A UHPLC-Q-Exactive Orbitrap MS Detection Method for Sophora japonica Gold and Silver Formula

Abstract

This invention provides a UHPLC-Q-Exactive Orbitrap MS method for detecting Sophora japonica and Lonicera japonica compound. This method is simple to operate, stable, reliable, scientific, and practical. This invention addresses common problems in existing UHPLC-MS methods for traditional Chinese medicine compound preparations. Generally, two different detection methods are needed for the detection of chemical components in traditional Chinese medicine compound preparations and serum. This is especially challenging given the dual challenges of the complex chemical composition of the original formula and the low content and significant interference of metabolites in serum. Determining the chemical components in both the traditional Chinese medicine compound preparation and serum leads to the shortcomings of the aforementioned detection methods, such as limited versatility and high detection costs.

Description

Technical Field

[0001] This invention belongs to the field of chemical component analysis and detection of traditional Chinese medicine, and in particular relates to a UHPLC-Q-Exactive Orbitrap MS detection method for Sophora japonica and Lysimachia christinae. Background Technology

[0002] Malignant melanoma, as the most aggressive malignant skin tumor, has seen a continuous increase in its global incidence over the past fifty years, with lesions in special locations such as mucous membranes and the uvea exhibiting extremely poor prognoses. Despite current clinical approaches employing surgical combined with targeted therapy or immunotherapy, key challenges remain, including drug resistance, insufficient treatment response rates, and severe toxic side effects. Commonly used chemotherapy drugs for malignant melanoma include dacarbazine, temozolomide, platinum-based drugs, vinblastine, paclitaxel, and formustin, with single-agent efficacy rates all below 20%. Dacarbazine and temozolomide are among the more effective chemotherapy drugs. Traditional Chinese medicine (TCM) considers this disease to fall under the categories of "black spots" and "gangrene," and clinical research has revealed its pathogenesis of "heat toxin stagnation," providing a treatment basis for clearing heat and detoxifying. Many TCM formulas for treating melanoma contain Sophora japonica and Lonicera japonica, exhibiting synergistic effects through multiple components, pathways, and targets, playing an important role in the treatment of various diseases. The Sophora japonica and honeysuckle formula was first recorded in *Medical Enlightenment*, and later in *Collection of Medical Enlightenment* by Zhai Liang of the Qing Dynasty, which states: "Sophora japonica and honeysuckle wine treats carbuncles, boils on the back, and all kinds of sores and boils, regardless of whether they are already formed or not, but those that cause burning pain." It is made by decocting Sophora japonica and honeysuckle with wine. The flavonoids such as rutin and quercetin, as well as active ingredients such as chlorogenic acid contained in the formula, have been shown to inhibit tumor angiogenesis and regulate apoptosis pathways. However, the overall pharmacodynamic material basis and the synergistic mechanism of the multi-component formula remain unclear.

[0003] The applicant has conducted a search and analysis of the relevant existing technology patents and journal articles of the Sophora japonica and Sophora chinensis formula. For example, Zhang Ming, Jiang Sanyuan, et al., Research on the Quality Standard of Compound Sophora japonica Granules, China Pharmacy, 2009-07-31, published a paper that uses ultraviolet spectrophotometry to determine the content of rutin in Sophora japonica flowers. The established standard can be used for the quality control of Compound Sophora japonica Granules. Pang Jing Lei Rong et al., Simultaneous Determination of 12 Components in Compound Honeysuckle Granules by HPLC, Food and Drug, 2025-07-31. This article describes the determination of the contents of 12 components in Compound Honeysuckle Granules by HPLC using acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase with gradient elution; detection wavelength of 320 nm; column temperature of 35℃. The contents of these components were determined as follows: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside B, forsythoside A, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, baicalin, norbaicalein, wogonin, and baicalein. Cao HaiyanChen Yiting et al., Food and Drug, 2025-03-20. In this paper, acetonitrile (A) and 0.2% acetic acid aqueous solution (B) were used as mobile phases, column temperature was 40℃, gradient elution was used, and detection wavelength was 355 nm. At the same time, four flavonoid components, namely rutin, quercetin, kaempferol and isorhamnetin, were determined in Sophora japonica buds and flowers. Shi Yiwei , Wang Hongling "Etc., Simultaneous Determination of Seven Components in the Classical Formula Huaihua San by UPLC-MS / MS, Journal of Pharmaceutical Analysis, 2023-02-28. This article uses 0.1% formic acid water (A)-methanol (B) as the mobile phase for gradient elution, and employs electrospray ionization (ESI) source in positive and negative ion mode with a nebulizer gas flow rate of 3 L·min⁻¹." -1 The heating gas flow rate is 10 L·min -1 The residence time was 45 ms, the ion source temperature was 300℃, the heating block temperature was 400℃, the desolvation tube temperature was 250℃, and quantitative analysis was performed using multiple reaction monitoring (MRM) mode to determine the content of seven active ingredients in the classic formula Huaihua San: rutin, menthol, hesperidin, naringin, quercetin, neohesperidin, and quercetin.

[0004] CN106770829A discloses a method for determining the metabolites of honeysuckle in rat serum. In this method, the mobile phase is an acetonitrile-0.1% formic acid aqueous solution gradient elution. The gradient elution program is: 0-8 min, 5-60% acetonitrile; 8-10 min, 60-90% acetonitrile; followed by a 7 min run time. The mass spectrometry conditions are: ESI source, negative ion mode detection, multiple reaction monitoring (MRM), electrospray voltage 4000 V, ion source temperature 325°C, drying gas flow rate 12.0 mL / min, and nebulizer gas pressure 35 psi. A full scan of eight compounds in MRM mode revealed the following eight chemical components: 1-strychnine, 2-chlorogenic acid, 3-caffeic acid, 4-rutin, 5-hyperoside, 6-isochlorogenic acid C, 7-quercetin, and 8-baicalin. Given the numerous shortcomings of the aforementioned detection methods, especially the scarcity of research on the chemical components of the Sophora japonica and Lonicera japonica compound traditional Chinese medicine, the development of a simple and easy-to-use method for detecting chemical components is particularly urgent. Summary of the Invention

[0005] This invention provides a UHPLC-Q-Exactive Orbitrap MS method for detecting Sophora japonica and Lonicera japonica in traditional Chinese medicine formulas. This method is simple to operate, stable, reliable, scientific, and practical. This invention addresses the common problems of existing UHPLC-MS methods for traditional Chinese medicine formulas. Generally, two different detection methods are needed for the detection of chemical components in traditional Chinese medicine formulas and serum. This is especially challenging given the dual challenges of the complex chemical composition of the original formula and the low content and significant interference of metabolites in serum. Determining the chemical components in both traditional Chinese medicine formulas and serum leads to the shortcomings of the aforementioned detection methods, such as limited versatility and high detection costs.

[0006] This invention creatively establishes an integrated detection method for the simultaneous and efficient analysis of the original formula of Sophora japonica and Lonicera japonica extract and its serum metabolites. By combining an optimized segmented gradient elution procedure with high-resolution time-of-flight mass spectrometry (HTMS) in both positive and negative ion modes, this invention achieves comprehensive characterization of hundreds of prototype components of various structural types, ranging from organic acids to flavonoids, in a single analysis. It also enables high-sensitivity capture and identification of major metabolites and their transformation pathways in serum after in vivo metabolism. This detection method overcomes the limitations of previous techniques that only targeted a few components of a single herb, significantly improving the overall coverage and analytical efficiency of the compound chemical composition and in vivo metabolomics. It provides crucial technical support for elucidating the pharmacodynamic material basis and in vivo processes of Sophora japonica and Lonicera japonica extract.

[0007] This invention analyzes the components of the Sophora japonica and Lonicera japonica extract using UPLC-MS / MS, identifying a total of 39 components. Among these, 31 components can be attributed to Lonicera japonica, including chlorogenic acid, quercetin, kaempferol, aloin, benzaldehyde, arbutin, rutin, gentianin, and quinic acid. 12 components can be attributed to Sophora japonica, including rutin, quercetin, kaempferol, soybean saponin I, chlorogenic acid, betulin, gentianin, genistein, sophoretic acid, soybean saponin B, limonene, and lignin.

[0008] This invention also analyzed the blood components of Sophora japonica and Lysimachia christinae formula using UPLC-MS / MS, identifying a total of 58 serum precursor components, including: Spanish salsa ketone, palmitoylethanolamine, phosphatidylcholine, vitamin D3, sphingomyelin, kaempferol-3-gentiobiglycoside, methyl cypermethrin, polygalactosyl A, phlorizin, 15-hydroxyeicosatetraenoic acid, 1,6-di-O-galloyl glucose, 3-hydroxyhexadecanoic acid, 2-hydroxystearic acid, D-tryptophan, nervonic acid, apigenin, transisooleic acid, swertiamarin, tripterygium lactone, chebulic acid, and other chemical components. Among them, there are 8 flavonoid components (kaempferol-3-gentiobiglycoside, phlorizin, apigenin, isorhamnetin-3-glucoside, kaempferol-3-O-rutin, rutin, gentiopicrin, and glycyrrhizin), including apigenin and rutin, which have been preliminarily identified as the direct pharmacodynamic material basis of this compound.

[0009] The technical solution of this invention patent application is as follows:

[0010] A UHPLC-Q-Exactive Orbitrap MS method for detecting Sophora japonica 'Gold and Silver Formula', the method comprising the following steps:

[0011] (1) Sample preparation: Weigh the Sophora japonica and Lycium barbarum powder, dissolve it in water, add ethanol, sonicate for 8-12 min, let stand for 10-14 h, centrifuge for 8-12 min, take the supernatant, blow the ethanol dry with nitrogen to obtain the purified solution, add ultrapure water to make up the volume, filter the solution through a 0.22 μm filter membrane, and set aside for later use.

[0012] (2) Chromatographic conditions:

[0013] Column C 18 The column temperature was 35–45℃, and the flow rate was 0.2–0.4 mL / min. The mobile phases were: A: 0.1% formic acid aqueous solution, B: 0.1% formic acid in acetonitrile solution. The gradient elution program was as follows: 0–1 min, 0% B; 1–2 min, B linearly changed from 0% to 30%; 2–12 min, B linearly changed from 30% to 50%; 12–21 min, B linearly changed from 50% to 100%; 21–26 min, B maintained at 100%; 26–26.1 min, B linearly changed from 100% to 0%; 26.1–30 min, B maintained at 0%.

[0014] (3) Mass spectrometry acquisition conditions are as follows:

[0015] Ion spray voltage ESI: positive ion (+) and negative ion (-) modes, capillary voltage +5500 to -4500 V, temperature: 480 to 520 °C, curtain gas: 42 to 47 psi; declustering voltage: 50 to 70 V, time-of-flight mass spectrometry: 8 to 12 V, scanning collision energy: 30 to 50 V, collision energy broadening: 15 to 25 V;

[0016] Mass spectrometry acquisition time: 20-40 min; TofMS scan range: 50-1500 m / z; Production scan range: 50-1500 m / z.

[0017] (4) Data processing and component analysis:

[0018] The acquired raw mass spectrometry data were processed using MSDIAL software for peak alignment, retention time correction, and peak area extraction. Metabolite structure identification parameters were set as follows: mass tolerance < 0.01 Da for primary spectrum matching, mass tolerance < 0.02 Da for secondary spectrum matching, and a secondary mass spectrometry matching score > 70%.

[0019] The components of the Sophora japonica and Lonicera japonica extract were analyzed by UPLC-MS / MS, and a total of 39 components were identified, of which 31 components belonged to Lonicera japonica and 12 components belonged to Sophora japonica.

[0020] Preferably, in step (1) of the detection method, the ultrasonic treatment is performed for 10 minutes, the standing time is 12 hours, and the centrifugation is performed for 10 minutes.

[0021] Preferably, the chromatographic column used in step (2) of the detection method is ACQUITYUPLC®HSST3.

[0022] Preferably, the chromatographic column in step (2) of the detection method has a size of 2.1 × 100 mm and a diameter of 1.8 µm.

[0023] Preferably, the column temperature in step (2) of the detection method is 40°C.

[0024] Preferably, the flow rate in step (2) of the detection method is 0.3 mL / min.

[0025] Preferably, the mass spectrometry acquisition conditions for the detection method (3) are: temperature: 500℃, curtain gas: 45psi; declustering voltage: 60V, time-of-flight mass spectrometry: 10V, scanning collision energy: 40V, and collision energy broadening: 20V.

[0026] Preferably, the mass spectrometry acquisition conditions for the detection method (3) are: mass spectrometry acquisition time: 30 min.

[0027] Preferably, in step (1) of the detection method, the test sample can also be serum containing the drug.

[0028] Preferably, the sample processing method for step (1) of the detection method is as follows: take serum containing Sophora japonica and Lysimachia christinae, add methanol, shake for 1 min, sonicate on ice for 20 min, let stand for 1 h, centrifuge for 20 min, take the supernatant, blow dry with nitrogen, add pre-cooled 50% methanol to reconstitute, centrifuge for 10 min, take the supernatant, filter, and set aside.

[0029] 1.1 Chemical composition of Sophora japonica and Lysimachia christinae

[0030] UPLC-MS / MS analysis of the extract of Sophora japonica and Lonicera japonica revealed 39 components, of which 31 were attributed to Lonicera japonica (quercetin, kaempferol, aloin, benzaldehyde, arbutin, rutin, sennain, quinic acid, 4,5-dicaffeoylquinic acid, chlorogenic acid, 3-O-methylquercetin, astragalin, strychnos nux-vomica glycoside, sucrose, oleanolic acid, p-coumaric acid, luteolin). Palmitic acid, succinic acid, caffeic acid, ursolic acid, linolenic acid, dibutyl phthalate, zeaxanthin, myristic acid, strychnine, apigenin, ethyl linoleate, vitamin E succinate, capric acid, strychnine, 12 components belonging to Sophora japonica (quercetin, kaempferol, soybean saponin I, rutin, chlorogenic acid, betulin, gentiopicrin, genistein, sophoretic acid, soybean saponin B, limonene).

[0031] 1.2 Blood-entering components of Sophora japonica and Lonicera japonica formula

[0032] Analysis of the blood components of Sophora japonica and Lysimachia christinae formula using UPLC-MS / MS identified 58 serum precursor components (including Spanish sage, palmitoylethanolamine, phosphatidylcholine (16:0 / 18:2), vitamin D3, phosphatidylcholine (14:0 / 16:0), sphingomyelin (d18:2 / 24:0), sphingomyelin (d18:1 / 16:1), kaempferol-3-gentiobiglycoside, methyl styracoside, polygalactosyl glycoside A, phlorizin, 15-hydroxyeicosatetraenoic acid, 1,6-di-O-galloyl glucose, 3-hydroxyhexadecanoic acid, 2-hydroxystearic acid, D-tryptophan, nervonic acid, apigenin, transisooleic acid, swertiamarin, and leucine). Chloramphenicol lactone, Terminalia chebulic acid, dibutyl sebate, costunolide, abscisic acid, vitamin E succinate, retinic acid, digitoxin, dihydro-gamma-linolenic acid, hexadecylamide, phosphatidylcholine (16:0 / 18:1), hexamethylcyclotrisiloxane, phosphatidylcholine (15:0 / 15:0), isorhamnetin-3-glucoside, dimethyl phthalate, soybean saponins (bg), phosphatidylcholine (18:1 / 18:1).

[0033] Phosphatidylcholine (20:5 / 16:0), γ-guanidinobutyric acid, emodin methyl ether, vendolidine, soybean saponin I, reserpine, 7-hydroxycaprolactone, bufotalin, astragaloside, rutin, kaempferol-3-O-rutinoside, glycyrrhizic acid, glycyrrhizin, hawthorn acid, ricinoleic acid, monganin, lysophosphatidylethanolamine 16:0, lysophosphatidylethanolamine 18:2, 16-methylheptadecanoic acid, palmitic acid, raffinose, including 8 flavonoid components (kaempferol-3-gentiobiglycoside, phlorizin, apigenin, isorhamnetin-3-glucoside, kaempferol-3-O-rutinoside, rutin, monganin, glycyrrhizin), including apigenin and rutin, which are preliminarily identified as the direct pharmacodynamic material basis of this compound.

[0034] The beneficial effects of the technical solution of this invention patent:

[0035] (1) Through extensive experimentation and trial, the optimal chromatographic and mass spectrometry parameters were determined in this invention: ① Chromatographic mobile phase A: 0.1% formic acid aqueous solution, B: 0.1% formic acid in acetonitrile solution. The chromatographic gradient elution program is as follows: 0–1 min, 0% B; 1–2 min, B linearly changes from 0% to 30%; 2–12 min, B linearly changes from 30% to 50%; 12–21 min, B linearly changes from 50% to 100%; 21–26 min, B remains at 100%; 26–26.1 min, B linearly changes from 100% to 0%; 26.1–30 min, B remains at 0%; ② Mass spectrometry acquisition conditions are: ion spray voltage ESI: positive ion (+) and negative ion (-) modes, capillary voltage +5500–-4500V, temperature: 480–520℃, curtain gas: 42–47℃. psi; declustering voltage: 50-70V, time-of-flight mass spectrometry: 8-12V, scanning collision energy: 30-50V, collision energy broadening: 15-25V; mass spectrometry acquisition time: 20-40min, TofMS scan range: 50-1500m / z, Production scan range: 50-1500m / z.

[0036] Given the lack of literature on the simultaneous "blood-chemical" dual-dimensional characterization of this formula, this invention creatively establishes an integrated detection strategy during method optimization that enables simultaneous and efficient analysis of the original Sophora japonica and Lonicera japonica formula and its serum metabolites. Addressing the dual challenges of the complex chemical composition of the original formula and the low concentration and significant interference of metabolites in serum, this invention combines an optimized segmented gradient elution procedure with high-resolution time-of-flight mass spectrometry (HTMS) in both positive and negative ion modes. This allows for the comprehensive characterization of hundreds of prototype components of various structural types, ranging from organic acids to flavonoids, in a single analysis, while also enabling the high-sensitivity capture and identification of major metabolites and their transformation pathways after in vivo metabolism in serum. This method overcomes the limitations of previous techniques that only target a few components of a single herb, significantly improving the overall coverage and analytical efficiency of the compound chemical composition and in vivo metabolomics, providing crucial technical support for elucidating the pharmacodynamic material basis and in vivo processes of traditional Chinese medicine compound formulas.

[0037] (2) The present invention uses UPLC-MS / MS to analyze the components of Sophora japonica and Lonicera japonica extract. A total of 39 components were detected, of which 33 components, including chlorogenic acid, were attributed to Lonicera japonica, and 12 components, including rutin, were attributed to Sophora japonica.

[0038] This invention also analyzed the blood components of the Sophora japonica and Lysimachia christinae formula using UPLC-MS / MS, identifying 58 original serum components, including 8 flavonoids: kaempferol-3-gentiobiglycoside, phlorizin, apigenin, isorhamnetin-3-glucoside, kaempferol-3-O-rutinoside, rutin, gentiopicrin, and glycyrrhizin. Among these, kaempferol-3-gentiobiglycoside exhibits antitumor activity. Phlorizin promotes apoptosis in cancer cells and inhibits tumor growth. Apigenin inhibits tumor cells, and isorhamnetin-3-glucoside reduces oxidative stress-induced cell damage. Kaempferol-3-O-rutinoside inhibits invasion, induces apoptosis, and exerts antitumor effects. Rutin has antiviral activity and inhibits aldose reductase activity. Gentianopicrin has strong antitumor biological activity, and glycyrrhizin inhibits tumor cell proliferation and metastasis, thus exerting antitumor effects. The aforementioned pharmacological effects are consistent with the efficacy of the Sophora japonica and Lonicera japonica formula. Therefore, these components are preliminarily identified as the direct pharmacodynamic material basis of this traditional Chinese medicine compound, providing a scientific reference for further elucidating the mechanism of this compound in treating diseases.

[0039] (3) The UPLC-MS / MS detection method established in this invention exhibits significant advantages in terms of ease of operation, analytical efficiency, cost-effectiveness, and component coverage. Operationally, this method employs unified pretreatment and chromatographic-mass spectrometry conditions, enabling simultaneous analysis of the original extract and serum samples without the need for multiple methods for different matrices, greatly simplifying the operational process. Regarding detection time and throughput, the optimized 30-minute gradient elution program combined with high-resolution mass spectrometry full scan enables efficient separation and identification of complex systems in a single run, with short analysis time and high throughput, significantly outperforming conventional methods that require multiple rounds and long-duration segmented analyses. In terms of detection cost, this method, with its high throughput and high information content, can acquire massive amounts of chemical information in a single analysis, avoiding the material and time costs of repeated experiments to identify different categories of components, demonstrating outstanding economic efficiency. The most critical advantage lies in the completeness of component detection: this method not only comprehensively identified 39 chemical components from the original formula, but also successfully captured and identified 58 prototype components from serum, including 8 key flavonoid pharmacological substances. This enabled the simultaneous and systematic characterization of the compound's "in vitro chemical composition - in vivo direct-acting substances", providing unprecedented and complete data support for fully elucidating the pharmacological material basis and mechanism of action of the compound.

[0040] (4) This study employed a research strategy combining UPLC-MS / MS technology, network pharmacology analysis, and in vitro experimental validation to systematically explore the pharmacodynamic material basis and mechanism of action of Sophora japonica and Lycium barbarum formula in combating melanoma. The results clearly showed that flavonoids in Sophora japonica and Lycium barbarum formula are the key components responsible for its anti-melanoma effect. By constructing a "drug-component-target" network diagram and performing network topology analysis, the following six key flavonoids were successfully screened: quercetin, luteolin, apigenin, kaempferol, and genistein. These six key flavonoids are the active and effective substances in Sophora japonica and Lycium barbarum formula for direct anti-tumor activity. Attached Figure Description

[0041] Figure 1. UPLC-Q-TOF-MS / MS chromatograms of Sophora japonica in positive ion (A) and negative ion (B) modes;

[0042] Figure 2. UPLC-Q-TOF-MS / MS chromatograms of serum components in rats treated with Sophora japonica and Lysimachia christinae. (A) Blank serum in positive ion mode, (B) Blank serum in negative ion mode, (C) Serum sample of rats after drug administration (positive ion mode), (D) Serum sample of rats after drug administration (negative ion mode).

[0043] Figure 3 A classification diagram of the medicinal components of the Sophora japonica and Lonicera japonica formula;

[0044] Figure 4The composition of the traditional Chinese medicine group, the blank control group, and the blood-entering components were compared with those of Wayne.

[0045] Figure 5 (A) PPI protein interaction diagram of Sophora japonica and honeysuckle formula (B) GO functional enrichment analysis of common target of Sophora japonica and honeysuckle formula and melanoma (C) "Drug-component-target" network diagram of Sophora japonica and honeysuckle formula.

[0046] Figure 6 The effect of core components on A375 cell viability Detailed Implementation

[0047] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0048] Example 1: Construction of the detection method of the present invention

[0049] 1. Instruments and Materials

[0050] 1.1 Instruments

[0051] SQP electronic analytical balance (Sartorius GmbH, Germany); Alpha 1-2LD plus vacuum freeze dryer (Boxin Instruments Chengdu Branch); VRD-4 two-stage rotary vane vacuum pump (Shenzhen Haoli Technology Co., Ltd.); TripleTOF6600 mass spectrometer (ABSciex); NexeraX2 (UHPLC) ultra-high pressure liquid chromatograph (Shimadzu); Bioruotor ultrasonic system (Diagenode); Centrifuge 5424R centrifuge (Eppendorf); Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA); Multiskan Skyhigh full-wavelength microplate reader (Thermo Fisher Scientific); NX-1R high-speed benchtop centrifuge (Beijing Dinghaoyuan Technology Co., Ltd.).

[0052] 1.2 Medicinal Materials and Reagents

[0053] 1.06007.4008 Methanol (Millipore, USA); 1.00030.4008 Acetonitrile (Millipore, USA); 111670 Formic Acid (Millipore, USA) USA); XK13-011-14001 Ethanol (Tianjin Fuyu Fine Chemical Co., Ltd.); CHB230310 Rutin (98.0%), CHB230329 Quercetin (98.0%), 24011908 Kaempferol (98.0%), CHB230813 Kaempferol (98.0%), CHB230220 Chlorogenic Acid (98.0%), CHB230302 Luteolin (98.0%), CHB240204 Apigenin (98.0%), CHB240330 Genistein (98.0%) (Chengdu Kloma Biotechnology Co., Ltd.); 20221001 Honeysuckle, 20220701 Sophora japonica (Shaanxi Xingshengde Pharmaceutical Co., Ltd.).

[0054] 2 Methods

[0055] 2.1 Chemical composition analysis and blood-entry component analysis of Sophora japonica and Lycium barbarum formula

[0056] 2.1.1 Preparation of Sophora japonica and Lonicera japonica freeze-dried powder

[0057] Take 88g of Sophora japonica flowers and 18.45g of Lonicera japonica flowers, soak them in 1000mL of 30% ethanol for 30min, extract them by heating and reflux for 2h to obtain the extract, then concentrate it by rotary evaporation, filter the precipitate, freeze dry it to obtain Sophora japonica and Lonicera japonica freeze-dried powder, and store it in a drying oven for subsequent experiments.

[0058] 2.1.2 Preparation of Sophora japonica and Lonicera japonica serum

[0059] Ten male SD rats were randomly divided into a control group and a treatment group, with five rats in each group. Rats were fasted for 12 hours before administration, but had free access to water. The treatment group was administered 2.4 g / kg of Sophora japonica and Lonicera japonica extract (twice the clinically equivalent dose) by gavage, while the control group received an equal volume of physiological saline. Administration was twice daily for 7 consecutive days. After the last administration, all rats were anesthetized with isoflurane, and blood was collected from the abdominal aorta. The blood was centrifuged (3000 r / min, 15 min), and the supernatant was collected and stored at -80℃ for later use.

[0060] 2.1.3 UHPLC-Q-Exactive Orbitrap MS Analysis

[0061] Traditional Chinese Medicine: Weigh 10 mg of the powder and dissolve it in 1 mL of water. Add 3 mL of ethanol, sonicate for 10 min, let stand at 4℃ for 12 h, centrifuge at 4000 g at 4℃ for 10 min, collect the supernatant, and dry the ethanol with nitrogen to obtain the purified solution. For mass spectrometry detection, add ultrapure water to make up the volume to 1 mL. Filter the solution through a 0.22 μm filter membrane for later use. Inject 10 μL of the mass spectrometer sample, and repeat the technique 3 times.

[0062] Serum: Add 100 μL of serum to 400 μL of pre-cooled pure methanol, shake for 1 min, sonicate on ice for 20 min, let stand for 1 h, centrifuge at 16000g at 4℃ for 20 min, take the supernatant, blow dry with nitrogen, add 100 μL of pre-cooled 50% methanol to reconstitute, centrifuge at 20000g at 4℃ for 10 min, take 50 μL of supernatant for later use, inject 10 μL of each sample into the mass spectrometer, and repeat the technique 3 times.

[0063] 2.1.4 Chromatographic conditions

[0064] Throughout the analysis, the sample was placed in an autosampler at 4°C. The sample was analyzed using a SHIMADZU-LC30 ultra-high performance liquid chromatography system with an ACQUITYUPLC® HSST3 (2.1×100mm, 1.8µm) column. The column temperature was 40°C, and the flow rate was 0.3 mL / min. The mobile phases were A: 0.1% formic acid aqueous solution and B: 0.1% formic acid in acetonitrile. The gradient elution program was as follows: 0–1 min, 0% B; 1–2 min, B linearly changed from 0% to 30%; 2–12 min, B linearly changed from 30% to 50%; 12–21 min, B linearly changed from 50% to 100%; 21–26 min, B maintained at 100%; 26–26.1 min, B linearly changed from 100% to 0%; 26.1–30 min, B maintained at 0%.

[0065] 2.1.5 Mass Spectrometry Acquisition

[0066] Each sample was detected using electrospray ionization (ESI) in both positive (+) and negative (-) modes. After UPLC separation, the samples were analyzed by mass spectrometry using a TripleTOF 6600 (ABSciex). The source gas parameters are as follows:

[0067]

[0068] Mass spectrometry acquisition settings are as follows: Acquisition time: 30 min. TofMS scan range: 50-1500 m / z. Production scan range: 50-1500 m / z. IDA trigger rule: The first 18 monovalent charged ions with an intensity greater than 100 cps in the TofMS scan. Dynamic background subtraction is enabled.

[0069] 2.1.6 Data Processing

[0070] The acquired raw mass spectrometry data were used for peak alignment, retention time correction, and peak area extraction using MSDIAL software. Metabolite structure identification parameters: mass tolerance for primary spectrum matching < 0.01 Da, mass tolerance for secondary spectrum matching < 0.02 Da, and a secondary mass spectrometry matching score greater than 70%.

[0071] 3.1 Chemical composition of Sophora japonica and Lonicera japonica

[0072] UPLC-MS / MS analysis of the components of the honeysuckle extract from Sophora japonica flower revealed 39 components (results are shown in Table 1). Among them, 31 components, including chlorogenic acid, were attributed to honeysuckle (quercetin, kaempferol, aloin, benzaldehyde, arbutin, rutin, quinic acid, 4,5-dicaffeoylquinic acid, chlorogenic acid, 3-O-methylquercetin, astragalin, strychnos nux-vomica glycoside, sucrose, oleanolic acid, p-coumaric acid, ... Twelve components, including luteolin, palmitic acid, succinic acid, caffeic acid, ursolic acid, linolenic acid, dibutyl phthalate, zeaxanthin, myristic acid, strychnic acid, apigenin, ethyl linoleate, vitamin E succinate, capric acid, strychnine, and rutin, are attributed to Sophora japonica (quercetin, kaempferol, soybean saponin I, rutin, chlorogenic acid, betulin, gentiopicrin, genistein, sophoretic acid, soybean saponin B, limonene).

[0073]

[0074] 3.2 Blood-entering components of Sophora japonica and Lonicera japonica formula

[0075] The blood-entering components of the Sophora japonica and Lonicera japonica formula were analyzed by UPLC-MS / MS. A total of 571 substances were detected in both positive and negative modes, including 451 herbal components and 58 blood-entering components (see instruction manual). Figure 3The 58 serum precursor components (Table 2) specifically include the following: Spanish sage, palmitoylethanolamine, phosphatidylcholine (16:0 / 18:2), vitamin D3, phosphatidylcholine (14:0 / 16:0), sphingomyelin (d18:2 / 24:0), sphingomyelin (d18:1 / 16:1), kaempferol-3-gentiobiglycoside, methyl sagein, polygalactosyl glycoside A, phlorizin, 15-hydroxyeicosatetraenoic acid, 1,6-di-O-galloyl glucose, 3-hydroxyhexadecanoic acid, 2-hydroxystearic acid, D-tryptophan, nervonic acid, apigenin, transisooleic acid, swertiamarin, and leucine. Chloramphenicol lactone, Terminalia chebulic acid, dibutyl sebate, costunolide, abscisic acid, vitamin E succinate, retinic acid, digitoxin, dihydro-gamma-linolenic acid, hexadecylamide, phosphatidylcholine (16:0 / 18:1), hexamethylcyclotrisiloxane, phosphatidylcholine (15:0 / 15:0), isorhamnetin-3-glucoside, dimethyl phthalate, soybean saponins (bg), phosphatidylcholine (18:1 / 18:1). Phosphatidylcholine (20:5 / 16:0), γ-guanidinobutyric acid, emodin methyl ether, vendolidine, soybean saponin I, reserpine, 7-hydroxycaprolactone, bufotalin, astragaloside, rutin, kaempferol-3-O-rutinoside, glycyrrhizic acid, glycyrrhizin, hawthorn acid, ricinoleic acid, monganin, lysophosphatidylethanolamine 16:0, lysophosphatidylethanolamine 18:2, 16-methylheptadecanoic acid, palmitic acid, raffinose, among which there are 8 flavonoid components (kaempferol-3-gentiobiglycoside, phlorizin, apigenin, isorhamnetin-3-glucoside, kaempferol-3-O-rutinoside, rutin, monganin, glycyrrhizin), including apigenin and rutin, which are preliminarily identified as the direct pharmacodynamic material basis of this compound. The composition of the herbal medicine group, blank control group, and blood-entering component group of this invention are shown in the appendix. Figure 4 .

[0076]

[0077] 3.3 Network Pharmacology of Sophora japonica and Lonicera japonica Formula

[0078] 3.3.1 Common targets of the core components of Sophora japonica and honeysuckle formula and melanoma

[0079] Chemical composition analysis under section "3.1" revealed 39 components in the Sophora japonica and Lysimachia christinae formula, resulting in 479 target sites after deduplication. Among these, 4670 targets were related to melanoma. Intersecting these targets yielded 254 co-target genes for the drug and disease, as shown below. Figure 5 (A).

[0080] 3.3.2 PPI Network Construction and Core Target Gene Screening

[0081] The 254 intersection target points obtained were imported into the STRING database, with the filtering criteria set to high confidence (highconfidence 0.7). They were then analyzed using Cytoscape 3.9.1 software. Free nodes were removed, resulting in a target PPI network diagram. The PPI network diagram was then plotted. (See attached image.) Figure 5 (B). Among the core targets, there are 10 targets with a Degree value greater than twice the mean. The top 10 targets in terms of Degree value are TP53 (cell tumor antigen p53), AKT1 (serine / threonine kinase 1), STAT3 (signal transduction transcription activator 3), EGFR (epidermal growth factor receptor), HSP90AA1 (heat shock protein 90), SRC (non-receptor tyrosine kinase), IL6 (interleukin-6), TNF (tumor necrosis factor), MAPK1 (mitogen-activated protein kinase 1), and PIK3CA (p110 catalytic subunit of phosphatidylinositol-3-kinase).

[0082] 3.3.3 Construction of the "Drug-Component-Target" Network

[0083] Targets with a Degree value greater than the median were selected from the PPI network, resulting in 131 core target information. A "drug-component-target" network diagram was constructed, connecting the two traditional Chinese medicines, 34 chemical components, and their intersection with the disease in HHJYF. (See...) Figure 5 (C). Among them, 15 chemical components had a degree value greater than twice the mean, namely, the top 15 in terms of degree value are alfalfa extract, genistein, apigenin, senna extract, kaempferol, luteolin, quercetin, sophoridine, 3-methylquercetin, caffeic acid, linoleic acid, dibutyl phthalate, ursolic acid, oleanolic acid, and vitamin E. Among them, alfalfa extract, genistein, apigenin, senna extract, kaempferol, luteolin, quercetin, and sophoridine showed the strongest correlation with melanoma inhibition, with a degree value greater than 30.

[0084] GO functional enrichment analysis was performed on 131 common targets of SLE-melanoma using DAVID software, yielding 293 BP, 115 CC, and 229 MF. Biological processes (BP) mainly participate in signal transduction, protein phosphorylation, negative regulation of apoptosis, response to exogenous stimuli, inflammatory responses, and positive regulation of cell proliferation. Cellular components (CC) are mainly enriched in the cytoplasm, nucleus, mitochondria, nucleoplasm, and cell membrane. Molecular functions (MF) mainly involve enzyme binding, identical protein binding, and protein serine / threonine / tyrosine kinase activity. The top 10 biological processes in each of the three dimensions were selected, and bar charts were created to visualize the enriched BP, CC, and MF based on P-values. Figure 5 (A)

[0085] 3.3.5 KEGG pathway enrichment analysis

[0086] KEGG enrichment analysis was performed on 130 common targets of Sophora japonica and Lonicera japonica in melanoma using DAVID software, resulting in 169 signaling pathways. These pathways were sorted according to their count values, and a bubble chart was plotted using the top 20 pathways. (See attached image) Figure 5 (B) Enrichment results showed that the active ingredients of Sophora japonica and Lonicera japonica extract have a preventive and therapeutic effect on melanoma mainly concentrated in cancer-related pathways, proteoglycans in cancer, chemical carcinogenesis-receptor activation, chemical carcinogenesis-reactive oxygen species, microRNAs in cancer, and prostate cancer. Classical signal transduction pathways: PI3K-Akt signaling pathway, MAPK signaling pathway, Ras signaling pathway, Rap1 signaling pathway, and neurotrophic protein signaling pathway. Viral infection and host interaction: human cytomegalovirus infection, Kaposi's sarcoma-associated herpesvirus infection, hepatitis B, human immunodeficiency virus type 1 infection, and bacterial dysentery. Cellular microenvironment and structure: focal adhesion. Metabolism and chronic diseases: lipids and atherosclerosis, endocrine resistance, and Alzheimer's disease. These pathways are closely related to cancer, signal transduction, and inflammatory responses.

[0087] 3.4 Effects of core components on A375 cell viability

[0088] The effects of different concentrations of gentianin, quercetin, luteolin, apigenin, kaempferol, and genistein on the proliferation of A375 cells were evaluated using a CCK-8 assay kit. Figure 6It can be seen that the IC50 of ginsenoside is 17.80 μM, luteolin is 18.80 μM, quercetin is 40.87 μM, kaempferol is 50.51 μM, genistein is 91.97 μM, and apigenin is 124.86 μM.

[0089] 4 Results and Discussion

[0090] This study employed a combined approach of UPLC-MS / MS, network pharmacology analysis, and in vitro experimental validation to systematically explore the pharmacodynamic material basis and mechanism of action of Sophora japonica and Lonicera japonica formula in anti-melanoma therapy. The results clearly showed that flavonoids in Sophora japonica and Lonicera japonica formula are the key components responsible for its anti-melanoma effect. By constructing a "drug-component-target" network diagram and performing network topology analysis, we successfully screened six key flavonoids: senna extract, quercetin, luteolin, apigenin, kaempferol, and genistein. To verify the direct antitumor activity of these components, we performed cell viability assays using A375 melanoma cells. The results showed that all key flavonoid compounds effectively inhibited tumor cell proliferation, but their inhibitory efficacy varied significantly. Among them, senna extract exhibited the strongest in vitro antitumor activity, with an IC50 value of [missing information]. 50 The activity level was 17.80 μM, slightly better than luteolin, while the activities of quercetin, kaempferol, genistein, and apigenin decreased in that order. This activity gradient corroborates the result of the highest activity level of luteolin in the network pharmacology analysis, further confirming its core position in the formula.

[0091] Regarding pharmacokinetic characteristics, blood entry analysis showed that apigenin could enter the bloodstream as a component of the formulation. This finding contrasts with its relatively weak direct cytotoxicity, suggesting that it may exert a synergistic therapeutic effect in vivo through other pathways or metabolites. Further mechanistic studies, through molecular docking analysis, revealed that apigenin has an optimal binding energy to the core target STAT3, which is highly consistent with its strongest in vitro antitumor activity. STAT3, as an important tumor-associated transcription factor, plays a crucial role in the development and progression of melanoma. Studies have shown that gentianin can significantly alter the composition of immune cells in the melanoma microenvironment and exert its anti-melanoma effect directly by inhibiting the STAT3 signaling pathway. Luteolin, on the other hand, showed a dual inhibitory effect on the Src / STAT3 signaling pathway in A375 and B16F10 melanoma models, inhibiting STAT3 activation and promoting STAT3 protein degradation. Moreover, its anti-tumor effect was weakened by excessive STAT3 activation, further confirming the central role of the STAT3 pathway in its mechanism of action. Apigenin was also shown to effectively inhibit STAT3 phosphorylation, reduce STAT3 nuclear localization, and thus inhibit STAT3 transcriptional activity, which may be an important molecular mechanism by which it inhibits melanoma cell migration and invasion. In addition, other flavonoid components also exert their anti-tumor effects through their own unique pathways: Quercetin mainly inhibits the migration and invasion of melanoma cells in vitro by suppressing the important tumor metastasis-related signaling pathway HGF / c-Met, and effectively prevents lung metastasis of melanoma cells in vivo; Kaempferol exhibits multi-pathway action, inhibiting tumor proliferation on the one hand by promoting the expression of gap junction proteins and enhancing gap junction communication function, and on the other hand by disrupting the binding of hexokinase 2 and voltage-dependent anion channels on mitochondria, blocking the aerobic glycolysis process of melanoma cells, thereby inhibiting tumor metastasis; Gentian root exhibits a unique concentration-dependent bidirectional regulatory effect—high concentrations significantly inhibit cell adhesion and migration, while lower concentrations promote cell invasion and migration. This complex characteristic suggests that precise dosage control is required in clinical applications. Notably, when genistein combines with metastases to form a hydrogel formulation, its ability to penetrate melanoma spheroids is significantly enhanced, and this formulation exhibits excellent physical and chemical stability, making it a potentially ideal dosage form for treating cutaneous melanoma. These findings collectively provide a scientific basis for the multi-component, multi-target anti-melanoma effects of Sophora japonica and Lonicera japonica formula.

Claims

1. A UHPLC-Q-Exactive Orbitrap MS method for detecting Sophora japonica 'Gold and Silver Formula', characterized in that, The detection method includes the following steps: (1) Sample preparation: Weigh the Sophora japonica and Lycium barbarum powder, dissolve it in water, add ethanol, sonicate for 8-12 min, let stand for 10-14 h, centrifuge for 8-12 min, take the supernatant, blow the ethanol dry with nitrogen to obtain the purified solution, add ultrapure water to make up the volume, filter the solution through a 0.22 μm filter membrane, and set aside for later use. (2) Chromatographic conditions: Column C 18 The column temperature was 35–45℃, and the flow rate was 0.2–0.4 mL / min. The mobile phases were A: 0.1% formic acid aqueous solution and B: 0.1% formic acid in acetonitrile solution. The gradient elution program was as follows: 0–1 min, 0% B; 1–2 min, B linearly changed from 0% to 30%; 2–12 min, B linearly changed from 30% to 50%; 12–21 min, B linearly changed from 50% to 100%; 21–26 min, B maintained at 100%; 26–26.1 min, B linearly changed from 100% to 0%. 26.1–30 min, B remained at 0%; (3) Mass spectrometry acquisition conditions are as follows: Ion spray voltage ESI: positive ion (+) and negative ion (-) modes, capillary voltage +5500 to -4500 V, temperature: 480 to 520 °C, curtain gas: 42 to 47 psi; declustering voltage: 50 to 70 V, time-of-flight mass spectrometry: 8 to 12 V, scanning collision energy: 30 to 50 V, collision energy broadening: 15 to 25 V; Mass spectrometry acquisition time: 20-40 min; TofMS scan range: 50-1500 m / z; Production scan range: 50-1500 m / z. (4) Data processing and component analysis: The acquired raw mass spectrometry data were processed using MSDIAL software for peak alignment, retention time correction, and peak area extraction. Metabolite structure identification parameters were set as follows: mass tolerance < 0.01 Da for primary spectrum matching, mass tolerance < 0.02 Da for secondary spectrum matching, and a secondary mass spectrometry matching score > 70%. The components of the Sophora japonica and Lonicera japonica extract were analyzed by UPLC-MS / MS, and a total of 39 components were detected, of which 33 were detected in Lonicera japonica and 12 were detected in Sophora japonica.

2. The detection method as described in claim 1, characterized in that, The detection method includes the following steps: (1) ultrasonic treatment for 10 minutes, standing for 12 hours, and centrifugation for 10 minutes.

3. The detection method as described in claim 1, characterized in that, The chromatographic column used in step (2) of the detection method is ACQUITYUPLC®HSST3.

4. The detection method as described in claim 1, characterized in that, The chromatographic column specifications for step (2) of the detection method are 2.1×100mm and 1.8µm.

5. The detection method as described in claim 1, characterized in that, The column temperature in step (2) of the detection method is 40℃.

6. The detection method as described in claim 1, characterized in that, The flow rate in step (2) of the detection method is 0.3 mL / min.

7. The detection method as described in claim 1, characterized in that, The mass spectrometry acquisition conditions for the detection method (3) are as follows: Temperature: 500℃, curtain gas: 45psi; declustering voltage: 60V, time-of-flight mass spectrometry: 10V, scanning collision energy: 40V, collision energy broadening: 20V.

8. The detection method as described in claim 1, characterized in that, The detection method (3) has the following mass spectrometry acquisition conditions: mass spectrometry acquisition time: 30 min.

9. The detection method as described in claim 1, characterized in that, The test sample in step (1) of the detection method can also be serum containing the drug.

10. The detection method as described in claim 9, characterized in that, The detection method step (1) is as follows: take serum containing Sophora japonica and Lysimachia christinae, add methanol, shake for 1 min, sonicate on ice for 20 min, let stand for 1 h, centrifuge for 20 min, take the supernatant, blow dry with nitrogen, add pre-cooled 50% methanol to reconstitute, centrifuge for 10 min, take the supernatant, filter, and set aside.

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