A method for constructing characteristic maps of lily medicinal materials of different origins and their preparations and a method for identifying the same

Abstract

The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically provides a method for constructing characteristic spectra of lily medicinal materials of different origins and preparations thereof and an identification method thereof, comprising the following steps: (1) preparing a lily test solution; and (2) taking the lily test solution and detecting it by high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler, a mobile phase comprising acetonitrile and a 0.05-0.2vt% phosphoric acid aqueous solution, and gradient elution, wherein the gradient elution program comprises: 0→10 minutes→15 minutes→23 minutes→30 minutes→35 minutes, and the volume percentage of acetonitrile in the mobile phase is 6%→12%→18%→18%→40%→70%. The present invention establishes characteristic spectra of lily medicinal materials of different origins and preparations thereof, which can be used to identify and distinguish lily medicinal materials and preparations thereof of lily dandelion origin, lily medicinal materials and preparations thereof of lily dandelion origin, and lily medicinal materials and preparations thereof of lily dandelion origin.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing characteristic maps of lily medicinal materials of different origins and preparations thereof and a method for identifying the same. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia stipulates that lily medicinal materials are derived from the dried fleshy scales of Lilium lancifolium Thunb., Lilium brownii FEBrown var. viridulum Baker, or Liliumpumilum DC., all members of the Liliaceae family. Currently, the medicinal lilies on the market are primarily Lilium lancifolium, followed by Lilium brevifolium. Lilium brevifolium is less commonly available. Lily medicinal materials based on Lilium brevifolium, Lilium lancifolium, and Lilium brevifolium vary significantly in terms of active ingredient content and efficacy. Lily slices from different sources have similar properties, making accurate identification difficult for inexperienced personnel. The Chinese Pharmacopoeia's lily medicinal material quality evaluation is based solely on thin-layer identification of properties and the content of water-soluble extracts. There is no identification method for specific varieties, making it difficult to ensure the accuracy of medicinal use. This method is also not applicable to standard decoctions or lily formula granules based on Lilium lancifolium, Lilium brevifolium, and Lilium brevifolium.

[0003] Traditional Chinese medicine formula granules and standard decoctions are common dosage forms for lily. Formulated granules are made by extracting Chinese herbal medicine slices with water and using a process that includes extraction, drying, and granulation. Their clinical efficacy should be consistent with that of the decoction. Standard decoctions serve as a material benchmark for determining whether formula granules are essentially identical to decoctions. However, both standard decoctions and granules lack the identifying characteristics of the original medicinal material, meaning they cannot be inspected and identified based on its shape, size, texture, and other characteristics. Summary of the Invention

[0004] Therefore, the present invention solves the problem that the methods in the prior art cannot accurately identify lily medicinal materials and preparations based on Fritillaria lily, Lilium spp. and Lilium tenuifolium, and provides a method for constructing characteristic maps of lily medicinal materials and preparations based on different origins and an identification method. Characteristic maps of lily medicinal materials and preparations based on different origins are established to identify and differentiate lily medicinal materials and preparations based on Fritillaria lily from lilium and lily medicinal materials and preparations based on Lilium tenuifolium, providing a rapid and reliable detection method for interspecies identification of lily medicinal materials and preparations.

[0005] The present invention provides a method for constructing characteristic maps of lily medicinal materials of different origins and preparations thereof, comprising the following steps:

[0006] (1) Preparation of lily test solution;

[0007] (2) The lily sample solution was tested by high performance liquid chromatography using octadecylsilane bonded silica gel as the filler, the mobile phase comprising acetonitrile and 0.05-0.2vt% phosphoric acid aqueous solution, and gradient elution. The gradient elution program included: 0→10 min→15 min→23 min→30 min→35 min, and the volume percentage of acetonitrile in the mobile phase was 6%→12%→18%→18%→40%→70%.

[0008] Furthermore, step (1) includes:

[0009] 1) Take a lily sample and extract it with water to obtain an extract;

[0010] 2) separating the extract into solid and liquid, drying the extract, and then extracting with an alcohol-water solution to obtain an extract;

[0011] 3) separating the solid and liquid of the extract and taking the liquid as the test solution; or, step (1) includes:

[0012] 1) Take a lily sample and extract it with an alcohol-water solution to obtain an extract;

[0013] 2) Separate the solid and liquid of the extract and take the liquid, which is the test solution.

[0014] Furthermore, step (1) also satisfies any one or more of the following A and F:

[0015] A. The water extraction is performed by adding water and then heating under reflux for at least 20 minutes, preferably 20-60 minutes;

[0016] B. The mass-to-volume ratio of the lily sample to water is 0.5-2:50, and the mass-to-solvent ratio is g / mL;

[0017] C. The alcohol-water extraction is performed by adding an alcohol-water solution and then performing reflux extraction or ultrasonic extraction, and the extraction time is 10 min-5 h;

[0018] D. The alcohol aqueous solution is selected from 30% to 70% by volume of ethanol aqueous solution or methanol aqueous solution;

[0019] E. The solid-liquid separation is centrifugation or filtration;

[0020] F. The ratio of the mass of the lily test sample to the volume of the alcohol-water solution is 0.2-2:20, and the ratio of the mass to the solvent is g / mL.

[0021] Furthermore, in step (2), the detection wavelength is 203-208 nm before 14-20 min, and the detection wavelength is switched to 308-313 nm after 14-20 min, the flow rate is 0.6-1.0 ml / min, the column temperature is 22-28° C., and the injection volume is 1-20 μl; and / or, an Ultimate XB-C18 chromatographic column with a specification of 4.6x250 mm, 5 μm is used.

[0022] Furthermore, the construction method also includes the step of preparing a reference solution using at least one of the reference substances of lirioside A, lirioside H, and 2-acetyllirioside A, and the step of detecting the reference solution by high performance liquid chromatography according to any of the above construction methods to obtain a reference spectrum of the reference substance; optionally, the solvent used for the reference solution is a 30-70vt% methanol aqueous solution; optionally, the concentration of the reference solution is 10-100μg / mL.

[0023] Furthermore, the lily medicinal materials and preparations thereof of different origins are: lily medicinal materials and preparations thereof of Lilium camara origin, lily medicinal materials and preparations thereof of Lilium camara origin or lily medicinal materials and preparations thereof of Lilium camara origin.

[0024] In the present invention, the term "Lily medicinal materials and preparations derived from Lilium chinense" and "Lily (Lilium chinense) and its preparations" refers to Lily medicinal materials, medicinal pieces or preparations derived from Lilium chinense. The term "Lily medicinal materials and preparations derived from Lilium chinense" and "Lily (Lilium chinense) and its preparations" refers to Lily medicinal materials, medicinal pieces or preparations derived from Lilium chinense. The term "Lily medicinal materials and preparations derived from Lilium truncatum" and "Lily (Lilium truncatum) and its preparations" refers to Lily medicinal materials, medicinal pieces or preparations derived from Lilium truncatum. Preparations can be in conventional dosage forms such as, but are not limited to, powders, formulated granules, and the like. Lily (Lilium truncatum) is referred to as Lilium truncatum. Lily (Lilium truncatum) is referred to as Lilium truncatum.

[0025] Furthermore, the characteristic spectrum of the lily medicinal material and its preparation has a total of 9 characteristic peaks, the peak corresponding to the peak of the reference substance of lilyoside A is the S peak, and the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), 1.46 (peak 9);

[0026] The lily medicinal material of the base of Lilium and its preparation have 10 characteristic peaks, the peak corresponding to the peak of the reference substance of lilyoside A is the S peak, and the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), 1.46 (peak 9); a chromatographic peak (peak 10) appears at the corresponding position of the chromatographic peak of the reference substance of 2-acetyllilyoside A (peak 10);

[0027] The lily medicinal material and preparation thereof based on the lily of tenuifolia have 11 characteristic peaks, the peak corresponding to the peak of the reference substance of lilyoside A is the S peak, and the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), 1.46 (peak 9), 1.49 (peak 11), and 1.50 (peak 12).

[0028] In the present invention, the "peak corresponding to the peak of the reference substance of the Wanglioside A standard" is a characteristic peak in the characteristic spectrum with an RSD of less than 5%, less than 3% or 1% of the retention time of the peak of the reference substance of the Wanglioside A standard, and / or a characteristic peak with a degree of overlap with the peak of the reference substance of the Wanglioside A standard of not less than 50%.

[0029] Furthermore, if a chromatographic peak appears at the corresponding position of the chromatographic peak of the 2-acetyllilyoside A reference substance, it is the genin of Lilium, and if there is no chromatographic peak, it is the genin of Lilium or Lilium tenuifolium.

[0030] It also includes the use of the fingerprint similarity evaluation software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee to generate a control characteristic spectrum. Through the identification and identification of characteristic peaks, the obtained HPLC characteristic spectrum of lily medicinal materials has a total of 9 to 11 chromatographic peaks.

[0031] The present invention also provides a method for identifying lily medicinal materials of different origins and preparations thereof, comprising the step of obtaining a characteristic spectrum of the lily product to be identified according to any of the above-mentioned construction methods.

[0032] If the lily product to be identified has a chromatographic peak at the corresponding position of the chromatographic peak of the 2-acetyl lilyoside A reference substance, it is Lilium genin. If the lily product to be identified has no chromatographic peak at the corresponding position of the chromatographic peak of the 2-acetyl lilyoside A reference substance and the relative peak area of ​​Peak 9 and Peak 4 is less than 0.23, it is Lilium genin. If there is no chromatographic peak and the relative peak area of ​​Peak 9 and Peak 4 is greater than or equal to 0.23, it is Lilium genin.

[0033] The technical solution of the present invention has the following advantages:

[0034] 1. The present invention provides a method for constructing characteristic maps of lily medicinal materials of different origins and preparations thereof. Through investigation of chromatographic conditions, the present invention found that when octadecylsilane bonded silica gel is used as a filler, the mobile phase includes acetonitrile and 0.05-0.2vt% phosphoric acid aqueous solution, gradient elution, and the gradient elution program includes: 0→10 minutes→15 minutes→23 minutes→30 minutes→35 minutes, and the volume percentage of acetonitrile in the mobile phase is 6%→12%→18%→18%→40%→70%, complete separation of 9-11 common characteristic peaks including lilyside A and lilyside H, and for Lutein radicals, also including 2-acetyllilyside A, is achieved, with good peak shape, stable baseline, and short detection time. Characteristic maps of lily medicinal materials of different origins and preparations thereof are constructed, which fully demonstrate the chemical component characteristics of lilies of different origins, provide a basis for identifying and distinguishing lily medicinal materials of different origins and preparations thereof, and the method is stable, with high precision, good stability, strong characteristic, and rich chromatographic information.

[0035] 2. The method for identifying lily medicinal materials and preparations of different origins provided by the present invention can be used to identify and differentiate lily medicinal materials and preparations of three different origins: lily medicinal materials and preparations of Lilium camara origin, lily medicinal materials and preparations of Lilium camara origin, and lily medicinal materials and preparations of Lilium truncatum origin. By positioning the 2-acetyllilyoside A reference substance or combining the relative peak area, the results displayed by the spectrum are extremely easy to determine, and can be directly determined without calculating the similarity with the reference characteristic spectrum. This overcomes the problem of difficulty in determining Lilium camara and Lilium camara origin in the prior art, and provides a rapid and reliable detection method for interspecies identification of lily medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 1 This is the characteristic spectrum of the Lilium (Lilium) medicinal material test sample in Example 1;

[0038] Figure 2 It is the characteristic spectrum of the lily (lily) slice test sample in Example 2;

[0039] Figure 3 This is the characteristic spectrum of the lyophilized powder test sample of the lily (Lily) standard decoction in Example 3;

[0040] Figure 4 The characteristic spectrum of the medicinal material of 18 batches of lily (lily) in Example 4;

[0041] Figure 5 is the control characteristic spectrum of the medicinal material Lilium (Lilium) in Example 4;

[0042] Figure 6 It is the characteristic spectrum of 18 batches of lily (lily) slices in Example 5;

[0043] Figure 7 The control characteristic maps in Examples 4-6 are, from bottom to top, the control characteristic maps of medicinal materials, the control characteristic maps of decoction pieces, and the control characteristic maps of standard decoctions;

[0044] Figure 8 The characteristic spectra of lily medicinal materials of different origins in Example 7, S1: 2-acetyl lilyoside A reference solution; S2: lily (lily) reference medicinal material; S3-S8 are 6 batches of lily (Lithuania liliifolia) medicinal materials, S9: lily (Lithuania liliifolia) reference medicinal material;

[0045] Figure 9 The characteristic spectra of lily medicinal materials of different origins in Example 7, S1: lily (lily) control medicinal material; S2-S3 are two batches of lily (lilium ulmoides) medicinal materials;

[0046] Figure 10 The characteristic spectra of the freeze-dried powder of the lily standard decoction of different origins in Example 8, S1: 2-acetyl lilyoside A; S2: freeze-dried powder of lily (Lilium); S3-S8 are 6 batches of freeze-dried powder of lily (Littoral Lilium) standard decoction respectively; S9: control medicinal material of lily (Littoral Lilium);

[0047] Figure 11 The characteristic spectra of the freeze-dried powders of the lily standard decoction of different origins in Example 8 are shown as follows: S1: freeze-dried powder of lily (Lilium bulbiferum); S2-S3 are two batches of freeze-dried powders of the lily (Lilium ulmoides) standard decoction;

[0048] Figure 12 This is the characteristic spectrum obtained by the Agilent SB AQ chromatographic column in Example 9;

[0049] Figure 13 This is the characteristic spectrum obtained by the YMC-Pack ODS-AQ chromatographic column in Example 9;

[0050] Figure 14 This is the characteristic spectrum obtained by the Ultimate XB-C18 chromatographic column in Example 9;

[0051] Figure 15 This is the characteristic spectrum obtained by gradient elution procedure 1 in Example 9;

[0052] Figure 16 This is the characteristic spectrum obtained by gradient elution procedure 2 in Example 9;

[0053] Figure 17 This is the characteristic spectrum obtained by gradient elution procedure 3 in Example 9;

[0054] Figure 18 This is the characteristic spectrum obtained by gradient elution procedure 4 in Example 9;

[0055] Figure 19 This is the characteristic spectrum obtained by gradient elution procedure 5 in Example 9;

[0056] Figure 20 This is the characteristic spectrum obtained by gradient elution procedure 6 in Example 9;

[0057] Figure 21 This is the characteristic spectrum obtained by gradient elution procedure 7 in Example 9;

[0058] Figure 22 The characteristic spectrum of lily medicinal material and the ultraviolet absorption spectrum of each characteristic peak are shown from top to bottom: the characteristic spectrum of lily medicinal material RK21050703 and the ultraviolet absorption spectra of lilyside H (left) and lilyside A (right) in the test sample;

[0059] Figure 23 These are the chromatograms and ultraviolet absorption spectra of the reference substances. From top to bottom, they are the chromatogram of the reference substance of lilyoside H, the chromatogram of the reference substance of lilyoside A, and the ultraviolet absorption spectra of the reference substances lilyoside H (left) and lilyoside A (right). DETAILED DESCRIPTION

[0060] 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.

[0061] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0062] The experimental instruments and reagents used in the embodiments and experimental examples of the present invention are as follows.

[0063] High performance liquid chromatograph 1: Waters E2695, including G7104A quaternary pump, 132-position autosampler, G7116B column oven, and diode array detector.

[0064] High performance liquid chromatograph 2: Agilent 1260, including G7111AQuat Pump VL; G7129A1260Vialsamplar; G7114A1260VWD.

[0065] High performance liquid chromatograph 3: Thermo U3000 chromatography system, including quaternary solvent manager (Pμmp), autosampler (Autosampler), original imported chromatography column oven (Colμmn Compartment), diode array ultraviolet detector (Detector), and Chromeleon chromatography management system.

[0066] Electronic analytical balances: MS205DU (Mettler), FA2004 (Shanghai Sunny Optical), and LD610-2 (Shenyang Longteng Electronics).

[0067] Ultrasonic cleaning machine: BILON30-720, Shanghai Bilan Instrument Manufacturing Co., Ltd.

[0068] Column 1: Agilent SB AQ, 4.6 x 250 mm, 5 μm.

[0069] Column 2: YMC–Pack ODS–AQ, 4.6 x 250 mm, 5 μm.

[0070] Column 3: Hypersil GOLD AQ C18, 4.6 x 250 mm, 5 μm.

[0071] Reagents: Acetonitrile was of chromatographic grade, water was ultrapure water; other reagents such as phosphoric acid were of analytical grade.

[0072] Wangbaiheoside A reference substance, batch number: 114420-66-5, was purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0073] Lily control medicinal material, batch number: 121100-201906, was purchased from China Food and Drug Inspection Institute.

[0074] Wangbaisuoside H reference substance, batch number: PS012362, purity ≥98.0%, Chengdu Pusi Biotechnology Co., Ltd.

[0075] Standard decoction freeze-dried powder can be prepared by conventional technical means in the field. In the present invention, the lily (lily) standard decoction freeze-dried powder, the lily (Tiger Lily) standard decoction freeze-dried powder, and the lily (Lilium ulmoides) standard decoction freeze-dried powder are respectively prepared using lily (lily) medicinal materials, lily (Tiger Lily) medicinal materials, and lily (Lilium ulmoides) medicinal materials according to the following methods.

[0076] Take lily medicinal materials and process them (processing process: lily, remove impurities) into lily slices that meet the requirements according to the relevant provisions of the 2020 edition of the Chinese Pharmacopoeia. Take about 150g of lily slices, place them in a sand pot, add 10 times the weight of water and soak for 30 minutes, first boil over high heat, then simmer for 30 minutes, filter while hot through a 200-mesh filter cloth, add 8 times the weight of water to the residue, boil over high heat, then simmer for 25 minutes, filter while hot through a 200-mesh filter cloth, and combine the filtrate; the filtrate is concentrated under reduced pressure (65°C) to a fluid extract with a relative density of about 1.02; freeze-dry, take out, mix, package, and seal to obtain the lily standard decoction freeze-dried powder.

[0077] Example 1

[0078] This embodiment provides a method for constructing a characteristic spectrum of lily medicinal material, comprising the following steps:

[0079] (1) Preparation of test solution: Lilium bulgurii (Lilium) was used as the test sample. 1 g of the test sample was placed in a stoppered conical flask, 50 ml of water was added, and the solution was heated under reflux for 30 minutes. The solution was filtered and the filtrate was evaporated to dryness. The residue was ultrasonically treated with 20 ml of 50% methanol (250 W, 40 kHz) for 30 minutes. The solution was cooled, mixed, filtered, and the filtrate was used as the test solution.

[0080] (2) High performance liquid chromatography: Accurately pipette 10 μl of the sample solution and inject it into the liquid chromatograph for determination. The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), the chromatographic column is Yuexu Ultimate XB-C18, 4.6 mm × 250 mm, 5 μm column; acetonitrile is used as mobile phase A, 0.1% phosphoric acid is used as mobile phase B, and gradient elution is performed according to the provisions in the table below; the flow rate is 0.8 ml per minute; the column temperature is 25°C; the detection wavelength is 205 nm, and the wavelength is cut to 310 nm after 15 minutes. The number of theoretical plates calculated based on Wangliuside A should be no less than 3000.

[0081]

[0082] The results are shown in the following table.

[0083] Table 1 Peak results of lily medicinal materials

[0084]

[0085] The results are shown in the table above and Figure 1 As shown, there are 9 characteristic peaks in the characteristic spectrum of Lilium (Lilium) medicinal material. Taking peak 4 as the reference peak, the relative retention times of peaks 1 to 3 and peaks 5 to 9 and peak 4 are 0.50, 0.62, 0.91, 1.02, 1.03, 1.30, 1.44 and 1.47, respectively.

[0086] Example 2

[0087] This embodiment provides a method for constructing a characteristic spectrum of lily slices, comprising the following steps:

[0088] (1) Preparation of test solution: Take lily medicinal material, remove impurities and prepare lily (lily) slices. Take lily (lily) slices as the test sample, take 1g of the test sample, place it in a stoppered conical flask, add 50ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, and treat the residue with 20ml of 50% methanol by ultrasonic treatment (250W, 40KHz) for 30 minutes, let it cool, mix well, filter, and take the filtrate as the test solution.

[0089] (2) Preparation of reference solution: Take appropriate amounts of lirioside H, lirioside A, and 2-acetyllirioside A reference substances, accurately weigh them, add 50% methanol to make a mixed solution containing 40 μg of lirioside H, 40 μg of lirioside A, and 40 μg of 2-acetyllirioside per 1 ml, shake well, and use as the reference solution;

[0090] (3) High performance liquid chromatography: Accurately aspirate 10 μl of the test solution and the reference solution, inject them into the liquid chromatograph, and measure. The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile is used as mobile phase A, 0.1% phosphoric acid is used as mobile phase B, and gradient elution is performed according to the provisions in the table below; the flow rate is 0.8 ml per minute; the column temperature is 25°C; the detection wavelength is 205 nm, and the wavelength is cut to 310 nm after 15 minutes. The theoretical plate number calculated based on lilyside A should be not less than 3000.

[0091]

[0092] The results are shown in the following table.

[0093] Table 2 Peak results of lily slices

[0094]

[0095] The results are shown in the table above and Figure 2 As shown in the figure, there are 9 characteristic peaks in the characteristic spectrum of lily (lily) slices. Taking peak 4 as the reference peak, the relative retention times of peaks 1 to 3 and peaks 5 to 9 to peak 4 are 0.50, 0.62, 0.91, 1.02, 1.03, 1.30, 1.44 and 1.46 respectively. Among them, peaks 3 and 4 are lilyside H peaks and lilyside A peaks, and there is no 2-acetyllilyside A peak.

[0096] Example 3

[0097] This embodiment provides a method for constructing a characteristic spectrum of lychee standard decoction freeze-dried powder, comprising the following steps:

[0098] (1) Preparation of test solution: Preparation of test solution: Take lily (lily) standard decoction freeze-dried powder as the test sample, batch number 21051801 (corresponding medicinal material is RK21050703), take 0.2g of the test sample, accurately weigh it, put it in a stoppered conical flask, add 20ml of 50% methanol, weigh it, and treat it with ultrasound (250W, 40KHz) for 30 minutes. Let it cool, make up the lost weight with 50% methanol, mix it, filter it, and take the filtrate to obtain the solution.

[0099] (2) Preparation of reference solution: same as in Example 2.

[0100] (3) High performance liquid chromatography: same as in Example 2.

[0101] Table 3 Peak results of lily standard decoction freeze-dried powder

[0102]

[0103] The results are shown in the table above and Figure 3 As shown in the figure, there are 9 characteristic peaks in the characteristic spectrum of lily (lily) slices. Taking peak 4 as the reference peak, the relative retention times of peaks 1 to 3 and peaks 5 to 9 relative to peak 4 are 0.45, 0.58, 0.90, 1.02, 1.03, 1.25, 1.46 and 1.50, respectively. Among them, peaks 3 and 4 are the peaks of lilyside H and lilyside A, and there is no 2-acetyllilyside A peak.

[0104] Example 4

[0105] This embodiment provides a method for constructing a characteristic spectrum of lily medicinal material, comprising the following steps:

[0106] (1) Preparation of test solution: 18 batches of lily (Lilium) medicinal materials were used as test samples, and the test solution was prepared according to the method of Example 1;

[0107] (2) Preparation of reference solution: Take about 1 g of Lilium bulbiferum (Lilium bulbiferum) as a reference medicinal material, place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of 50% methanol to the residue, sonicate (250 W, 40 kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Take appropriate amounts of Wangliuside H and Wangliuside A reference substances, accurately weigh them, and add 50% methanol to make a solution containing 40 μg of each reference substance per 1 ml.

[0108] (3) High performance liquid chromatography: Accurately pipette 10 μl of the sample solution and inject it into a liquid chromatograph for determination. The chromatographic conditions are the same as those in Example 1.

[0109] The characteristic spectra of the 18 samples (such as Figure 4 As shown in Figure 2), the fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate a reference characteristic spectrum. By identifying and specifying the characteristic peaks, the HPLC characteristic spectrum of lily medicinal material obtained had a total of 9 chromatographic peaks, and the chromatographic peaks were arranged in order of sequence (as shown in Figure 2). Figure 5 (as shown). Based on the results of peak identification and reference substance positioning, Peak 3 was determined to be wanglioside H and Peak 4 to be wanglioside A. Based on the peak identification and peak selection results for characteristic peaks in lily medicinal materials, the reference substance peak with a larger response and better stability was wanglioside A, which served as the indicator component for content determination. In summary, wanglioside A was designated as the S peak of this characteristic spectrum. The relative retention times of the characteristic peaks were calculated, with wanglioside A as the S peak. The relative peak areas of the peaks and the S peaks were also calculated.

[0110] According to the peaks of the control medicinal materials and 18 batches of medicinal materials corresponding to the peak of the reference material (Wang Bailioside A), the relative retention times of each characteristic peak and the S peak were calculated, and they were all within the range of ±10% of the mean. Therefore, the characteristic spectrum standard of lily medicinal materials was established, that is, 9 characteristic peaks should be presented in the characteristic spectrum of the test sample, and the relative retention times should correspond to the 9 characteristic peaks in the chromatogram of the control medicinal material reference material. The peak corresponding to the Wang Bailioside A reference material peak was taken as the S peak, and the relative retention times of peaks 1 to 3, peaks 5 to 9 and the S peak were calculated. It is stipulated that the relative retention time of each characteristic peak should be within the range of ±10% of the specified value, and the specified values ​​are: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), and 1.46 (peak 9).

[0111] The peak areas of the characteristic spectra of 18 batches of medicinal materials vary greatly. The relative peak areas of peaks in multiple batches of medicinal materials compared with peak 1 of wanglioside A range from 0.254 to 0.801, the relative peak areas of peak 2 range from 0.348 to 0.980, the relative peak areas of peak 3 range from 0.109 to 0.158, the relative peak areas of peak 5 range from 0.093 to 0.275, the relative peak areas of peak 6 range from 0.036 to 0.208, the relative peak areas of peak 7 range from 0.022 to 0.096, the relative peak areas of peak 8 range from 0.158 to 0.490, and the relative peak areas of peak 9 range from 0.331 to 0.525. Combined with the results of characteristic spectrum identification, peak 3 is wanglioside H and peak 4 is wanglioside A. Combined with the characteristic spectra of different sources, the relative peak areas of peak 9 and peak 4 are tentatively determined.

[0112] Table 4 Relative retention time of characteristic peaks

[0113]

[0114]

[0115] Table 5 Relative peak areas of characteristic peaks

[0116]

[0117]

[0118] Example 5

[0119] This embodiment provides a method for constructing a characteristic spectrum of lily slices, comprising the following steps:

[0120] (1) Preparation of test solution: 18 batches of lily (lily) slices were used as test samples, and the test solution was prepared according to the method of Example 2;

[0121] (2) High Performance Liquid Chromatography: Accurately pipette 10 μl of the sample solution and inject it into a liquid chromatograph for determination. The chromatographic conditions are the same as those in Example 2.

[0122] The characteristic spectra of the 18 samples (such as Figure 6 As shown in Figure 2), the fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate a reference characteristic spectrum. By identifying and specifying the characteristic peaks, the HPLC characteristic spectrum of lily medicinal material obtained had a total of 9 chromatographic peaks, and the chromatographic peaks were arranged in order of sequence (as shown in Figure 2). Figure 7 The results are shown in the table below. Taking the peak corresponding to the Wang Bailioside A reference peak as the S peak, the relative retention times of peaks 1 to 3, peaks 5 to 9, and the S peak in the characteristic spectra of 18 batches of lily (lily) slices are all within the range of ±10% of the specified values. The specified values ​​are: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), and 1.46 (peak 9), all of which meet the requirements. The relative peak area of ​​peak 9 and peak 4 is not less than 0.23.

[0123] Table 6 Relative retention time of characteristic peaks of decoction pieces

[0124]

[0125]

[0126] Table 7 Relative peak areas of characteristic peaks of decoction pieces

[0127]

[0128]

[0129] Example 6

[0130] This embodiment provides a method for constructing a characteristic spectrum of lychee standard decoction freeze-dried powder, comprising the following steps:

[0131] (1) Preparation of test solution: 18 batches of lily (Lilium) medicinal materials were used as the test sample to obtain freeze-dried powder of standard decoction, and the test solution was prepared according to the method of Example 3;

[0132] (2) High Performance Liquid Chromatography: Accurately pipette 10 μl of the sample solution and inject it into a liquid chromatograph for determination. The chromatographic conditions are the same as those in Example 3.

[0133] The characteristic spectra of the 18 samples were obtained using the fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee to generate a reference characteristic spectrum. By identifying and assigning characteristic peaks, the obtained HPLC characteristic spectrum of lily medicinal material had a total of 9 chromatographic peaks, and the chromatographic peaks were numbered 1 to 9 according to the order of the chromatographic peaks (such as Figure 7 As shown in the figure, the peak corresponding to the peak of the reference substance Wang Bailioside A is designated as the S peak. The relative retention times of peaks 1 to 3, 5 to 9, and the S peak in the characteristic spectrum of 18 batches of lily (lily) standard decoction freeze-dried powder are all within the range of ±10% of the specified values. The specified values ​​are: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), and 1.46 (peak 9), all of which meet the requirements. In addition, the relative peak area of ​​peak 9 to peak 4 is not less than 0.23.

[0134] Table 8 Relative retention time results of characteristic peaks of 18 batches of lily (lily) standard decoction (lyophilized powder)

[0135]

[0136]

[0137] Table 9 Results of relative peak areas of characteristic spectra of 18 batches of lily (lily) standard decoction (lyophilized powder)

[0138]

[0139] Example 7

[0140] This embodiment provides a method for identifying lily medicinal materials of different origins, comprising the following steps:

[0141] (1) Preparation of test solution: 6 batches of Lilium (Lilium spp.) and 2 batches of Lilium (Lilium ulmoides) were used as test samples to prepare test solution according to the following method: 1 g of the test sample was placed in a stoppered conical flask, 50 ml of water was added, and the solution was heated under reflux for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was ultrasonically treated with 20 ml of 50% methanol (250 W, 40 kHz) for 30 minutes, cooled, mixed, filtered, and the filtrate was used as the test solution.

[0142] (2) Preparation of reference solution: Take approximately 1 g of Lilium (Liu Dan) reference medicinal material (provided by the China Food and Drug Administration) and Lilium (Lily) reference medicinal material (provided by the China Food and Drug Administration), place in a stoppered conical flask, add 50 ml of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of 50% methanol to the residue, ultrasonically treat (250 W, 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Take an appropriate amount of 2-acetyl lilyoside A reference substance, accurately weigh it, and add 50% methanol to make a solution containing 40 μg of 2-acetyl lilyoside A per 1 ml.

[0143] (3) High performance liquid chromatography: Accurately aspirate 10 μl of the test solution and reference solution respectively, inject them into the liquid chromatograph, and measure. The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile is used as mobile phase A, 0.1% phosphoric acid is used as mobile phase B, and gradient elution is performed according to the provisions in the table below; the flow rate is 0.8 ml per minute; the column temperature is 25°C; the detection wavelength is 205 nm, and the wavelength is cut to 310 nm after 15 minutes. The theoretical plate number calculated based on lilyside A should be not less than 3000.

[0144]

[0145] The relative peak areas of the characteristic spectrum of Lilium (Lilium) medicinal materials were compared with those of 18 batches of Lilium (Lilium) medicinal materials, and the results are shown in the following table.

[0146] Table 10 Relative peak areas of characteristic spectrum of Lilium chinense

[0147]

[0148] Compared with Lilium (Lilium) medicinal materials, the relative peak areas of the characteristic spectrum of Lilium (Lilium ulmoides) medicinal materials were compared with 18 batches of Lilium (Lilium) medicinal materials, and the results are shown in the following table.

[0149] Table 11 Relative peak areas of characteristic spectrum of Lilium tenuifolium medicinal materials

[0150]

[0151]

[0152] See the results Figure 8 and 9 As shown in the above table, compared with lily (lily) medicinal materials, the characteristic peak response value of peak 9 in the characteristic spectrum of lily (frigate) medicinal materials and lily (lilium) medicinal materials is extremely low, which is significantly lower than that of lily (lily) medicinal materials. For example, when the relative peak area of ​​peak 9 and peak 4 is less than 0.23, it is lily (frigate or lilium) medicinal materials; when the relative peak area of ​​peak 9 and peak 4 is greater than or equal to 0.23, it is lily (lily) medicinal materials.

[0153] Moreover, the chromatographic peak of 2-acetyllilyoside A does not exist in the characteristic spectra of Lilium (Lilium) and Lilium (Lilium ulmoides) medicinal materials, but peaks 11 and 12 are obviously present in Lilium (Lilium) medicinal materials, while they are absent in Lilium (Lilium) medicinal materials and Lilium (Lilium ulmoides) medicinal materials.

[0154] The lily medicinal material of the origin of Lilium scutellariae has 10 characteristic peaks, the peak corresponding to the peak of the reference substance of lilyoside A is the S peak, and the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), and 1.46 (peak 9); a chromatographic peak appears at the corresponding position of the chromatographic peak of the reference substance 2-acetyllilyoside A (peak 10);

[0155] The lily medicinal material of the lily of tenuifolia origin has 11 characteristic peaks, and the peak corresponding to the peak of the reference substance of lilyoside A is the S peak. The relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), 1.46 (peak 9), 1.49 (peak 11), and 1.50 (peak 12).

[0156] Example 8

[0157] This embodiment provides a method for identifying freeze-dried powders of lily standard decoctions of different origins, comprising the following steps:

[0158] (1) Preparation of test solution: 6 batches of freeze-dried powder of standard decoction of lily (Littoria dasyphylla) and 2 batches of freeze-dried powder of standard decoction of lily (Lilium ulmoides) were used as test samples. About 0.2 g of test sample powder was accurately weighed and placed in a stoppered conical flask. 20 ml of 50% methanol was accurately added and weighed. Ultrasonic treatment (250 W, 40 kHz) was performed for 30 minutes. The solution was cooled and weighed again. The lost weight was supplemented with 50% methanol. The solution was mixed and filtered. The filtrate was obtained.

[0159] (2) Preparation of reference solution: Take approximately 1 g of Lilium lily (Liu Dan) reference medicinal material (provided by the China Food and Drug Administration), place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of 50% methanol to the residue, ultrasonically treat (250W, 40 kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Take an appropriate amount of 2-acetyl lilyoside A reference substance, accurately weigh it, and add 50% methanol to make a solution containing 40 μg of 2-acetyl lilyoside A per 1 ml.

[0160] (3) High performance liquid chromatography: same as in Example 7.

[0161] The results are as follows Figure 10 and 11 As shown in the table below, there is a chromatographic peak of 2-acetyl lilyoside A in the characteristic spectrum of the freeze-dried powder of the standard decoction of Lilium (Lilium spp.), but there is no chromatographic peak of 2-acetyl lilyoside A in the freeze-dried powder of the standard decoction of Lilium (Lilium spp.) and Lilium tiliaceum. There is no chromatographic peak of 2-acetyl lilyoside A in the characteristic spectrum of the freeze-dried powder of Lilium tiliaceum, but there are obvious chromatographic peaks 11 and 12.

[0162] The relative peak areas of the characteristic spectrum of the freeze-dried powder of the standard decoction of Lilium (Liriodendron dasyphyllum) were compared with those of 18 batches of freeze-dried powder of the standard decoction of Lilium (Liriodendron dasyphyllum) and the results are shown in the following table.

[0163] Table 12 Relative peak areas of characteristic spectrum of standard decoction of Lilium leucophyllum

[0164]

[0165]

[0166] The relative peak areas of the characteristic spectrum of the freeze-dried powder of the standard decoction of Lilium (Lilium ulmoides) were compared with those of 18 batches of freeze-dried powder of the standard decoction of Lilium (Lilium ulmoides). The results are shown in the following table.

[0167] Table 13 Relative peak areas of characteristic spectrum of standard decoction of Lilium truncatum

[0168]

[0169] See the results Figure 10 and 11As shown in the table above, the relative peak area of ​​peak 8 in 18 batches of lily (Lily) standard decoction freeze-dried powder ranges from 0.19 to 0.28, with an average of 0.24, and the relative peak area of ​​peak 9 ranges from 0.315 to 0.44, with an average of 0.37. The peak area of ​​peak 8 in lily of the genus Lilium freeze-dried powder ranges from 0.00 to 0.01, and the relative peak area of ​​peak 9 ranges from 0.04 to 0.07. Peak 8 and peak 9 in lily (Liriodendron) standard decoction freeze-dried powder are difficult to integrate. Based on comprehensive considerations, the relative peak area of ​​Peak 9 of the freeze-dried powder of the standard decoction of Lilium is regulated, and it is recommended to follow the regulation of -30% of the lower limit of the actual measurement range of multiple batches (Peak 9: 0.315*0.7=0.2205, rounded to 0.23). That is, when the relative peak area of ​​Peak 9 and Peak 4 in the freeze-dried powder of the standard decoction of Lilium to be identified is greater than or equal to 0.23, it is freeze-dried powder of Lilium (Lilium); when it is less than 0.23, it is freeze-dried powder of Lilium (Littoral Lilium) or Lilium (Lilium ulmoides) standard decoction; and when the chromatographic peak of 2-acetyllioside A is present, it is freeze-dried powder of Lilium (Littoral Lilium) standard decoction.

[0170] The freeze-dried powder of the standard decoction of Lilium sulcatum has 10 characteristic peaks, the peak corresponding to the peak of the reference substance of lilyoside A is the S peak, and the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, which is: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), and 1.46 (peak 9); a chromatographic peak (peak 10) appears at the corresponding position of the chromatographic peak of the reference substance of 2-acetyllilyoside A (peak 10);

[0171] The freeze-dried powder of the lily standard decoction based on lily of tenuifolia has 11 characteristic peaks, and the peak corresponding to the peak of the reference substance of lilyoside A is the S peak. The relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 0.50 (peak 1), 0.62 (peak 2), 0.91 (peak 3), 1.02 (peak 5), 1.04 (peak 6), 1.30 (peak 7), 1.43 (peak 8), 1.46 (peak 9), 1.49 (peak 11), and 1.50 (peak 12).

[0172] Example 9

[0173] 1. Instruments:

[0174] High performance liquid chromatograph 1: Waters E2695, including G7104A quaternary pump, 132-position autosampler, G7116B column oven, and diode array detector.

[0175] High performance liquid chromatograph 2: Agilent 1260, including G7111A Quat Pump VL; G7129A 1260 Vialsamplar; G7114A 1260 VWD.

[0176] Electronic analytical balance: METTLER TOLEDO (Mettler, Switzerland) ME36S, XS204, XS205, XSE205 (1 / 100,000); SK5200H Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0177] Column 1: Agilent SB AQ, 4.6 x 250 mm, 5 μm;

[0178] Column 2: YMC–Pack ODS–AQ, 4.6 x 250 mm, 5 μm;

[0179] Column 3: Ultimate XB-C18, 4.6 x 250 mm, 5 μm;

[0180] 2. Reagents and test drugs:

[0181] Reagents: Acetonitrile was of chromatographic grade, water was ultrapure water; other reagents such as phosphoric acid were of analytical grade.

[0182] Wangliuside A reference substance (batch number: 114420-66-5, purchased from Chengdu Pusi Biotechnology Co., Ltd.)

[0183] Lilium control medicinal material (batch number: 121100-201906, purchased from China Food and Drug Inspection Institute).

[0184] Preparation method of test solution: same as Example 7.

[0185] 3. Experimental study on chromatographic conditions

[0186] (1) Screening experiment of chromatographic columns

[0187] The above test solution was tested using the following three different chromatographic columns.

[0188] Chromatographic conditions 1: Agilent SB AQ, 4.6 x 250 mm, 5 μm column, acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, elution according to gradient 1; flow rate, 0.8 ml / min; column temperature, 25°C; detection wavelength, 205 nm, with a wavelength cut to 310 nm at 15 minutes.

[0189] Chromatographic Conditions 2: YMC–Pack ODS–AQ, 4.6 x 250 mm, 5 μm column, mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid, gradient 1; flow rate: 0.8 ml / min; column temperature: 25°C; detection wavelength: 205 nm, cutoff: 310 nm at 15 minutes. Chromatographic Conditions 3: Ultimate XB-C18, 4.6 x 250 mm, 5 μm column, mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid, gradient 1; flow rate: 0.8 ml / min; column temperature: 25°C; detection wavelength: 205 nm, cutoff: 310 nm at 20 minutes.

[0190] Test results see Figure 12-14 As shown in the figure, the Ultimate XB-C18 column has better separation of characteristic chromatographic peaks than the YMC–Pack ODS–AQ and YMC–Pack ODS–AQ columns and has more target peaks. Therefore, the Ultimate XB-C18 4.6x250mm 5μm column was selected for further investigation.

[0191] (2) Gradient Optimization

[0192] The sample solution was tested using the following gradient elution program: acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, and gradient elution program 1; the flow rate was 0.8 ml / min; the column temperature was 25°C; the detection wavelength was 205 nm, and gradients 1 to 3 were 20 minutes with a cutoff wavelength of 310 nm. Gradients 4 to 7 were 15 minutes with a cutoff wavelength of 310 nm.

[0193] Table 14 Gradient elution program 1

[0194]

[0195] Table 15 Gradient elution program 2

[0196]

[0197] Table 16 Gradient elution program 3

[0198]

[0199]

[0200] Table 17 Gradient elution program 4

[0201]

[0202] Table 18 Gradient elution program 5

[0203]

[0204] Table 19 Gradient elution program 6

[0205]

[0206] Table 20 Gradient elution program 7

[0207]

[0208] Table 21 Peak results

[0209]

[0210]

[0211] The results are shown in the table above and Figure 15-21 As shown, compared with gradient elution programs 1-5, the optimized gradient elution program 7 of the present invention has 9 characteristic peaks, and each characteristic peak is well separated, and the separation degree of each characteristic peak reaches above 1.5, so the gradient elution program 7 is tentatively selected for the next investigation.

[0212] (3) Different instruments

[0213] According to the above method, samples were injected on chromatographs of different brands (Waters E2695, Agilent 1290Ⅱ and Thermo U3000), and the rest of the chromatographic conditions were the same as in Example 1. The durability of different instruments was investigated. The results showed that the RSDs of the relative retention times of the nine characteristic peaks were within ±10% of the specified values, indicating that the optimized method had good instrument durability.

[0214] 4. Characteristic peak identification

[0215] Preparation of each reference solution: Take appropriate amount of lirioside H, lirioside A, and 2-acetyllirioside A reference substances, accurately weigh them, add 50% methanol to make a mixed solution containing 40 μg of lirioside H, 40 μg of lirioside, and 40 μg of 2-acetyllirioside per 1 ml, shake well, and use as reference solution;

[0216] The reference solution and the test solution (prepared by the method of Example 1) were respectively aspirated and tested under the chromatographic conditions of Example 1 to obtain a characteristic spectrum. Detection was performed using ultraviolet chromatography.

[0217] like Figure 22 and 23 As shown, by comparing with the chromatogram of the reference solution, it was determined that peak 3 in the characteristic spectrum of lily (lily) medicinal material is lilyside H; peak 4 is lilyside A; and there is no chromatographic peak of 2-acetyllilyside A.

[0218] Example 10 Methodology Verification

[0219] 1. Precision

[0220] (1) Instrument precision

[0221] About 1 g of lily (Lilium bulgur) was accurately weighed and a test solution was prepared according to the method of Example 1. The sample was injected 6 times continuously, and the chromatogram was recorded. The RSD% of the relative retention time and relative peak area of ​​the 9 characteristic peaks of the present invention was calculated. The results showed that the RSD% of the relative retention time and relative peak area of ​​the 9 characteristic peaks were all less than 3%. Comprehensive judgment showed that the precision of the method was good and met the requirements of the characteristic spectrum.

[0222] Take about 0.2g of the standard decoction of lily (lily) slices (lyophilized powder), accurately weigh it, prepare the test solution according to the method of Example 3, inject it 6 times continuously, record the chromatogram, calculate the relative retention time and relative peak area RSD% of the 9 characteristic peaks of the present invention, and the results show that the relative retention time and relative peak area RSD% of the 9 characteristic peaks are all less than 2%. Comprehensive judgment shows that the precision of the method is good and meets the requirements of the characteristic spectrum.

[0223] (2) Repeatability experiment

[0224] About 1 g of lily (Lilium bulgurii) from the same batch was accurately weighed and 6 test solutions were prepared in parallel according to the method of Example 1. The samples were injected and analyzed, and the chromatograms were recorded. The relative retention times and relative peak areas RSD% of the 9 characteristic peaks of the present invention were calculated. The results showed that the relative retention times and relative peak areas RSD% of the 9 characteristic peaks were all less than 2%. Overall judgment showed that the precision of the method was good and met the requirements of the characteristic spectrum.

[0225] Take about 0.2g of the standard decoction (lyophilized powder) of lily (lily) slices from the same batch, accurately weigh it, prepare 6 test solutions in parallel according to the method of Example 3, inject the sample for analysis, record the chromatogram, and calculate the relative retention time and relative peak area RSD% of the 9 characteristic peaks of the present invention. The results show that the relative retention time and relative peak area RSD% of the 9 characteristic peaks are all less than 2%. Comprehensive judgment shows that the precision of the method is good and meets the requirements of the characteristic spectrum.

[0226] (3) Different personnel (intermediate precision)

[0227] Using the same batch of lily (Lilium bulgur) medicinal materials, independent experimenters A, B, and C prepared test solutions according to the method of Example 1. The samples were injected and analyzed under the chromatographic conditions of Example 1. The chromatograms were recorded and the relative retention times and relative peak areas of the nine characteristic peaks of the present invention were calculated. The results showed that the relative retention times and relative peak areas of the nine characteristic peaks had an RSD% of less than 3%. Overall, the intermediate precision (different experimenters) of the method was good and met the requirements of the characteristic spectrum.

[0228] The same batch of lily (lily) slices standard decoction (lyophilized powder) was taken and independently operated by experimenters A, B, and C. The test solution was prepared according to the method of Example 3, and the sample was injected and analyzed under the chromatographic conditions of Example 3. The chromatogram was recorded and the relative retention time and relative peak area RSD% of the 9 characteristic peaks of the present invention were calculated. The results showed that the relative retention time and relative peak area RSD% of the 9 characteristic peaks were less than 2%. Comprehensive judgment showed that the intermediate precision (different personnel) of the method was good and met the requirements of the characteristic spectrum.

[0229] 2. Stability

[0230] A test solution of the same lily (Lilium bulbiferum) medicinal material prepared according to the method of Example 1 was measured at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h under the chromatographic conditions of Example 1. The chromatogram was recorded, and the relative retention time and relative peak area RSD% of each characteristic peak were calculated to investigate the stability of the test solution. The results showed that the relative retention time and relative peak area RSD% of the nine characteristic peaks were less than 2%. Overall, the method was considered to be stable and met the requirements of the characteristic spectrum.

[0231] Take the same lily (lily) slice standard decoction (lyophilized powder) test solution prepared by the method of Example 3, and measure it according to the chromatographic conditions of Example 3 at 0h, 4h, 8h, 12h, 16h, and 24h, record the chromatogram, and calculate the relative retention time and relative peak area RSD% of each characteristic peak to investigate the stability of the test solution. The results show that the relative retention time and relative peak area RSD% of the 9 characteristic peaks are less than 2%. Comprehensive judgment shows that the stability of the method is good and meets the requirements of the characteristic spectrum.

[0232] 3. Exclusivity

[0233] Take about 1g of lily (lily) medicinal material, accurately weigh it, prepare the test solution according to the method of Example 1, take the test sample and use 50% methanol as the extraction solvent, accurately draw 10μL of the test sample solution and negative control solution (50% methanol), respectively, inject them into the high performance liquid chromatograph, and test according to the method of Example 1. The results show negative and no interference.

[0234] Take about 0.2 g of the standard decoction of lily (lily) slices (lyophilized powder), accurately weigh it, and prepare the test solution according to the method of Example 3. The test sample uses 50% methanol as the extraction solvent. Accurately draw 10 μL of the test solution and the negative control solution (50% methanol), respectively, and inject them into the high performance liquid chromatograph. The test is carried out according to the method of Example 3. The results show that the results are negative and there is no interference.

[0235] 4. Durability

[0236] (1) Different flow rates

[0237] Take the same lily (lily) medicinal material test solution prepared by the method of Example 1, and test it at different flow rates of 0.75mL / min, 0.80mL / min and 0.85mL / min, respectively. The other conditions are the same as in Example 1. Record the chromatogram and calculate the relative retention time and relative peak area RSD% of each characteristic peak. The results show that the relative retention time RSD% of the 9 characteristic peaks is 0% to 3.7%, and the relative peak area RSD% is between 0% and 8.6%. It can be seen from the figure that the flow rate fluctuation has little effect on the characteristic peaks.

[0238] Take the same portion of the test solution of the standard decoction of lily (lily) slices (lyophilized powder) prepared by the method of Example 3, and test it at different flow rates of 0.75mL / min, 0.80mL / min and 0.85mL / min, respectively. The other conditions are the same as those in Example 1. Record the chromatogram and calculate the relative retention time and relative peak area RSD% of each characteristic peak. The results are as follows. The results show that the relative retention time RSD% of the 9 characteristic peaks is 0% to 3.6%, and the relative peak area RSD% of the 9 characteristic peaks is between 0% and 5.4%. As can be seen from the figure, different flow rates have little effect on the characteristic peaks.

[0239] (2) Different column temperatures

[0240] The same lily (lily) medicinal material test solution prepared by the method of Example 1 was tested at 22°C, 25°C and 28°C, respectively. The other conditions were the same as in Example 1. The chromatogram was recorded and the relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results showed that the relative retention time RSD% of the 9 characteristic peaks was between 0% and 7.9%, and the relative peak area RSD% of the 9 characteristic peaks was between 0% and 9.7%. It can be seen from the figure that different column temperature conditions have little effect on the characteristic peaks.

[0241] Take the same lily (lily) slice standard decoction (lyophilized powder) test solution prepared by the method of Example 3 and test at 22 ℃, 25 ℃ and 28 ℃ respectively. The other conditions are the same as those in Example 1. Record the chromatogram and calculate the relative retention time and relative peak area RSD% of each characteristic peak. The results are as follows. The results show that the relative retention time RSD% of the 9 characteristic peaks is between 0% and 7.8%, and the relative peak area RSD% of the 9 characteristic peaks is between 0% and 5.7%. As can be seen from the figure, the influence of different column temperature conditions on the characteristic peaks is small.

[0242] (3) Different instruments

[0243] The same tester took the same lily (Lilium bulbiferum) medicinal material test solution prepared according to the method of Example 1 at different times and tested it on different instruments of Agilent and Waters respectively. The other conditions were the same as those in Example 1. The chromatograms were recorded and the RSD% of the relative retention time and relative peak area of ​​each characteristic peak were calculated. The results showed that the RSD of the relative retention time of the 9 characteristic peaks was between 0% and 8.3%, which was within the range of ±10% of the specified value.

[0244] 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 a lily medicinal material of the base of Lilium and its preparation, characterized in that: The following steps are included: (1) Preparation of a lily test sample solution; comprising: 1) taking a lily test sample, extracting it with water to obtain an extract; 2) subjecting the extract to solid-liquid separation, drying, and extracting it with an alcohol-water solution to obtain an extract; 3) subjecting the extract to solid-liquid separation, and obtaining a liquid, which is the test sample solution; or comprising: 1) taking a lily test sample, extracting it with an alcohol-water solution to obtain an extract; 2) subjecting the extract to solid-liquid separation, and obtaining a liquid, which is the test sample solution; (2) The lily test solution and the reference solution were detected by high performance liquid chromatography using an Ultimate XB-C18 column with a specification of 4.6x250mm and 5μm. The detection wavelength was 203-208nm before 14-20min and switched to 308-313nm after 14-20min. The mobile phase A was acetonitrile and the mobile phase B was 0.05-0.2vt% phosphoric acid aqueous solution. The gradient elution program was: 0→10min→15min→23min→30min→35min. The volume percentage of acetonitrile in the mobile phase was 6%→12%→18%→18%→40%→70%. Reference solutions were prepared using oxaliside A, oxaliside H, and 2-acetyloxaliside A reference substances.

2. The construction method according to claim 1, characterized in that Step (1) also satisfies any one or more of the following A and F: A. The water extraction is performed by adding water and then heating under reflux for at least 20 minutes; B. The mass-to-volume ratio of the lily sample to water is 0.5-2:50, and the mass-to-solvent ratio is g / mL; C. The alcohol-water extraction is performed by adding an alcohol-water solution and then performing reflux extraction or ultrasonic extraction, and the extraction time is 10 min-5 h; D. The alcohol aqueous solution is selected from an ethanol aqueous solution or a methanol aqueous solution with a volume percentage of 30%-70%; E. The solid-liquid separation is centrifugation or filtration; F. The ratio of the mass of the lily test sample to the volume of the alcohol-water solution is 0.2-2:20, and the ratio of the mass to the solvent is g / mL.

3. The construction method according to claim 2, characterized in that The water extraction is performed by adding water and then heating under reflux for 20-60 minutes.

4. The construction method according to any one of claims 1 to 3, characterized in that The flow rate is 0.6-1.0 ml / min, the column temperature is 22-28°C, and the injection volume is 1-20 μl.

5. The construction method according to claim 1, characterized in that The solvent used for the reference solution is 30-70vt% methanol aqueous solution.

6. The construction method according to claim 5, characterized in that: The concentration of the reference solution is 10-100µg / mL.

7. The construction method according to claim 1, characterized in that The lily medicinal material of the base of Lilium and its preparation have 10 characteristic peaks, the peak corresponding to the peak of the reference substance of Wangliuside A is the S peak, the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified values ​​of peak 1-peak 3 and peak 5-peak 9 are: 0.50, 0.62, 0.91, 1.02, 1.04, 1.30, 1.43, 1.46; chromatographic peaks appear at the corresponding positions of the chromatographic peak of 2-acetyl lilyoside A reference substance.

8. A method for identifying lily medicinal materials of different origins and their preparations, characterized in that: The method comprises the steps of constructing a characteristic spectrum of the lily product to be identified according to the construction method described in any one of claims 1 to 7, wherein if the lily product to be identified has a chromatographic peak at a position corresponding to the chromatographic peak of the 2-acetyl lilyoside A reference substance, the lily product to be identified is Lilium genistein; if the lily product to be identified has no chromatographic peak at a position corresponding to the chromatographic peak of the 2-acetyl lilyoside A reference substance, the lily product to be identified is Lilium genistein or Lilium genistein.

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