Ingredient determination method of premna microphylla leaf water extract

Abstract

The invention discloses a method for determining the components of a premna microphylla leaf water extract, which is characterized in that a content detection method of the premna microphylla leaf water extract is established by adopting a liquid chromatography-mass spectrometry method, original data acquired by mass spectrometry is called by using software, a mass spectrometry information database is combined for comparative analysis, and the chemical components of the premna microphylla leaf water extract are identified. Finally, 18 chemical components in the water extract are detected, and 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 radix scutellariae, and in particular to a method for determining the components of a water extract of radix scutellariae 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 the composition of water extract.

[0003] There are few methods for detecting the chemical components of P. styracifolia, especially the methods for determining the relevant components of the aqueous extract of P. styracifolia leaves have not been reported in the literature. Due to the lack of clarity of the effective components of P. styracifolia, it is difficult to comprehensively evaluate the intrinsic quality of P. styracifolia. Therefore, it is of great significance to establish a method that can quickly, efficiently and accurately determine the main chemical components of P. styracifolia to achieve comprehensive control of the quality of this substance and provide a more sufficient basis for the quality evaluation of P. styracifolia. 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 water extract of the leaves of the herb. The method can be used to qualitatively analyze the chemical components in the water extract of the leaves of the herb, overcoming the problems of difficulty in detecting the chemical components of the herb 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 aqueous extract of a leaf of Psoralea corylifolia comprises the following steps:

[0007] a. Preparation of aqueous extract of Herba Lysimachiae leaves: Dry and crush the Herba Lysimachiae leaves, weigh 30-50g of powder and place it in a 1000mL stoppered conical flask, add deionized water to dissolve it in a solid-liquid ratio of 1:30-50, soak it for 10-14h, ultrasonically extract it for 1.5-2.5h, filter it with hot gauze after ultrasonication, filter the supernatant by suction, vacuum freeze-dry it, and seal it for storage;

[0008] b. Preparation of the test solution: Dissolve the sealed and preserved lyophilized powder of the water 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, calling the raw data collected by the mass spectrometry using Thermo Xcalibur Qual Browser software, and comparing and analyzing it with the mass spectrometry information database to identify the chemical components of the aqueous extract of the leaves of the herb.

[0011] In the above step a, the leaves of the Herba Lysimachiae are dried and crushed to pass through a 35-45 mesh sieve, 35-45 g of the powder is weighed and placed in a 1000 mL stoppered conical flask, deionized water is added to dissolve it at a solid-liquid ratio of 1:35-45, and then soaked for 11-13 hours, and then ultrasonically extracted for 1.8-2.2 hours, with an ultrasonic power of 200 W, a frequency of 300 Hz, and an ultrasonic temperature condition of 90-110° C. After ultrasonication, the mixture is filtered through gauze while hot, and the supernatant is filtered and vacuum freeze-dried, and sealed for storage.

[0012] Specifically, in the above step a, the leaves of the Herba Lycopodii are dried and crushed and passed through a 40-mesh sieve, 40 g of the powder is weighed and placed in a 1000 mL stoppered conical flask, deionized water is added at a solid-liquid ratio of 1:40 to dissolve, and then soaked for 12 hours, and ultrasonically extracted for 2 hours, with an ultrasonic power of 200 W, a frequency of 300 Hz, and an ultrasonic temperature of 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 above step b, the sealed and preserved water extract freeze-dried powder 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 preserved water extract freeze-dried powder 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 min, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 100 ° C. After ultrasonication, shake and vortex for 30 s, pass through a 0.22 μm microporous filter membrane, and take the filtrate and store it 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 adopts 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 (organic phase C) and water containing 0.1% formic acid (aqueous phase B), and the elution time and organic phase ratio are: 0-2min (5% C + 95% B); 42-47min (95% C + 5% B); 47.1-50min (5% C + 95% B), the flow rate is 0.3mL / min, and the column temperature is 40°C. The specific parameters of the above experiments are obtained after several 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 body odor firewood cannot be detected or the detection is inaccurate.

[0026] (3) The method for detecting the chemical components of the herb can quickly, accurately and sensitively detect 18 chemical components of the water extract of the leaves of the herb, including sucrose, fenugreek hydrochloride, adenosine, L-leucine, L-phenylalanine, p-methoxybenzaldehyde, loganinic acid, inosinate, picroside III, schaftoside, plantagoside, honeysuckle glycoside, verbascoside, akebia phenylethanol glycoside B, coniferyl aldehyde, hesperidin, chelerythrine and 6-gingerol, providing guidance for the evaluation of the intrinsic quality of the herb. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : ESI ion mode diagram of chemical components in the water extract of the leaves of Psoralea corylifolia (A is positive ion mode, B is negative ion mode);

[0028] Figure 2 ; The structure, molecular weight and secondary mass spectrum of the chemical components of sucrose and trigonelline hydrochloride in the aqueous extract of the leaves of Psoralea corylifolia;

[0029] Figure 3 ; Structure, molecular weight and secondary mass spectrum of adenosine, L-leucine and L-phenylalanine in the aqueous extract of the leaves of Psoralea corylifolia;

[0030] Figure 4 ; The structure, molecular weight and secondary mass spectrum of p-anisaldehyde, loganin and cynaroside in the aqueous extract of the leaves of Psoralea corylifolia;

[0031] Figure 5 ; Structure, molecular weight and secondary mass spectrum of picroside III, schaftoside and plantain glycosides from the aqueous extract of the leaves of Psoralea corylifolia;

[0032] Figure 6 ; Structure, molecular weight and secondary mass spectrum of honeysuckle glycoside, verbascoside and akebia phenylethanoid glycoside B in the aqueous extract of the leaves of Psoralea corylifolia;

[0033] Figure 7 ; Structure, molecular weight and secondary mass spectrum of coniferaldehyde, hesperidin and chelerythrine in the aqueous extract of the leaves of Psoralea corylifolia;

[0034] Figure 8 ; Structure, molecular weight and secondary mass spectrum of 6-gingerol from the aqueous extract of the leaves of Psoralea corylifolia.

[0035] 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

[0036] Embodiment 1:

[0037] The method for determining the components of the water extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0038] a. Preparation of aqueous extract of leaves of Psoralea corylifolia: The leaves of Psoralea corylifolia were dried and crushed through a 40-mesh sieve, 40 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, deionized water was added at a solid-liquid ratio of 1:40 to dissolve, and then soaked for 12 h, and then ultrasonically extracted at 100 ° C for 2 h (200 W, 300 Hz), filtered through hot gauze after ultrasonication, and the supernatant was filtered and vacuum freeze-dried and sealed for storage;

[0039] b. Preparation of the test solution: Dissolve the sealed and preserved lyophilized powder of the water 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 later use;

[0040] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0041] (1) The chromatographic detection conditions are:

[0042] Liquid chromatography-mass spectrometry system and ESI ion source were used to detect the chemical components of the water extract of Psoralea corylifolia.

[0043] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0044] The liquid phase system was: Thermo Fisher Ultimate 3000RSLC (HPG);

[0045] 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;

[0046] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0047] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0048] (2) The mass spectrometer system was Thermo Scientific Q Exactive Focus, with a full mass spectrometer scan range of 100–1500 m / z, a resolution of 17500–70000, a 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;

[0049] d. The mass spectrometry system of step c was used to set the ion source parameters for qualitative detection to obtain mass spectrometry information, and the raw data collected by the mass spectrometry was called by Thermo Xcalibur Qual Browser software, and compared and analyzed with the mass spectrometry information database to identify the chemical components of the aqueous extract of the leaves of the herb;

[0050] 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 purge gas was 0 arb.

[0051] Embodiment 2:

[0052] The method for determining the components of the water extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0053] a. Preparation of aqueous extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed through a 35-mesh sieve, 30 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, deionized water was added at a solid-liquid ratio of 1:30 to dissolve, and then soaked for 10 h, and ultrasonically extracted at 110 ° C for 1.5 h (200 W, 300 Hz), filtered through hot gauze after ultrasonication, and the supernatant was filtered and vacuum freeze-dried and sealed for storage;

[0054] b. Preparation of the test solution: Dissolve the sealed and preserved lyophilized powder of the water 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;

[0055] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0056] (1) The chromatographic detection conditions are:

[0057] Liquid chromatography-mass spectrometry system and ESI ion source were used to detect the chemical components of the water extract of Psoralea corylifolia.

[0058] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0059] The liquid phase system was: Thermo Fisher Ultimate 3000RSLC (HPG);

[0060] 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;

[0061] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0062] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0063] (2) The mass spectrometer system was Thermo Scientific Q Exactive Focus, with a full mass spectrometer scan range of 100–1500 m / z, a resolution of 17500–70000, a 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;

[0064] d. The mass spectrometry system of step c was used to set the ion source parameters for qualitative detection to obtain mass spectrometry information, and the raw data collected by the mass spectrometry was called by Thermo Xcalibur Qual Browser software, and compared and analyzed with the mass spectrometry information database to identify the chemical components of the aqueous extract of the leaves of the herb;

[0065] 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 purge gas was 0 arb.

[0066] Embodiment 3:

[0067] The method for determining the components of the water extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0068] a. Preparation of aqueous extract of Herba Lysimachiae leaves: The Herba Lysimachiae leaves were dried and crushed through a 45-mesh sieve, 50 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, deionized water was added at a solid-liquid ratio of 1:50 to dissolve, and then soaked for 12 h, and ultrasonically extracted at 90 ° C for 2.5 h (200 W, 300 Hz), filtered through hot gauze after ultrasonication, and the supernatant was filtered and vacuum freeze-dried and sealed for storage;

[0069] b. Preparation of the test solution: Dissolve the sealed and preserved lyophilized powder of the water 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;

[0070] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0071] (1) The chromatographic detection conditions are:

[0072] Liquid chromatography-mass spectrometry system and ESI ion source were used to detect the chemical components of the water extract of Psoralea corylifolia.

[0073] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0074] The liquid phase system was: Thermo Fisher Ultimate 3000RSLC (HPG);

[0075] 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;

[0076] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0077] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0078] (2) The mass spectrometer system was Thermo Scientific Q Exactive Focus, with a full mass spectrometer scan range of 100–1500 m / z, a resolution of 17500–70000, a 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;

[0079] d. The mass spectrometry system of step c was used to set the ion source parameters for qualitative detection to obtain mass spectrometry information, and the raw data collected by the mass spectrometry was called by Thermo Xcalibur Qual Browser software, and compared and analyzed with the mass spectrometry information database to identify the chemical components of the aqueous extract of the leaves of the herb;

[0080] 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 purge gas was 0 arb.

[0081] Embodiment 4:

[0082] The method for determining the components of the water extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0083] a. Preparation of aqueous extract of leaves of Psoralea corylifolia: The leaves of Psoralea corylifolia were dried and crushed through a 40-mesh sieve, 35 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, deionized water was added to dissolve it in a solid-liquid ratio of 1:45, and then soaked for 12 h, and then ultrasonically extracted at 100 ° C for 2.2 h (200 W, 300 Hz), filtered through hot gauze after ultrasonication, and the supernatant was filtered and vacuum freeze-dried and sealed for storage;

[0084] b. Preparation of the test solution: Dissolve the sealed and preserved lyophilized powder of the water 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 11 min (200 W, 300 Hz), shake and vortex for 28 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 5°C for standby use;

[0085] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0086] (1) The chromatographic detection conditions are:

[0087] Liquid chromatography-mass spectrometry system and ESI ion source were used to detect the chemical components of the water extract of Psoralea corylifolia.

[0088] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0089] The liquid phase system was: Thermo Fisher Ultimate 3000RSLC (HPG);

[0090] 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;

[0091] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0092] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0093] (2) The mass spectrometer system was Thermo Scientific Q Exactive Focus, with a full mass spectrometer scan range of 100–1500 m / z, a resolution of 17500–70000, a 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;

[0094] d. The mass spectrometry system of step c was used to set the ion source parameters for qualitative detection to obtain mass spectrometry information, and the raw data collected by the mass spectrometry was called by Thermo Xcalibur Qual Browser software, and compared and analyzed with the mass spectrometry information database to identify the chemical components of the aqueous extract of the leaves of the herb;

[0095] 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 purge gas was 0 arb.

[0096] Embodiment 5:

[0097] The method for determining the components of the water extract of the leaves of the Herba Lysimachiae comprises the following steps:

[0098] a. Preparation of aqueous extract of leaves of Psoralea corylifolia: The leaves of Psoralea corylifolia were dried and crushed through a 40-mesh sieve, 45 g of powder was weighed and placed in a 1000 mL conical flask with a stopper, deionized water was added to dissolve the mixture in a solid-liquid ratio of 1:35, and then soaked for 13 h, and then ultrasonically extracted at 100 ° C for 2 h (200 W, 300 Hz), filtered through hot gauze after ultrasonication, and the supernatant was filtered and vacuum freeze-dried and sealed for storage;

[0099] b. Preparation of the test solution: Dissolve the sealed and preserved lyophilized powder of the water 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 9 min (200 W, 300 Hz), shake and vortex for 32 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3.5°C for standby use;

[0100] c. Use the UPLC-Q-Orbitrap HRMS liquid chromatography mass spectrometry system to separate and detect the components of the test solution;

[0101] (1) The chromatographic detection conditions are:

[0102] Liquid chromatography-mass spectrometry system and ESI ion source were used to detect the chemical components of the water extract of Psoralea corylifolia.

[0103] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm);

[0104] The liquid phase system was: Thermo Fisher Ultimate 3000RSLC (HPG);

[0105] 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;

[0106] The elution process was: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A;

[0107] The flow rate was 0.3 mL / min and the column temperature was 40 °C;

[0108] (2) The mass spectrometer system was Thermo Scientific Q Exactive Focus, with a full mass spectrometer scan range of 100–1500 m / z, a resolution of 17500–70000, a 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;

[0109] d. The mass spectrometry system of step c was used to set the ion source parameters for qualitative detection to obtain mass spectrometry information, and the raw data collected by the mass spectrometry was called by Thermo Xcalibur Qual Browser software, and compared and analyzed with the mass spectrometry information database to identify the chemical components of the aqueous extract of the leaves of the herb;

[0110] 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 purge gas was 0 arb.

[0111] 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:

[0112] 1. Preparation of Extract from the Leaves of Psoralea corylifolia

[0113] 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.

[0114] Dry and crush the leaves of the Herba Lysimachiae, weigh 40g of the powder and place it in a 1000mL stoppered conical flask, add deionized water at a solid-liquid ratio of 1:40 to dissolve and soak. Ultrasonic extraction was performed after 12 hours, and the supernatant was filtered through gauze while hot, and then vacuum freeze-dried and sealed for storage.

[0115] 2. Preparation of test solution

[0116] The sealed and preserved freeze-dried powder of the water 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, 30°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.

[0117] 3. Liquid phase detection conditions

[0118] Chromatographic column: Waters Acquity uplc hss T3 (100*2.1mm*1.8μm, waters).

[0119] The liquid phase system was Thermo Fisher 3000 RSLC (HPG).

[0120] Mobile phase: The gradient elution solvent is acetonitrile solution containing 0.1% formic acid (organic phase C) and aqueous solution containing 0.1% formic acid (aqueous phase B). The elution time and organic phase ratio are: 0-2min (5% C + 95% B); 42-47min (95% C + 5% B); 47.1-50min (5% C + 95% B), the flow rate is 0.3mL / min, and the column temperature is 40°C.

[0121] 4. Mass spectrometry detection conditions

[0122] The mass spectrometer system was Thermo Scientific Q Exactive Focus, with a full mass spectrometer scan range of 100-1500 m / z, a resolution of 17500-70000, a Full MS-MS / MS of 50-100 ms, an isolation window 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 exclusion is 5s.

[0123] 5. Ion source settings

[0124] The parameters of the ion source were set as follows: the ionization mode was electrospray ion source ESI, positive and negative ions were collected simultaneously, and its 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.

[0125] 6. Qualitative chemical composition

[0126] The mass spectrometry system was Thermo Scientific Q Exactive Focus. The raw data collected by the mass spectrometry were called by Compound Discovery software and compared with the compound mass spectrum information database for analysis. Eighteen chemical components of the water extract of the leaves of the herb were identified, including: sucrose (1), trigonelline hydrochloride (2), adenosine (3), L-leucine (4), L-phenylalanine (5), p-methoxybenzaldehyde (6), loganinic acid (7), corydaline (8), picroside III (9), schaftoside (10), plantagoside (11), honeysuckle (12), verbascoside (13), akebia phenylethanol glycoside B (14), coniferaldehyde (15), hesperidin (16), chelerythrine (17), 6-gingerol (18), including 2 alkaloids, 2 amino acids, 4 flavonoids, 3 glycosides, and 7 other components (see Figure 1 and Table 1.

[0127] Table 1 Identification results of substances in water extract of Psoralea corylifolia

[0128]

[0129]

Claims

1. A method for determining the components of an aqueous extract of a leaf of Psoralea corylifolia, characterized in that: The following steps are involved: a. Preparation of aqueous extract of Herba Lysimachiae leaves: Dry and crush the Herba Lysimachiae leaves, weigh 30-50g of powder and place it in a 1000mL stoppered conical flask, add deionized water to dissolve it in a solid-liquid ratio of 1:30-50, soak it for 10-14h, ultrasonically extract it for 1.5-2.5h, filter it with hot gauze after ultrasonication, filter the supernatant by suction, vacuum freeze-dry it, and seal it for storage; b. Preparation of the test solution: Dissolve the sealed and preserved lyophilized powder of the water 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, calling the raw data collected by the mass spectrometry using Thermo Xcalibur Qual Browser software, and comparing and analyzing it with the mass spectrometry information database to identify the chemical components of the aqueous extract of the leaves of the herb.

2. The method for determining the components of the aqueous extract of the leaves of Psoralea corylifolia according to claim 1, wherein: In the step a, the leaves of the Herba Lysimachiae are dried and crushed to pass through a 35-45 mesh sieve, 35-45 g of the powder is weighed and placed in a 1000 mL stoppered conical flask, deionized water is added to dissolve the powder at a solid-liquid ratio of 1:35-45, and the powder is soaked for 11-13 hours, and then ultrasonic extraction is performed for 1.8-2.2 hours, wherein the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature condition is 90-110° C. After ultrasonic extraction, the powder is filtered through gauze while hot, and the supernatant is filtered and vacuum freeze-dried, and the powder is sealed and stored.

3. The method for determining the components of the aqueous extract of the leaves of Psoralea corylifolia according to claim 2, wherein: In the step a, the leaves of the Herba Lysimachiae are dried and crushed to pass through a 40-mesh sieve, 40 g of the powder is weighed and placed in a 1000 mL stoppered conical flask, deionized water is added at a solid-liquid ratio of 1:40 to dissolve, and then soaked for 12 hours, and then ultrasonically extracted for 2 hours, with an ultrasonic power of 200 W, a frequency of 300 Hz, and an ultrasonic temperature condition of 100° C. After ultrasonication, the mixture is filtered through gauze while hot, and the supernatant is filtered and vacuum freeze-dried, and sealed for storage.

4. The method for determining the components of the aqueous extract of the leaves of Psoralea corylifolia according to claim 1, wherein: In the step b, the sealed and preserved water extract freeze-dried powder 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 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 aqueous extract of the leaves of Psoralea corylifolia according to claim 4, characterized in that: In the step b, the sealed and preserved water extract freeze-dried powder 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.

6. The method for determining the components of the aqueous 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 aqueous 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 aqueous 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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