An identification method and fingerprint detection method of Tripterygium wilfordii medicinal material derived from Orchidaceae

Abstract

The present invention relates to the technical field of medicinal material quality detection, and in particular to an identification method and fingerprint detection method of a Herba Lycoris Radiatae medicinal material. The present invention provides an identification method of a Herba Lycoris Radiatae medicinal material, using at least one compound of Bletilla striata glycosides, Dactylorhin A or Pleionoid A, and at least one compound of Pleionoid B or Pleionoid C as markers for identifying Herba Lycoris Radiatae medicinal materials, and by optimizing the extraction method, chromatographic conditions, pretreatment method and thin layer development conditions, the qualitative and quantitative analysis of Herba Lycoris Radiatae medicinal materials is successfully achieved, providing a research basis for the formulation of Herba Lycoris Radiatae quality standards, the rational development and utilization of resources, etc. The present invention also provides a fingerprint detection method of Herba Lycoris Radiatae medicinal materials, which has strong specificity, is simple and feasible, and can meet the requirements of drug analysis.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal material quality detection, and in particular to an identification method and a fingerprint spectrum detection method of a Tripterygium wilfordii medicinal material derived from Orchidaceae. Background Art

[0002] Pleione bulbocodioides (Franch.) Rolfe is one of the origins of Trillium gracile medicinal materials. It has the effects of clearing away heat and detoxifying, resolving phlegm and dispersing nodules. It is often used in the treatment of various tumors clinically. It can also be used in combination with other medicinal flavors. It has the effects of enhancing efficacy and reducing toxicity, and treating urinary retention. However, the current 2020 edition of the "Chinese Pharmacopoeia" only includes its microscopic identification under Trillium gracile, without systematic qualitative and quantitative standards. This has also led to the existence of many counterfeit Trillium gracile products on the market, which has brought great safety risks to the clinical application of Trillium gracile and its preparations. Therefore, the identification and quality control of the authenticity of Trillium gracile products have become particularly important. Summary of the invention

[0003] In view of the above technical problems, the present invention provides an identification method for the medicinal material of Cymbidium dulcis, which completes the identification of the authenticity of the medicinal material of Cymbidium dulcis by identifying specific markers; the present invention also provides a fingerprint spectrum detection method for the medicinal material of Cymbidium dulcis, which is highly specific, simple and feasible, and can meet the requirements of drug analysis.

[0004] In order to solve the above technical problems, the present invention provides a method for identifying the medicinal material of Pleionoides sutchuenensis, using at least one compound of Bletilla striata glycosides, Dactylorhin A or Pleionoid A, and at least one compound of Pleionoid B or Pleionoid C as markers for identifying the medicinal material of Pleionoides sutchuenensis; wherein the chemical structural formulas of Pleionoid A, Pleionoid B and Pleionoid C are shown in Formula I, Formula II and Formula III, respectively:

[0005]

[0006] The inventors found in a large number of experimental studies that compounds Pleionoid A, Pleionoid B and Pleionoid C are unique ingredients contained in the medicinal material of D. edulis, and they, Bletilla striata glycosides and Dactylorhin A are succinic acid bibenzyl ester glycoside compounds contained in the medicinal material of D. edulis, while other counterfeit products of Psoralea corylifolia do not meet the condition of containing the five compounds at the same time. Therefore, the inventors used the above five ingredients or some of the ingredients as markers to identify the authenticity of the medicinal material of D. edulis.

[0007] Among them, compounds Pleionoid A, Pleionoid B and Pleionoid C are compounds named independently by the inventors, and their chemical structures correspond to Formula I, Formula II and Formula III, respectively.

[0008] Preferably, any two compounds of Bletilla striata glycosides, Dactylorhin A or Pleionoid A, and Pleionoid B or Pleionoid C are used as markers.

[0009] More preferably, Bletilla striata glycosides, Dactylorhin A and Pleionoid A, and Pleionoid B and / or Pleionoid C compounds are used as markers.

[0010] The second aspect of the present invention provides a method for identifying the medicinal material of Psoralea corylifolia, using ultra-high performance liquid chromatography, comprising the following steps:

[0011] Prepare a reference solution by taking at least one of Bletilla striata glycosides, Dactylorhin A or Pleionoid A, and Pleionoid B or Pleionoid C; prepare a test solution by pre-treating the medicinal material to be identified;

[0012] Respectively aspirate the reference substance mixed solution and the test substance solution, inject them into the high performance liquid chromatograph, record the reference chromatogram and the test substance chromatogram; compare the test substance chromatogram with the reference chromatogram;

[0013] The specific chromatographic conditions include:

[0014] Chromatographic column: C18 column;

[0015] Mobile phase: Mobile phase A is water, and mobile phase B is a mixture of methanol and acetonitrile in a volume ratio of 1:1;

[0016] Flow rate: 0.27~0.33mL / min;

[0017] Column temperature: 25-35°C;

[0018] Injection volume: 0.5~3μL;

[0019] Detection wavelength: 210~254nm;

[0020] The gradient elution method was used, and the elution procedure was as follows:

[0021] Time (min) Mobile phase A Mobile phase B 0～10 80％～74％ 20％～26％ 10～20 74％ 26％ 20～30 74％～58％ 26％～42％ 30～45 58％ 42％ 45～55 58％～20％ 42％～80％ .

[0022] The inventors took the above-mentioned markers as the starting point and explored a method for identifying the medicinal material of D. edulis. The method has high sensitivity, good repeatability and reliable results, which provides an important basis for the quality control and evaluation of D. edulis.

[0023] This identification method can simultaneously determine the contents of the compounds Bletilla striata glycoside, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C in the medicinal material of Dactylorhinus odoratus.

[0024] Preferably, the C18 column is selected from Waters Acquity HSS T3, Welch XB-C18 or Thermo Scientific HypersilGold TM For any one of the Vanquish, the specifications of the C18 column are 2.1×100 mm and 1.8 μm.

[0025] Preferably, the flow rate is 0.30 mL / min.

[0026] Preferably, the column temperature is 30°C.

[0027] Preferably, the injection volume is 2 μL.

[0028] Preferably, the detection wavelength is 221 nm.

[0029] In combination with the second aspect, the test solution is obtained by extracting the pseudobulbs of the Orchidaceae with an alcohol solution, and the alcohol solution includes any one of methanol, ethanol, methanol-water solution and ethanol-water solution.

[0030] For example, ultrasonic extraction can be used to extract succinic acid dibenzyl ester glycoside compounds in the pseudobulbs of Orchidaceae. The extraction can be performed in a ratio of 1g of Orchidaceae pseudobulb dry powder to 20-150mL of extract, such as 1g:50mL or 1g:100mL.

[0031] Preferably, the extraction is performed in a ratio of 1 g of dried powder of Orchidaceae pseudobulbs to 50 mL of extract.

[0032] In some embodiments, the ultrasonic time of the extraction process can be 15 to 60 minutes, preferably 30 minutes. After ultrasonic extraction, the extract can be filtered using a 0.22 μm microporous filter membrane.

[0033] In some embodiments, the alcohol solution used for the extracting solution can be anhydrous methanol, 70% methanol, 50% methanol, 10% methanol, anhydrous ethanol, 70% ethanol, 50% ethanol or 10% ethanol, preferably 50% ethanol as the extracting solution.

[0034] Preferably, the content of the succinic acid bibenzyl ester glycoside compound can be determined by an external standard method or a one-measurement-multiple-evaluation method.

[0035] Illustratively, the one-measurement-multiple-evaluation method determines the content of Bletilla striata glycoside in the medicinal material of Dactylorhinus aviculare, and then calculates the contents of Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C in the test solution, and the corresponding relative correction factors are 1.241, 0.946, 0.919 and 0.921, respectively; the external standard method uses the above-mentioned ultra high performance liquid chromatography to respectively determine the contents of blank solution, reference solution and test solution, and calculates the contents of Bletilla striata glycoside, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C in the test solution by the external standard method.

[0036] Illustratively, when the external standard method is used, the concentration range of the reference stock solution of the Bletilla striata glycoside compound is 0.00485-1.94000 mg / mL, the concentration range of the reference stock solution of Dactylorhin A is 0.00505-2.02000 mg / mL, the concentration range of the reference stock solution of Pleionoid A is 0.00200-0.20000 mg / mL, the concentration range of the reference stock solution of Pleionoid B is 0.00190-0.19000 mg / mL, and the concentration range of the reference stock solution of Pleionoid C is 0.00260-0.20800 mg / mL.

[0037] In combination with the second aspect, the identification method of the medicinal material of the Herba Lycoris Radiatae can also adopt a thin layer identification method, comprising the following steps:

[0038] Prepare a reference solution by taking at least one of Bletilla striata glycosides, Dactylorhin A or Pleionoid A, and Pleionoid B; prepare a test solution by pre-treating the medicinal material to be identified;

[0039] Pipette the reference substance mixture and the test substance solution separately and develop them on a thin layer chromatogram, and compare the test substance thin layer chromatogram with the reference substance thin layer chromatogram;

[0040] The developing solvent used for identification is a mixture of chloroform-ethyl acetate-methanol-formic acid-water in a volume ratio of 3:5:3:1.5:0.25.

[0041] Specifically, in the above thin layer identification method, the detection wavelength used is ultraviolet light and white light of 254nm; the silica gel plate used is Merck HPTLC 60F254 plate, Yantai Huanghai HPTLC GF254 plate, Qingdao Ocean HPTLC GF254 plate or Crester HPTLC GF254 plate, preferably Merck HPTLC 60F254 plate; the development distance is 70cm; the color developer is 0.1% vanillin-10% ethanol sulfate, 10% ethanol sulfate or 10% phosphomolybdic acid ethanol, preferably 0.1% vanillin-10% ethanol sulfate.

[0042] In some embodiments, appropriate heating is performed after spraying the developer, and the heating time is 5 min, 10 min or 15 min at a heating temperature of 100° C., 105° C. or 110° C., preferably 10 min at 105° C.

[0043] In some embodiments, after the test solution of the medicinal material of D. edulis is pre-treated by SPE (polyamide), the spotting volume of the test solution obtained by eluting with 20% methanol and the test solution obtained by eluting with methanol can be 5 μL, 10 μL, 20 μL or 25 μL.

[0044] Preferably, the spotting volume of the test solution obtained by eluting with 20% methanol is 10 μL, and the spotting volume of the test solution obtained by eluting with methanol is 20 μL.

[0045] In some embodiments, the spotted amount of the standard substances Dactylorhin A, Bletilla striata glycosides, Pleionoid A, and Pleionoid B can be 5 μg, 10 μg, 15 μg, or 20 μg.

[0046] Preferably, the spotting amount of the standard substances Dactylorhin A, Bletilla striata glycosides, Pleionoid A and Pleionoid B is 15 μg.

[0047] The third aspect of the present invention provides a method for detecting the fingerprint of the medicinal material of Pleionoides uniflora, which comprises constructing a fingerprint containing Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C. The fingerprint can be used to identify the authenticity of Pleionoides uniflora, providing a strong guarantee for the quality control of Pleionoides uniflora.

[0048] In conjunction with the third aspect, the detection method adopts ultra-high performance liquid chromatography, wherein the chromatographic conditions include:

[0049] Chromatographic column: C18 column;

[0050] Mobile phase: Mobile phase A is water, and mobile phase B is a mixture of methanol and acetonitrile in a volume ratio of 1:1;

[0051] Flow rate: 0.27~0.33mL / min;

[0052] Column temperature: 25-35°C;

[0053] Injection volume: 0.5~3μL;

[0054] Detection wavelength: 210~254nm;

[0055] The gradient elution method was used, and the elution procedure was as follows:

[0056] Time (min) Mobile phase A Mobile phase B 0～10 80％～74％ 20％～26％ 10～20 74％ 26％ 20～30 74％～58％ 26％～42％ 30～45 58％ 42％ 45～55 58％～20％ 42％～80％ .

[0057] The fingerprint detection method of the medicinal material of Dactylorhinus edulis provided by the present invention can be used to detect or separate the components in the medicinal material of Dactylorhinus edulis, and the components in the medicinal material of Dactylorhinus edulis include one or more of Dactylorhin E, Dactylorhin A, Bletilla striata glycoside, Pleionoside M, Pleionoid A, Pleionoid B and Pleionoid C.

[0058] In combination with the third aspect, in the fingerprint spectrum, the white glycoside peak is the S peak, and there are characteristic peaks at one or more of the following relative retention times: at least one of 0.371, 0.565, 0.605, 1.000, 1.216, 1.276, 1.368, 1.561, 1.583, 1.742 and 1.816, the absolute retention time of the S peak is 23.18±0.03min, and the error of the relative retention time is within ±5%.

[0059] As for the absolute retention time, in the fingerprint spectrum, there are characteristic peaks at one or more of the following absolute retention times: 9.279±0.013min, 14.130±0.017min, 15.147±0.020min, 25.028±0.021min, 30.432±0.012min, 31.937±0.010min, 34.231±0.024min, 39.076±0.011min, 39.626±0.019min, 43.604±0.024min and 45.467±0.034min.

[0060] Exemplarily, when the fingerprint detection method is used in the detection of the medicinal material of Herba Lycoris Radiatae, the following steps are included:

[0061] Take the test solution of the Herba Lysimachiae to be tested, and use the above-mentioned ultra-high performance liquid chromatography method to obtain a corresponding liquid chromatogram;

[0062] The obtained liquid chromatogram is evaluated for similarity with the fingerprint product.

[0063] The fingerprint provided by the present invention can also be used to produce products in different forms, such as electronic products with computer storage as the medium, or paper instructions obtained by drawing or printing. These products can be used as standard fingerprints for the detection and quality control of the medicinal material of Dictyophora indica.

[0064] The beneficial effects of the present invention are as follows: the present invention respectively provides an identification method for the medicinal material of Glechoma longituba and a detection method for the fingerprint spectrum of the medicinal material of Glechoma longituba, these methods are highly specific, simple and easy to operate, can meet the requirements of drug analysis, and have the characteristics of accurate results and convenient operation; in addition, by optimizing the extraction method, chromatographic conditions, pretreatment method and thin-layer development conditions, the content determination, similarity evaluation and thin-layer identification of the above five components in the medicinal material of Glechoma longituba are successfully realized, which provides a research basis for the formulation of the quality standard of the medicinal material, the rational development and utilization of resources, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 For the compound Pleionoid A 1 H-NMR spectrum;

[0066] Figure 2 For the compound Pleionoid A 13 C-NMR spectrum;

[0067] Figure 3 is the HMBC spectrum of compound Pleionoid A;

[0068] Figure 4 is the HSQC spectrum of compound Pleionoid A;

[0069] Figure 5 For the compound Pleionoid A 1 H- 1 H COSY spectrum;

[0070] Figure 6 For the compound Pleionoid B 1 H-NMR spectrum;

[0071] Figure 7 For the compound Pleionoid B 13 C-NMR spectrum;

[0072] Figure 8 is the HMBC spectrum of compound Pleionoid B;

[0073] Fig. 9DEPT 135° spectrum of compound Pleionoid B;

[0074] Fig.10 For the compound Pleionoid B 1 H- 1 H COSY spectrum;

[0075] Fig.11 is the HSQC spectrum of compound Pleionoid B;

[0076] Fig.12 For the compound Pleionoid C 1 H-NMR spectrum;

[0077] Fig.13 For the compound Pleionoid C 13 C-NMR spectrum;

[0078] Fig.14 is the HMBC spectrum of compound Pleionoid C;

[0079] Fig.15 DEPT 135° spectrum of compound Pleionoid C;

[0080] Fig.16 For the compound Pleionoid C 1 H- 1 H COSY spectrum;

[0081] Fig.17 is the HSQC spectrum of compound Pleionoid C;

[0082] Fig.18 The fingerprints and reference maps of 14 batches of Orchidaceae in Example 4;

[0083] Fig.19 is the control fingerprint obtained in Example 4;

[0084] Fig. 20 It is a comparison diagram of the chromatogram of the genuine and fake products in Example 4 and the chromatogram of the Orchidaceae;

[0085] Fig.21 The fingerprint peak chromatogram in Example 6;

[0086] Fig. 22 UPLC chromatograms corresponding to different chromatographic columns in Example 6;

[0087] Fig.23 UPLC chromatograms corresponding to different chromatographic column temperatures in Example 6;

[0088] Fig.24 UPLC chromatograms corresponding to different mobile phase flow rates in Example 6;

[0089] Fig.25 This is the UPLC chromatogram obtained using an Agilent 1290 Infinity II instrument in Example 6;

[0090] Fig.26 This is the UPLC chromatogram obtained using a Thermo Vanquish Scientific instrument in Example 6;

[0091] Fig. 27 The Waters Acquity TM UPLC chromatogram obtained by H-class instrument;

[0092] Fig.28 The thin layer chromatograms corresponding to different pre-treatment methods in Example 7, wherein Figure a1 is a thin layer chromatogram developed with a color developer, and Figure a2 is a thin layer chromatogram under 254 nm ultraviolet light irradiation;

[0093] Fig.29 are thin layer chromatograms obtained when different types of developing systems are used in Example 8, wherein Figure a1, Figure b1, Figure c1, and Figure d1 are thin layer chromatograms developed with corresponding developing agents and then developed with color developing agents, and Figure a2, Figure b2, Figure c2, and Figure d2 are thin layer chromatograms developed with corresponding developing agents and then irradiated with 254 nm ultraviolet light;

[0094] Fig.30 are thin layer chromatograms obtained when using different proportions of developing agents in Example 8, wherein Figure a1, Figure b1, Figure c1, Figure d1, and Figure e1 are thin layer chromatograms developed with developing agents in corresponding proportions and then developed with color developing agents, and Figure a2, Figure b2, Figure c2, Figure d2, and Figure e2 are thin layer chromatograms developed with developing agents in corresponding proportions and then irradiated with 254 nm ultraviolet light;

[0095] Fig.31 The thin layer chromatograms obtained when different types of color developers are used in Example 8, wherein Figure a, Figure b, and Figure c are thin layer chromatograms after color development using corresponding color developers, respectively;

[0096] Fig.32The thin layer chromatograms obtained when different sample amounts are applied in Example 8, wherein Figure a1, Figure b1, and Figure c1 are thin layer chromatograms of the corresponding components applied with different sample amounts and developed with a color developer, and Figure a2, Figure b2, and Figure c2 are thin layer chromatograms of the corresponding components applied with different sample amounts and developed under 254 nm ultraviolet light irradiation;

[0097] Fig.33 are thin layer chromatograms obtained under different humidity conditions in Example 8, wherein Figure a1, Figure b1, and Figure c1 are thin layer chromatograms of the developing agent containing each component after being developed under the corresponding humidity conditions and then developed by the color developer, and Figure a2, Figure b2, and Figure c2 are thin layer chromatograms of the developing agent containing each component after being developed under the corresponding humidity conditions and then irradiated with 254 nm ultraviolet light;

[0098] Fig.34 are thin layer chromatograms obtained under different temperature conditions in Example 8, wherein Figure a1 and Figure b1 are thin layer chromatograms of the developing agent containing each component after being developed at the corresponding temperature and then developed by the color developer, and Figure a2 and Figure b2 are thin layer chromatograms of the developing agent containing each component after being developed at the corresponding temperature and then irradiated with 254 nm ultraviolet light;

[0099] Fig.35 The thin layer chromatograms obtained by using different thin layer plates in Example 8, wherein Figure a1, Figure b1, Figure c1, and Figure d1 are thin layer chromatograms of the thin layer plates containing the respective components after being developed by the developer and then developed by the developer, and Figure a2, Figure b2, Figure c2, and Figure d2 are thin layer chromatograms of the thin layer plates containing the respective components after being developed by the developer and then developed by the developer under 254 nm ultraviolet light;

[0100] Fig.36 These are the thin layer chromatograms of the thin layer chromatogram for thin layer identification of the authenticity of Psoralea corylifolia in Example 9, wherein Figure a1 is a thin layer chromatogram photograph of the Psoralea corylifolia after being developed with a developing agent containing different components of authentic Psoralea corylifolia and then developed with a color developer, Figure b1 is a thin layer chromatogram photograph of the Psoralea corylifolia after being developed with a developing agent containing different components of authentic Psoralea corylifolia and then developed with a color developer, and Figure b2 is a thin layer chromatogram photograph of the Psoralea corylifolia after being developed with a developing agent containing different components of authentic Psoralea corylifolia under 254nm ultraviolet light. DETAILED DESCRIPTION

[0101] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0102] The chromatographic conditions in the content determination and fingerprint method in the following Examples 1 to 6 are as follows: the chromatographic column is Waters Acquity HSS T3 (2.1*100mm, 1.8μm), mobile phase: water (A)-methanol:acetonitrile (1:1 volume ratio) (B), gradient elution (0-10min, 80%-74% A; 10-20min, 74% A; 20-30min, 74%-58% A; 30-45min, 58%-58% A; 45-55min, 58%-20% A), wavelength 221nm; column temperature 30℃; flow rate 0.3mL·min -1 , injection volume 2μL.

[0103] In the following examples, DSL-1 represents Pleionoid A, and DSL-2 represents Pleionoid B; the specific information of various medicinal materials used is shown in the following table:

[0104]

[0105]

[0106]

[0107] In addition, among the reference substances used in the following examples of the present invention, the reference substance Bletilla striata glycoside and the reference substance Dactylorhin A are both purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., wherein the content of the reference substance Bletilla striata glycoside is ≥98%, and the batch number is DSTDP002501, and the content of the reference substance Dactylorhin A is ≥98%, and the batch number is DSTDD023301; the reference substances of Pleionoid A, Pleionoid B and Pleionoid C are all obtained by the inventor by separating and enriching the extract of Pleionoid striata, and the specific method steps include:

[0108] 1) Extraction:

[0109] The medicinal material of Herba Sophorae Flavescentis was subjected to reflux extraction with 60% ethanol, wherein the amount of 60% ethanol used was 10 (v / m) times the amount of the medicinal material of Herba Sophorae Flavescentis, and the extraction was performed twice, each time for 1 hour; the two extracts were filtered, combined, and dried to obtain a Herba Sophorae Flavescentis dry extract; the extract was suspended in 2 times the amount of water, and extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol in sequence to obtain a n-butanol extract.

[0110] 2) Column chromatography:

[0111] The obtained n-butanol extract was subjected to polyamide column chromatography, and gradient elution was performed with water, 20% methanol, 40% methanol, 60% methanol and methanol in sequence, wherein the amount of water, 20% methanol, 40% methanol and 60% methanol was 3 times the column volume (v / v) (i.e., the volume of solvent used was 3 times the volume of the polyamide column), and a total of 13 components were obtained (including 1 methanol component and other 12 components, 12 components = 4 solvents × 3). Component 9 (i.e., the eluate collected when the amount of eluent used was the 9th column volume, i.e., the eluate collected when the amount of 40% methanol was 3 times the column volume) was collected and analyzed by ultra-high performance liquid chromatography. R =39min, 43min, 45min. The eluent (i.e. component 9) was concentrated under reduced pressure and separated by high-performance liquid chromatography (chromatographic conditions: injection volume: 5mL; column temperature: 25°C; stationary phase: C18 bonded silica gel; mobile phase: methanol (A)-water (B), gradient elution (0-30min, 40%-90% A; 30-45min, 100% A), detection wavelength 210nm; flow rate 70mL / min), and the component from 0 to 15min, i.e. component B, was collected and analyzed by ultra-high performance liquid chromatography. R =39min,43min,45min.

[0112] 3) HPLC preparation:

[0113] The component B obtained by column chromatography was prepared by HPLC (LC3050N high performance liquid chromatograph, chromatographic column model: Agilent Eclipse XDB-C18 (9.4*250mm, 5μm)); injection volume: 200μL; column temperature: 25°C; stationary phase: C18 bonded silica gel; mobile phase: 45% methanol aqueous solution; detection wavelength: 221nm; flow rate: 3mL / min); component C and the succinic acid bibenzyl ester compound shown in formula I were obtained: (2R)-1-{4-β-D-[(E)-3-cinnamoyl-pyranoglucopyranosyloxy]benzyloxy}-4-(4-β-D-pyranoglucopyranosyloxybenzyloxy)-2-O-β-D-pyranoglucopyranosyl-2-isosuccinate (t R =16.5min) (named as Pleionoid A).

[0114] The above component C was prepared by HPLC (LC3050N high performance liquid chromatograph, chromatographic column model: Prep C18 (19*250mm, 5μm); injection volume: 200μL; column temperature: 25℃; stationary phase: C18 bonded silica gel; mobile phase: 23% acetonitrile aqueous solution; detection wavelength: 221nm; flow rate: 10mL / min); get t R=13.2min and 15.5min of the succinic acid bibenzyl ester compounds shown in formula II and formula III: (2R)-1-{4-β-D-[(E)-6-cinnamoyl-pyranoglucopyranosyloxy]benzyloxy}-4-(4-β-D-pyranoglucopyranosyloxybenzyloxy)-2-O-β-D-pyranoglucopyranoside-2-isosuccinate (named Pleionoid B), (2R)-1-{4-β-D-[(E)-2-cinnamoyl-pyranoglucopyranosyloxy]benzyloxy}-4-(4-β-D-pyranoglucopyranosyloxybenzyloxy)-2-O-β-D-pyranoglucopyranoside-2-isosuccinate (named Pleionoid C).

[0115] The inventors also conducted structural identification and characterization of the isolated and enriched compounds Pleionoid A, Pleionoid B and Pleionoid C, including:

[0116] 1) Structural identification of compound Pleionoid A

[0117] ESI-MS m / z 1017.2293[MH] - , the molecular weight of compound Pleionoid A was determined to be 1018.

[0118]

[0119]

[0120] 1 H-NMR spectrum (such as Figure 1 The HMBC spectrum (as shown) shows a group of methylene signals δH 3.10 (1H, d, J = 17.4 Hz, H-3a) and 2.99 (1H, d, J = 17.4 Hz, H-3b), two methyl signals δH 0.77 (3H, d, J = 6.0 Hz, Me-7) and 0.84 (3H, d, J = 6.0 Hz, Me-8), and a methine signal δH 1.71 (1H, m, H-6). Figure 3 (as shown) shows that H-3 is related to the two carbonyl carbons δC 172.28 (C-1) and δC 170.08 (C-4) and the oxygen carbon δC79.62 (C-2), δH1.73 (1H, m, H-5a) and δH1.69 (1H, m, H-5b) are related to C-2, δC 23.89 (C-6), δC 23.28 (C-7), and δC 24.32 (C-8), thereby obtaining the fragment structure of 2-hydroxy-2-isobutylsuccinic acid.

[0121] 1H-NMR showed two groups of AA′BB′ signals: δH 7.31 (2H, d, J = 8.4 Hz, H-2′, H-6′), δH 7.27 (2H, d, J = 8.4 Hz, H-2″″, H-6″″), δH 7.06 (2H, d, J = 8.4 Hz, H-3′, H-5′), δH 7.02 (2H, d, J = 8.4 Hz, H-3″″, H-5″″); two groups of oxymethylene signals: δH 5.09 (1H, d, J = 12.0 Hz, H-7′a), δH HMBC showed that H2-7′ was correlated with δC 129.14 (C-1′) and δC 129.95 (C-2′ / C-6′), and H2-7″″ was correlated with δC 129.05 (C-1″″) and δC 130.06 (C-2″″ / C-6″″), confirming the presence of two 4-oxobenzyloxy fragments.

[0122] 1 H-NMR showed three terminal protons: δH5.09 (1H, d, J=7.8 Hz, H-1″), δH4.86 (1H, d, J=7.8 Hz, H-1″″′), δH4.67 (1H, d, J=7.8 Hz, H-1″″″). At the same time, according to the overlapping oxymethyl and oxymethylene peaks between δH2.90 and 5.07, it was indicated that there were three β-glucose structures. HMBC showed that the terminal protons H-1″ and H-1″″′ were respectively related to δC 157.21 (C-4′) and δC157.47 (C-4″″), indicating the presence of two gastrodin fragments. H2-7′ was related to C-1, and H2-7″″ was related to C-4. It can be determined that these two fragments are esterified with the parent nucleus isobutylsuccinic acid.

[0123] also, 1 H-NMR showed that there was also a monosubstituted benzene ring: δH7.74 (2H, m, H-2″′, H-6″′), δH7.44 (2H, m, H-3″′, H-5″′), δH7.44 (1H, m, H-4″′); a group of trans-coupled double bond hydrogen signals δH7.68 (1H, d, J=16.2 Hz, H-7″′) and δH6.68 (1H, d, J=16.2 Hz, H-8″′). HMBC showed that H-7″′ was related to δC 134.29 (C-1″′), and H-7″′ and H-8″′ were related to carbonyl carbon δC 165.94 (C-9″′), thus confirming the cinnamoyl structural fragment. Further combined with HMBC and HSQC (such as Figure 4 shown) and1 H- 1 HCOSY (such as Figure 5 Determine its connection position. 1 H- 1 HCOSY showed that H-2″ was correlated with H-3″, and H-2″ was correlated with H-1″; HMBC showed that δH5.07 (1H, m, H-3″) was correlated with δC100.05 (C-1″) and δC165.94 (C-9″′), thus confirming that the cinnamoyl structure was connected to the 3″ position of glucose connected to the benzene ring, which was consistent with the mass spectrometry fragmentation rule, thus confirming the planar structure of Formula I.

[0124] The absolute configuration of glucose was determined by sugar derivatization after hydrochloric acid hydrolysis: 5 mg of the compound was added to 1N HCl (2.0 mL), refluxed at 80°C for 6 h, the product was extracted 3 times with ethyl acetate (5 mL × 3 times), the aqueous phase was decompressed and dried, and the obtained sample was derivatized with 5 mg of D-glucose, L-glucose and D-galactose standards, that is, after being dissolved in anhydrous pyridine (1.0 mL), L-cysteine ​​methyl ester hydrochloride (3.0 mg) was added respectively, and derivatization reaction was carried out at 60°C for 1 h, phenyl isothiocyanate (5.0 μL) was added to the reaction product, and the reaction was carried out at 60°C for 1 h, the product was decompressed and dried, and then dissolved in 1 mL of methanol, and analyzed by HPLC, the detection wavelength was 250 nm, the mobile phase was 78% MeCN-22% H2O, and the flow rate was 1 mL / min. It was known that the sugar in the compound Pleionoid A was D-glucose. The compound is succinic acid bibenzyl ester glycoside. According to the biogenic pathway analysis of this type of compound, the absolute configuration of C-2 is determined to be R. Therefore, the stereostructure of the succinic acid bibenzyl ester glycoside compound shown in Formula I is as follows:

[0125]

[0126] 2) Structural identification of compound Pleionoid B

[0127] ESI-MS m / z 1017.2293[MH] - , the molecular weight of compound Pleionoid B was determined to be 1018.

[0128] Compound PleionoidA and compound PleionoidB are isomers. The NMR data of PleionoidB are similar to those of PleionoidA, with the main difference being that the chemical shifts of C-6″ and H-6″ shift downfield, suggesting that the cinnamoyl structure may be connected to the H-6″ position. HMBC (such as Figure 8The results of the above analysis show that δH4.44 (1H, d, J = 11.4 Hz, H-6"a), δH4.23 (1H, d, J = 11.4 Hz, H-6"b) are related to the carbonyl carbon δC 166.13 (C-9"'), which confirms that the phenylpropionic acid structure is connected to the 6-position of glucose. The method for determining the absolute configuration of glucose is the same as that of Pleionoid A, which is D-glucose. The compound is succinic acid bibenzyl ester glycoside. According to the analysis of the biogenic pathway of this type of compound, the absolute configuration of C-2 can be determined to be R. Therefore, the stereostructure of the succinic acid bibenzyl ester glycoside compound shown in Formula II is as follows:

[0129]

[0130] 3) Structural identification of compound Pleionoid C

[0131] ESI-MS m / z 1017.2685[MH] - , the molecular weight of the compound was determined to be 1018.

[0132] Compound Pleionoid C is an isomer of compound Pleionoid A and compound Pleionoid B. The NMR data of Pleionoid C are also similar to those of Pleionoid A, with the main difference being that H-2″ is significantly shifted to the downfield, suggesting that the cinnamoyl structure may be connected to the H-2″ position. HMBC (such as Fig.14 ) shows that δH4.94 (1H, m, H-2″) is related to the carbonyl carbon δC165.39 (C-9″′) and the terminal carbon δC 98.19 (C-1″′), indicating that the cinnamoyl structure is connected to the 2-position of glucose. The method for determining the absolute configuration of glucose is the same as that of Pleionoid B, which is D-glucose. The compound is succinic acid bibenzyl ester glycoside. According to the biogenic synthesis pathway of this type of compound, the absolute configuration of C-2 can be determined to be R. Therefore, the stereostructure of the succinic acid bibenzyl ester glycoside compound shown in formula III is as follows:

[0133]

[0134] After the structural identification of compounds PleionoidA, PleionoidB and PleionoidC, the inventors conducted a series of experiments to realize the application value of five succinate dibenzyl glycosides - Bletilla striata glycoside, Dactylorhin A, PleionoidA, Pleionoid B and Pleionoid C in the identification and quality control of Dactylorhinus officinalis medicinal materials.

[0135] Example 1

[0136] In order to determine the optimal extraction method and extraction effect of five succinate bibenzyl glycosides - Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C in the pseudobulbs of P. uniflora, the inventors conducted research from the following aspects, including:

[0137] (I) Selection of extraction method

[0138] Weigh 4 portions of 0.5g of Pseudobulb powder of Pseudobulb of Pseudobulb (passed through No. 3 sieve) accurately, and place them in stoppered conical bottles respectively. Extract the 4 portions of Pseudobulb powder of Pseudobulb by ultrasonic and reflux respectively, and each extraction method corresponds to 2 parallel samples; add 25mL of methanol with a volume concentration of 70% to the 4 samples respectively, weigh the weight, extract 2 portions of the samples by ultrasonic (power 250W, frequency 40kHz) for 1 hour, and extract 2 portions of the samples by heating reflux for 1 hour. After cooling, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. Take 2μL of the filtrate respectively and inject it into the ultra-high performance liquid chromatograph for determination, and determine according to the above chromatographic conditions. Take the average value of the 2 parallel samples. The contents of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C obtained by the two extraction methods are shown in the following table. It can be seen that there is no significant difference in the yield of each component in the extract obtained by ultrasonic extraction and reflux extraction, so the ultrasonic extraction method with simpler operation is selected in the present invention.

[0139]

[0140] (II) Selection of extraction solvent

[0141] 18 portions of 0.5 g of Pseudobulb powder of Pseudobulb of Pseudobulb (passed through No. 3 sieve) were accurately weighed and placed in stoppered conical bottles. The aforementioned 18 portions of Pseudobulb powder of Pseudobulb corresponded to nine solvents, and each solvent corresponded to two parallel samples. 25 mL of ethanol, 70% ethanol, 50% ethanol, 10% ethanol, methanol, 70% methanol, 50% methanol, 10% methanol and water were added to the solvents, and the weight was weighed. Ultrasonic (power 250 W, frequency 40 kHz) extraction was performed for 1 hour, and the mixture was cooled, and the weight lost was supplemented with the corresponding solvent, shaken, filtered, and the filtrate was obtained. 2 μL of the filtrate was injected into an ultra-high performance liquid chromatograph for determination. The above chromatographic conditions were used for determination, and the average value of the two parallel samples was taken. The contents of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C in the filtrate corresponding to each extraction solvent are shown in the following table. It can be seen that when 50% ethanol is used as the extraction solvent, the yield of each component is the highest and the extraction efficiency is the best, so 50% ethanol is selected as the extraction solvent.

[0142]

[0143] (III) Selection of extraction material-liquid ratio

[0144] Weigh 8 portions of 0.5g of Pseudobulb powder of Pseudobulb of Pseudobulb (passed through No. 3 sieve) accurately and place them in a stoppered conical bottle. The aforementioned 8 portions of Pseudobulb powder of Pseudobulb correspond to 4 liquid-to-solid ratios, and each liquid-to-solid ratio corresponds to 2 parallel samples. Add 50% ethanol at liquid-to-solid ratios of 1:20, 1:50, 1:100 and 1:150, weigh the weight, extract by ultrasound (power 250W, frequency 40kHz) for 1 hour, cool, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. Take 2μL of the filtrate and inject it into the ultra-high performance liquid chromatograph for determination. Take the average value of the 2 parallel samples. The contents of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C in the filtrate corresponding to each liquid-to-solid ratio are shown in the following table. It can be seen that when the solid-liquid ratio is 1:50, the yields of Dactylorhin A, Bletilla striata glycosides and Pleionoid A are the highest, and the contents of Pleionoid B and Pleionoid C are not much different from those when using other solid-liquid ratios. Therefore, the present invention selects a solid-liquid ratio of 1:50 for extraction.

[0145] (IV) Selection of extraction time

[0146] 8 portions of 0.5g of Pseudobulb powder of Pseudobulb of Pseudobulb (passed through No. 3 sieve) were accurately weighed and placed in a stoppered conical bottle. The aforementioned 8 portions of Pseudobulb powder of Pseudobulb corresponded to 4 extraction times, and each extraction time corresponded to 2 parallel samples. Each sample was added with 25mL of 50% ethanol at a liquid-to-solid ratio of 1:50, weighed, and ultrasonically extracted for 15min, 30min, 45min and 60min (power 250W, frequency 40kHz), cooled, supplemented with weight loss, shaken, filtered, and the filtrate was obtained. 2μL of the filtrate was injected into an ultra-high performance liquid chromatograph for determination, and the average value of the 2 parallel samples was taken. The contents of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C in the filtrate obtained by extraction at different extraction times are shown in the following table. It can be seen that when the extraction time is 30 minutes, Dactylorhin A, Bletilla striata glycosides, Pleionoid A and Pleionoid C are all completely extracted, and the yield of Pleionoid B in the extract at different extraction times is not much different. Therefore, from the perspective of energy saving, the extraction time of the present invention is selected as 30 minutes.

[0147]

[0148] Example 2

[0149] This embodiment provides an application of succinic acid bibenzyl glycoside compounds in the quality control of the medicinal material of Dactylorhinus sutchuenensis, and uses ultra-high performance liquid chromatography to determine the content of Bletilla striata glycoside compounds and / or Dactylorhin A compounds and / or Pleionoid A compounds and / or Pleionoid B compounds and / or Pleionoid C compounds in the test solution, and the chromatographic conditions used are the chromatographic conditions given above. The feasibility of the high performance liquid chromatography is verified in terms of wavelength selection, linearity, repeatability, stability, precision, accuracy and durability.

[0150] (I) Selection of detection wavelength

[0151] The absorbance of the standard solutions of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C was measured at 210nm, 221nm, 254nm, 285nm and 310nm wavelengths respectively. The results showed that the absorbance of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C was the highest at 221nm, so 221nm was selected as the detection wavelength of this method.

[0152] (II) Linear relationship

[0153] Take appropriate amount of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C reference substances, accurately weigh them, add methanol to prepare reference substance stock solutions of 5.075, 2.799, 3.000, 4.440 and 4.120 mg / ml respectively, and dilute them step by step according to experimental requirements to prepare mixed standard solutions of required concentrations. The concentration points and corresponding peak areas of each component are shown in the following table:

[0154]

[0155]

[0156] According to the determined liquid phase method, the samples were injected, and the concentrations (μg / mL) of the reference substances of Bletilla striata, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C were used as the horizontal coordinates and the peak area (mAU) was used as the vertical coordinates for linear regression to obtain the regression equation, correlation coefficient and linear range of each component. The concentration when the peak height signal-to-noise ratio S / N of each component was 3 was taken as the detection limit (LOD), and the concentration when the S / N of each component was 10 was taken as the quantification limit (LOQ). The results are shown in the following table. It can be seen that the linear relationship of each component is good within its respective range (r≥0.9994), and the linearity meets the requirements.

[0157]

[0158] (III) Repeatability experiment

[0159] Six portions of 0.5 g powder of the pseudobulbs of the same batch of Orchidaceae were weighed in parallel and ultrasonically extracted for 30 min using 25 mL of 50% ethanol solution. The extract was analyzed according to the above-mentioned chromatographic conditions to obtain the contents of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C. The results are shown in the table below.

[0160] It can be seen that the RSD values ​​of the contents of each compound are all less than 3.00%, indicating that the repeatability of this method is good.

[0161]

[0162]

[0163] (IV) Stability test

[0164] Weigh 0.5g of the pseudobulb powder of the single garlic orchid, and use 25mL of 50% ethanol solution to ultrasonically extract for 30min to obtain the test solution, and investigate its stability within 48h, and calculate the RSD values ​​of the contents of baiji glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C. The results are shown in the table below. It can be seen that the RSD values ​​of the contents of each compound are all less than 4.00%, indicating that the test solution obtained by this method has good stability within 48h.

[0165]

[0166] (V) Precision experiment

[0167] The reference solutions of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C were taken respectively, and the concentrations of the reference solutions were 0.194 mg / mL, 0.2 mg / mL, 0.02 mg / mL, 0.019 mg / mL and 0.021 mg / mL, respectively. According to the above chromatographic conditions, the samples were injected six times continuously on the same day for UPLC analysis, and the peak areas of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C were calculated respectively. The results are shown in the table below. It can be seen that the RSD values ​​of each component are less than 1.30%, indicating that the intra-day precision of the method is good.

[0168]

[0169]

[0170] The reference solutions of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C were taken respectively, and the concentrations of the reference solutions were 0.194 mg / mL, 0.2 mg / mL, 0.02 mg / mL, 0.019 mg / mL and 0.021 mg / mL, respectively. The samples were injected 6 times by different operators on different days under the above chromatographic conditions, and the peak areas of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C were calculated respectively. The results are shown in the table below. It can be seen that the RSD values ​​of each component are less than 0.40%, indicating that the day-to-day precision of this method is good.

[0171]

[0172] (VI) Spike recovery test

[0173] Five groups of 0.25g of the same batch of pseudobulb powder of Pseudobulb ...

[0174]

[0175]

[0176] (VII) Relative correction factor f k / s calculate

[0177] 0.5μL, 1.0μL, 1.5μL, 2.0μL, 2.5μL and 3.0μL of the reference solution of equal volume of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C were respectively drawn, and the concentrations of each reference solution were 0.507mg / mL, 0.280mg / mL, 0.038mg / mL, 0.011mg / mL and 0.007mg / mL, respectively. UPLC injection analysis was performed according to the above liquid phase method, and each injection volume was repeated twice. The relative correction factors of Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C were calculated respectively using the stable and easily available Bletilla striata glycosides as the internal reference, and the calculation results are shown in the table below.

[0178]

[0179]

[0180] (VIII) Relative correction factor f k / s Durability evaluation

[0181] 8.1. The durability of the correction factors obtained above was investigated at different volume flow rates. The effects of different volume flow rates (0.27 mL / min, 0.30 mL / min and 0.33 mL / min) on f k / sThe results are shown in the table below. It can be seen that the RSD values ​​of the relative correction factors of each component at different volume flow rates are all less than 1.00%, indicating that fine-tuning the volume flow rate has a significant effect on f k / s The impact is not significant.

[0182]

[0183] 8.2. The durability of the correction factors obtained above was investigated at different column temperatures. The effects of different column temperatures (25°C, 30°C, and 35°C) on f k / s The results are shown in the table below. It can be seen that the RSD values ​​of each component are less than 1.20%, indicating that different column temperatures have a significant effect on f k / s The impact is not significant.

[0184]

[0185] 8.3. The durability of different chromatographic columns was investigated using the correction factors obtained above. Three chromatographic columns with similar length and filler particle size were investigated (Waters Acquity HSS T3(2.1*100mm,1.8μm),Welch XB-C18(2.1*100mm,1.8μm), Thermo Scientific HypersilGold TM Vanquish (2.1*100mm, 1.8μm)) for f k / s The results are shown in the table below. It can be seen that the RSD values ​​of each component are less than 1.60%, indicating that different chromatographic columns have a significant effect on f k / s The impact is not significant.

[0186]

[0187] 8.4. The durability of different instruments was investigated for the calibration factors obtained above. Three brands of instruments (Agilent 1290InfinityⅡ, Thermo Vanquish Scientific, Waters Acquity TM H-class) k / s The results are shown in the table below. It can be seen that the RSD values ​​of each component are less than 3.00%, indicating that different brands of instruments have different effects on f k / s The impact is not significant.

[0188]

[0189] (IX) Positioning of chromatographic peaks of components to be measured

[0190] 9.1. Three columns with similar length and particle size were investigated (Waters Acquity HSS T3(2.1*100mm,1.8μm),Welch XB-C18(2.1*100mm,1.8μm), ThermoScientific HypersilGold TM The effect of Vanquish (2.1*100mm, 1.8μm) on the relative retention time is shown in the table below. TM The relative retention time measured by the Vanquish column is quite different from that of the other two columns. Therefore, it is not recommended to use the ThermoScientific HypersilGold TM Vanquish columns, Welch Ultimate XB-C18 and Waters Acquity HSS T3 column.

[0191]

[0192]

[0193] 9.2. Three brands of instruments (Agilent 1290 Infinity II, Thermo Vanquish Scientific, Waters Acquity TM The results are shown in the table below. The case can be closed. The RSD values ​​of each component are all less than 3.00%, indicating that different brands of instruments have little effect on the relative retention time.

[0194]

[0195] (X) Comparison of content determination results between QAMS and ESM

[0196] 14 samples of D. uniflora were accurately weighed, and ultrasonic extraction was performed for 30 minutes with 50% ethanol solution at a solid-liquid ratio of 1:50 to obtain a test solution. The above-mentioned chromatographic conditions were used for UPLC injection analysis, and the content of each component was calculated by the one-measurement-multiple-evaluation method and the external standard method, respectively. The results of the determination are shown in the table below, and the relative errors (RD) of the results of the two methods were calculated and T tests were performed. It can be seen that the RD values ​​of each component are less than 5%, and the P value is greater than 0.05, indicating that there is no significant difference in the content results of each component measured by the two methods, indicating that the one-measurement-multiple-evaluation method (QAMS) established by the present invention has good applicability in the determination of the content of each component of D. uniflora.

[0197]

[0198] Example 3

[0199] In this example, the above-mentioned ultra-high performance liquid chromatography method is used to determine the contents of Bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C in authentic and counterfeit products of Tripterygium wilfordii. The specific determination method is as follows:

[0200] Take authentic Trillium gracile and extract with 50% ethanol solution by volume for 30 minutes at a solid-liquid ratio of 1:50 to obtain the test solution. Prepare two parallel samples for each batch of samples and perform UPLC injection analysis according to the above chromatographic conditions. Each test solution is injected twice and the contents of the above five components are calculated by the external standard method. The determination results are shown in the table below.

[0201]

[0202]

[0203]

[0204] (Note: “-” indicates the content is lower than the detection limit of this method)

[0205] From the data in the table, we can see that Bletilla striata, Bletilla striata and Cymbidium orchid all contain Bletilla striata glycosides (content ranges are 5.12-20.43 mg / g, 19.65-59.26 mg / g, 1.76-6.57 mg / g respectively) and Dactylorhin A (content ranges are 3.33-14.73 mg / g, 3.75-15.42 mg / g, 0.04-3.67 mg / g respectively), and some batches of Bletilla striata and Cymbidium orchid contain Pleionoid A (content ranges are 0-0.50 mg / g, 0-0.25 mg / g respectively); Cymbidium orchid and Cibotium glycyrrhiza do not contain Dactylorhin A and Bletilla striata glycosides; Cibotium orchid does not contain Dactylorhin A, but some batches contain Bletilla striata glycosides (content ranges from 0-2.01 mg / g). Pleionoid B and Pleionoid C were only detected in D. edulis (the content range was 0.25-0.81 mg / g and 0.28-0.75 mg / g, respectively). It can be seen that it is difficult to distinguish D. edulis from Bletilla striata and D. tuberosa by measuring the content of Bletilla striata and Dactylorhin A alone, but it can be used to distinguish the authenticity of other Tripterygium wilfordii. Pleionoid B and Pleionoid C are unique components in D. edulis. By measuring the content of the above five components, it can effectively distinguish D. edulis from other Tripterygium wilfordii.

[0206] Example 4

[0207] This embodiment provides a method for establishing a fingerprint spectrum, specifically:

[0208] Take the sample of D. uniflora and prepare the sample according to the preparation method of the test solution. Import the chromatogram data in CDF format of 14 batches of D. uniflora samples into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2021.120723 Edition)" software, use the spectrum of the sample with batch number DSL-07-01 as the reference spectrum, use multi-point correction, median method, and set the time window to 0.1min for chromatographic peak matching. Fig.18 As shown, a control fingerprint is generated, such as Fig.19 As shown, the similarity between the sample and the control fingerprint was calculated (see the table below). The chromatograms of some genuine and fake products and the comparison diagram of the single garlic orchid are shown in Fig. 20 shown.

[0209]

[0210]

[0211] From the data in the table above, we can see that the similarity between the genuine and fake products of each Tripterygium wilfordii is: Du Suan Lan (14 batches) 0.945~1.000; Mao Cigu (10 batches) 0~0.853; Tuber-petal orchid (9 batches) 0.531~0.913; Bletilla striata (21 batches) 0.863~0.945; Shan Lan (4 batches) 0; Guang Cigu (4 batches) 0. Therefore, through similarity calculation, Du Suan Lan can be distinguished from the genuine and fake products of Tripterygium wilfordii except Bletilla striata and Tuber-petal orchid. The similarity of the 14 batches of Du Suan Lan samples is greater than 0.90, indicating that the quality of Du Suan Lan medicinal materials is relatively stable. A total of 11 common peaks have been identified, which need to be further compared with standard samples and identified by mass spectrometry.

[0212] Example 5

[0213] This embodiment provides a method for identifying common peaks in fingerprint spectra, specifically:

[0214] Take the sample of D. uniflora and prepare the sample according to the preparation method of the test solution. MassLynx 4.1 software was used to collect mass spectrometry data, extract the quasi-molecular ion peak and fragment peak information of each chromatographic peak, and combine the standard comparison with mass spectrometry analysis to identify 8 of the 11 common peaks. The results are shown in the table below.

[0215]

[0216]

[0217] Example 6

[0218] This example is a methodological verification of the fingerprint established above. The fingerprint peak chromatogram is as follows Fig.21 The precision, repeatability, stability and durability of the above fingerprint establishment methods were investigated respectively, as follows:

[0219] (I) Precision inspection

[0220] Take 2 samples of D. uniflora, each of which includes 6 parallel samples, prepare the samples according to the preparation method of the test solution, and perform UPLC injection analysis according to the above chromatographic conditions. Fig.21 Peak 4 (Baiji glycoside) in the above is used as the reference peak, and the relative retention time and relative peak area of ​​each common peak are calculated. The results are shown in the table below. It can be seen that the RSD values ​​of the relative retention time and relative peak area are both less than 1.70%, indicating that the instrument has good precision.

[0221]

[0222]

[0223] (II) Repeatability Study

[0224] Take 2 samples of D. uniflora, each of which includes 6 parallel samples, prepare the samples according to the preparation method of the test solution, and perform UPLC injection analysis according to the above chromatographic conditions. Fig.21 Peak 4 (Baiji glycoside) in the above is used as the reference peak, and the relative retention time and relative peak area of ​​each common peak are calculated. The results are shown in the table below. It can be seen that the RSD values ​​of the relative retention time and relative peak area are both less than 2.10%, indicating that the instrument has good precision.

[0225]

[0226]

[0227] (III) Stability investigation

[0228] Take 2 samples of D. uniflora and prepare the samples according to the preparation method of the test solution. According to the above chromatographic conditions, perform UPLC injection analysis at different times. Fig.21 Peak 4 (Baiji glycoside) in the above is used as the reference peak, and the relative retention time and relative peak area of ​​each common peak are calculated. The results are shown in the table below. It can be seen that the RSD values ​​of the relative retention time and relative peak area are both less than 2.00%, indicating that the instrument has good precision.

[0229]

[0230]

[0231] (IV) Durability inspection

[0232] 4.1. Investigation of different chromatographic columns

[0233] Take the sample of Orchidaceae, prepare the sample according to the preparation method of the test solution, perform UPLC injection analysis according to the above chromatographic conditions, and use different chromatographic columns for detection (Waters Acquity HSS T3(2.1*100mm,1.8μm),Welch XB-C18(2.1*100mm,1.8μm), Thermo Scientific HypersilGold TM Vanquish (2.1*100mm, 1.8μm), the results are as follows Fig. 22 As shown (the three chromatographic columns used are abbreviated as: Waters-T3C18, Welch-Ultimate XB-C18, Thermo-Gold C18). Fig. 22 It can be seen that the separation of the 11 common peaks obtained using each chromatographic column is good, indicating that this method does not need to rely on a specific chromatographic column.

[0234] 4.2. Investigation of different column temperatures

[0235] Take the sample of D. uniflora and prepare the sample according to the preparation method of the test solution. Perform UPLC injection analysis according to the above chromatographic conditions and use different column temperatures for detection. The results are as follows: Fig.23 As shown. Fig.23 It can be seen that when the column temperature is 25℃ and 30℃, the separation of the 11 common peaks is good, but when the column temperature is 35℃, the separation of peaks 1, 4, 5, 6 and the surrounding peaks is poor, indicating that this method is not suitable for testing at a chromatographic column temperature of 35℃.

[0236] 4.3. Investigation of different flow rates

[0237] Take a sample of Psoralea corylifolia and prepare it according to the preparation method of the test solution. Perform UPLC injection analysis according to the above chromatographic conditions and perform detection at different mobile phase flow rates. The results are as follows: Fig.24 As shown, it can be seen that the separation of the 11 common peaks obtained at different mobile phase flow rates is good, indicating that this method is not affected by slight fluctuations in the mobile phase flow rate.

[0238] 4.4. Investigation with different instruments

[0239] Take the sample of D. uniflora and prepare the sample according to the preparation method of the test solution. Perform UPLC injection analysis according to the above chromatographic conditions and use different instruments for detection (Agilent1290 InfinityⅡ, Thermo VanquishScientific, Waters Acquity TM H-class), the results are as follows Figures 25-27 As shown in the figure, it can be seen that the separation of the 11 common peaks in the chromatograms obtained by the three instruments is good, indicating that the method is less affected by the instrument.

[0240] Example 7

[0241] In addition to providing the above-mentioned ultra-high performance liquid chromatography method and the establishment of fingerprint, the present invention also provides a thin layer chromatography identification method. The following provides a selection process for the preparation method of the test solution in the thin layer chromatography identification method. According to the location of Bletilla striata glycosides, Dactylorhin A, Pleionoid A and Pleionoid B, the development effects of the samples without pretreatment and after three different pretreatment methods are investigated respectively:

[0242] (I) Samples without pre-treatment

[0243] After preparing the solution according to the test solution preparation method, the solution was decompressed and dried by spin drying, dissolved in 1 mL of methanol, filtered through a 0.45 μm microporous filter membrane to obtain the test solution without pretreatment, and stored at 4°C for use.

[0244] (ii) Samples were extracted with n-butanol

[0245] After preparing the solution according to the test solution preparation method, the solution was dried under reduced pressure and spun dry. After the extract was suspended in 10 mL of water, it was extracted with water-saturated n-butanol, 10 mL each time, and extracted 3 times. After the n-butanol portion was dried under reduced pressure, it was dissolved with 1 mL of methanol and filtered through a 0.45 μm microporous filter membrane to obtain the test solution extracted with n-butanol, which was stored at 4°C for use.

[0246] (III) The sample was extracted with dichloromethane and n-butanol in turn

[0247] After preparing the solution according to the test solution preparation method, the solution was dried under reduced pressure. After the extract was suspended in 10 mL of water, it was first extracted with dichloromethane, 10 mL each time, extracted 3 times, and the dichloromethane portion was discarded. After the water portion was dried under reduced pressure, 10 mL of water was added to the residue, and extracted with water-saturated n-butanol, 10 mL each time, extracted 3 times. After the n-butanol portion was dried under reduced pressure, it was dissolved in 1 mL of methanol and filtered through a 0.45 μm microporous filter membrane to obtain the test solution extracted with dichloromethane and n-butanol in sequence, which was stored at 4°C for use.

[0248] (IV) Sample pre-treatment by SPE (polyamide)

[0249] Guided by the contents of the above four quality markers in different elution parts, the SPE (polyamide) pretreatment method was finally determined by investigating the ratio of the sample load to the elution solvent. Specifically:

[0250] After preparing the solution according to the test solution preparation method, the solution was dried under reduced pressure and dissolved in 2 mL of 20% methanol. 1 mL of SPE (polyamide, 1 g / 6 mL) was accurately aspirated and eluted with 3 BV of 20% methanol and 1 BV of methanol in sequence. The two elution sites were dried under reduced pressure and dissolved in 1 mL of methanol respectively. After filtering with a 0.45 μm microporous filter membrane, the test solution eluted with 20% methanol solution and the test solution eluted with methanol were obtained, and stored at 4°C for use.

[0251] Using chloroform-ethyl acetate-methanol-formic acid-water (ratio: 3:5:3:1.5:0.25) as the developing agent and 0.1% vanillin-10% ethanolic sulfuric acid as the color developing agent, the above five different test solutions and two mixed standards were analyzed by thin layer detection. The results are as follows Fig.28As shown (wherein, 1 is the test solution obtained without pretreatment, 2 is the test solution obtained by extraction with n-butanol, 3 is the test solution obtained by extraction with dichloromethane and n-butanol in sequence, 4 is the test solution obtained by elution with 20% methanol solution after SPE pretreatment, 5 is the test solution obtained by elution with methanol after SPE pretreatment, 6 is a mixed standard of Bletilla striata glycosides and Dactylorhin A, and 7 is a mixed standard of Pleionoid A and Pleionoid B). Fig.28 It can be seen that due to the low content of Pleionoid A and the Rf value close to that of Bletilla striata glycosides, if the sample is extracted only by n-butanol or extracted by dichloromethane and n-butanol in sequence, Pleionoid B and Bletilla striata glycosides cannot be well separated or Pleionoid A is not clearly identified. After SPE (polyamide) pretreatment, Pleionoid B and Bletilla striata glycosides are respectively in different parts, and Pleionoid A and Pleionoid B are enriched. All four compounds are clearly identified on the thin layer plate. Therefore, it is determined that the sample is pretreated by SPE (polyamide).

[0252] Example 8

[0253] This example provides a methodological investigation and durability investigation test of the above-mentioned thin layer chromatography identification method, specifically:

[0254] 1. Methodological investigation

[0255] 1.1. Investigation of the developing agent system

[0256] Four development systems were investigated, namely chloroform-methanol, cyclohexane-ethyl acetate-methanol, chloroform-acetone-methanol-concentrated ammonia, and chloroform-ethyl acetate-methanol-formic acid-water. The corresponding thin layer chromatograms are shown in Figure 1. Fig.29 As shown. Fig.29 It can be seen that (where 1 is the test sample of Dactylorhinus edulis, 2 is Dactylorhin A, 3 is Bletilla striata glycoside, 4 is Pleionoid A, 5 is Pleionoid B, and 6 is Pleionoid C) that the development system of chloroform-ethyl acetate-methanol-formic acid-water is more suitable for the development of the above five quality markers.

[0257] 1.2. Study on the ratio of developing agent

[0258] Five developing solvent ratios of chloroform-ethyl acetate-methanol-formic acid-water system (4:3:4:1.5:0.25, 4:5:3:1.5:0.25, 4:7:3:1.5:0.25, 3:5:3:1.5:0.25, 3:5:3:1.5:0.5) were investigated respectively. Fig.30As shown (wherein 1 is the Dactylorhinus edulis sample, 2 is Dactylorhin A, 3 is Bletilla striata glycoside, 4 is Pleionoid A, 5 is Pleionoid B, and 6 is Pleionoid C), it can be seen that the developing system of chloroform-ethyl acetate-methanol-formic acid-water is more suitable for development when the ratio is 3:5:3:1.5:0.25. Since Pleionoid B and Pleionoid C are difficult to separate, only the more stable Pleionoid B is finally selected for identification.

[0259] 1.3. Study on colorimetric agent

[0260] The color development effects of 10% ethanolic sulfuric acid, 0.1% vanillin-10% ethanolic sulfuric acid, and 10% ethanolic phosphomolybdic acid were investigated respectively. Fig.31 As shown (where 1 is the Dactylorhin test sample, 2 is the mixed standard of Dactylorhin A, Bletilla striata glycosides, Pleionoid A and Pleionoid B). It can be seen that when 0.1% vanillin-10% ethanol sulfate is used as the color developer, the color development effect is better and the spots are clearer, so 0.1% vanillin-10% ethanol sulfate is selected as the color developer.

[0261] 1.4. Sample quantity inspection

[0262] Standard sample quantity inspection: inspect the clarity of the bands when 5μg, 10μg, 15μg, and 20μg of the above four standards are respectively inspected. Fig.32 As shown, each control substance was already clear when 15 μg was spotted, so the spotted amount was determined to be 15 μg.

[0263] Sample spotting volume inspection: inspect the clarity of the bands when 5μL, 10μL, 15μL, and 20μL are spotted at the two elution sites. Fig.32As shown (wherein, in Figures a1 to a2, 1 to 4 correspond to the spotting amounts of 5 μg, 10 μg, 15 μg, and 20 μg of Dactylorhin A, 5 to 8 correspond to the spotting amounts of 5 μg, 10 μg, 15 μg, and 20 μg of Bletilla striata glycosides, and 9 is the SPE 20% methanol elution site; in Figures b1 to b2, 1 to 4 correspond to the spotting amounts of 5 μg, 10 μg, 15 μg, and 20 μg of DSL-1, 5 to 8 correspond to the spotting amounts of 5 μg, 10 μg, 15 μg, and 20 μg of DSL-2, and 9 is the SPE methanol elution site; in Figures c1 to c2, 1 to 4 correspond to the spotting amounts of 5 μL, 10 μL, 15 μL, and 20 μL of SPE 20% methanol elution site, 5 to 8 correspond to the SPE methanol elution sites with the sample volume of 5μL, 10μL, 15μL, and 20μL, 9 is the mixed standard of Bletilla striata glycosides and Dactylorhin A, and 10 is the mixed standard of DSL-1 and DSL-2). It can be seen that the SPE 20% methanol elution site is clearer at 10μL, and the sample volume is determined to be 10μL; the SPE methanol elution site is clearer at 20μL, and the sample volume is determined to be 20μL.

[0264] (II) Durability inspection

[0265] 2.1 Humidity investigation

[0266] By preparing different supersaturated salt solutions, the effects of different humidity conditions on the development of thin layer chromatography were investigated. The different humidity conditions were: 40% RH (supersaturated magnesium chloride), 63% RH (air humidity), and 80% RH (supersaturated potassium chloride). Fig.33 As shown (where 1 is the test solution obtained by eluting with 20% methanol after SPE pretreatment, with a spotting volume of 10 μL; 2 is the test solution obtained by eluting with methanol after SPE pretreatment, with a spotting volume of 20 μl; 3 is a mixed standard of Bletilla striata glycosides and Dactylorhin A, with a spotting volume of 15 μg each; 4 is a mixed standard of DSL-1 and DSL-2, with a spotting volume of 15 μg each). It can be seen that humidity has no obvious effect on the development of thin layer chromatography.

[0267] 2.2 Temperature investigation

[0268] The effects of temperature conditions of 4°C and 25°C on the development of thin layer chromatography were investigated. Fig.34As shown (wherein, 1 is the test solution obtained by eluting with 20% methanol after SPE pretreatment, with a spotting amount of 10 μL; 2 is the test solution obtained by eluting with methanol after SPE pretreatment, with a spotting amount of 20 μL; 3 is a mixed standard of Bletilla striata glycosides and Dactylorhin A, with a spotting amount of 15 μg each; 4 is a mixed standard of DSL-1 and DSL-2, with a spotting amount of 15 μg each). It can be seen that temperature has no obvious effect on the development of thin layer chromatography.

[0269] 2.3. Thin layer plate inspection

[0270] The effects of four types of thin layer plates (Merck, Qingdao Ocean, Crest, and Yantai Huanghai) on the development of thin layer chromatography were investigated. Fig.35 As shown in the figure (where 1 is the test solution obtained by eluting with 20% methanol after SPE pretreatment, with a spotting volume of 10 μL; 2 is the test solution obtained by eluting with methanol after SPE pretreatment, with a spotting volume of 20 μL; 3 is the mixed standard of Bletilla striata glycosides and Dactylorhin A, with a spotting volume of 15 μg each; 4 is the mixed standard of DSL-1 and DSL-2, with a spotting volume of 15 μg each). It can be seen that different brands of thin layer plates have a greater impact on the development of thin layer chromatography, among which Merck thin layer plates have a better development effect, so Merck plates are recommended for thin layer plates.

[0271] Example 9

[0272] This embodiment provides an application of the above-mentioned thin layer chromatography identification method in the identification of the authenticity of Psoralea corylifolia, specifically:

[0273] Take the authentic products of Tripterygium wilfordii and perform TLC identification on multiple batches of authentic products of Tripterygium wilfordii according to the above TLC identification method. Fig.36 The results show that the bands of the four compounds were not detected in the other authentic and counterfeit products of Tripterygium wilfordii except Bletilla striata and Cyperus tuberosus. The bands of Bletilla striata and Dactylorhin A were detected in Bletilla striata, and the bands of Pleionoid A were also detected in some batches of Bletilla striata, and there was interference at the band of Pleionoid B; only the band of Bletilla striata was detected in Cyperus tuberosus. Therefore, the established TLC identification method can be used to distinguish the authenticity of other Tripterygium wilfordii except Bletilla striata, and other methods are still needed to distinguish Bletilla striata.

[0274] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of a medicinal material of Psoralea corylifolia derived from Psoralea corylifolia, characterized in that: The construction method comprises constructing a fingerprint containing bletilla striata glycosides, Dactylorhin A, Pleionoid A, Pleionoid B and Pleionoid C; wherein the chemical structural formulas of Pleionoid A, Pleionoid B and Pleionoid C are shown in Formula I, Formula II and Formula III respectively: The construction method adopts ultra-high performance liquid chromatography, wherein the chromatographic conditions include: Chromatographic column: C18 column; Mobile phase: Mobile phase A is water, and mobile phase B is a mixture of methanol and acetonitrile in a volume ratio of 1:1; Detection wavelength: 210~221nm; The gradient elution method was used, and the elution procedure was as follows: 。 2. The method for constructing the fingerprint of the medicinal material of Psoralea corylifolia derived from Psoralea corylifolia as claimed in claim 1, characterized in that: The construction method adopts ultra-high performance liquid chromatography, wherein the chromatographic conditions also include: Flow rate: 0.27~0.33mL / min; Column temperature: 25-35°C; Injection volume: 0.5~3μL.

3. The method for constructing the fingerprint of the medicinal material of Pseudobulbus Atractylodes lancea derived from Pseudobulbus Atractylodes as claimed in claim 1, characterized in that: In the fingerprint, the white glycoside peak is taken as the S peak, and the relative retention time of the characteristic peak includes at least one of 0.371, 0.565, 0.605, 1.000, 1.216, 1.276, 1.368, 1.561, 1.583, 1.742 and 1.816, the absolute retention time of the S peak is 23.18±0.03min, and the error of the relative retention time is within ±5%.

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