In Vivo Metabolism Study of Timosaponin BIII in Rat Using HPLC-QTOF-MS/MS

Timosaponin BIII, as one of the steroid saponins isolated from Anemarrhena asphodeloides Bge., was proved to have many pharmacological activities in recent years and became a natural active compound with good development prospect. In the present study, a simple and rapid method using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry was developed for the determination of the structures of timosaponin BIII and its metabolites in rats after administrating intragastrically at 300 mg kg⁻¹. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent compound, nine metabolites were detected and identified in urine, and eight in plasma as well as four in brain. It is also indicated that the deglycosylation and oxidation reactions were the main metabolic pathways in the biotransformation of timosaponin BIII in vivo and the structures of the nine metabolites were identified and proposed to be timosaponin BII(M1), the hydroxylated metabolite of TBII(M2), the hydroxylated metabolites of TBIII(M3 and M4), deglycosylation and monooxygenation product of TBIII(M5), the deglycosylation product of TBII(M6), timosaponin AIII(M8), the isomers of timosaponin AIII(M7 and M9).

     

Identification of major alkaloids and steroidal saponins in rat serum by HPLC-diode array detection-MS/MS following oral administration of Huangbai-Zhimu herb-pair Extract

Huangbai-Zhimu herb-pair (HBZMHP) is a widely used Chinese traditional medicine formula in treating various diseases; however, its active components have remained unknown. In this paper, serum chemistry and combined high-performance liquid chromatography (HPLC), diode-array detection and mass-spectrometry (MS) techniques were used to study the constituents of HBZMHP extract absorbed into rat serum after oral administration. A total of nine characteristic HPLC peaks in the TIC chromatograms were identified as magnoflorine (1), menisperine (2), palmatine (3), berberine (4), timosaponin N or timosaponin E1 (5), timosaponin D (6), timosaponin BIII, anemarsaponin C or xilingsaponin B (7) timosaponin BII (8) and timosaponin AIII (9). All of the identified peaks were constituents of HBZMHP extract. The results narrow the range of active compounds to be found in HBZMHP extract, and pave the way for the follow-up action mechanism research.     

Identification of major xanthones and steroidal saponins in rat urine by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technology following oral administration of Rhizoma Anemarrhenae decoction

Rhizoma Anemarrhenae (Zhimu in Chinese), the dried rhizome of Anemarrhena asphodeloides Bge. (Fam. Liliaceae), is a well-known traditional Chinese medicinal herb and has been used clinically in China for centuries to cure various diseases. However, like other traditional Chinese medicines, the effective constituents of this medicine, especially the assimilation and metabolites in vivo, which are very important to show their effects, have not been systematically studied. In this paper, solid-phase extraction and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technologies were used to study the constituents absorbed into rat urine and their metabolites after oral administration of Rhizoma Anemarrhenae decoction. A total of 11 compounds, including two xanthones, three of their metabolites and six steroidal saponins, were identified in rat urine sample. They were neomangiferin (1), glucuronide and monomethyl conjugate of mangiferin (2), mangiferin (3), monomethyl conjugate of mangiferin (4), dimethyl conjugate of mangiferin (5), timosaponin N or timosaponin E1 (6), timosaponin BII (7), timosaponin BIII (8), anemarrhenasaponin I or anemarrhenasaponin II (9), timosaponin AII (10) and timosaponin AIII (11). The results would efficaciously narrow the potentially active compounds range in Rhizoma Anemarrhenae decoction, and pave a helpful way for follow-up mechanism of action research.     

Preparation of highly purified timosaponin AIII from rhizoma anemarrhenae through an enzymatic method combined with preparative liquid chromatography

Timosaponin AIII (TAIII) exhibits extensive pharmacological activities and has been reported as a potent antitumour agent for various human cancers. In the present study, a potential industrial process for producing TAIII that involves biotransformation directly in the crude extract liquid of rhizoma anemarrhenae (RA) was developed. β-D-glycosidase was used to transform timosaponin BII (TBII) into TAIII, and monofactor experiments were conducted to optimise the enzymolysis conditions. In addition, AB-8 macroporous resin column chromatography, preparative liquid chromatography, and crystallisation technique were applied for yielding TAIII crystals with a purity > 97%. Approximately, 7 g of TAIII with a high purity of > 97% was obtained from 1 kg of RA through this five-step preparation method, which can be used to produce TAIII on a large scale.     

高效液相色谱法考察不同炮制方法对知母中5种主要化学成分的影响

建立了高效液相色谱(HPLC)同时测定知母不同炮制品中新芒果苷、芒果苷、知母皂苷BIII、知母皂苷I和知母皂苷AIII含量的方法,探讨了不同炮制方法对知母化学成分的影响,为通过采用不同炮制方法来改变知母药性提供了依据。采用AlltimaTM C18色谱柱(250 mm×4.6 mm, 5 μm)分离,所用流动相为乙腈(A)和0.1%甲酸水溶液(B),梯度洗脱,流速为0.8 mL/min;用紫外检测器检测新芒果苷与芒果苷,检测温度为室温,检测波长为265 nm。用蒸发光散射检测器检测知母皂苷BIII、知母皂苷I和知母皂苷AIII,漂移管温度为50 ℃,气体压力为179.1 kPa(26 psi)。知母经炮制后,5种化合物含量均发生了不同程度的改变,表明炮制方法的不同对知母化学成分的含量影响较大。该方法对进一步研究炮制方法对知母的药效学影响提供了一定的参考依据。     

The structural elucidation of two new artificial steroidal saponins

Two novel steroidal saponins(timosaponin BⅢ-a,timosaponin BⅢ-b) together with a known steroidal saponin(timosaponin BⅡ-b) were prepared by the dilute acid hydrolysis of timosaponin BⅢ.Their structures were elucidated by means of 1D,2D NMR and TOF-MS spectral analysis.     

A novel ultra high‐performance liquid chromatography‐tandem mass spectrometry method for the simultaneous determination of xanthones and steroidal saponins in crude and salt‐processed Anemarrhenae Rhizoma aqueous extracts

We established a rapid and sensitive ultra high‐performance liquid chromatography tandem mass spectrometry method for the simultaneous quantification of xanthones and steroidal saponins in rat plasma. Chromatographic separation was achieved on a C₁₈ column with a mobile phase comprising acetonitrile and 0.1% formic acid. The detection was performed by negative electrospray ionization in multiple reaction monitoring mode. The validated method showed good linearity within the tested range (r > 0.9945). The intra‐ and interday precision at high, medium, and low concentrations was less than 7.96%. The bias of accuracies ranged from −1.92 to 9.62%. The extraction recoveries of the compounds ranged from 84.78 to 88.69%, and the matrix effects ranged from 96.76 to 108.59%. This method was successfully applied to a pharmacokinetic comparison of crude and salt‐processed Anemarrhenae Rhizoma aqueous extracts after oral administration in rats. The maximum plasma concentration and area under concentration–time curve of timosaponin BIII and timosaponin AIII increased significantly (P < 0.05 or 0.01) and those of timosaponin BII decreased significantly (P < 0.05) after processing. These results could contribute to the clinical application of crude and salt‐processed Anemarrhenae Rhizoma and reveal the processing mechanism.     

Biotransformation of Timosaponin BII into Seven Characteristic Metabolites by the Gut Microbiota

Timosaponin BII is one of the most abundant Anemarrhena saponins and is in a phase II clinical trial for the treatment of dementia. However, the pharmacological activity of timosaponin BII does not match its low bioavailability. In this study, we aimed to determine the effects of gut microbiota on timosaponin BII metabolism. We found that intestinal flora had a strong metabolic effect on timosaponin BII by HPLC-MS/MS. At the same time, seven potential metabolites (M1–M7) produced by rat intestinal flora were identified using HPLC/MS-Q-TOF. Among them, three structures identified are reported in gut microbiota for the first time. A comparison of rat liver homogenate and a rat liver microsome incubation system revealed that the metabolic behavior of timosaponin BII was unique to the gut microbiota system. Finally, a quantitative method for the three representative metabolites was established by HPLC-MS/MS, and the temporal relationship among the metabolites was initially clarified. In summary, it is suggested that the metabolic characteristics of gut microbiota may be an important indicator of the pharmacological activity of timosaponin BII, which can be applied to guide its application and clinical use in the future.     

Aliphatic hydroxylation and epoxidation of capsaicin by cytochrome P450 CYP505X

Microbial cytochrome P450 enzymes (CYPs) are able to mimic the metabolism of human CYPs. One challenge is to identify the respective drug metabolites and to compare substrate specificities to those of the human enzymes. In this study, a class VIII self-sufficient CYP from Aspergillus fumigatus (CYP505X) and variants of this enzyme were heterologously expressed in E. coli. The substrate scope of the variants was determined using active pharmaceutical ingredients (APIs) and (hetero)cyclic compounds. Capsaicin – the active compound in chili peppers – was oxidized most efficiently (4.36?μM/min) in a whole cell mediated biotransformation. The products were isolated, purified and their structures elucidated by 1D and 2D NMR. The two major metabolites showed modifications on the lipophilic side chain. Specifically, capsaicin was hydroxylated at position 8 to give (E)-8-hydroxy-N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide and epoxidized at the double bond to give N-(4-hydroxy-3-methoxybenzyl)-5-(3-isopropyloxiran-2-yl)-pentanamide.     

In vivo and in vitro metabolism of a novel β2-adrenoceptor agonist, trantinterol: metabolites isolation and identification by LC-MS/MS and NMR

Trantinterol is a novel β₂-adrenoceptor agonist used for the treatment of asthma. The aim of this study is to identify the metabolites of trantinterol using liquid chromatography tandem mass spectrometry (LC-MS/MS), to isolate the main metabolites, and confirm their structures by nuclear magnetic resonance (NMR). Urine, feces, bile, and blood samples of rats were obtained and analyzed. Reference standards of six metabolites were achieved with the combination of chemical synthesis, microbial transformation, and the model systems of rats. Moreover, in order to investigate the phase I metabolism of trantinterol in humans and to study the species differences between rats and humans, incubations with liver microsomes were performed. The biotransformation by a microbial model Cunninghamella blakesleana AS 3.970 was also studied. A total of 18 metabolites were identified in vivo and in vitro together, 13 of which were newly detected. Three phase I metabolites were detected in vivo and in vitro as well as in the microbial model, including the arylhydroxylamine (M1), the tert-butyl hydroxylated trantinterol (M2) and the 1-carbonyltrantinterol (M3). Another important pathway in rats is glutathione conjugation and further catabolism and oxidation to form consecutive derivatives (M4 through M10). Other metabolites include glucuronide, glucoside, and sulfate conjugates. The results of in vitro experiments indicate no species difference exists among rats, humans, and C. blakesleana AS 3.970 on the phase I metabolism of trantinterol. Our study provided the most comprehensive picture for trantinterol in vivo and in vitro metabolism to this day, and may predict its metabolism in humans.     

Analysis of Arecoline in Rat Urine, and Identification of its Metabolites, by Liquid Chromatography–Tandem Mass Spectrometry

A simple and rapid high-performance liquid chromatographic–electrospray ionization (ESI) tandem mass spectrometric method has been developed for elucidation of the structures of the metabolites of arecoline in rat urine after administration of a single dose (20 mg kg^?1). The urine samples were purified on a C₁₈ solid-phase extraction cartridge and analysis was then performed on a reversed-phase C₁₈ column with 60:40 (v/v) methanol–0.01% triethylamine solution (2 mmol L^?1, adjusted to pH 3.5 with formic acid) as mobile phase and detection by on-line MS–MS. Identification of the metabolites and elucidation of their structures were performed by comparing molecular masses (ΔM), retention-times, and product ion spectra with those of the parent drug. The parent drug arecoline, four phase-I metabolites, and one phase-II metabolite were identified in rat urine.     

Structural elucidation of in vivo metabolites of phencynonate and its analogue thiencynonate in rats by HPLC-ESI-MS

The structural elucidation of the metabolites of phencynonate and its analogue thiencynonate in rats was performed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MSⁿ) in positive ion mode, by comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug. Phencynonate and thiencynonate were easily biotransformed in vivo by the pathways of N-demethylated, oxidative, hydroxylated and methoxylated to form seventeen metabolites that retained the some features of the two parent molecules. These metabolites included ten phencynonate metabolites (N-demethyphencynonate monoxide, N-demethyhydroxy phencynonate, phencynonate monoxide, hydroxyphencynonate, phencynonate dioxide, methoxyphencynonate, dihydroxyphencynonate, dihydroxyphencynonate, hydroxymethoxy phencynonate, trihydroxyphencynonate) and seven thiencynonate metabolites (N-demethy thiencynonate, N-demethythiencynonate monoxide, N-demethyhydroxythiencynonate, thiencynonate monoxide, hydroxythiencynonate, hydroxythiencynonate monoxide, dihydroxy thiencynonate). The described method has wide applicability to rapidly screen and provide structural information of these metabolites. The identifications of precise structures of these metabolites need to be confirmed by other techniques such as the ¹H and ¹³C NMR.     

Integrated rapid resolution liquid chromatography–tandem mass spectrometric approach for screening and identification of metabolites of the potential anticancer agent 3,6,7-trimethoxyphenanthroindolizidine in rat urine

An integrated approach combining data acquisition using MS^E and multi-period product ion scan (mpMS/MS), with high-resolution characteristic extracted ion chromatograms (hcXIC) as a data mining method, was developed for in vivo drug metabolites screening and identification. This approach is illustrated by analyzing metabolites of a potential anticancer agent, 3,6,7-trimethoxyphenanthroindolizidine (CAT) in rat urine based on rapid resolution liquid chromatography combined with tandem mass spectrometry (RRLC–MS/MS). Untargeted full-scan MS^E enabled the high-throughput acquisition of potential metabolites, and targeted mpMS/MS contributed to the sensitivity and specificity of the acquisition of molecules of interest. The data processing method hcXIC, based on the structure of CAT, was shown to be highly effective for the metabolite discovery. Through the double-filtering effect of the characteristic ion and accurate mass, conventional extracted ion chromatograms that contained a substantial number of false-positive peaks were simplified into chromatograms essentially free of endogenous interferences. As a result, 21 metabolites were detected in rat urine after oral administration of CAT. Based on the characteristic fragmentation patterns of the phenanthroindolizidine alkaloid, the structures of 9 metabolites were identified. Furthermore, the interpretation of the MS/MS spectra of these metabolites enabled the determination of demethylation position as well as the differentiation between N-oxidized and hydroxylated metabolites.     

Metabolic profile and pharmacokinetics of polyphyllin I,an anticancer candidate,in rats by UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS

Polyphyllin I (PPI), a natural steroidal saponin originating from rihzome of Paris polyphylla , is a potential anticancer candidate. Previous pharmacokinetics study showed that the oral bioavailability of PPI was very low, which suggested that certain amount of PPI might be metabolized in vivo . However, to date, information regarding the final metabolic fates of PPI is very limited. In this study, metabolites of PPI and their pharmacokinetics in rats were investigated using UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS. A total of seven putative metabolites, including six phase I and one phase II metabolites, were detected and identified with three exact structures by comparison with authentic standards for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. The pharmacokinetics of prosapogenin A, trillin and diosgenin, three deglycosylation metabolites of PPI with definite anticancer effects, were further studied, which suggested that the metabolites underwent a prolonged absorption and slower elimination after intragastric administration of PPI at the dose of 500 mg/kg. This study provides valuable and new information on the metabolic fate of PPI, which will be helpful in further understanding its mechanism of action.     

Analysis of Metabolites of Anthraquinones by Human Fecal Bacteria Using UPLC-Q-TOF-HRMS/MS

This study aimed to investigate the metabolism of anthraquinones, including chrysophanol (1), rhein (2), aloe-emodin (3), emodin (4), sennoside A (5) and sennoside B (6), by mixed human fecal bacteria to clarify the relationship between their chemical structural characteristics and intestinal metabolism. Six parent compounds were incubated with mixed human fecal bacteria in vitro to study the metabolic process. A highly sensitive and specific ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution tandem mass spectrometry (UPLC-Q-TOF-HRMS/MS) with MS^E technology and MetaboLynx software has been developed to analyze the metabolites of anthraquinones. With this method, a total of ten metabolites were identified, including 1,4,8-trihydroxy-3-hydroxymethylanthraquinone (M1), 2-methylrhein (M2), 7-methylrhein (M3), methyl-esterificated rhein (M4), 1,8-dihydroxy-3-hydroxymethyl-4-methylanthraquinone (M5), physcion (M6), sennidin A (M7), rhein (M8), sennidin B (M9) and rhein (M10), six (M1–M6) of which were first detected on the basis of the exact mass by mixed human fecal bacteria in this work. The metabolism of anthraquinones occurred via hydroxylation, oxidation, methylation, deglycosylation and esterification. In particular, the methyl-esterificated rhein (M4) was first identified as one of the metabolites of rhein, whose metabolic pathway (esterification) is also reported for the first time. The presence of human fecal bacteria played a vital role in the metabolism of anthraquinones and the substitutional groups determined the different metabolic reactions for anthraquinones, which will be useful for the investigation of the study of anthraquinones in vivo.

     

Beam-foil study of doubly and singly excited states in BIII

Doubly excited⁴ L and² L systems of BIII have been studied by beam-foil spectroscopy. Spectra of boron (λ: 28.7–50.1 nm) have been recorded at Bochum with a resolution superior to that obtained previously. Eight new lines are classified in the⁴ L system and one in the² L system. The wavelength accuracy of most of the transitions already reported in the core-excited systems of BIII is greatly improved. On the basis of these results, six and two unidentified lines appearing in spectra recorded at Liège (λ: 50–200 nm) are classified in the⁴ L and² L core-excited systems, respectively. Where comparison with theoretical predictions by Chung et al. [1] is possible, excellent agreement is found for all the assigned lines. The first energy values are reported for seven terms in the doubly excited quartet system. Moreover, seven new lines are identified in the singly excited system of BIII.     

Gas chromatography–tandem mass spectrometry analysis of 52 monohydroxylated metabolites of polycyclic aromatic hydrocarbons in hairs of rats after controlled exposure

A method based on gas chromatography–tandem mass spectrometry after derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide was developed for the analysis of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in hair. The method focused on 52 target compounds corresponding to two- to six-ring monohydroxylated metabolites of polycyclic aromatic hydrocarbons (PAHs). The limits of quantification ranged from 0.2 to 50 pg mg^?1. The method was then applied to the analysis of hair samples collected from rats exposed to 12 PAHs at 0.01, 0.1, and 1 mg kg^?1, by intraperitoneal injection, for 28 days. The results of this study confirm that these metabolites can be incorporated in hair after intraperitoneal administration of the corresponding parent compound. Only 20 of the 52 metabolites were actually detected in hair samples and corresponded to nine parent PAHs. The mean concentrations of OH-PAHs in rat hair samples exposed to PAHs at 1 mg kg^?1 ranged from 0.6?±?0.2 pg mg^?1 for 8-hydroxybenzo[b]fluoranthene to 6.7?±?1.0 pg mg^?1 for 1-hydroxypyrene. The results also demonstrated that hair pigmentation has no influence on the concentration of most OH-PAHs. This animal experiment confirmed the incorporation of PAH metabolites in hair and demonstrated that the method was sufficiently sensitive to detect low levels of exposure to PAHs. These results confirmed the usefulness of hair analysis in the biomonitoring of human exposure to PAHs.

Structural identification of mouse urinary metabolites of pterostilbene using liquid chromatography/tandem mass spectrometry

Pterostilbene, the dimethoxy derivative of resveratrol, has drawn much attention recently due to its potential beneficial health effects. The metabolic fate of pterostilbene, however, is not well understood. In the present study, we identified nine novel mouse urinary pterostilbene metabolites, pterostilbene glucuronide, pterostilbene sulfate, mono‐demethylated pterostilbene glucuronide, mono‐demethylated pterostilbene sulfate, mono‐hydroxylated pterostilbene, mono‐hydroxylated pterostilbene glucuronide, mono‐hydroxylated pterostilbene sulfate, and mono‐hydroxylated pterostilbene glucuronide sulfate, using liquid chromatography/atmospheric pressure chemical ionization and electrospray ionization tandem mass spectrometry. The structures of these metabolites were confirmed by analyzing the MSⁿ (n = 1–3) spectra. To our knowledge, this is the first report of the identification of urinary metabolites of pterostilbene in mice. Copyright © 2010 John Wiley & Sons, Ltd.     

Aspochalasin H1: A New Cyclic Aspochalasin from Hawaiian Plant-Associated Endophytic Fungus Aspergillus sp. FT1307

Aspergillus is one of the most diverse genera, and it is chemically profound and known to produce many biologically active secondary metabolites. In the present study, a new aspochalasin H1 (1), together with nine known compounds (2–10), were isolated from a Hawaiian plant-associated endophytic fungus Aspergillus sp. FT1307. The structures were elucidated using nuclear magnetic resonance (NMR) (¹H, ¹H-¹H COSY, HSQC, HMBC, ROESY and 1D NOE), high-resolution electrospray ionization mass spectroscopy (HRESIMS), and comparisons with the reported literature. The absolute configuration of the new compound was established by electronic circular dichroism (ECD) in combination with NMR calculations. The new compound contains an epoxide moiety and an adjacent trans-diol, which has not been reported before in the aspochalasin family. The antibacterial screening of the isolated compounds was carried out against pathogenic bacteria (Staphylococcus aureus, Methicillin-resistant S. aureus and Bacillus subtilis). The antiproliferative activity of compounds 1–10 was evaluated against human breast cancer cell lines (MCF-7 and T46D) and ovarian cancer cell lines (A2780).     

Facile Synthesis and Antitumor Activities of Timosaponin AIII and Its Analogs

An efficient synthesis of timosaponin AIII (TAIII) and five structurally modified analogs via a one-pot sequential glycosylation strategy is described. A partially protected D-galactopyranosyl thioglycoside was employed for regioselective glycosylation to facilitate the target synthesis. The antitumor activities of the synthetic saponins against human epithelial cervical cancer cell (HeLa) were preliminarily evaluated by means of CCK-8 assay. An L-rhamnosyl analog was discovered to display stronger inhibitory activity (IC₅₀ 3.3 μM) than TAIII (IC₅₀ 10.7 μM) to HeLa cell. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Carbohydrate Chemistry to view the free supplemental file.