The pharmacological effects of flavonoids in Oroxylum indicum (L.) Kurz against inflammation, bacterial, and oxidation have been well-documented. Additionally, it is commonly consumed as tea. However, the in vivo mechanism of its main compounds has not been well elucidated. In this study, a highly selective and sensitive UHPLC-Q-TOF-MS method combined with Mass Spectrum-based Orthogonal Projection (MSOP) theory and four-step analytical strategy was established and validated to identify metabolites in rats following oral administration Oroxylum indicum (L.) Kurz extract. Furthermore, a sensitive LC-MS/MS method was developed and validated for the first time to analyze the pharmacokinetics of ten main flavonoids in rats. Notably, a total of 47 metabolites were identified in blood, bile, urine, and feces samples. The maximum plasma concentration (Cmax) values for oroxin A, oroxin B, baicalin, chrysin, baicalein, scutellarein, apigenin, quercetin oroxylin A and isorhamnetin were 2945.1 ± 11.23 ng/mL, 3123.9 ± 16.37 ng/mL, 130.40 ± 27.52 ng/mL, 117.20 ± 28.54 ng/mL, 64.12 ± 19.33 ng/mL, 97.22 ± 24.27 ng/mL, 145.22 ± 29.92 ng/mL, 45.19 ± 18.84 ng/mL, 67.32 ± 15.78 ng/mL and 128.44 ± 26.42 ng/mL. A double peak was observed in the drug-time curve of apigenin, due to enterohepatic recirculation. This study demonstrated that MSOP method provided more technical support for the identification of flavonoid metabolites in complex system than traditional methods.

目的:建立快速、准确、可同时测定矮地茶中10个成分(3个儿茶素类、4个黄酮、2个酚酸和1个异香豆素类)的HPLC-ESI-MS/MS分析方法,并用于不同于产地矮地茶药材的分析.方法:采用Diamonsil-C18(150 mm×4.6 mm,5 μm,Dikma)色谱柱,以甲醇-0.02％甲酸水为流动相,梯度洗脱,流速0.8 mL·min-1;采用ESI负离子化监测,源喷射电压(IS)-4 500 V,离子源温度650℃,雾化气(Gasl)413 kPa,加热气(Gas2)448 kPa,帘气172 kPa.由MRM-IDA-EPI模式对矮地茶中没食子酸、表儿茶素、岩白菜素、原儿茶酸、表没食子儿茶素没食子酸酯、表儿茶素没食子酸酯、杨梅苷、芦丁、杨梅素、槲皮素10个成分的结构进行确证,采用MRM扫描模式建立测定其含量的方法,用所建立的方法对25批不同产地的矮地茶药材进行定量测定,将不同组分的含量,应用SPSS软件经过标准化转换后做散点图进行分析.结果:10个化学成分的峰面积与质量浓度在测定范围内均具有良好的线性关系,检测限、定量限、精密度和稳定性均符合要求,加样回收率范围为97.7％～103.6％,RSD为0.43％～3.3％.样品测定结果表明,不同产地矮地茶有效成分的含量差异较大,矮地茶的道地药材(来自于湖南、湖北和广东)与对照药材趋于一致.结论:本法简便、快速,灵敏度高,专属性好,可用于矮地茶中10个成分的含量测定,可为矮地茶药材质量控制提供强有力的工具.

Galangin, a naturally available flavonoid, induces a variety of pharmacological activities and biological effects via several mechanisms. However, in vivo metabolism of galangin has not been fully explored, which means knowledge of its pharmacodynamics and application potential is limited. The objective of this study was to establish an ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry method for the rapid profiling and identification of galangin metabolites in vitro and in vivo using unique online information-dependent acquisition with multiple mass defect filtering combined with dynamic background subtraction in positive ion mode. A total of 27 metabolites were detected and characterized, among which eight metabolites in liver microsomes and four metabolites in intestinal microflora were characterized, and 27 metabolites from rat plasma, bile, urine, feces, and a number of different tissue samples were characterized. Thirteen major metabolic pathways including hydrogenation, hydroxylation, glycosylation, methylation, acetylation, glucuronidation, and sulfation were observed to be attributable to the biotransformation of the metabolites. This study provides evidence for the presence of in vitro and in vivo metabolites and the pharmacokinetic mechanism of galangin. Moreover, the study promotes the further development and utilization of galangin and the plant from which it is derived, Alpinia officinarum Hance.

Ginkgolide B is a dietary diterpene with multiple pharmacological activities. However, current research on ginkgolide B is not comprehensive. The current study analyzed the metabolic profile of ginkgolide B in vivo and in vitro using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. To detect and identify the different metabolites in ginkgolide B, a novel data processing method was used as an assistant tool. A total of 53 different metabolites of ginkgolide B (38 phase I metabolites and 15 phase II metabolites) were detected relative to blank samples. The biotransformation route of ginkgolide B was identified as oxidation, dehydroxylation, hydrogenation, decarbonylation, demethylation, sulfate conjugation, glucose conjugation, methylation, and acetylation. The current study demonstrated a method for rapidly detecting and identifying metabolites and provided useful information to further characterize the pharmacology and mechanism of ginkgolide B. A method for the analysis of other diterpene metabolic components in vivo and in vitro was also established.

Introduction: Phillyrin, the main pharmacological component of Forsythia suspensa, exhibits a wide variability of therapeutic activities, such as anti-oxidative stress, free radical clearing, antibacterial activity, hepatic protection, restoration of endothelial glycocalyx damage, prevention of bone loss, attenuation of inflammatory responses, and so on. Previous research has found that phillyrin is not easily absorbed by the body and is rarely excreted into bile, excrement and urine, suggesting that phillyrin circulates primarily in the form of metabolites. Materials and Methods: In the present study, HPLC-ESI-MS/MS method was used for the simultaneous detection of phillyrin and its three metabolites in rat bile, excrement and urine samples. Liquid-liquid extraction with ethyl acetate was carried out for the pretreatment of bile and urine samples, while excrement samples were subjected to ultrasonic pretreatment with acetone. Chromatographic separation was performed on a C18 column with gradient elution. A tandem mass system coupled with a TurboIonSpray interface operating in simultaneous positive and negative ion multiple reaction monitoring modes was employed for the simultaneous detection of the analytes. Results: The proposed method demonstrated excellent accuracy and repeatability. Conclusion: This method was successfully applied for the pharmacokinetic evaluation of phillyrin and its three metabolites simultaneously.

Celastrol has attracted great attention owing to its anti-arthritis, antioxidant, and anticancer activities. Nevertheless, its metabolism in vivo (rats) and in vitro (rat liver microsomes and intestinal flora) has not been comprehensively characterized. In this study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry was used as a rapid and sensitive approach for studying the metabolism of celastrol in vivo and in vitro. A total of 43 metabolites were identified and characterized. These include 26 metabolites in vivo, and 28 metabolites in vitro (nine metabolites in rat liver microsomes and 24 metabolites in rat intestinal flora). Additionally, the celastrol-biotransformation capacity of the intestinal tract was confirmed to exceed that of the liver. Furthermore, the metabolic profile of celastrol is summarised. The information obtained from this study may provide a basis for understanding the pharmacological mechanisms of celastrol and will be beneficial for clinical applications.

Oroxin B, a flavonoid, is a major bioactive component form Oroxylum indicum (L.) Vent. with enormous anti-hepatoma effects. To data, the oroxin B metabolism studies remain underexplored. This study was designed to characterize oroxin B metabolism in vivo and in vitro by ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). Consequently, 30 metabolites in rats, 8 metabolites in liver microsomes and 18 metabolites in intestinal bacteria were identified, and 9 metabolites were recognized by comparison with standards. The biotransformation processes involved ketone, acetylation, loss of C12H20O10, and loss of C6H10O5. And baicalein and oroxin A were generated after loss of C12H20O10, and loss of C6H10O5, respectively, and further went through some other reactions, such as oxidation, methylation, internal hydrolysis, hydrogenation, loss of O, ketone, glycine conjugation, glucuronide conjugation and their composite reactions. The results provide valuable evidence for elucidation the potential mechanism of oroxin B pharmacological action, and offer reasonable guidelines for further investigations of oroxin B safety and efficacy.

Quality control and pharmacokinetics of Buddleja lindleyana Fort by HPLC-MS/MS.

Background: Spinosin is one of the major bioactive constituents among the total flavonoids in semen ziziphi spinosae, which has sedation and hypnosis actions. Methods: A simple and rapid high-resolution ultra-high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was developed and validated for predicting the structures of its spinosin metabolic products. This paper presents the first research focused on the metabolites of spinosin in human liver microsomes. Results: Based on the analytical strategy, 8 spinosin metabolites were detected in human liver microsome incubation samples, and the metabolic pathways required to generate these metabolites were proposed. However, no phase II metabolites were found. The cytochrome P450 enzyme is the main metabolic enzyme involved in drug metabolism, accounting for approximately 75% of the total number of different metabolic reactions. Conclusion: The in vitro metabolism of spinosin was proposed. These results allow us to learn about spinosin metabolism, leading to a better understanding of drug biotransformation and providing a basis for clinical applications. Moreover, this study laid the foundation for developing new pharmaceutical drugs.

Abstract Hinokiflavone (HF) is a natural biflavonoid extracted from medicinal plants such as Selaginella tamariscina and Platycladus orientalis. HF plays a crucial role in the treatment of several cancers. However, its poor solubility, instability, and low bioavailability have limited its use. In this study, soluplus/d-α-tocopherol acid polyethylene glycol 1000 succinate (TPGS)/dequalinium (DQA) was applied to improve the solubilization efficiency and stability of HF. HF hybrid micelles were prepared via thin-film hydration method. The physicochemical properties of micelles, including particle size, zeta potential, encapsulation efficiency, drug loading, CMC value, and stability were investigated. The in vitro cytotoxicity assay showed that the cytotoxicity of the HF hybrid micelles was higher than that of free HF. In addition, the HF hybrid micelles improved anticancer efficacy and induced mitochondria-mediated apoptosis, which is associated with the high levels of ROS inducing decreased mitochondrial membrane potential, promoting apoptosis of tumor cells. Furthermore, in vivo tumor suppression, smaller tumor volume and increased expression of pro-apoptotic proteins were found in nude mice treated with HF hybrid micelles, suggesting that HF hybrid micelles had stronger tumor suppressive activity compared with free HF. In summary, HF hybrid micelles developed in this study enhanced antitumor effect, which may be a potential drug delivery system for the treatment of lung adenocarcinoma.

Bilobetin, a natural compound extracted from Ginkgo biloba, has various pharmacological activities such as antioxidation, anticancer, antibacterial, antifungal, anti-inflammatory, antiviral, and promoting osteoblast differentiation. However, few studies have been conducted and there are no reports on its metabolites owing to its low content in nature. In addition, it has been reported to have potential liver and kidney toxicity. Therefore, this study aimed to identify the metabolites of bilobetin in vitro and in vivo. Bilobetin was incubated with liver microsomes to determine metabolites in vitro, and faeces and urine were collected after oral administration to rats to determine metabolites in vivo. After the samples were processed, they were measured using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. As a result, a total of 21 and 9 metabolites were detected in vivo and in vitro, respectively. Demethylation, demethylation and loss of water, demethylation and hydrogenation, demethylation and glycine conjugation, oxidation, methylation, oxidation and methylation, and hydrogenation were the main metabolic pathways. This study is the first to identify the metabolites of bilobetin and provides a theoretical foundation for the safe use of bilobetin in clinical application and the development of new drugs.

Background: Commelina communis Linn (Commelinae Herba) is a traditional Chinese medicine that can be used both as food and as medicine. It has been used to treat a variety of disorders, including a cold, high fever, sore throat, edema and oliguria for many years. Two pairs of isomeric flavonoid glycosides are the main active components in Commelina communis Linn, and they have a high content. Objective: The objective of this study was to determine the pharmacodynamic and pharmacological effects of Commelina communis Linn. Method: A sensitive, efficient, and rapid LC-MS/MS method was developed to simultaneously identify two pairs of isomeric flavonoid glycosides in rats. Chromatographic separation was carried out on a Wonda Cract ODS-2 C18 column (150 mm x 4.6 mm, 5 μm) using a mobile phase composed of 0.1% formic acid (aqueous solution) and methanol at a flow rate of 0.8 mL/min. The detection of the four analytes and the internal standard (IS) sulfamethoxazole was performed with multiple reaction monitoring (MRM) in negative electrospray ionization mode. All the analytes were eluted within 20 min. Results: This method was successfully applied for simultaneous identification of the concentrations of the four compounds in the plasma after the oral administration of 10 mL/kg Commelina communis Linn extract to rats. The pharmacokinetic study indicated that analytes reached their Cmax in approximately 15 min and could be detected until 12 h. Conclusion: The method complies with the State Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, extraction recovery and stability. This is the first report on the pharmacokinetics of Commelina communis Linn. The information gained from this research may be valuable for the preclinical and clinical applications of Commelina communis Linn.

Eriocitrin is one of the major active constituents of lemon fruit, and it possesses strong antioxidant, lipid-lowering, anticancer and anti-inflammatory activities and has long been used in food, beverages and wine. In this study, for the first time, a rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry method (LC/MS/MS) with protein precipitation was developed and validated for the analysis of eriocitrin in rat plasma. Chromatographic separation was achieved using a mobile phase, comprising 0.1% formic acid aqueous solution and acetonitrile eluted at a flow rate of 0.8 mL min-1. In multiple reaction monitoring (MRM) modes, eriocitrin and internal standard (IS) were quantified using precursor-to-product ion transitions of m/z 595.4 → 287.1 and m/z 252.0 → 155.9, respectively. The intra- and inter-day precision (RSD) were below 6.79% in plasma, while accuracy (RE) was within ±7.67%. The matrix effect, recovery and stability were also demonstrated to be within acceptable limits. This method was successfully employed in the pharmacokinetic study on rats after the oral administration of eriocitrin. The pharmacokinetic parameters show that the maximum plasma concentration (C max) of eriocitrin was 299.833 ± 16.743 μg L-1, while the corresponding time to reach C max(T max) was 0.094 ± 0.019 h, and the half-time (T 1/2) was 1.752 ± 0.323 h. The present results would be valuable for further research and development of eriocitrin.

Objective To establish a specific and rapid ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS) method for measuring ticarcillin and clavulanate levels in rat plasma. Methods A Waters ACQUITY BEH C18 column (50 mm × 2.1 mm, 1.7 μm) and SCIEX QTRAP® LC-MS/MS System were used. Analyses were conducted to optimize the chromatographic and MS conditions, and the pharmacokinetic parameters of ticarcillin and clavulanate were assessed. Results Linear relationships were observed in the ranges of 10 to 10,000 ng/mL for ticarcillin R (r2 = 0.9967) 30 to 10,000 ng/mL for ticarcillin S (r2 = 0.9961), and 30 to 10,000 ng/mL for clavulanate (r2 = 0.9981). The average extraction recoveries of all compounds ranged from 86.9% to 96.4%. The pharmacokinetic parameters of the ticarcillin R and S isomers in rats were distinctive. The ticarcillin R and S isomers and clavulanate were rapidly absorbed in vivo. Ticarcillin S and clavulanate had similar elimination rates, whereas that of ticarcillin R was slower. Conclusion A UPLC-ESI-MS/MS method was developed and validated for the determination of ticarcillin and clavulanate in rat plasma.

Eupatorin, a bioactive compound extracted from Java tea (Orthosiphon stamineus), possesses potent anti-cancer, anti-inflammatory and vasodilation activities. To date, no pharmacokinetics studies on eupatorin have yet been performed. Here, we established and validated a sensitive and selective LC-MS/MS (liquid chromatography-tandem mass spectrometry) approach for determining plasma eupatorin in rats. Chromatographic fractionation was conducted on a Wonda Cract ODS-2 C18 Column (4.6 mm × 150 mm, 5 μm) with a mobile phase containing aqueous 0.1% formic acid and acetonitrile using a flow rate of 0.8 ml min-1. In multiple reaction monitoring mode, precursor-to-product ion transitions for quantification of eupatorin and the internal standard were set at 343.1 → 328.1 and 252.0 → 155.9, respectively. The intra- and inter-day precision and accuracy were found to be below 6.72% and within ±8.26% in rat plasma, respectively. Meanwhile, all values of the matrix effect, recovery and stability were within the accepted ranges. Furthermore, we carried out the pharmacokinetic analysis using the developed method. The pharmacokinetic study revealed that while the C max (maximum plasma concentration) of eupatorin and time for reaching the C max (T max) were 974.886 ± 293.898 μg L-1 and 0.25 h, respectively, the half-life was 0.353 ± 0.026 h. This study will be of great significance to the research on the pharmacology, clinical pharmacy and drug action mechanism of eupatorin.

At present, cancer is one of the most lethal diseases in the world, and researchers are committed to developing effective anticancer drugs. Isoginkgetin (IGG) is a kind of biflavone with the potential to treat cancer due to the features of altering the cell cycle and inhibiting tumor cell infiltration. However, its solubility, absorbability and bioavailability are poor, so in this study, IGG was prepared into mixed nanomicelles and evaluated in vitro and in vivo. After condition optimization, IGG-loaded TPGS/soluplus mixed nanomicelles with particle size of 62.34 ± 1.10 nm, entrapment efficiency of 96.92 ± 0.66% and drug loading of 2.42 ± 0.02% were successfully prepared. The physicochemical properties of the nanomicelles were stable within 60 days, and the cytotoxicity of the nanomicelles was significantly higher than that of IGG. The metabolism results showed that 32 kinds of metabolites of IGG and 21 kinds of IGG-loaded nanomicelles were detected. The metabolites of IGG can only be detected in feces of rats, while the metabolites of IGG-loaded nanomicelles can be detected in plasma, bile, urine and feces. All these indicated that after prepared into nanomicelles, the stability, solubility, cytotoxicity and bioavailability of IGG were increased significantly, which provided a new choice for the development of new drugs.

Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.

Acacetin, a dietary component, is abundant in acacia honey and has superior anticancer activities. To date, no research on the metabolism of acacetin has been reported. In the current research, an online detection strategy of ultra-high-performance liquid chromatography connected to a quadrupole time-of-flight mass spectrometer (UHPLC-Q-TOF-MS/MS) was utilized for metabolite identification in vivo (rat plasma, bile, urine, and feces) and in vitro (rat liver microsomes). A total of 31 metabolites were structurally characterized in rats, and 25 metabolites were detected in rat liver microsomes, among which, 4 metabolites were compared with standards. Oxidation, the loss of CH2, reduction, hydrolysis, glucuronide conjugation, sulfate conjugation, methylation, and N-acetylation were the main metabolic pathways of acacetin. This study is the first to characterize acacetin metabolites in vivo and in vitro, and the results of this study offer novel and valuable evidence for a comprehensive understanding of the safety and efficacy of acacetin.

Biochanin A is a dietary isoflavone with multiple biological functions. Owing to a lack of comprehensive studies of biochanin A metabolism, this study was designed to further clarify the processes involved in biochanin A metabolism. In this study, ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was utilized to characterize the metabolism of biochanin A in vivo and in vitro. As a result, 43 metabolites in rats, 22 metabolites in liver microsomes, and 18 metabolites in intestinal flora were elucidated, and 5 metabolites were identified by comparison with standards. Oxidation, demethylation, hydrogenation, internal hydrolysis, conjugation (e.g., glucuronidation, sulfonation, glucose conjugation, methylation, and acetylation), and their composite reactions were determined to be major processes involved in biochanin A biotransformation. The results contribute to a better understanding of the pharmacological mechanism of biochanin A and provide a basis for comprehension of the safety and toxicity of biochanin A.