Component determination method of premna microphylla leaf alcohol extract

Abstract

The invention discloses a method for determining the components of a premna microphylla leaf alcohol extract, which is characterized in that a liquid chromatography-mass spectrometry method is adopted to establish a premna microphylla leaf alcohol extract content detection method, original data acquired by mass spectrometry is called by software, a mass spectrometry information database is combined for comparative analysis, and the chemical components of the premna microphylla leaf alcohol extract are identified. And finally, 44 chemical components in the alcohol extract are detected, so that a basis is provided for the quality and quality evaluation of the premna microphylla medicinal material.

Description

Field of the Invention

[0001] The invention relates to a method for determining the components of ophiopogon wiltshire thorn, and particularly relates to a method for determining the components of an ethanol extract of ophiopogon wiltshire thorn leaves. Background Art

[0002] Premna puberula Pamb. is a perennial shrub of the Verbenaceae family, mainly distributed in Guizhou, Yunnan, Sichuan and other places in my country. Its leaves contain a variety of nutrients such as protein, pectin, flavonoids, peroxidase, etc., and can be used in food, chemical industry, medicine, etc. At present, the research on pharmacological effects is mainly focused on the same genus plant Premnamicrophylla Turcz. The extract has significant anti-inflammatory, tumor prevention, analgesic and other functions, which are closely related to its components. Many scholars have extracted the flavonoid components in Turcz by microwave, ultrasound and other methods, and found that it has high content of flavonoids and polyphenols. However, there are few reports on the components of Premna puberula, especially no relevant literature on alcohol extracts.

[0003] There are few methods for detecting the chemical components of P. odoratum, especially the determination method of the relevant components of the alcohol extract of P. odoratum leaves has not been reported in the literature. Due to the lack of clarity of the effective components of P. odoratum, it is difficult to comprehensively evaluate the intrinsic quality of P. odoratum. Therefore, it is of great significance to establish a method that can quickly, efficiently and accurately determine the main chemical components of P. odoratum to achieve comprehensive control of the quality of this substance and provide a more sufficient basis for the quality evaluation of P. odoratum. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for determining the components of the alcohol extract of the leaves of the Chinese yew vine. The method can be used to qualitatively analyze the chemical components in the alcohol extract of the leaves of the Chinese yew vine, overcoming the problems of difficulty in detecting the chemical components of the Chinese yew vine and uncertainty of the effective components in the prior art.

[0005] The present invention is implemented by the following technical solutions:

[0006] A method for determining the components of an alcohol extract of a leaf of Psoralea corylifolia comprises the following steps:

[0007] a. Preparation of alcohol extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed, 15-25g of powder was weighed and placed in a 1000mL conical flask with a stopper, 65-75% ethanol was added to dissolve, ultrasonic extraction was performed for 1.8-2.5h, hot gauze was filtered after ultrasonication, the supernatant was filtered, vacuum freeze-dried, and sealed for storage;

[0008] b. Preparation of the test solution: Dissolve the sealed and stored lyophilized powder of the alcohol extract in 15-25 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 80-100:10, ultrasonically treat for 8-12 min, shake and vortex for 25-35 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3-5 ° C for standby use;

[0009] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0010] d. Using the mass spectrometry system of step c, setting the ion source parameters for qualitative detection to obtain mass spectrometry information, using Thermo Xcalibur Qual Browser software to compare and analyze the raw data collected by the mass spectrometry, combined with the mass spectrometry information database, to identify the chemical components of the alcohol extract of the leaves of the Herba Lysimachiae.

[0011] In the above step a, the leaves of the Herba Lysimachiae are dried and crushed through a 30-50 mesh sieve, 18-22 g of the powder is weighed and placed in a 1000 mL stoppered conical flask, 65-75% ethanol is added to dissolve, and ultrasonic extraction is performed for 1.2-2.2 h, with an ultrasonic power of 200 W, a frequency of 300 Hz, and an ultrasonic temperature condition of 90-110° C. After ultrasonication, the extract is filtered through hot gauze, the supernatant is filtered, vacuum freeze-dried, and sealed for storage.

[0012] Specifically, in the aforementioned step a, the leaves of the Herba Lycopodii are dried and crushed through a 40-mesh sieve, 20 g of the powder is weighed and placed in a 1000 mL stoppered conical flask, 70% ethanol is added to dissolve, and ultrasonic extraction is performed for 2 h. The ultrasonic power used is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 100°C. After ultrasonication, the mixture is filtered through gauze while hot, the supernatant is filtered, vacuum freeze-dried, and sealed for storage.

[0013] In the aforementioned step b, the sealed and stored lyophilized powder of the alcohol extract is dissolved in 18-22 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 85-95:10, and ultrasonic treatment is performed for 9-11 min, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 90-110° C. After ultrasonication, it is shaken and vortexed for 28-32 s, passed through a 0.22 μm microporous filter membrane, and the filtrate is stored at 3.5-4.5° C. for standby use.

[0014] Specifically, in the aforementioned step b, the sealed and stored lyophilized powder of the alcohol extract is dissolved in 20 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 90:10, and ultrasonic treatment is performed for 10 minutes, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 100° C. After ultrasonication, it is shaken and vortexed for 30 seconds, passed through a 0.22 μm microporous filter membrane, and the filtrate is stored at 4° C. for standby use.

[0015] The chromatographic detection conditions of the aforementioned step c are:

[0016] Chromatographic column: Waters Acquity uplc hss T3, specification: 100*2.1mm*1.8μm;

[0017] The liquid phase system was: Thermo Scientific Ultimate 3000RSLC, HPG;

[0018] Mobile phase: Gradient elution mobile phase A is acetonitrile solution containing 0.1% formic acid, and mobile phase B is 0.1% formic acid in water;

[0019] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0020] The flow rate was 0.3 mL / min and the column temperature was 40 °C.

[0021] In the above step c, the mass spectrometry conditions are as follows: the mass spectrometry system is Thermo Scientific Q Exactive Focus, the full mass spectrometry scan range is 100-1500 m / z, the resolution is 17500-70000, the Full MS-MS / MS is 50-100 ms, the separation width is 1.5 m / z, and the threshold is set to 1e 6 and 2e 5 , the intensity threshold is 1.6e 5 , the minimum AGC target is set to 8e 3 , dynamic emission reduction is 5s.

[0022] In the above step d, the parameters of the ion source are set as follows: the ionization mode is electrospray ion source ESI, detection is in positive and negative ion mode respectively, the atomization voltage is 3.0 kV+ / 2.5 kV-, the capillary temperature is 320°C, the probe heating temperature is 350°C, the sheath gas is 35 arb, the auxiliary gas is 10 arb, and the purge gas is 0 arb.

[0023] Beneficial effects of the present invention:

[0024] (1) It overcomes the shortcomings of the chemical composition detection methods of B. odorata, unclear effective ingredients, and difficulty in evaluating the intrinsic quality of the medicinal material.

[0025] (2) The present invention uses a liquid chromatography-mass spectrometry system (Thermo Fisher Scientific, USA) and a HESI-I1 ion source, and the gradient elution solvent is an acetonitrile solution containing 0.1% formic acid (mobile phase A) and an aqueous solution containing 0.1% formic acid (mobile phase B), and the elution time and organic phase ratio are: 0-2min (5% A + 95% B); 42-47min (95% A + 5% B); 47.1-50min (5% A + 95% B), the flow rate is 0.3mL / min, and the column temperature is 40°C. The specific parameters of the above experiment were obtained after more than two months of condition exploration. If the experimental parameters such as the ratio of the mobile phase, the ion source, and the mass spectrometry conditions change, the chemical components in the leaves of the herb cannot be detected or the detection is inaccurate.

[0026] (3) The method for detecting the chemical components of the present invention can quickly, accurately and sensitively detect 44 chemical components of the alcohol extract of the leaves of the herbaceous herbaceous herb: lactose (1), sucrose (2), fenugreek hydrochloride (3), L-proline (4), adenosine (5), L-leucine (6), caffeic acid (7), schaftoside (8), plantagoside (9), honeysuckle (10), hyperoside (11), akebia phenylethanol glycoside B (12), artemisia glycoside (13), verbascoside (14), dioscorea glycoside (15), hesperidin (16), luteolin (17), picroside I (18), saponin (19), montanol (20), luteolin (21), phellin The results showed that the main ingredients of the antioxidant enzymes were 1, 2-dimethoate (22), dianthin (23), isorhamnetin (24), irisin (25), hydroxygenkanthin (26), isorhamnetin (27), cyanidin (28), sweet orange flavonoids (29), isorhamnetin (30), nobiletin (31), acetoin B (32), acacetin (33), strychnine (34), strychnine (35), 6-gingerol (36), isorhamnetin (37), aflatin (38), glycyrrhetinic acid (39), gardeniaflavin B (40), demethylnobiletin (41), echinocystic acid (42), ursolic acid (43), and α-linolenic acid (44), which provided guidance for the evaluation of the intrinsic quality of B. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : ESI ion mode diagram of chemical components in alcohol extract of Herba Lysimachiae (A is positive ion mode, B is negative ion mode);

[0028] Figure 2 ; Structure, molecular weight and secondary mass spectrum of chemical components of lactose, sucrose and trigonelline hydrochloride in the alcohol extract of the leaves of the herb.

[0029] Figure 3 ; Structure, molecular weight and secondary mass spectrum of L-proline, adenylation and L-leucine in the alcohol extract of Herba Lysimachiae;

[0030] Figure 4 ; Structure, molecular weight and secondary mass spectrum of caffeic acid, schaftoside and plantagoside in alcohol extract of Herba Lysimachiae;

[0031] Figure 5 ; Structure, molecular weight and secondary mass spectrum of honeysuckle glycoside, hyperoside and akebia phenylethanol glycoside B extracted from the leaves of the herbaceous plant Herba Lysimachiae;

[0032] Figure 6 ; The structure, molecular weight and secondary mass spectrum of the alcohol extracts of Codonopsis pilosula, Verbascoside and Dioscorea chinensis;

[0033] Figure 7 ; Structure, molecular weight and secondary mass spectrum of hesperidin, luteolin and picroside I of the alcohol extract of the leaf of Herba Lysimachiae;

[0034] Figure 8 ; The structure, molecular weight and secondary mass spectrum of saponin, linalool and luteolin from the alcohol extract of the leaves of the Herba Lysimachiae;

[0035] Fig. 9 ; Structure, molecular weight and secondary mass spectrum of mulberry yellow, dioscorea linalool and isorhamnetin extracted from the leaves of Psoralea corylifolia;

[0036] Fig.10 ; Structure, molecular weight and secondary mass spectrum of irisin, hydroxygenkwain and isoeupatorium from the alcohol extract of the leaves of Herba Lysimachiae;

[0037] Fig.11 ; Structure, molecular weight and secondary mass spectrum of brown cyanidin, sweet orange flavonoids and isoeupatoria flavescens from the alcohol extract of the leaves of the herbaceous plant Herba Lysimachiae;

[0038] Fig.12 ; The structure, molecular weight and secondary mass spectrum of nobiletin, acetogenin and acacetin extracted from the leaves of the herb Herba Lysimachiae;

[0039] Fig.13 ; The structure, molecular weight and secondary mass spectrum of the chemical components of strychnine, strychnine and 6-gingerol in the alcohol extract of the leaves of Psoralea corylifolia;

[0040] Fig.14 ; Structure, molecular weight and secondary mass spectrum of asiatic acid, edible flavonoids and glycyrrhetinic acid in the alcohol extract of Herba Lysimachiae;

[0041] Fig.15 ; The structure, molecular weight and secondary mass spectrum of gardeniaflavin B, demethylnobiletin and echinocystic acid extracted from the leaves of the herb Herba Lysimachiae;

[0042] Fig.16 ; The structure, molecular weight and secondary mass spectrum of ursolic acid and α-linolenic acid in the alcohol extract of the leaves of Herba Lysimachiae.

[0043] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments, but the embodiments are not intended to limit the present invention. DETAILED DESCRIPTION

[0044] Embodiment 1:

[0045] The method for determining the components of the alcohol extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0046] a. Preparation of alcohol extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed through a 40-mesh sieve, 20 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, 70% ethanol was added to dissolve, ultrasonic extraction was performed at 100 ° C for 2 h (200 W, 300 Hz), filtered through hot gauze, the supernatant was filtered, vacuum freeze-dried, and sealed for storage;

[0047] b. Preparation of the test solution: Dissolve the sealed and stored lyophilized powder of the alcohol extract in 20 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 90:10, ultrasonically treat at 100°C for 10 min (200 W, 300 Hz), shake and vortex for 30 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 4°C for standby use;

[0048] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0049] The chromatographic detection conditions are:

[0050] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0051] The liquid phase system was: Thermo Scientific Ultimate 3000RSLC (HPG);

[0052] Mobile phase: Gradient elution mobile phase A is acetonitrile solution containing 0.1% formic acid, and mobile phase B is 0.1% formic acid in water;

[0053] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0054] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0055] The mass spectrometry conditions were as follows: the mass spectrometry system was Thermo Scientific Q Exactive Focus, the full mass spectrometry scan range was 100-1500 m / z, the resolution was 17500-70000, the Full MS-MS / MS was 50-100 ms, the separation width was 1.5 m / z, and the threshold was set to 1e 6 and 2e 5 , the intensity threshold is 1.6e 5 , the minimum AGC target is set to 8e 3 , dynamic emission reduction is 5s;

[0056] d. Use a mass spectrometer system and set ion source parameters for qualitative analysis;

[0057] Mass spectrometry conditions Same as step c ;

[0058] The parameters of the ion source were set as follows: the ionization mode was electrospray ion source ESI, detection was in positive and negative ion modes, respectively, the nebulizer voltage was 3.0 kV (+) / 2.5 kV (-), the capillary temperature was 320 °C, the probe heating temperature was 350 °C, the sheath gas was 35 arb, the auxiliary gas was 10 arb, and the sweep gas was 0 arb;

[0059] The original data collected by mass spectrometry were called by Thermo Xcalibur Qual Browser software and compared with the mass spectrometry information database for analysis and identification of the chemical components of the ethanol extract of the leaves of Psoralea corylifolia.

[0060] Embodiment 2:

[0061] The method for determining the components of the alcohol extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0062] a. Preparation of alcohol extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed, 15 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, 65-75% ethanol was added to dissolve, ultrasonic extraction was performed at 110 ° C for 1.5 h (200 W, 300 Hz), filtered through hot gauze, the supernatant was filtered, vacuum freeze-dried, and sealed for storage;

[0063] b. Preparation of the test solution: Dissolve the sealed and stored lyophilized powder of the alcohol extract in 15 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 80:10, ultrasonically treat at 110°C for 8 min (200 W, 300 Hz), shake and vortex for 25 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3°C ​​for standby use;

[0064] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0065] The chromatographic detection conditions are:

[0066] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0067] The liquid phase system was: Thermo Scientific Ultimate 3000RSLC (HPG);

[0068] Mobile phase: Gradient elution mobile phase A is acetonitrile solution containing 0.1% formic acid, and mobile phase B is 0.1% formic acid in water;

[0069] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0070] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0071] The mass spectrometry conditions were as follows: the mass spectrometry system was Thermo Scientific Q Exactive Focus, the full mass spectrometry scan range was 100-1500 m / z, the resolution was 17500-70000, the Full MS-MS / MS was 50-100 ms, the separation width was 1.5 m / z, and the threshold was set to 1e 6 and 2e 5 , the intensity threshold is 1.6e 5 , the minimum AGC target is set to 8e 3 , dynamic emission reduction is 5s;

[0072] d. Use a mass spectrometer system and set ion source parameters for qualitative analysis;

[0073] Mass spectrometry conditions Same as step c ;

[0074] The parameters of the ion source were set as follows: the ionization mode was electrospray ion source ESI, detection was in positive and negative ion modes, respectively, the nebulizer voltage was 3.0 kV (+) / 2.5 kV (-), the capillary temperature was 320 °C, the probe heating temperature was 350 °C, the sheath gas was 35 arb, the auxiliary gas was 10 arb, and the sweep gas was 0 arb;

[0075] The original data collected by mass spectrometry were called by Thermo Xcalibur Qual Browser software and compared with the mass spectrometry information database for analysis and identification of the chemical components of the ethanol extract of the leaves of Psoralea corylifolia.

[0076] Embodiment 3:

[0077] The method for determining the components of the alcohol extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0078] a. Preparation of alcohol extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed, 25 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, 75% ethanol was added to dissolve, ultrasonic extraction was performed at 90 ° C for 2.5 h (200 W, 300 Hz), hot gauze was filtered, the supernatant was filtered, vacuum freeze-dried, and sealed for storage;

[0079] b. Preparation of the test solution: Dissolve the sealed and stored lyophilized powder of the alcohol extract in 25 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 100:10, ultrasonically treat at 90°C for 12 min (200 W, 300 Hz), shake and vortex for 35 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3-5°C for standby use;

[0080] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0081] The chromatographic detection conditions are:

[0082] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0083] The liquid phase system was: Thermo Scientific Ultimate 3000RSLC (HPG);

[0084] Mobile phase: Gradient elution mobile phase A is acetonitrile solution containing 0.1% formic acid, and mobile phase B is 0.1% formic acid in water;

[0085] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0086] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0087] The mass spectrometry conditions were as follows: the mass spectrometry system was Thermo Scientific Q Exactive Focus, the full mass spectrometry scan range was 100-1500 m / z, the resolution was 17500-70000, the Full MS-MS / MS was 50-100 ms, the separation width was 1.5 m / z, and the threshold was set to 1e 6 and 2e 5 , the intensity threshold is 1.6e5 , the minimum AGC target is set to 8e 3 , dynamic emission reduction is 5s;

[0088] d. Use a mass spectrometer system and set ion source parameters for qualitative analysis;

[0089] Mass spectrometry conditions Same as step c ;

[0090] The parameters of the ion source were set as follows: the ionization mode was electrospray ion source ESI, detection was in positive and negative ion modes, respectively, the nebulizer voltage was 3.0 kV (+) / 2.5 kV (-), the capillary temperature was 320 °C, the probe heating temperature was 350 °C, the sheath gas was 35 arb, the auxiliary gas was 10 arb, and the sweep gas was 0 arb;

[0091] The original data collected by mass spectrometry were called by Thermo Xcalibur Qual Browser software and compared with the mass spectrometry information database for analysis and identification of the chemical components of the ethanol extract of the leaves of Psoralea corylifolia.

[0092] Embodiment 4:

[0093] The method for determining the components of the alcohol extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0094] a. Preparation of alcohol extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed, 18 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, 65% ethanol was added to dissolve, ultrasonic extraction was performed at 100 ° C for 2.5 h (200 W, 300 Hz), hot gauze was filtered, the supernatant was filtered, vacuum freeze-dried, and sealed for storage;

[0095] b. Preparation of the test solution: Dissolve the sealed and stored lyophilized powder of the alcohol extract in 22 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 95:10, ultrasonically treat at 100°C for 12 min (200 W, 300 Hz), shake and vortex for 25 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 5°C for standby use;

[0096] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0097] The chromatographic detection conditions are:

[0098] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0099] The liquid phase system was: Thermo Scientific Ultimate 3000RSLC (HPG);

[0100] Mobile phase: Gradient elution mobile phase A is acetonitrile solution containing 0.1% formic acid, and mobile phase B is 0.1% formic acid in water;

[0101] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0102] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0103] The mass spectrometry conditions were as follows: the mass spectrometry system was Thermo Scientific Q Exactive Focus, the full mass spectrometry scan range was 100-1500 m / z, the resolution was 17500-70000, the Full MS-MS / MS was 50-100 ms, the separation width was 1.5 m / z, and the threshold was set to 1e 6 and 2e 5 , the intensity threshold is 1.6e 5 , the minimum AGC target is set to 8e 3 , dynamic emission reduction is 5s;

[0104] d. Use a mass spectrometer system and set ion source parameters for qualitative analysis;

[0105] Mass spectrometry conditions Same as step c ;

[0106] The parameters of the ion source were set as follows: the ionization mode was electrospray ion source ESI, detection was in positive and negative ion modes, respectively, the nebulizer voltage was 3.0 kV (+) / 2.5 kV (-), the capillary temperature was 320 °C, the probe heating temperature was 350 °C, the sheath gas was 35 arb, the auxiliary gas was 10 arb, and the sweep gas was 0 arb;

[0107] The original data collected by mass spectrometry were called by Thermo Xcalibur Qual Browser software and compared with the mass spectrometry information database for analysis and identification of the chemical components of the ethanol extract of the leaves of Psoralea corylifolia.

[0108] Embodiment 5:

[0109] The method for determining the components of the alcohol extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0110] a. Preparation of alcohol extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed, 22 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, 75% ethanol was added to dissolve, ultrasonic extraction was performed at 100 ° C for 1.5 h (200 W, 300 Hz), hot gauze was filtered, the supernatant was filtered, vacuum freeze-dried, and sealed for storage;

[0111] b. Preparation of the test solution: Dissolve the sealed and stored lyophilized powder of the alcohol extract in 18 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 80-100:10, ultrasonically treat at 100 ° C for 8 min (200 W, 300 Hz), shake and vortex for 35 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3 ° C for standby use;

[0112] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0113] The chromatographic detection conditions are:

[0114] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0115] The liquid phase system was: Thermo Scientific Ultimate 3000RSLC (HPG);

[0116] Mobile phase: Gradient elution mobile phase A is acetonitrile solution containing 0.1% formic acid, and mobile phase B is 0.1% formic acid in water;

[0117] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0118] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0119] The mass spectrometry conditions were as follows: the mass spectrometry system was Thermo Scientific Q Exactive Focus, the full mass spectrometry scan range was 100-1500 m / z, the resolution was 17500-70000, the Full MS-MS / MS was 50-100 ms, the separation width was 1.5 m / z, and the threshold was set to 1e 6 and 2e 5 , the intensity threshold is 1.6e 5 , the minimum AGC target is set to 8e 3 , dynamic emission reduction is 5s;

[0120] d. Use a mass spectrometer system and set ion source parameters for qualitative analysis;

[0121] Mass spectrometry conditions Same as step c ;

[0122] The parameters of the ion source were set as follows: the ionization mode was electrospray ion source ESI, detection was in positive and negative ion modes, respectively, the nebulizer voltage was 3.0 kV (+) / 2.5 kV (-), the capillary temperature was 320 °C, the probe heating temperature was 350 °C, the sheath gas was 35 arb, the auxiliary gas was 10 arb, and the sweep gas was 0 arb;

[0123] The original data collected by mass spectrometry were called by Thermo Xcalibur Qual Browser software and compared with the mass spectrometry information database for analysis and identification of the chemical components of the ethanol extract of the leaves of Psoralea corylifolia.

[0124] The present invention has done a lot of analysis and verification experiments, and the following are the results of the experimental research of the present invention:

[0125] 1. Preparation of Alcohol Extract from the Leaves of Psoralea corylifolia

[0126] Experimental materials P.puberula were collected from Wuchuan Gelao and Miao Autonomous County, Guizhou Province. The research team has studied and established its seedling breeding technology. In early 2019, the two-year-old seedlings of P.puberula were planted at the scientific research base of Guizhou University (26°25'54"N, 106°40′53″E, with a subtropical humid monsoon climate, an average altitude of 1178m, an average annual temperature of 20.21℃, and an average annual precipitation of 1318.90mm). Leaves were collected on July 30, 2023 for related research.

[0127] Dry and crush the leaves of the Herba Lysimachiae, weigh 20g of the powder and place it in a 1000mL stoppered conical flask, add 70% ethanol to dissolve, perform ultrasonic extraction for 2h, filter through gauze while hot, take out the supernatant by suction, perform vacuum freeze drying, and seal for storage.

[0128] 2. Preparation of test solution

[0129] The sealed and stored freeze-dried powder of the alcohol extract was dissolved in 20 mL of acetonitrile: 0.1% formic acid aqueous solution (10:90, v / v), ultrasonically treated for 10 min (300 Hz, 100°C), shaken and vortexed for 30 s, filtered through a 0.22 μm microporous filter membrane, and the filtrate was stored at 4°C for later use.

[0130] 3. Liquid phase detection conditions

[0131] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm).

[0132] The liquid phase system was Thermo Scientific Ultimate 3000RSLC (HPG).

[0133] Mobile phase: The gradient elution solvents were acetonitrile solution containing 0.1% formic acid (organic phase A) and aqueous solution containing 0.1% formic acid (aqueous phase B). The elution time and organic phase ratio were: 0-2 min (5% A + 95% B); 42-47 min (95% A + 5% B); 47.1-50 min (5% A + 95% B), the flow rate was 0.3 mL / min, and the column temperature was 40°C.

[0134] 4. Mass spectrometry detection conditions

[0135] The mass spectrometer system was Thermo Scientific Q Exactive Focus, with a full mass spectrometry scan range of 100-1500 m / z and a resolution of 70,000; a secondary fragmentation scan resolution of 17,500, Full MS-MS / MS of 50-100 ms, a separation width of 1.5 m / z, and a threshold of 1e 6 and 2e 5 , the intensity threshold is 1.6e 5 , the minimum AGC target is set to 8e 3 , dynamic emission reduction is 5s.

[0136] 5. Ion source settings

[0137] The parameters of the ion source were set as follows: the ionization mode was electrospray ion source ESI, and the detection was in positive and negative ion modes respectively. The parameters were set as follows: atomization voltage 3.0 kV (+) / 2.5 kV (-), capillary temperature 320 °C, probe heating temperature 350 °C, sheath gas 35 arb, auxiliary gas 10 arb, and purge gas 0 arb.

[0138] 6. Qualitative chemical composition

[0139] The mass spectrometry system was Thermo Scientific Q Exactive Focus. The raw data collected by the mass spectrometry were called by ThermoFish Compound Discovery software and compared with the compound mass spectrometry information database for analysis. 44 chemical components of the alcohol extract of the leaves of Psoralea corylifolia were identified. Figure 1-Figure 16 and Table 1.

[0140] Table 1 Detailed information of alcohol extract of Herba Lysimachiae

[0141]

[0142]

Claims

1. A method for determining the components of an ethanol extract of a leaf of Psoralea corylifolia, characterized in that: The following steps are involved: a. Preparation of alcohol extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed, 15-25g of powder was weighed and placed in a 1000mL conical flask with a stopper, 65-75% ethanol was added to dissolve, ultrasonic extraction was performed for 1.5-2.5h, hot gauze was filtered after ultrasonication, the supernatant was filtered, vacuum freeze-dried, and sealed for storage; b. Preparation of the test solution: Dissolve the sealed and stored lyophilized powder of the alcohol extract in 15-25 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution is 80-100:10, ultrasonically treat for 8-12 min, shake and vortex for 25-35 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3-5 ° C for standby use; c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution; d. Using the mass spectrometry system of step c, setting the ion source parameters for qualitative detection to obtain mass spectrometry information, using Thermo Xcalibur Qual Browser software to compare and analyze the raw data collected by the mass spectrometry, combined with the mass spectrometry information database, to identify the chemical components of the alcohol extract of the leaves of the Herba Lysimachiae.

2. The method for determining the components of the alcohol extract of the leaf of Psoralea corylifolia according to claim 1, wherein: In the step a, the leaves of the Herba Lysimachiae are dried and crushed through a 30-50 mesh sieve, 18-22 g of the powder is weighed and placed in a 1000 mL stoppered conical flask, 65-75% ethanol is added to dissolve, ultrasonic extraction is performed for 1.8-2.2 h, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 90-110° C. After ultrasonication, the mixture is filtered through gauze while hot, the supernatant is filtered, vacuum freeze-dried, and sealed for storage.

3. The method for determining the components of the alcohol extract of the leaf of Psoralea corylifolia according to claim 2, wherein: In the step a, the leaves of the Herba Lysimachiae are dried and crushed through a 40-mesh sieve, 20 g of the powder is weighed and placed in a 1000 mL stoppered conical flask, 70% ethanol is added to dissolve, and ultrasonic extraction is performed for 2 h, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 100° C. After ultrasonication, the mixture is filtered through gauze while hot, the supernatant is filtered, vacuum freeze-dried, and sealed for storage.

4. The method for determining the components of the alcohol extract of the leaf of Psoralea corylifolia according to claim 1, wherein: In the step b, the sealed and stored lyophilized powder of the alcohol extract is dissolved in 18-22 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of the 0.1% formic acid aqueous solution to the acetonitrile solution is 85-95:10, and ultrasonic treatment is performed for 9-11 minutes, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 90-110° C. After ultrasonication, it is shaken and vortexed for 28-32 seconds, passed through a 0.22 μm microporous filter membrane, and the filtrate is stored at 3.5-4.5° C. for standby use.

5. The method for determining the components of the alcohol extract of the leaves of Psoralea corylifolia according to claim 4, characterized in that: In the step b, the sealed and stored lyophilized powder of the alcohol extract is dissolved in 20 mL of 0.1% formic acid aqueous solution: acetonitrile, the volume ratio of the 0.1% formic acid aqueous solution to the acetonitrile solution is 90:10, and ultrasonic treatment is performed for 10 minutes, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 100° C. After ultrasonication, it is shaken and vortexed for 30 seconds, passed through a 0.22 μm microporous filter membrane, and the filtrate is stored at 4° C. for standby use.

6. The method for determining the components of the alcohol extract of the leaves of Psoralea corylifolia according to claim 1, characterized in that: The chromatographic detection conditions of step c are: Chromatographic column: Waters Acquity uplc hss T3, specification: 100*2.1mm*1.8μm; The liquid phase system was: Thermo Scientific Ultimate 3000RSLC, HPG; Mobile phase: Gradient elution mobile phase A is acetonitrile solution containing 0.1% formic acid, and mobile phase B is 0.1% formic acid in water; The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A; The flow rate was 0.3 mL / min and the column temperature was 40 °C.

7. The method for determining the components of the alcohol extract of the leaves of Psoralea corylifolia according to claim 1, characterized in that: In step c, the mass spectrometry conditions are as follows: the mass spectrometry system is Thermo Scientific Q Exactive Focus, the full mass spectrometry scanning range is 100-1500 m / z, the resolution is 17500-70000, the Full MS-MS / MS is 50-100 ms, the separation width is 1.5 m / z, and the threshold is set to 1e 6 and 2e 5 , the intensity threshold is 1.6e 5 , the minimum AGC target is set to 8e 3 , dynamic emission reduction is 5s.

8. The method for determining the components of the alcohol extract of the leaves of Psoralea corylifolia according to claim 1, characterized in that: In the step d, the parameters of the ion source are set as follows: the ionization mode is electrospray ion source ESI, detection is in positive and negative ion mode respectively, the atomization voltage is 3.0kV+ / 2.5kV-, the capillary temperature is 320°C, the probe heating temperature is 350°C, the sheath gas is 35arb, the auxiliary gas is 10arb, and the purge gas is 0arb.

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