A method for determining the components of a water extract of isodon japonicus leaf

Abstract

The application discloses a component determination method of a water extract of Rhus copallinum L.var latifolia Engelm leaves, and adopts a liquid chromatography-mass spectrometry method to establish a content detection method of the water extract of Rhus copallinum L.var latifolia Engelm, calls original data collected by mass spectrometry by using software, and compares and analyzes by combining a mass spectrometry information database to identify chemical components of the water extract of Rhus copallinum L.var latifolia Engelm leaves. Finally, 18 chemical components in the water extract are detected, and a basis is provided for quality and property evaluation of Rhus copallinum L.var latifolia Engelm medicinal materials.

Description

Invention Field

[0001] This invention relates to a method for determining the components of *Clerodendrum trichotomum*, specifically a method for determining the components of an aqueous extract of *Clerodendrum trichotomum* leaves. Background Technology

[0002] *Premna puberula* Pamb., a perennial shrub belonging to the genus *Premna* in the family Verbenaceae, is mainly distributed in Guizhou, Yunnan, and Sichuan provinces of my country. Its leaves contain various nutrients such as protein, pectin, flavonoids, and peroxidase, and can be used in food, chemical, and pharmaceutical industries. Current research on its pharmacological effects mainly focuses on the related plant *Premna microphylla* Turcz., whose extracts have shown significant anti-inflammatory, anti-tumor, and analgesic functions, which are closely related to its components. Several scholars have extracted flavonoids from *Premna microphylla* using various methods such as microwave and ultrasound, finding high levels of flavonoids and polyphenols. However, reports on the components of *Premna puberula* are scarce, especially regarding the composition of its water extracts, which is currently unavailable.

[0003] There are few methods for detecting the chemical components of *Bromus japonica*, especially for determining the components of its water extracts, which are not reported in the literature. Because the effective components of *Bromus japonica* are not clearly defined, it is difficult to comprehensively evaluate its intrinsic quality. Therefore, establishing a rapid, efficient, and accurate method for determining the main chemical components of *Bromus japonica*, enabling comprehensive control over the quality of these substances, and providing a more sufficient basis for the quality evaluation of *Bromus japonica*, is of great significance. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for determining the components of an aqueous extract of *Clerodendrum trichotomum* leaves. This method can perform qualitative analysis of the chemical components in the aqueous extract of *Clerodendrum trichotomum* leaves, overcoming the difficulties in detecting the chemical components of *Clerodendrum trichotomum* and the uncertainty of the effective components in existing technologies.

[0005] This invention is achieved using the following technical solution:

[0006] A method for determining the components of an aqueous extract of *Clerodendrum trichotomum* leaves includes the following steps:

[0007] a. Preparation of aqueous extract of *Clerodendrum trichotomum* leaves: Dry and pulverize *Clerodendrum trichotomum* leaves, weigh 30-50g of powder and place it in a 1000mL stoppered conical flask, add deionized water at a material-to-liquid ratio of 1:30-50 to dissolve and soak for 10-14h, then ultrasonically extract for 1.5-2.5h, filter with gauze while hot after ultrasonication, collect the supernatant by vacuum filtration, freeze-dry under vacuum, and store in a sealed container;

[0008] b. Preparation of test solution: Dissolve the sealed and preserved lyophilized water extract powder in 15-25 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution of 80-100:10. Sonicate for 8-12 min, shake well and vortex for 25-35 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3-5℃ for later use.

[0009] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

[0010] d. Using the mass spectrometry system of step c, set the ion source parameters for qualitative detection to obtain mass spectrometry information. Use Thermo Xcalibur Qual Browser software to retrieve the raw data acquired by mass spectrometry and compare and analyze it with the mass spectrometry information database to identify the chemical composition of the water extract of *Clerodendrum trichotomum* leaves.

[0011] In step a above, the leaves of *Clerodendrum trichotomum* are dried, pulverized, and passed through a 35-45 mesh sieve. 35-45g of powder is weighed and placed in a 1000mL stoppered conical flask. Deionized water is added at a material-to-liquid ratio of 1:35-45 to dissolve the powder, and the mixture is soaked for 11-13 hours. Then, ultrasonic extraction is performed for 1.8-2.2 hours. The ultrasonic power is 200W, the frequency is 300Hz, and the ultrasonic temperature is 90-110℃. After ultrasonic extraction, the mixture is filtered through gauze while still hot. The supernatant is then collected by vacuum freeze-drying and sealed for storage.

[0012] Specifically, in step a above, the leaves of *Clerodendrum trichotomum* are dried, pulverized, and passed through a 40-mesh sieve. 40g of powder is weighed and placed in a 1000mL stoppered conical flask. Deionized water is added at a material-to-liquid ratio of 1:40 to dissolve the powder, and the mixture is soaked for 12 hours. Then, it is ultrasonically extracted for 2 hours. The ultrasonic power is 200W, the frequency is 300Hz, and the ultrasonic temperature is 100℃. After ultrasonic extraction, the mixture is filtered through gauze while still hot. The supernatant is then collected by vacuum freeze-drying and sealed for storage.

[0013] In step b above, the sealed and preserved freeze-dried water extract powder is dissolved in 18-22 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 85-95:10. The mixture is ultrasonically treated for 9-11 min at a power of 200 W, a frequency of 300 Hz, and a temperature of 90-110 °C. After ultrasonication, the mixture is shaken and vortexed for 28-32 s. The solution is then filtered through a 0.22 μm microporous membrane, and the filtrate is stored at 3.5-4.5 °C for later use.

[0014] Specifically, in step b above, the sealed and preserved freeze-dried water extract powder is dissolved in 20 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 90:10. The mixture is ultrasonically treated for 10 min at a power of 200 W, a frequency of 300 Hz, and a temperature of 100 °C. After ultrasonication, the mixture is shaken and vortexed for 30 s. The solution is then filtered through a 0.22 μm microporous membrane, and the filtrate is stored at 4 °C for later use.

[0015] The chromatographic detection conditions for step c above are as follows:

[0016] Chromatographic column: Waters Acquity UPLC HSS T3, dimensions 100*2.1mm*1.8μm;

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

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

[0019] The elution process is as follows: 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 step c above, the mass spectrometry conditions are as follows: the mass spectrometry system is a Thermo Scientific Q Exactive Focus, the full mass spectrum scan range is 100-1500 m / z, the resolution is 17500-70000, the Full MS-MS / MS interval is 50-100 ms, the separation width is 1.5 m / z, and the threshold is set to 1 e. 6 and 2e 5 The intensity threshold is 1.6e. 5 The minimum AGC target is set to 8e. 3 The dynamic emission reduction is 5 seconds.

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

[0023] Beneficial effects of this invention:

[0024] (1) It overcomes the shortcomings of the limited chemical composition detection methods of *Bungarus odoratus*, unclear effective components, and difficulty in evaluating the intrinsic quality of medicinal materials.

[0025] (2) This invention employs a liquid chromatography-mass spectrometry (LC-MS) system (Thermo Fisher Scientific, USA) and a HESI-I1 ion source. 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). The elution times and organic phase ratios are: 0-2 min (5% C + 95% B); 42-47 min (95% C + 5% B); 47.1-50 min (5% C + 95% B). The flow rate is 0.3 mL / min, and the column temperature is 40℃. The specific parameters of the above experiments were obtained after several months of trial and error. If the experimental parameters such as the mobile phase ratio, ion source, and mass spectrometry conditions change, the chemical components in the leaves of *Clerodendrum trichotomum* will not be detected or will be detected inaccurately.

[0026] (3) The chemical composition detection method of *Clerodendrum trichotomum* described in this invention can quickly, accurately and sensitively detect 18 chemical components in the water extract of *Clerodendrum trichotomum* leaves, including sucrose, trigonelline hydrochloride, adenosine, L-leucine, L-phenylalanine, p-methoxybenzaldehyde, loganic acid, styracin, gentianin, berberine III, shampoosin, plantago asiatica glycoside, honeysuckle glycoside, verbascoside, akebia phenylethanol glycoside B, coniferaldehyde, hesperidin, chelidonine, and 6-gingerol, providing guidance for the assessment of the intrinsic quality of *Clerodendrum trichotomum*. Attached Figure Description

[0027] Figure 1 ESI ion pattern diagram of chemical components in the water extract of *Bambusa textilis* leaves (A is positive ion pattern, B is negative ion pattern);

[0028] Figure 2 Structure, molecular weight, and secondary mass spectrum of sucrose and trigonelline hydrochloride from the aqueous extract of *Bromus odoratus* leaves;

[0029] Figure 3 Structure, molecular weight, and secondary mass spectrum of adenosine, L-leucine, and L-phenylalanine extracted from the leaves of *Bambusa textilis*;

[0030] Figure 4 Structure, molecular weight, and secondary mass spectrum of p-methoxybenzaldehyde, loganic acid, and strychnine from the aqueous extract of *Bambusa textilis* leaves;

[0031] Figure 5 Structure, molecular weight, and secondary mass spectrum of the aqueous extracts of *Bletilla striata* leaves containing berberine III, saftoside, and psyllium glycoside.

[0032] Figure 6 Structure, molecular weight, and secondary mass spectrum of the aqueous extracts of *Bambusa textilis* leaves, including honeysuckle glycoside, verbascoside, and akebia phenylethanol glycoside B.

[0033] Figure 7 Structure, molecular weight, and secondary mass spectrum of the water extracts of *Bambusa textilis* leaves containing coniferaldehyde, hesperidin, and celandine;

[0034] Figure 8 Structure, molecular weight, and secondary mass spectrum of 6-gingerol, an aqueous extract of *Bromhidrosis pilosula* leaves.

[0035] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention. Detailed Implementation

[0036] Example 1:

[0037] A method for determining the components of water extracts from leaves of *Clerodendrum trichotomum* includes the following steps:

[0038] a. Preparation of aqueous extract of *Clerodendrum trichotomum* leaves: Dry and pulverize *Clerodendrum trichotomum* leaves and pass them through a 40-mesh sieve. Weigh 40g of powder and place it in a 1000mL stoppered conical flask. Add deionized water at a material-to-liquid ratio of 1:40 to dissolve the powder and soak for 12 hours. Then, extract the powder by ultrasonication at 100℃ for 2 hours (200W, 300Hz). After ultrasonication, filter the powder through gauze while it is still hot. After vacuum filtration, collect the supernatant and freeze-dry it under vacuum. Store it in a sealed container.

[0039] b. Preparation of test solution: Dissolve the sealed and preserved lyophilized water extract powder in 20 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 90:10 between the 0.1% formic acid aqueous solution and the acetonitrile solution. Sonicate at 100℃ for 10 min (200 W, 300 Hz), shake well and vortex for 30 s. Filter through a 0.22 μm microporous membrane. Store the filtrate at 4℃ for later use.

[0040] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

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

[0042] The chemical components of the water extract of *Bromhidrosis pilosula* were detected using liquid chromatography-mass spectrometry and ESI ion source, respectively.

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

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

[0045] Mobile phase: Gradient elution mobile phase A is an acetonitrile solution containing 0.1% formic acid, and mobile phase B is an aqueous solution of 0.1% formic acid;

[0046] The elution process is as follows: 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 spectrometry system was a Thermo Scientific Q Exactive Focus with a full mass spectrum scanning range of 100-1500 m / z, a resolution of 17500-70000, a Full MS-MS / MS range of 50-100 ms, a separation width of 1.5 m / z, and a threshold set to 1 e. 6 and 2e 5 The intensity threshold is 1.6e. 5 The minimum AGC target is set to 8e. 3 Dynamic emission reduction is 5 seconds;

[0049] d. Using the mass spectrometry system of step c, set the ion source parameters to perform qualitative detection and obtain mass spectrometry information. Use Thermo Xcalibur Qual Browser software to retrieve the raw data acquired by mass spectrometry and compare and analyze it with the mass spectrometry information database to identify the chemical composition of the water extract of the leaves of *Clerodendrum trichotomum*.

[0050] The ion source parameters are set as follows: ionization mode is electrospray ionization (ESI), detection is performed in positive and negative ion modes respectively, atomization voltage is 3.0kV(+) / 2.5kV(-), capillary temperature is 320℃, probe heating temperature is 350℃, sheath gas is 35arb, auxiliary gas is 10arb, and purge gas is 0arb.

[0051] Example 2:

[0052] A method for determining the components of water extracts from leaves of *Clerodendrum trichotomum* includes the following steps:

[0053] a. Preparation of aqueous extract of *Clerodendrum trichotomum* leaves: Dry and pulverize *Clerodendrum trichotomum* leaves and pass them through a 35-mesh sieve. Weigh 30g of powder and place it in a 1000mL stoppered conical flask. Add deionized water at a material-to-liquid ratio of 1:30 to dissolve the powder and soak for 10 hours. Then, extract the extract by ultrasonication at 110℃ for 1.5 hours (200W, 300Hz). After ultrasonication, filter the extract through gauze while it is still hot. After vacuum filtration, collect the supernatant and freeze-dry it under vacuum. Store it in a sealed container.

[0054] b. Preparation of test solution: Dissolve the sealed and preserved lyophilized water extract powder in 15 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 80:10. Sonicate at 110℃ for 8 min (200W, 300Hz), shake well and vortex for 25 s. Filter through a 0.22 μm microporous membrane. Store the filtrate at 3℃ for later use.

[0055] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

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

[0057] The chemical components of the water extract of *Bromhidrosis pilosula* were detected using liquid chromatography-mass spectrometry and ESI ion source, respectively.

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

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

[0060] Mobile phase: Gradient elution mobile phase A is an acetonitrile solution containing 0.1% formic acid, and mobile phase B is an aqueous solution of 0.1% formic acid;

[0061] The elution process is as follows: 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 spectrometry system was a Thermo Scientific Q Exactive Focus with a full mass spectrum scanning range of 100-1500 m / z, a resolution of 17500-70000, a Full MS-MS / MS range of 50-100 ms, a separation width of 1.5 m / z, and a threshold set to 1 e. 6 and 2e 5 The intensity threshold is 1.6e. 5 The minimum AGC target is set to 8e. 3 Dynamic emission reduction is 5 seconds;

[0064] d. Using the mass spectrometry system of step c, set the ion source parameters to perform qualitative detection and obtain mass spectrometry information. Use Thermo Xcalibur Qual Browser software to retrieve the raw data acquired by mass spectrometry and compare and analyze it with the mass spectrometry information database to identify the chemical composition of the water extract of the leaves of *Clerodendrum trichotomum*.

[0065] The ion source parameters are set as follows: ionization mode is electrospray ionization (ESI), detection is performed in positive and negative ion modes respectively, atomization voltage is 3.0kV(+) / 2.5kV(-), capillary temperature is 320℃, probe heating temperature is 350℃, sheath gas is 35arb, auxiliary gas is 10arb, and purge gas is 0arb.

[0066] Example 3:

[0067] A method for determining the components of water extracts from leaves of *Clerodendrum trichotomum* includes the following steps:

[0068] a. Preparation of aqueous extract of *Clerodendrum trichotomum* leaves: Dry and pulverize *Clerodendrum trichotomum* leaves and pass them through a 45-mesh sieve. Weigh 50g of powder and place it in a 1000mL stoppered conical flask. Add deionized water at a material-to-liquid ratio of 1:50 to dissolve the powder and soak for 12 hours. Then, extract the powder by ultrasonication at 90℃ for 2.5 hours (200W, 300Hz). After ultrasonication, filter the powder through gauze while it is still hot. After vacuum filtration, collect the supernatant and freeze-dry it under vacuum. Store it in a sealed container.

[0069] b. Preparation of test solution: Dissolve the sealed and preserved lyophilized water extract powder in 25 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution of 100:10. Sonicate at 90℃ for 12 min (200W, 300Hz), shake well and vortex for 35 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3-5℃ for later use.

[0070] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

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

[0072] The chemical components of the water extract of *Bromhidrosis pilosula* were detected using liquid chromatography-mass spectrometry and ESI ion source, respectively.

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

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

[0075] Mobile phase: Gradient elution mobile phase A is an acetonitrile solution containing 0.1% formic acid, and mobile phase B is an aqueous solution of 0.1% formic acid;

[0076] The elution process is as follows: 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 spectrometry system was a Thermo Scientific Q Exactive Focus with a full mass spectrum scanning range of 100-1500 m / z, a resolution of 17500-70000, a Full MS-MS / MS range of 50-100 ms, a separation width of 1.5 m / z, and a threshold set to 1 e. 6 and 2e 5 The intensity threshold is 1.6e. 5 The minimum AGC target is set to 8e. 3 Dynamic emission reduction is 5 seconds;

[0079] d. Using the mass spectrometry system of step c, set the ion source parameters to perform qualitative detection and obtain mass spectrometry information. Use Thermo Xcalibur Qual Browser software to retrieve the raw data acquired by mass spectrometry and compare and analyze it with the mass spectrometry information database to identify the chemical composition of the water extract of the leaves of *Clerodendrum trichotomum*.

[0080] The ion source parameters are set as follows: ionization mode is electrospray ionization (ESI), detection is performed in positive and negative ion modes respectively, atomization voltage is 3.0kV(+) / 2.5kV(-), capillary temperature is 320℃, probe heating temperature is 350℃, sheath gas is 35arb, auxiliary gas is 10arb, and purge gas is 0arb.

[0081] Example 4:

[0082] A method for determining the components of water extracts from leaves of *Clerodendrum trichotomum* includes the following steps:

[0083] a. Preparation of aqueous extract of *Clerodendrum trichotomum* leaves: Dry and pulverize *Clerodendrum trichotomum* leaves and pass them through a 40-mesh sieve. Weigh 35g of powder and place it in a 1000mL stoppered conical flask. Add deionized water at a material-to-liquid ratio of 1:45 to dissolve the powder and soak for 12 hours. Then, extract the powder by ultrasonication at 100℃ for 2.2 hours (200W, 300Hz). After ultrasonication, filter the powder through gauze while it is still hot. After vacuum filtration, collect the supernatant and freeze-dry it under vacuum. Store it in a sealed container.

[0084] b. Preparation of test solution: Dissolve the sealed and preserved lyophilized water extract powder in 22 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution of 95:10. Sonicate at 100℃ for 11 min (200W, 300Hz), shake well and vortex for 28 s. Filter through a 0.22 μm microporous membrane. Store the filtrate at 5℃ for later use.

[0085] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

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

[0087] The chemical components of the water extract of *Bromhidrosis pilosula* were detected using liquid chromatography-mass spectrometry and ESI ion source, respectively.

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

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

[0090] Mobile phase: Gradient elution mobile phase A is an acetonitrile solution containing 0.1% formic acid, and mobile phase B is an aqueous solution of 0.1% formic acid;

[0091] The elution process is as follows: 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 spectrometry system was a Thermo Scientific Q Exactive Focus with a full mass spectrum scanning range of 100-1500 m / z, a resolution of 17500-70000, a Full MS-MS / MS range of 50-100 ms, a separation width of 1.5 m / z, and a threshold set to 1 e. 6 and 2e 5 The intensity threshold is 1.6e. 5 The minimum AGC target is set to 8e. 3 Dynamic emission reduction is 5 seconds;

[0094] d. Using the mass spectrometry system of step c, set the ion source parameters to perform qualitative detection and obtain mass spectrometry information. Use Thermo Xcalibur Qual Browser software to retrieve the raw data acquired by mass spectrometry and compare and analyze it with the mass spectrometry information database to identify the chemical composition of the water extract of the leaves of *Clerodendrum trichotomum*.

[0095] The ion source parameters are set as follows: ionization mode is electrospray ionization (ESI), detection is performed in positive and negative ion modes respectively, atomization voltage is 3.0kV(+) / 2.5kV(-), capillary temperature is 320℃, probe heating temperature is 350℃, sheath gas is 35arb, auxiliary gas is 10arb, and purge gas is 0arb.

[0096] Example 5:

[0097] A method for determining the components of water extracts from leaves of *Clerodendrum trichotomum* includes the following steps:

[0098] a. Preparation of aqueous extract of *Clerodendrum trichotomum* leaves: Dry and pulverize *Clerodendrum trichotomum* leaves and pass them through a 40-mesh sieve. Weigh 45g of powder and place it in a 1000mL stoppered conical flask. Add deionized water at a material-to-liquid ratio of 1:35 to dissolve the powder and soak for 13 hours. Then, extract the powder by ultrasonication at 100℃ for 2 hours (200W, 300Hz). After ultrasonication, filter the powder through gauze while it is still hot. After vacuum filtration, collect the supernatant and freeze-dry it under vacuum. Store it in a sealed container.

[0099] b. Preparation of test solution: Dissolve the sealed and preserved lyophilized water extract powder in 18 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution of 80-100:10. Sonicate at 100℃ for 9 min (200W, 300Hz), shake well and vortex for 32 s. Filter through a 0.22 μm microporous membrane. Store the filtrate at 3.5℃ for later use.

[0100] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

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

[0102] The chemical components of the water extract of *Bromhidrosis pilosula* were detected using liquid chromatography-mass spectrometry and ESI ion source, respectively.

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

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

[0105] Mobile phase: Gradient elution mobile phase A is an acetonitrile solution containing 0.1% formic acid, and mobile phase B is an aqueous solution of 0.1% formic acid;

[0106] The elution process is as follows: 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 spectrometry system was a Thermo Scientific Q Exactive Focus with a full mass spectrum scanning range of 100-1500 m / z, a resolution of 17500-70000, a Full MS-MS / MS range of 50-100 ms, a separation width of 1.5 m / z, and a threshold set to 1 e. 6 and 2e 5 The intensity threshold is 1.6e. 5 The minimum AGC target is set to 8e. 3 Dynamic emission reduction is 5 seconds;

[0109] d. Using the mass spectrometry system of step c, set the ion source parameters to perform qualitative detection and obtain mass spectrometry information. Use Thermo Xcalibur Qual Browser software to retrieve the raw data acquired by mass spectrometry and compare and analyze it with the mass spectrometry information database to identify the chemical composition of the water extract of the leaves of *Clerodendrum trichotomum*.

[0110] The ion source parameters are set as follows: ionization mode is electrospray ionization (ESI), detection is performed in positive and negative ion modes respectively, atomization voltage is 3.0kV(+) / 2.5kV(-), capillary temperature is 320℃, probe heating temperature is 350℃, sheath gas is 35arb, auxiliary gas is 10arb, and purge gas is 0arb.

[0111] This invention has undergone extensive analytical verification experiments, and the following are the results of the experimental research of this invention:

[0112] 1. Preparation of extract from leaves of *Clerodendrum trichotomum* (a type of shrub).

[0113] The experimental material, *P. puberula*, was collected from Wuchuan Gelao and Miao Autonomous County, Guizhou Province. The research group has established its seedling propagation technology. In early 2019, two-year-old seedlings of *P. puberula* were planted at the Guizhou University Research Base (26°25'54"N, 106°40′53″E, located in a subtropical humid monsoon climate zone with 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] The leaves of *Clerodendrum trichotomum* were dried and pulverized. 40g of the powder was placed in a 1000mL stoppered conical flask, and deionized water was added at a ratio of 1:40 to dissolve the powder. After soaking for 12 hours, ultrasonic extraction was performed. The mixture was filtered through gauze while still hot, and the supernatant was collected by vacuum freeze-drying and then sealed for storage.

[0115] 2. Preparation of the test solution

[0116] Dissolve the sealed, freeze-dried water extract powder in 20 mL of acetonitrile: 0.1% formic acid aqueous solution (10:90, v / v), sonicate for 10 min (300 Hz, 30 °C), shake well and vortex for 30 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 4 °C for later use.

[0117] 3. Liquid Chromatography Detection Conditions

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

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

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

[0121] 4. Mass spectrometry detection conditions

[0122] The mass spectrometry system was a Thermo Scientific Q Exactive Focus with a full mass spectrum scan range of 100–1500 m / z, a resolution of 17500–70000 m / z, a Full MS-MS / MS time of 50–100 ms, an isolation window of 1.5 m / z, and a threshold set to 1 eΩ. 6 and 2e 5 The intensity threshold is 1.6e. 5 The minimum AGC target is set to 8e. 3 Dynamic exclusion takes 5 seconds.

[0123] 5. Ion source settings

[0124] The ion source parameters are set as follows: the ionization mode is electrospray ionization (ESI), with simultaneous collection of positive and negative ions. The parameters are set as follows: atomization voltage 3.0kV(+) / 2.5kV(-), capillary temperature 320℃, probe heating temperature 350℃, sheath gas 35arb, auxiliary gas 10arb, and purge gas 0arb.

[0125] 6. Qualitative chemical composition

[0126] The mass spectrometry system used was Thermo Scientific Q Exactive Focus. The raw data acquired by mass spectrometry was retrieved using Compound Discovery software and compared with a compound mass spectrometry database. Eighteen chemical components were identified in the water extract of *Clerodendrum trichotomum* leaves, including: sucrose (1), trigonelline hydrochloride (2), adenosine (3), L-leucine (4), L-phenylalanine (5), p-methoxybenzaldehyde (6), loganic acid (7), styracin (8), berberine III (9), shampooside (10), plantago asiatica glycoside (11), honeysuckle glycoside (12), verbascoside (13), akebia phenylethanol glycoside B (14), coniferaldehyde (15), hesperidin (16), chelidonine (17), and 6-gingerol (18). Among these, there were 2 alkaloids, 2 amino acids, 4 flavonoids, 3 glycosides, and 7 other components. (See details...) Figure 1 See Table 1.

[0127] Table 1. Identification results of the water extract of *Bromhidrosis pilosula*.

[0128]

[0129]

Claims

1. A method for determining the components of an aqueous extract of *Clerodendrum trichotomum* leaves, characterized in that: Includes the following steps: a. Preparation of aqueous extract of *Clerodendrum trichotomum* leaves: Dry and pulverize *Clerodendrum trichotomum* leaves, weigh 30-50g of powder and place it in a 1000mL stoppered conical flask, add deionized water at a material-to-liquid ratio of 1:30-50 to dissolve and soak for 10-14h, then ultrasonically extract for 1.5-2.5h, filter with gauze while hot after ultrasonication, collect the supernatant by vacuum filtration, freeze-dry under vacuum, and store in a sealed container; b. Preparation of test solution: Dissolve the sealed and preserved lyophilized water extract powder in 15-25 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 0.1% formic acid aqueous solution to acetonitrile solution of 80-100:

10. Sonicate for 8-12 min, shake well and vortex for 25-35 s, filter through a 0.22 μm microporous membrane, and store the filtrate at 3-5℃ for later use. c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system. The chromatographic detection conditions are as follows: Chromatographic column: Waters Acquity UPLC HSS T3, dimensions 100*2.1mm*1.8μm; The liquid phase system is: Thermo Scientific Ultimate 3000 RSLC, HPG; Mobile phase: Gradient elution mobile phase A is an acetonitrile solution containing 0.1% formic acid, and mobile phase B is an aqueous solution of 0.1% formic acid; The elution process is as follows: 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. The mass spectrometry conditions were as follows: the mass spectrometry system was a Thermo Scientific Q Exactive Focus; the full mass spectrum scan range was 100–1500 m / z; the resolution was 17500–70000 m / z; the Full MS-MS / MS frequency was 50–100 ms; the separation width was 1.5 m / z; and the threshold was set to 1 eΩ. 6 and 2e 5 The intensity threshold is 1.6e. 5 The minimum AGC target is set to 8e. 3 Dynamic emission reduction is 5 seconds; d. Using the mass spectrometry system of step c, set the ion source parameters to perform qualitative detection and obtain mass spectrometry information. Use Thermo Xcalibur Qual Browser software to retrieve the raw data acquired by mass spectrometry and compare and analyze it with the mass spectrometry information database to identify the chemical composition of the water extract of the leaves of *Clerodendrum trichotomum*. The parameters of the ion source are set as follows: the ionization mode is electrospray ionization (ESI), with detection in positive and negative ion modes respectively, the atomization voltage is 3.0kV+ / 2.5kV-, the capillary temperature is 320℃, the probe heating temperature is 350℃, the sheath gas is 35arb, the auxiliary gas is 10arb, and the purge gas is 0arb. The aqueous extract of *Clerodendrum trichotomum* leaves includes sucrose, trigonelline hydrochloride, adenosine, L-leucine, L-phenylalanine, p-methoxybenzaldehyde, loganic acid, styracin, berberine, styracin, styracin, physalisin, honeysuckle glycoside, verbascoside, akebia phenylethanol glycoside B, coniferaldehyde, hesperidin, celandine, and 6-gingerol.

2. The method for determining the components of the water extract of *Clerodendrum trichotomum* leaves according to claim 1, characterized in that: In step a, the leaves of *Clerodendrum trichotomum* are dried, pulverized, and passed through a 35-45 mesh sieve. 35-45g of powder is weighed and placed in a 1000mL stoppered conical flask. Deionized water is added at a material-to-liquid ratio of 1:35-45 to dissolve the powder, and the mixture is soaked for 11-13 hours. Then, ultrasonic extraction is performed for 1.8-2.2 hours. The ultrasonic power is 200W, the frequency is 300Hz, and the ultrasonic temperature is 90-110℃. After ultrasonic extraction, the mixture is filtered through gauze while still hot. The supernatant is then collected by vacuum freeze-drying and sealed for storage.

3. The method for determining the components of the aqueous extract of *Clerodendrum trichotomum* leaves according to claim 2, characterized in that: In step a, the leaves of *Clerodendrum trichotomum* are dried, pulverized, and passed through a 40-mesh sieve. 40g of powder is weighed and placed in a 1000mL stoppered conical flask. Deionized water is added at a ratio of 1:40 to dissolve the powder, and the mixture is soaked for 12 hours. Then, ultrasonic extraction is performed for 2 hours. The ultrasonic power is 200W, the frequency is 300Hz, and the ultrasonic temperature is 100℃. After ultrasonic extraction, the mixture is filtered through a gauze while still hot. The supernatant is then collected by vacuum freeze-drying and sealed for storage.

4. The method for determining the components of the water extract of *Clerodendrum trichotomum* leaves according to claim 1, characterized in that: In step b, the sealed and preserved freeze-dried water extract powder is dissolved in 18-22 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 85-95:

10. The mixture is ultrasonically treated for 9-11 min at a power of 200 W, a frequency of 300 Hz, and a temperature of 90-110 °C. After ultrasonication, the mixture is shaken and vortexed for 28-32 s. The solution is then filtered through a 0.22 μm microporous membrane, and the filtrate is stored at 3.5-4.5 °C for later use.

5. The method for determining the components of the water extract of *Clerodendrum trichotomum* leaves according to claim 4, characterized in that: In step b, the sealed and preserved freeze-dried water extract powder is dissolved in 20 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 90:

10. The mixture is ultrasonically treated for 10 min at a power of 200 W, a frequency of 300 Hz, and a temperature of 100 °C. After ultrasonication, the mixture is shaken and vortexed for 30 s. The solution is then filtered through a 0.22 μm microporous membrane, and the filtrate is stored at 4 °C for later use.

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