A method for determining the components of an alcohol extract of *Clerodendrum trichotomum* leaves.

Abstract

This invention discloses a method for determining the components of an ethanol extract of *Clerodendrum trichotomum* leaves. A liquid chromatography-mass spectrometry (LC-MS) method was established to detect the content of the ethanol extract. Raw data acquired by mass spectrometry was retrieved using software and compared with a mass spectrometry database to identify the chemical components of the ethanol extract. Ultimately, 44 chemical components were detected in the ethanol extract, providing a basis for the quality and efficacy evaluation of *Clerodendrum trichotomum* 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 alcohol 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 its alcohol extract, which lacks relevant literature.

[0003] There are few methods for detecting the chemical components of *Clerodendrum trichotomum*, especially for determining the components of the ethanol extract of its leaves, which lacks published literature. Because the effective components of *Clerodendrum trichotomum* 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 *Clerodendrum trichotomum*, enabling comprehensive control over the quality of these substances, and providing a more sufficient basis for the quality evaluation of *Clerodendrum trichotomum*, 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 ethanol extract of *Clerodendrum trichotomum* leaves. This method can perform qualitative analysis of the chemical components in the ethanol 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 alcohol extract of *Clerodendrum trichotomum* leaves includes the following steps:

[0007] a. Preparation of alcohol extract from leaves of *Clerodendrum trichotomum*: Dry and pulverize leaves of *Clerodendrum trichotomum*, weigh 15-25g of powder and place it in a 1000mL stoppered conical flask, add 65-75% ethanol to dissolve, and extract by ultrasonication for 1.8-2.5h. After ultrasonication, filter with gauze while hot, filter the supernatant, freeze-dry under vacuum, and store in a sealed container.

[0008] b. Preparation of test solution: Dissolve the sealed, lyophilized ethanol extract powder in 15-25 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio 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 compare and analyze the raw data acquired by mass spectrometry with the mass spectrometry information database to identify the chemical components of the alcohol extract of *Clerodendrum trichotomum* leaves.

[0011] In step a above, the leaves of *Clerodendrum trichotomum* are dried and pulverized through a 30-50 mesh sieve. 18-22g of the powder is weighed and placed in a 1000mL stoppered conical flask. 65-75% ethanol is added to dissolve the powder, and ultrasonic extraction is performed for 1.2-2.2 hours. The ultrasonic power is 200W, the frequency is 300Hz, and the ultrasonic temperature is 90-110℃. After ultrasonic extraction, the powder 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 and pulverized through a 40-mesh sieve. 20g of powder is weighed and placed in a 1000mL stoppered conical flask, dissolved in 70% ethanol, and extracted by ultrasonication for 2 hours. The ultrasonic power is 200W, the frequency is 300Hz, and the ultrasonic temperature is 100℃. After ultrasonication, 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 lyophilized ethanol 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 lyophilized ethanol 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 sonicated for 10 min at a power of 200 W, a frequency of 300 Hz, and a temperature of 100 °C. After sonication, 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 solvents are acetonitrile solution containing 0.1% formic acid (mobile phase A) and aqueous solution containing 0.1% formic acid (mobile phase B). The elution times and organic phase ratios are: 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 is 0.3 mL / min, and the column temperature is 40℃. The specific parameters of the above experiments were obtained after more than two months of condition exploration. 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* cannot be detected or the detection is inaccurate.

[0026] (3) The chemical component detection method of *Clerodendrum trichotomum* described in this invention can quickly, accurately, and sensitively detect 44 chemical components of the alcohol extract of *Clerodendrum trichotomum* leaves: lactose (1), sucrose (2), trigonelline hydrochloride (3), L-proline (4), adenosine (5), L-leucine (6), caffeic acid (7), shampooside (8), plantagoside (9), honeysuckle glycoside (10), hyperoside (11), akebia phenylethanol glycoside B (12), styracin (13), verbascoside (14), geraniol (15), hesperidin (16), luteolin (17), piperidine I (18), saponin (19), buddleja glycoside (20), luteolin (21), and *Phellinus linteus*. The following compounds were identified: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 30, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 ... 48, 49, 40, 41, Attached Figure Description

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

[0028] Figure 2 Structure, molecular weight, and secondary mass spectrum of the chemical components of lactose, sucrose, and trigonelline hydrochloride extracted from the leaves of *Bletilla striata*;

[0029] Figure 3 Structure, molecular weight, and secondary mass spectrum of L-proline, adenosine, and L-leucine in the extract of *Bletilla striata* leaf;

[0030] Figure 4 Structure, molecular weight, and secondary mass spectrum of caffeic acid, shampooside, and psyllium glycoside extracted from *Bletilla striata* leaves;

[0031] Figure 5 Structure, molecular weight, and secondary mass spectrum of the extracts of *Bambusa textilis* leaves, including lonicotinic acid, hyperotinic acid, and akebia phenylethanol glycoside B.

[0032] Figure 6 Structure, molecular weight, and secondary mass spectrum of the extracts of *Bambusa textilis* leaves, including ginsenoside, verbascoside, and geraniol;

[0033] Figure 7 Structure, molecular weight, and secondary mass spectrum of hesperidin, luteolin, and berberine I extracted from the leaves of *Bambusa textilis*;

[0034] Figure 8 Structure, molecular weight, and secondary mass spectrum of saponins, buddleja glycosides, and luteolin extracted from the leaves of *Bromhidrosis pilosula*;

[0035] Figure 9 Structure, molecular weight, and secondary mass spectrum of morin, geraniol, and isorhamnetin extracted from the leaves of *Bambusa textilis*;

[0036] Figure 10 Structure, molecular weight, and secondary mass spectrum of iris flavonoids, hydroxygenistein, and isozygoflavonoids extracted from the leaves of *Bambusa textilis*;

[0037] Figure 11 Structure, molecular weight, and secondary mass spectrum of brown cyanidin, sweet orange flavonoids, and isozygoflavin extracted from the leaves of *Bromus odoratus*;

[0038] Figure 12 Structure, molecular weight, and secondary mass spectrum of the extracts of *Bromhidrosis pilosula* leaves, including hesperidin, kaempferol, and acaciatin;

[0039] Figure 13 Structure, molecular weight, and secondary mass spectra of the chemical components of *Bromus odoratus* leaf extract, including strychnine, crocin, and 6-gingerol;

[0040] Figure 14 Structure, molecular weight, and secondary mass spectrum of asiatic acid, ethionine, and glycyrrhetinic acid extracted from the leaves of *Bambusa textilis*;

[0041] Figure 15 Structure, molecular weight, and secondary mass spectrum of genistein B, demethylhesperidin, and styracidin extracted from the leaves of *Bambusa textilis*;

[0042] Figure 16 Structure, molecular weight, and secondary mass spectrum of ursolic acid and α-linolenic acid extracted from the leaves of *Bambusa textilis*.

[0043] 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

[0044] Example 1:

[0045] The method for determining the components of the alcohol extract of *Clerodendrum trichotomum* leaves includes the following steps:

[0046] a. Preparation of alcohol extract from leaves of *Clerodendrum trichotomum*: Dry and pulverize leaves of *Clerodendrum trichotomum* through a 40-mesh sieve. Weigh 20g of powder and place it in a 1000mL stoppered conical flask. Add 70% ethanol to dissolve the powder and extract it by ultrasonic extraction at 100℃ for 2h (200W, 300Hz). 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.

[0047] b. Preparation of test solution: Dissolve the sealed, lyophilized ethanol extract powder in 20 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 90:10. 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.

[0048] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

[0049] The chromatographic detection conditions are as follows:

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

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

[0052] 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;

[0053] The elution process is as follows: 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 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;

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

[0057] Mass spectrometry conditions Same as step c ;

[0058] 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.

[0059] The raw data acquired by mass spectrometry was retrieved using Thermo Xcalibur Qual Browser software and compared with a mass spectrometry information database to identify the chemical components of the alcohol extract of *Clerodendrum trichotomum* leaves.

[0060] Example 2:

[0061] The method for determining the components of the alcohol extract of *Clerodendrum trichotomum* leaves includes the following steps:

[0062] a. Preparation of alcohol extract from leaves of *Clerodendrum trichotomum*: Dry and pulverize leaves of *Clerodendrum trichotomum*, weigh 15g of powder and place it in a 1000mL stoppered conical flask, add 65-75% ethanol to dissolve, and extract by ultrasonication at 110℃ for 1.5h (200W, 300Hz). Filter with gauze while hot, collect the supernatant by vacuum filtration, freeze-dry under vacuum, and store in a sealed container.

[0063] b. Preparation of test solution: Dissolve the sealed, lyophilized ethanol 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.

[0064] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

[0065] The chromatographic detection conditions are as follows:

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

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

[0068] 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;

[0069] The elution process is as follows: 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 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;

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

[0073] Mass spectrometry conditions Same as step c ;

[0074] 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.

[0075] The raw data acquired by mass spectrometry was retrieved using Thermo Xcalibur Qual Browser software and compared with a mass spectrometry information database to identify the chemical components of the alcohol extract of *Clerodendrum trichotomum* leaves.

[0076] Example 3:

[0077] The method for determining the components of the alcohol extract of *Clerodendrum trichotomum* leaves includes the following steps:

[0078] a. Preparation of alcohol extract of leaves of *Clerodendrum trichotomum*: Dry and pulverize leaves of *Clerodendrum trichotomum*, weigh 25g of powder and place it in a 1000mL stoppered conical flask, add 75% ethanol to dissolve, and extract by ultrasonication at 90℃ for 2.5h (200W, 300Hz). Filter with gauze while hot, and after vacuum filtration, freeze dry the supernatant and store it in a sealed container.

[0079] b. Preparation of test solution: Dissolve the sealed, lyophilized ethanol 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. Store the filtrate at 3-5℃ for later use.

[0080] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

[0081] The chromatographic detection conditions are as follows:

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

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

[0084] 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;

[0085] The elution process is as follows: 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 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;

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

[0089] Mass spectrometry conditions Same as step c ;

[0090] 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.

[0091] The raw data acquired by mass spectrometry was retrieved using Thermo Xcalibur Qual Browser software and compared with a mass spectrometry information database to identify the chemical components of the alcohol extract of *Clerodendrum trichotomum* leaves.

[0092] Example 4:

[0093] The method for determining the components of the alcohol extract of *Clerodendrum trichotomum* leaves includes the following steps:

[0094] a. Preparation of alcohol extract from leaves of *Clerodendrum trichotomum*: Dry and pulverize leaves of *Clerodendrum trichotomum*, weigh 18g of powder and place it in a 1000mL stoppered conical flask, add 65% ethanol to dissolve, and extract by ultrasonication at 100℃ for 2.5h (200W, 300Hz). Filter with gauze while hot, collect the supernatant by vacuum filtration, freeze dry under vacuum, and store in a sealed container.

[0095] b. Preparation of test solution: Dissolve the sealed, lyophilized ethanol extract powder in 22 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 95:10. Sonicate at 100℃ for 12 min (200 W, 300 Hz), shake well and vortex for 25 s. Filter through a 0.22 μm microporous membrane. Store the filtrate at 5℃ for later use.

[0096] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

[0097] The chromatographic detection conditions are as follows:

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

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

[0100] 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;

[0101] The elution process is as follows: 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 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;

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

[0105] Mass spectrometry conditions Same as step c ;

[0106] 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.

[0107] The raw data acquired by mass spectrometry was retrieved using Thermo Xcalibur Qual Browser software and compared with a mass spectrometry information database to identify the chemical components of the alcohol extract of *Clerodendrum trichotomum* leaves.

[0108] Example 5:

[0109] The method for determining the components of the alcohol extract of *Clerodendrum trichotomum* leaves includes the following steps:

[0110] a. Preparation of alcohol extract of leaves of *Clerodendrum trichotomum*: Dry and pulverize leaves of *Clerodendrum trichotomum*, weigh 22g of powder and place it in a 1000mL stoppered conical flask, add 75% ethanol to dissolve, and extract by ultrasonication at 100℃ for 1.5h (200W, 300Hz). Filter with gauze while hot, and after vacuum filtration, freeze dry the supernatant and store it in a sealed container.

[0111] b. Preparation of test solution: Dissolve the sealed, lyophilized ethanol extract powder in 18 mL of 0.1% formic acid aqueous solution: acetonitrile, with a volume ratio of 80-100:10. Sonicate at 100℃ for 8 min (200W, 300Hz), shake well and vortex for 35 s. Filter through a 0.22 μm microporous membrane. Store the filtrate at 3℃ for later use.

[0112] c. The components of the test solution were separated and detected using a liquid chromatography-mass spectrometry (UPLC-Q-OrbitrapHRMS) system.

[0113] The chromatographic detection conditions are as follows:

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

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

[0116] 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;

[0117] The elution process is as follows: 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 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;

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

[0121] Mass spectrometry conditions Same as step c ;

[0122] 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.

[0123] The raw data acquired by mass spectrometry was retrieved using Thermo Xcalibur Qual Browser software and compared with a mass spectrometry information database to identify the chemical components of the alcohol extract of *Clerodendrum trichotomum* leaves.

[0124] This invention has undergone extensive analytical verification experiments, and the following are the results of the experimental research of this invention:

[0125] 1. Preparation of alcohol extract from leaves of *Clerodendrum trichotomum*

[0126] 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.

[0127] Dry and pulverize the leaves of *Clerodendrum trichotomum*, weigh 20g of the powder and place it in a 1000mL stoppered conical flask, then dissolve it in 70% ethanol. Extract by ultrasonication for 2 hours, filter while hot through gauze, collect the supernatant by vacuum freeze-drying, and store in a sealed container.

[0128] 2. Preparation of the test solution

[0129] Dissolve the sealed, lyophilized ethanol extract powder in 20 mL of acetonitrile: 0.1% formic acid aqueous solution (10:90, v / v), sonicate for 10 min (300 Hz, 100 °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.

[0130] 3. Liquid Chromatography Detection Conditions

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

[0132] The liquid phase system is: 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 times and organic phase ratios 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 spectrometry system was a Thermo Scientific Q Exactive Focus, with a full mass spectrum scanning range of 100–1500 m / z and a resolution of 70,000; the secondary fragmentation scanning resolution was 17,500; the Full MS-MS / MS range was 50–100 ms; the separation width was 1.5 m / z; and the threshold was 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.

[0136] 5. Ion source settings

[0137] The ion source parameters are set as follows: the ionization mode is electrospray ionization (ESI), and detection is performed in both positive and negative ion modes. 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.

[0138] 6. Qualitative chemical composition

[0139] The mass spectrometry system used was Thermo Scientific Q Exactive Focus. The raw data acquired by mass spectrometry was retrieved using ThermoFish Compound Discovery software and compared with a compound mass spectrometry database. Forty-four chemical components were identified in the ethanol extract of *Clerodendrum trichotomum* leaves. (See attached image.) Figures 1-16 See Table 1.

[0140] Table 1. Detailed information on the alcohol extract of *Bromhidrosis pilosula*.

[0141]

[0142]

Claims

1. A method for determining the components of an alcohol extract of Isodon japonica leaf, characterized by: Comprising the following steps: a. Preparation of the alcohol extract of the leaves of Rhus copallinum: Dry and crush the leaves of Rhus copallinum, take 15-25 g of the powder and place it in a 1000 mL conical flask with a plug, add 65-75% ethanol to dissolve, ultrasonic extraction for 1.5-2.5 h, filter with a hot gauze after ultrasonic extraction, and then perform vacuum freeze-drying on the supernatant, and seal for storage; b. Preparation of the test solution: Dissolve the alcohol extract freeze-dried powder stored in a sealed container in 15-25 mL of 0.1% formic acid solution:acetonitrile, the volume ratio of 0.1% formic acid solution to acetonitrile solution is 80-100:10, ultrasonic treatment for 8-12 min, shake well and vortex for 25-35 s, pass through a 0.22 μm microporous filter membrane, store the filtrate at 3-5°C, and reserve for use; c. Use the liquid chromatography-mass spectrometry system UPLC-Q-Orbitrap HRMS to separate and detect the components of the test solution; The chromatographic detection conditions are: The chromatographic column is Waters Acquity uplc hss T3, with a size of 100*2.1mm*1.8 μm; The liquid phase system is Thermo Scientific Ultimate 3000 RSLC, HPG; The mobile phase is: the gradient elution mobile phase A is 0.1% formic acid acetonitrile solution, and the mobile phase B is 0.1% formic acid aqueous solution; The elution process is: 0-2 min, 5% A; 42-47 min, 95% A; 47.1-50 min, 5% A; The flow rate is 0.3 mL / min, and the column temperature is 40°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 value is set to 1e 6 and 2e 5 , the intensity threshold value is 1.6e 5 , the minimum AGC target is set to 8e 3 , and the dynamic reduction is 5 s. d. Use the mass spectrometry system of step c to set the ion source parameters for qualitative detection to obtain mass spectrometry information, use Thermo Xcalibur Qual Browser software to compare and analyze the original data collected by mass spectrometry, and identify the chemical components of the alcohol extract of the leaves of Rhus copallinum; The ion source parameter settings are as follows: the ionization mode is electrospray ion source ESI, detected in positive and negative ion modes 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; The alcohol extract of the leaves of Rhus copallinum includes lactose, sucrose, trigonelline hydrochloride, L-proline, adenosine, L-leucine, caffeic acid, shatavari glycoside, plantain glycoside, honeysuckle glycoside, hypericin glycoside, akebone glycoside B, zingiberol glycoside, verbascoside, hinoxylin, orange glycoside, osmanthus glycoside, picraline glycoside I, saponin acid, mongolian glycoside, osmanthus glycoside, morin glycoside, hinoxylin, isorhamnetin, wild iris flavonoids, hydroxyeugenol, isomulin, brown carthamus glycoside, sweet orange flavonoids, isomulin, naringenin, hinoxylin, glycyrrhizic acid, gardenia flavonoids B, nor-naringenin, stellacyanin, ursolic acid, and alpha-linolenic acid.

2. The method of claim 1, wherein the constituents of the Isodon japonicus leaf ethanol extract are determined. In the step a, the leaves of the bushy St. John's-wort are dried and crushed to 30-50 mesh, 18-22 g of the powder is weighed and placed in a 1000 mL conical flask with a stopper, 65-75% ethanol is added for dissolution, 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 is 90-110℃, after ultrasonic extraction, hot gauze filtration is performed, the supernatant is obtained by suction filtration, vacuum freeze-drying is performed, and the product is sealed and stored.

3. The method of claim 2, wherein the constituents of the Isodon japonicus leaf ethanol extract are identified. In the step a, the leaves of the bushy St. John's-wort are dried and crushed to 40 mesh, 20 g of the powder is weighed and placed in a 1000 mL conical flask with a stopper, 70% ethanol is added for dissolution, ultrasonic extraction is performed for 2 h, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature is 100℃, after ultrasonic extraction, hot gauze filtration is performed, the supernatant is obtained by suction filtration, vacuum freeze-drying is performed, and the product is sealed and stored.

4. The method of claim 1, wherein the constituents of the Isodon japonicus leaf ethanol extract are identified. In the step b, the freeze-dried powder of the alcohol extract stored in a sealed state 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, ultrasonic treatment is performed for 9-11 min, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature is 90-110℃, after ultrasonic extraction, the solution is shaken and vortexed for 28-32 s, 0.22 μm microporous filter membrane is used for filtration, and the filtrate is stored at 3.5-4.5℃ for storage and standby.

5. The method of claim 4, wherein the constituents of the Isodon japonicus leaf ethanol extract are identified. In the step b, the freeze-dried powder of the alcohol extract stored in a sealed state 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, ultrasonic treatment is performed for 10 min, the ultrasonic power is 200 W, the frequency is 300 Hz, and the ultrasonic temperature is 100℃, after ultrasonic extraction, the solution is shaken and vortexed for 30 s, 0.22 μm microporous filter membrane is used for filtration, and the filtrate is stored at 4℃ for storage and standby.

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