column

[0283] The test solution prepared in Example 6 was subjected to HPLC testing using the following different chromatographic columns (1, Dikma Platisil ODS; 2, HEXI Morphing WD-C18; 3, Welch Ultimate XB-C18) to investigate the effect of different chromatographic columns on the separation of the index components. The other chromatographic conditions were the same as in Example 6. The results showed that the system adaptability parameters and separation effect of the chromatographic peak of chlorogenic acid measured under different chromatographic column conditions were slightly affected, so there was no need to change the chromatographic column.

[0284]

[0285] (5) Selection of chromatograph

[0286] The test solution prepared in Example 6 was subjected to HPLC testing using the following different chromatographs (1, Dionex; 2, Agilent; 3, Waters) to investigate the effect of different chromatographs on the separation of the index components. The other chromatographic conditions were the same as in Example 6. The results showed that the system adaptability parameters and separation effect of the chromatographic peak of chlorogenic acid measured under different chromatographic column conditions were slightly affected, so there was no need to change the chromatographic column.

[0287]

[0288] (6) Investigation of ultrasonic power

[0289] The effect of different ultrasonic powers (100W, 200W, and 250W) on the content of chlorogenic acid, the indicator component, in the test sample was investigated. The remaining process conditions were the same as in Example 6. According to the chromatographic conditions of Example 6, two parallel samples were prepared for each group and tested. The average values ​​of the powers of 100W, 200W, and 250W were 14.7 mg / g, 14.8 mg / g, and 14.8 mg / g, respectively. The relative mean deviations of the groups were 0.34%, 0.14%, and 0.20%, respectively.

[0290] The test results show that the results of the content of the freeze-dried powder of standard decoction of nardostachys galanga are basically the same under different ultrasonic powers, indicating that the ultrasonic power has little effect on the content determination. Therefore, the commonly used 250W is preferred as the ultrasonic power for the content determination of the freeze-dried powder of standard decoction of nardostachys galanga.

[0291] (7) Selection of extraction solvent

[0292] The extraction effects of different extraction solvents (water, methanol, ethanol, 50% methanol and 50% ethanol) on the chlorogenic acid content in the standard decoction of Nardostachys rapa slices were investigated, and the other process conditions were the same as in Example 6. The chromatographic conditions of Example 6 were used for detection. Two parallel samples were prepared for each group and tested. The average values ​​of water, ethanol, methanol, 50% ethanol and 50% methanol were 14.4 mg / g, 11.3 mg / g, 12.7 mg / g, 14.3 mg / g and 14.7 mg / g, respectively. The relative average deviations of the groups were 0.38%, 0.93%, 1.74%, 1.09% and 0.31%, respectively.

[0293] The results showed that different types of extraction solvents had a certain impact on the content determination of lyophilized powder of standard decoction of nardostachys grandiflora. The content was highest when 50% methanol was used, so 50% methanol was the preferred extraction solvent for the content determination of lyophilized powder of standard decoction of nardostachys grandiflora.

[0294] (8) Selection of solvent dosage

[0295] The effects of different extraction solvent volumes (10, 25, and 50 mL) on the chlorogenic acid content in a standard decoction of Nardostachys rapa slices were investigated. All other process conditions were the same as in Example 6, and the chromatographic conditions used for testing were the same. Two parallel samples were prepared for each group and tested. The average values ​​for the 10, 25, and 50 mL volumes were 14.4 mg / g, 14.7 mg / g, and 14.7 mg / g, respectively, with relative mean deviations of 0.69%, 0.31%, and 0.17%, respectively. The results demonstrate that all three extraction volumes completely extract the chlorogenic acid content, with similar extraction efficiencies. Therefore, 25 mL was the preferred extraction volume for this experiment.

[0296] (9) Investigation of ultrasound time

[0297] The extraction effects of 15, 30, and 45 minutes of extraction time on the chlorogenic acid content in the standard decoction of Nardostachys chinensis slices were investigated respectively. The remaining process conditions were the same as in Example 6, and the chromatographic conditions of Example 6 were used for detection. Two parallel samples were prepared for each group for testing. The average values ​​at 15, 30, and 45 minutes were 14.5 mg / g, 14.7 mg / g, and 14.7 mg / g, respectively, and the relative average deviations of the groups were 0.24%, 0.31%, and 0.17%, respectively. The results showed that when the ultrasonic time was 15-45 minutes, the content results were equivalent, and the ultrasonic time for the content determination of the freeze-dried powder of the standard decoction of Nardostachys chinensis was preferably 30 minutes.

[0298] (10) Sampling quantity investigation

[0299] The extraction effects of 0.05g, 0.1g, and 0.2g of sample sizes on the chlorogenic acid content in the standard decoction of Nardostachys rapa slices were investigated. The remaining process conditions were the same as in Example 6, and the chromatographic conditions of Example 6 were used for detection. Two parallel samples were prepared for each group and tested. The average values ​​of 0.05g, 0.1g, and 0.2g were 14.7mg / g, 14.7mg / g, and 14.6mg / g, respectively, with relative mean deviations of 0.24%, 0.31%, and 0.07% for each group.

[0300] The test results show that the extraction efficiency is close and the extraction is complete when the sampling size is between 0.05g and 0.1g, so the sample size of 0.1g is selected as the sampling size for this test.

[0301] Example 9 Investigation of the Determination Method of Deoxycandol A Content

[0302] (1) Investigation of mobile phase composition

[0303] The test solution prepared in Example 7 was subjected to HPLC testing, using the following mobile phases: ①: 0.1% formic acid-acetonitrile (volume ratio 65:35), ②: 0.1% formic acid-acetonitrile (volume ratio 67:33), and ③: 0.1% formic acid-acetonitrile (volume ratio 70:30). The effects of different mobile phases on the separation of index components were investigated, and the remaining chromatographic conditions were the same as in Example 7. The results showed that the baseline was more stable in gradient condition 3, so gradient 3 (0.1% formic acid-acetonitrile (volume ratio 70:30)) was ultimately preferred for subsequent studies.

[0304]

[0305] (2) Selection of mobile phase components

[0306] The test solution prepared in Example 7 was subjected to HPLC testing, using the following mobile phases: ① 0.1% formic acid-acetonitrile (volume ratio 70:30) ② 0.1% acetic acid-acetonitrile (volume ratio 70:30) ③ water-acetonitrile (volume ratio 70:30) ④ 0.1% formic acid-methanol (volume ratio 70:30) to investigate the effects of different mobile phases on the separation of index components. The remaining chromatographic conditions were the same as in Example 7. The results showed that the methanol mobile phase could not produce a peak, and different water phases had little effect on the separation of index components. Therefore, the method selected the mobile phase system as water-acetonitrile.

[0307]

[0308] (3) Selection of flow rate

[0309] The test solution prepared in Example 7 was subjected to HPLC testing, using flow rates of 0.8, 1.0, and 1.2 ml / min, respectively, to investigate the effect of different flow rates on the separation of the index components. The other chromatographic conditions were the same as in Example 7. The results showed that when the flow rate was 1.0 ml / min, the separation effect of the index components and the system adaptability parameters were relatively good, so the flow rate of 1.0 ml / min was selected.

[0310]

[0311] (4) Selection of column temperature

[0312] The test solution prepared in Example 7 was subjected to HPLC testing, and the column temperatures of 20°C, 25°C, and 30°C were used to investigate the effect of different column temperatures on the separation of the index components. The other chromatographic conditions were the same as in Example 7. The results showed that when the column temperature was 25°C, the separation effect of the index components and the system adaptability parameters were relatively good, so the column temperature was determined to be 25°C.

[0313]

[0314] (5) Selection of chromatographic column

[0315] The test solution prepared in Example 7 was subjected to HPLC testing using the following different chromatographic columns (1. Water HSS T3 (4.6×250 mm, 5 μm); 2. Agilent TC-C18 (4.6×250 mm, 5 μm); 3. Shimadzu Inrtsil ODS-3 (4.6×250 mm, 5 μm)) to investigate the effects of different chromatographic columns on the separation of the index components. The remaining chromatographic conditions were the same as in Example 7. The results showed that the chromatographic peak system adaptability parameters and separation effect of deoxygalactopyranol A measured under different chromatographic column conditions had little effect, so there was no need to change the chromatographic column.

[0316]

[0317] (6) Selection of chromatograph

[0318] The test solution prepared in Example 7 was subjected to HPLC testing using the following different chromatographs (1, Dionex; 2, Agilent; 3, Waters) to investigate the effect of different chromatographs on the separation of the index components. The remaining chromatographic conditions were the same as in Example 7. The results showed that the chromatographic peak system adaptability parameters and separation effect of deoxygalactonol A measured under different chromatographic column conditions were slightly affected, so there was no need to change the chromatographic column.

[0319]

[0320] (7) Selection of extraction solvent

[0321] The extraction effects of different extraction solvents (water, methanol, ethanol, 50% methanol and 50% ethanol) on the content of deoxynarconol A in the standard decoction of Nardostachys rapa slices were investigated, and the remaining process conditions were the same as in Example 7. The chromatographic conditions of Example 7 were used for detection. Two parallel samples were prepared for each group and tested. The average values ​​of water, ethanol, methanol, 50% ethanol and 50% methanol were 18.2 mg / g, 17.3 mg / g, 18.5 mg / g, 18.8 mg / g and 18.7 mg / g, respectively. The relative mean deviations of the groups were 0.33%, 0.63%, 0.22%, 0.53% and 0.19%, respectively.

[0322] The results showed that there was no significant difference in the content of lyophilized powder of standard decoction of nardostachys grandiflora using different extraction solvents. The content of deoxynardol A in the test solution was slightly higher when 70% methanol was used as the extraction solvent, so 70% methanol was the preferred extraction solvent.

[0323] (8) Investigation of ultrasound time

[0324] The extraction effect of 15, 30, and 45 minutes of extraction time on the content of deoxynarconol A in the standard decoction of naringa slices was investigated respectively. The remaining process conditions were all as in Example 7, and the chromatographic conditions of Example 7 were detected. Two parallel samples were prepared in each group for testing, and the average values ​​of 15, 30, and 45 minutes were 18.7 mg / g, 18.8 mg / g, and 18.8 mg / g, respectively, with relative average deviations of 0.11%, 0.53%, and 0.13% in each group. The results showed that when the ultrasonic time was 15-45 minutes, the content results were comparable, and the ultrasonic time for determining the content of the freeze-dried powder of the standard decoction of naringa was preferably 30 minutes.

[0325] (9) Sampling quantity investigation

[0326] The extraction effects of 0.05 g, 0.1 g, and 0.2 g of the sample amount on the content of deoxynarconol A in the standard decoction of naringa slices were investigated respectively. The remaining process conditions were the same as those in Example 7, and the chromatographic conditions of Example 7 were used for detection. Two parallel samples were prepared for each group and tested. The average values ​​of 0.05 g, 0.1 g, and 0.2 g were 18.8 mg / g, 18.8 mg / g, and 18.7 mg / g, respectively, and the relative average deviations of the groups were 0.45%, 0.53%, and 0.51%.

[0327] The test results show that the extraction efficiency is close and the extraction is complete when the sampling size is between 0.05g and 0.1g. The preferred sampling size is 0.1g as the sampling size for this test.

[0328] (10) Sampling quantity investigation

[0329] The effects of different extraction solvent dosages (5 μL, 10 μL, and 15 μL) on the content of deoxynarconol A in the standard decoction of Nardostachys rapa slices were investigated. The remaining process conditions were the same as in Example 7, and the chromatographic conditions of Example 7 were used for detection. Two parallel samples were prepared for each group and tested. The average values ​​of 5 μL, 10 μL, and 15 μL were 18.8 mg / g, 18.8 mg / g, and 18.7 mg / g, respectively. The relative mean deviations of the groups were 0.19%, 0.53%, and 0.27%, respectively.

[0330] The test results show that the extraction efficiency is similar and the extraction is complete when the sampling solvent dosage is 5μL, 10μL, and 15μL, and 10μL is preferred as the injection volume for this test.

[0331] Example 10 Methodology Verification

[0332] Methodological verification was performed on the methods of Examples 6 and 7, respectively.

[0333] 1. Specificity inspection

[0334] Take the blank sample solution (methanol) and the test solution and reference solution prepared in Example 6, perform HPLC analysis according to the chromatographic conditions under Example 6, record the chromatogram, and see the results. Figure 36 and 37 As shown, there was no interference in the negative control experiment.

[0335] Take the blank sample solution (methanol) and the test solution and reference solution prepared in Example 7, perform HPLC analysis according to the chromatographic conditions under Example 7, record the chromatogram, and see the results. Figure 36 and 37 As shown, there was no interference in the negative control experiment.

[0336] 2. Linear relationship investigation

[0337] Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, and add methanol to make a solution containing 2.651μg, 5.303μg, 10.61μg, 21.21μg, 53.03μg, 106.1μg, and 212.1μg per 1mL. Accurately pipette 10μl of the above 7 different concentrations of chlorogenic acid reference substances and inject them into the liquid chromatograph. Determine the peak area according to the chromatographic conditions under Example 6. Use the chlorogenic acid injection mass concentration as the abscissa and the peak area integral value as the ordinate to draw a standard curve. The measurement results are shown in the table below, the standard curve is shown in the figure below, and the regression equation is: y = 0.8531x - 1.5069, R 2 =0.9993. The experimental results showed that the linear relationship was good in the range of chlorogenic acid injection concentration from 2.651 μg / ml to 212.1 μg / ml.

[0338] Take an appropriate amount of deoxycandol A reference substance and add methanol to prepare solutions containing 432.2 μg, 216.1 μg, 108.1 μg, 43.2 μg, 21.6 μg, and 4.32 μg per 1 mL, respectively. Accurately pipette 10 μL of the above deoxycandol A reference substance solutions of different concentrations into a liquid chromatograph. Determine the peak area according to the chromatographic conditions under Example 7. Plot a standard curve with the injected mass concentration of deoxycandol A as the abscissa and the peak area integral as the ordinate. The results are shown in the table below. The regression equation is: y = 0.3697x + 2.0155, R 2 =0.9994. The experimental results showed that the linear relationship was good in the injection concentration range of deoxycandol A from 4.32 μg / ml to 432.2 μg / ml.

[0339] 3. Precision test

[0340] (1) Instrument precision test

[0341] The same sample solution of the standard decoction of Nardostachys glutinosa (batch number: A1) prepared in Example 6 was taken and the sample was injected 6 times according to the chromatographic conditions under Example 6. The chlorogenic acid peak area was measured. The peak area RSD value was 0.29%, indicating that the instrument precision of this method was good.

[0342] The same sample solution of the standard decoction of Nardostachys radix serrata (batch number: A1) prepared in Example 7 was taken and the sample was injected 6 times according to the chromatographic conditions under Example 7. The peak area of ​​deoxynarconol A was measured. The peak area RSD value was 0.24%, indicating that the instrument precision of this method was good.

[0343] (2) Investigation of the intermediate precision of different personnel

[0344] The same batch of standard decoction of Nardostachys chinensis slices (batch number: A1) was independently operated by experimenters A, B, and C, and processed according to the method of Example 6. The RSD of the chlorogenic acid content was determined to be 0.80%. The results showed that the method had good precision among different operators.

[0345] The same batch of standard decoction of Nardostachys chinensis slices (batch number: A1) was taken and operated independently by experimenters A, B, and C. The deoxynarconol A content was determined according to the method of Example 7. The RSD was 0.28%, which showed that the method had good precision among different operators.

[0346] (3) Repeatability study

[0347] Take 6 portions of the same batch of standard decoction of Nardostachys radix spicata (batch number: A1) and process them according to the method of Example 6. Determine and calculate the content RSD%. The results are shown in the table below. The RSD is 0.82%, indicating that the method has good repeatability.

[0348] Take 6 portions of the same batch of standard decoction of Nardostachys radix (batch number: A1), prepare the test solution according to the preparation method of Example 7, perform HPLC analysis according to the chromatographic conditions under Example 7, and determine and calculate the content results RSD%. The results are shown in the table below, and the RSD is 0.33%, indicating that the method has good repeatability.

[0349] (4) Accuracy assessment

[0350] That is, the sample addition recovery test, accurately weighed 6 portions of standard decoction of Gansong decoction pieces (batch number: A1) sample (the chlorogenic acid content of the accompanying sample was 14.86 mg / g) about 0.05g, with 50% methanol to prepare chlorogenic acid reference solution (0.0155 mg / mL, 0.0309 mg / mL, 0.0464 mg / mL) accurately added 25mL, weighed, ultrasonically treated (power 250W, frequency 40kHz) for 30 minutes, removed, cooled, supplemented with 50% methanol to reduce the weight loss, filtered, and the filtrate was obtained. HPLC analysis was performed according to the chromatographic conditions under Example 6. The average recovery rate was 99.2%, and the RSD value was 1.2%. The experimental results showed that the method was accurate.

[0351] Accurately weigh about 0.05 g of 6 samples of standard decoction of ganjiangmai slices (batch number: A1) (the content of deoxyganjiangmai alcohol A in the accompanying sample is 18.7 mg / g), and 20 mL of chlorogenic acid reference solution (0.0267 mg / mL, 0.0534 mg / mL, 0.08005 mg / mL) are accurately added to each sample with 70% methanol. The weight is determined, and ultrasonic treatment (power 250 W, frequency 40 kHz) is performed for 30 minutes. The sample is taken out, cooled, and the weight loss is supplemented with 70% methanol. The sample is filtered and the filtrate is obtained. HPLC analysis was performed according to the chromatographic conditions under Example 7. The average recovery rate was 99.0%, and the RSD value was 0.83%. The experimental results show that the method is accurate.

[0352] (5) Solution stability

[0353] The same sample solution of the standard decoction of Nardostachys radix serrata (Batch No. A1) prepared in Example 6 was injected into a liquid chromatograph at 0, 2, 4, 8, 10, 12, 18, and 24 hours, respectively. HPLC analysis was performed according to the chromatographic conditions of Example 6 to measure the chlorogenic acid peak area and calculate the peak area RSD value. The results are shown in the table below. The RSD value is 0.60%, indicating that the sample solution is stable within 24 hours and can meet the measurement requirements.

[0354] The same sample solution of the standard decoction of Nardostachys radix serrata (batch number: A1) prepared in Example 7 was injected into a liquid chromatograph at 0, 2, 4, 8, 12, and 24 hours, respectively. HPLC analysis was performed according to the chromatographic conditions under Example 7 to determine the peak area of ​​deoxynarconol A and calculate the peak area RSD value. The results are shown in the table below. The RSD value is 0.67%, indicating that the sample solution is stable within 24 hours and can meet the measurement requirements.

[0355] (6) Durability inspection

[0356] ① Durability inspection at different flow rates

[0357] The same test solution of the standard decoction of Nardostachys glutinosa (batch number: A1) prepared in Example 6 was tested at different flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, and the rest were analyzed by HPLC according to the chromatographic conditions under Example 6. The RSD of the chlorogenic acid content was 0.44%. The results showed that different flow rates had little effect on the content of the index component, indicating that the method has good durability for flow rate.

[0358] The same test solution of the standard decoction of Nardostachys glutinosa slices (batch number: A1) prepared in Example 7 was tested at different flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, and the rest were subjected to HPLC analysis according to the chromatographic conditions under Example 7. The RSD of the deoxynarconol A content was 0.88%. The results showed that different flow rates had little effect on the content of the index component, indicating that the method has good durability for flow rate.

[0359] ② Investigation of different column temperatures

[0360] The same test solution of the standard decoction of Nardostachys glutinosa (Batch No.: A1) prepared in Example 6 was tested at different column temperatures of 23°C, 25°C, and 27°C, and the rest were subjected to HPLC analysis according to the chromatographic conditions under Example 6. The RSD of the chlorogenic acid content was 0.83%. The results showed that different column temperatures had little effect on the content of the index component.

[0361] The same test solution of the standard decoction of Nardostachys glutinosa slices (batch number: A1) prepared in Example 7 was tested at different column temperatures of 23°C, 25°C, and 27°C, and the rest was subjected to HPLC analysis according to the chromatographic conditions under Example 7. The RSD of the deoxynarconol A content was 0.61%, indicating that different column temperatures had little effect on the content of the index component.

[0362] ③Investigation on the durability of different instruments

[0363] The same sample solution of the standard decoction of Nardostachys chinensis (Batch No. A1) prepared in Example 6 was injected using different instruments (1. Waters e2695; 2. Agilent 1260; 3. Dionex U3000). HPLC analysis was performed according to the chromatographic conditions described in Example 6 to determine the chlorogenic acid content. The results showed that the chlorogenic acid content measured under the different column types was similar, with an RSD of 0.80%, indicating that the method has good flow rate robustness.

[0364] The same sample solution of the standard decoction of Nardostachys radix serrata (Batch No. A1) prepared in Example 7 was injected using different instruments (1, Waters; 2, Agilent; 3, Dionex). HPLC analysis was performed according to the chromatographic conditions in Example 7 to determine the content of deoxygenated galactosidol A. The results showed that the deoxygenated galactosidol A contents measured under the conditions of different chromatographic columns were relatively close, with an RSD of 0.72%, indicating that the method has good robustness to flow rate.

[0365] ④Investigation of different chromatographic columns

[0366] The same sample solution of the standard decoction of Nardostachys chinensis slices (Batch No. A1) prepared in Example 6 was injected using different chromatographic columns (1, Dikma; 2, HEXI; 3, Welch). HPLC analysis was performed according to the chromatographic conditions in Example 6 to determine the chlorogenic acid content. The results showed that the chlorogenic acid content measured under the different column types was relatively close, with an RSD of 0.40%, indicating that the method is robust for different chromatographic columns.

[0367] The same sample solution of the standard decoction of Nardostachys grandiflora (Batch No. A1) prepared in Example 7 was injected using different types of chromatographic columns (1, Water T3; 2, Agilent TC-C18; 3, Shimadzu Inrtsil ODS-3). HPLC analysis was performed according to the chromatographic conditions in Example 7 to determine the content of deoxynarconol A. The results showed that the deoxynarconol A content measured under the conditions of different types of chromatographic columns was relatively close, with an RSD of 0.58%, indicating that the method has good robustness to flow rate.

[0368] (7) Determination of the content of freeze-dried powder of standard decoction of nardus

[0369] Eighteen batches of standard decoctions (lyophilized powder) of Nardostachys rapa were collected and processed according to the method in Example 6 to determine the chlorogenic acid content of each batch of lyophilized powder and calculate the transfer rate. The results showed that the average transfer rate of chlorogenic acid in each batch of standard decoctions ranged from 25.7% to 42.2%, with a mean of 33.0% and a SD of 4.7%. The fluctuation range of the mean ±30% was 23.1% to 42.8%. The transfer rate of chlorogenic acid content in the standard decoction of Gansong slices is stipulated to be in the range of 25.0% to 42.0%. Combined with the actual paste yield, the average chlorogenic acid content of the freeze-dried powder is converted to 7.6-16.2 mg / g, with a mean of 10.2 mg / g, SD of 2.4 mg / g, and a range of mean ±30% of 7.2-13.3 mg / g. According to the theoretical value, the chlorogenic acid content of the freeze-dried powder of the standard decoction of Gansong slices is stipulated to be in the range of 7.6-16.0 mg / g, which complies with the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules".

[0370] Eighteen batches of standard decoction (lyophilized powder) samples of Nardostachys grandiflora were taken and processed according to the method in Example 7 to determine the content of deoxynarconol A in each batch of lyophilized powder and calculate the transfer rate. The results showed that the measured average transfer rate of deoxynarconol A in each batch of standard decoction ranged from 77.6% to 95.2%, with a mean of 87.9%, a SD of 5.6%, and a fluctuation range of 61.5% to 114.3% within the mean ±30%. The transfer rate of deoxycanol A content in the standard decoction of galangal slices is stipulated to be in the range of 77.5% to 95.0%. Combined with the actual paste yield, the average deoxycanol A content in the freeze-dried powder is converted to 13.9-32.8 mg / g, with a mean of 21.2 mg / g, an SD of 6.0 mg / g, and a mean ±30% range of 14.9-27.6 mg / g. According to the theoretical value, the content of deoxycanol A in the freeze-dried powder of the standard decoction of galangal slices is stipulated to be in the range of 14.0-33.0 mg / g, which complies with the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules".

[0371] Based on the above results, the content of the standard decoction of Nardostachys chinensis slices is stipulated as follows: Each 1g of this product contains 1.0%-3.5% (ml / g) of volatile oil, 14.0mg-33.0mg of deoxynapthol A (C12H16O2), and 7.6mg~16.0mg of chlorogenic acid (C16H18O9).

[0372] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of nard and its preparation, characterized in that: The following steps are included: (1) Preparation of a test solution; comprising the following steps: 1) extracting the test sample into an extraction solvent to obtain an extract; 2) subjecting the extract to solid-liquid separation and obtaining the liquid to obtain the test solution; the extraction solvent comprises methanol or a methanol-water solution; (2) The test solution and the reference solution were respectively taken and tested by high performance liquid chromatography to obtain the characteristic spectrum of the test sample. The chromatographic conditions included: octadecylsilane bonded silica gel as the filler, mobile phase A was acetonitrile, mobile phase B was an aqueous solution containing formic acid, gradient elution was performed, the gradient elution program was: 0→5min→40min→50min, the volume percentage of acetonitrile in the mobile phase was: 10%→10%→35%→50%; the detection wavelength was 254nm; Chlorogenic acid, cryptochlorogenic acid, deoxygalactone A, galactone diol and galactone A were used as reference substances and solvents were added to prepare reference substance solutions.

2. The construction method according to claim 1, characterized in that In step (2), the column temperature is 30-40°C; and / or, the flow rate is 0.8-1.2 mL / min; and / or, the gradient elution program further includes: 50→56-60 min, the volume percentage of acetonitrile in the mobile phase is: 50%→90%; and / or, the volume percentage of formic acid in the formic acid-containing aqueous solution is 0.05-0.2%; and / or, the injection volume is 5µL-20µL.

3. The construction method according to claim 2, characterized in that Step (1) satisfies any one or more of the following AD: A. In step 1), the mass-to-volume ratio of the test sample to the extraction solvent is 0.1-0.4:10-50; the mass-to-volume ratio is g / mL; B. The extraction solvent is 50% methanol aqueous solution; C. In step 1), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 15min-45min; D. In step 2), the solid-liquid separation is performed by centrifugation or filtration.

4. The construction method according to claim 1, characterized in that The solvent used in the preparation of the reference solution is a methanol aqueous solution with a volume percentage of more than 70%.

5. The construction method according to claim 1, characterized in that Each 1 mL of reference solution contains 0.04-0.2 mg of each reference substance.

6. The construction method according to any one of claims 1 to 3, characterized in that: The construction method also includes extracting the solid obtained by adding water to the reference medicinal material of Nardostachys rapa, filtering, and drying, extracting it with an extraction solvent, separating the solid and liquid, and taking the liquid to obtain a reference medicinal material solution, and detecting the reference medicinal material solution by high performance liquid chromatography according to the construction method described in any one of claims 1 to 3 to obtain a reference medicinal material reference atlas.

7. The construction method according to any one of claims 1 to 3, characterized in that: The characteristic spectrum of the described spikenard and its preparation has 9 common characteristic peaks, peak 2 and peak 7 correspond to the retention times of the chlorogenic acid reference substance and the deoxysapranthenol A reference substance peak, respectively. The peak corresponding to the chlorogenic acid reference substance is the S1 peak, and the peak corresponding to the deoxysapranthenol A reference substance is the S2 peak. The relative retention times of peak 1, peaks 3 to 5 and S1 peak are within the range of ±10% of the specified values. The specified values ​​of peak 1, peaks 3 to 5 and S1 peak are as follows: 0.47, 1.06, 1.31, 1.63; the relative retention times of peaks 6, 8~9 and S2 are within ±10% of the specified values. The specified values ​​of peaks 6, 8~9 and S2 are as follows: 0.92、1.14、1.30。 8. The construction method according to claim 7, characterized in that: The relative peak area of ​​Peak 5 and Peak 2 is not less than 0.13; the relative peak area of ​​Peak 7 and Peak 2 is not less than 1.

5.

9. The construction method according to any one of claims 1 to 3, characterized in that: The characteristic spectrum of the described spikenard and its preparation has 9 common characteristic peaks, wherein peak 2 is chlorogenic acid; peak 3 is cryptochlorogenic acid; peak 7 is deoxysapranol A; peak 8 is spikenardone diol; and peak 9 is spikenardone A.

10. A quality detection method for nard and its preparation products, characterized in that: The method comprises the steps of obtaining a characteristic spectrum of the product to be tested according to the construction method described in any one of claims 1 to 9.

11. The quality detection method of nard and its preparation products according to claim 10, characterized in that: The method also includes the step of determining the content of deoxynarconol A and / or chlorogenic acid in the spikenard and its preparation products.

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