Alkaloids In Rats Research Articles

Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged −7.09–11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

The toxicokinetic and metabolism of structurally diverse pyrrolizidine alkaloids in rats

Ethnopharmacological relevancePyrrolizidine alkaloids (PAs) are a group of phytotoxins present in about 3% of flowering plants worldwide. Ingestion of PA-containing herbal products may lead to hepatotoxicity. Notably, the toxicokinetic (TK) behaviors, especially pyrrole-protein adducts (PPAs) having the same structure but generated from metabolic activation of different PAs, significantly affect the toxicity of structurally diverse PAs, therefore studying them in their pure form is preferable to extracts to stratify toxic potency of different PAs co-existing in herbal extracts. However, previous studies mainly focus on the establishment of TK profiles of the intact PAs, revealing less or no kinetic information on the main PA metabolites (PA N-oxides) and PPAs which mediate PA-induced hepatotoxicity. In this study, PPA was measured as the biomarker of PA exposure and PA-induced toxicity. Aim of studyThis study aims to investigate the TK difference between structurally diverse PAs of retronecine-type PAs: retrorsine (RTS) and monocrotaline (MCT), and otonecine-type PA: clivorine (CLI), and their toxicity-related metabolite PPAs and PA N-oxides, the main metabolite of retronecine-type PAs, for the establishment of a more accurate risk assessment of PAs exposure. Materials and methodsThe TK studies were conducted using rats through intravenous (i.v.) or oral (p.o.) administration of PAs at 20 mg/kg. The main TK parameters of PAs and PA N-oxides were determined from plasma concentration-time profiles, and the kinetic profiles of PPAs were assessed from both plasma and erythrocyte concentration-time profiles. ResultsMCT demonstrated the slowest but the highest extent of absorption among the three PAs, while RTS demonstrated a similar absorption rate with a lower extent than CLI. For elimination, MCT demonstrated a similar elimination rate as RTS but the lowest extent of elimination among the three PAs, and CLI exhibited significantly faster elimination than MCT and RTS. Moreover, the formation of PA N-oxide, which only occurs in retronecine-type PAs, was remarkably less in MCT-treated rats compared to RTS-treated ones. Of note, the retronecine-type RTS and MCT induced more PPAs via p.o. than i.v. administration route, whereas the otonecine-type CLI showed the opposite trend. ConclusionDramatic TK differences, including not only PAs but also PA N-oxides and the derived protein adduct PPAs, were found among structurally diverse PAs in rats, laying the basis for varied hepatotoxic potencies induced by different PA-containing herbal products. Notably, our findings for the first time uncovered that oral administration of retronecine-type PAs might cause severer toxicity compared with the intravenous route, which warrants further in-depth exploration.

Pharmacokinetics and tissue distribution of Ramulus Mori (Sangzhi) alkaloids in rats and its effects on liver enzyme activity.

Ramulus Mori (Sangzhi) alkaloids (SZ-A) derived from twigs of mulberry (Morus alba L., genus Morus in the Moraceae family) was approved by the National Medical Products Administration in 2020 for the treatment of type 2 diabetes mellitus. In addition to excellent hypoglycemic effect, increasing evidence has confirmed that SZ-A exerts multiple pharmacological effects, such as protecting pancreatic ß-cell function, stimulating adiponectin expression, and alleviating hepatic steatosis. Importantly, a specific distribution of SZ-A in target tissues following oral absorption into the blood is essential for the induction of multiple pharmacological effects. However, there is a lack of studies thoroughly exploring the pharmacokinetic profiles and tissue distribution of SZ-A following oral absorption into the blood, particularly dose-linear pharmacokinetics and target tissue distribution associated with glycolipid metabolic diseases. In the present study, we systematically investigated the pharmacokinetics and tissue distribution of SZ-A and its metabolites in human and rat liver microsomes, and rat plasma, as well as its effects on the activity of hepatic cytochrome P450 enzymes (CYP450s). The results revealed that SZ-A was rapidly absorbed into the blood, exhibited linear pharmacokinetic characteristics in the dose range of 25-200mg/kg, and was broadly distributed in glycolipid metabolism-related tissues. The highest SZ-A concentrations were observed in the kidney, liver, and aortic vessels, followed by the brown and subcutaneous adipose tissues, and the heart, spleen, lung, muscle, pancreas, and brain. Except for the trace oxidation products produced by fagomine, other phase I or phase II metabolites were not detected. SZ-A had no inhibitory or activating effects on major CYP450s. Conclusively, SZ-A is rapidly and widely distributed in target tissues, with good metabolic stability and a low risk of triggering drug-drug interactions. This study provides a framework for deciphering the material basis of the multiple pharmacological functions of SZ-A, its rational clinical use, and the expansion of its indications.

Pharmacokinetic, acute toxicity, and pharmacodynamic studies of semen strychni total alkaloid microcapsules

Purpose: To investigate the safety and effectiveness of semen strychni total alkaloid microcapsules (SSTAM), compared with semen strychni total alkaloids (SSTA).
 Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to assess pharmacokinetics of brucine and strychnine in rats. Acute toxicity was investigated in pre-test and formal experiments in mice. The pharmacodynamics of SSTAM and SSTA were evaluated by their analgesic and anti-inflammatory activities.
 Results: With respect to brucine, the half-life of SSTA group (1.6 mg/kg), low-dose SSTAM group (6 mg/kg) and high-dose SSTAM group (10 mg/kg) was 5.723, 9.321 and 9.025 h, respectively. With respect to strychnine, the half-life of SSTA group, low-dose SSTAM group and high-dose SSTAM group was 4.065, 8.819 and 8.654 h, respectively. The LD50 values of SSTAM group and SSTA group were 236.59 and 30.27 mg/kg, respectively. The pain inhibition rates of SSTAM groups (25 and 50 mg/kg) were higher than that of SSTA group (p < 0.05) while the pain threshold values of the SSTAM groups (25 and 50 mg/kg) were higher than that of blank control (p < 0.01) and SSTA groups (p < 0.01) at 60 min and 120 min. The inhibition rates of the SSTAM groups (25 and 50 mg/kg) were higher than that of SSTA group based on ear swelling and cotton ball granulation tests. Compared with blank control and SSTA groups, the absorbance values of SSTAM groups (25 and 50 mg/kg) were lower (p < 0.01).
 Conclusion: SSTAM increases the dosage of administration but reducea the toxicity of the alkaloids in rats, and is thus a potentially safe and effective drug delivery system.

Traditional Chinese herbal medicine Astragalus Radix and its effects on intestinal absorption of aconite alkaloids in rats

ObjectiveAstragalus Radix (AR, Huangqi in Chinese) has been widely used as a qi (energy) restoring herb that is thought to act through reinvigorating the spleen and lung. Aconite is used to rebalance the body temperature during illness and played an irreplaceable role in disease control since ancient times, but it is limited by its strong neuro and cardiotoxicity. Since the Song Dynasty (1227), the two herbs have been commonly used as herbal pairs including in the famous Qifu Decotion, from the “Wei's Family Prescription”. However, many ancient texts also record that they are not compatible using together, suggesting they can have negative outcomes when mixed. This study investigated whether Astragali Radix had either positive or negative effects on absorption of six different active alkaloids derived from aconite. MethodsSingle intestinal perfusion model was used to study the effects of Astragali Radix on aconite alkaloids absorption. Response of ABC transporters and distribution of three tight junction proteins on the surface of intestinal enothelium were assessed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blot and immunofluorescence microscopy, respectively. ResultsThe results showed that aconite alkaloids absorption could be inhibited, and different concentrations of Astragali Radix considerably increased the expression levels of the ABC transporters and tight junction proteins with Astragali Radix treatment. ConclusionThese results suggest that Astragali Radix can block absorption of aconite alkaloids through the upregulation expression of ATP-binding cassette transporters (ABC transporters) and tight junction proteins. It demonstrates that co-administration of Astragali Radix with other drugs might change the absorption profile of the second drug which is important to know in clinic therapy.

Toxicokinetics of 11 Gelsemium Alkaloids in Rats by UPLC-MS/MS.

Gelsemium elegans (Gardn. & Champ.) Benth. is a plant belonging to the genus Gelsemium (family Gelsemiaceae), and its main components are alkaloids. It is a Chinese traditional medicinal plant and notoriously known as a highly toxic medicine. However, a method has not yet been found for the simultaneous detection of 11 Gelsemium alkaloids in rat plasma, and the toxicokinetics of 11 Gelsemium alkaloids after intravenous administration has not been reported. In this work, we have developed a sensitive and rapid method of ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) for the detection of 11 Gelsemium alkaloids in rat plasma. The toxicokinetic behavior was also investigated, so as to provide a reference of the scientific properties of Gelsemium elegans and improve the efficacy and safety of drugs. Sixty-six Sprague-Dawley rats were randomly divided into 11 groups, six rats in each group. Each group was intravenously given one alkaloid (0.1 mg/kg), respectively. A Waters UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) was used for chromatographic separation. Methanol and water (containing 0.1% formic acid) were used for the mobile phase with gradient elution. Multiple reactions were monitored, and positive electrospray ionization was used for quantitative analysis. The precision was less than 16%, and the accuracy was between 86.9% and 113.2%. The extraction efficiency was better than 75.8%, and the matrix effects ranged from 88.5% to 107.8%. The calibration curves were in the range of 0.1–200 ng/mL, with a correlation coefficient (R2) greater than 0.995. The UPLC-MS/MS method was successfully applied to the toxicokinetics of 11 Gelsemium alkaloids in rats after intravenous administration (0.1 mg/kg for each alkaloid). The results of the toxicokinetics provide a basis for the pharmacology and toxicology of Gelsemium alkaloids and scientific evidence for the clinical use of Gelsemium alkaloids.

Pharmacokinetic Studies of Three Alkaloids in Rats After Intragastrical Administration of Lycopodii Herba Extract by LC-MS/MS.

Lycopodii Herba is a widely used traditional medicinal herb, and contains diverse fascinating alkaloids. In this study, a fast and sensitive LC-MS/MS method for the simultaneous determination of lycodoline, α-obscurine, and N-demethyl-α-obscurine from Lycopodii Herba in rat plasma and brain tissue was developed and validated. Biological samples were extracted via a protein precipitation procedure using methanol as the extraction solvent and Huperzine B as the internal standard. Chromatographic separation was carried out using a Thermo Syncronis-C18 column (50 mm × 2.1 mm, 5 μm) and a gradient mobile phase containing methanol and water with 0.05% formic acid. The three alkaloids were detected by positive electrospray ionization in selective reaction monitoring mode. The selectivity, crosstalk, carryover effect, linearity, accuracy, precision, extraction recovery, matrix effect, and stability of the current method were validated. Then, using the validated method, the plasma pharmacokinetics and brain tissue distribution of the alkaloids in rats were investigated after intragastrical administration of Lycopodii Herba extract. The three alkaloids were shown to be rapidly absorbed into the blood (Tmax, 0.79–1.58 h), and then also eliminated rapidly (t1/2, 1.27–2.24 h). All of them could pass through the blood–brain barrier. The method provides a new research approach to expand preclinical studies of Lycopodii Herba.

Profiling and Pharmacokinetic Studies of Alkaloids in Rats After Oral Administration of Zanthoxylum nitidum Decoction by UPLC-Q-TOF-MS/MS and HPLC-MS/MS.

Zanthoxylum nitidum (Roxb.) DC (Rutaceae), called as “liangmianzhen” in China, is well known for its anti-inflammation and analgesic effect. Alkaloids are its main active constituents. However, little has been known about the absorption of main alkaloids in vivo. In this study, an ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry was employed for identification of absorbed alkaloids in rats after oral administration of Z. nitidum decoction. By analyzing the fragmentation patterns, a total of nineteen alkaloids were exactly or tentatively identified in rat plasma after treatment, of which magnoflorine, α-allocryptopine, and skimmianine are dominant. Moreover, a high performance liquid chromatography coupled mass spectrometry method was developed for simultaneous quantification of magnoflorine, α-allocryptopine, and skimmianine, and successfully applied to pharmacokinetic study in rats after oral administration of Z. nitidum decoction. The research would contribute to comprehensive understanding of the material basis and function mechanism of Z. nitidum decoction.

Pyrrolizidine alkaloids in commercial feedstuffs for horses.

Pyrrolizidine alkaloids are secondary plant metabolites with hepatotoxic effect in humans and several animal species. In recent studies, foods such as herbal teas and honey have been found to be contaminated with pyrrolizidine alkaloids. The aim of this study was to identify and assess pyrrolizidine alkaloids in compound feeds manufactured for horses and containing either alfalfa or a blend of herbs. Forty-eight feed products for horses were included in the study. The feedstuffs were analysed for 28 selected pyrrolizidine alkaloids by liquid chromatography-mass spectrometry. The concentrations of the individual pyrrolizidine alkaloids were summed to calculate the total pyrrolizidine alkaloid content. In 7 of 48 samples, pyrrolizidine alkaloid concentrations were below the limit of quantification of 1-5 μg/kg. The median of 41 out of 48 samples was 58 μg/kg, and the 25 and 75th percentiles were 8 and 151 μg/kg. The highest observed pyrrolizidine alkaloid concentrations, 1306 and 1222 μg/kg, were found in two alfalfa-based feed products, followed by 836 μg/kg in an herb-containing feed product. Lycopsamine, seneciphylline, seneciphylline-N-oxide, senecionine and senecionine-N-oxide were the most frequently detected alkaloids. Risk assessment was based on no-observed-adverse-effect-level for pyrrolizidine alkaloids in rats and humans. The specific susceptibility of horses to pyrrolizidine alkaloids remains unknown. According to our risk assessment, pyrrolizidine alkaloid contamination should be limited to <90 μg/kg in equine compound feeds. We showed a high rate of pyrrolizidine alkaloids contamination in feed products for horses. In 43% of the analysed samples, pyrrolizidine alkaloid levels exceeded the calculated maximum tolerable levels. There is a need to introduce measures to reduce pyrrolizidine contamination in equine feedstuffs. The Summary is available in Portuguese - see Supporting Information.

Simultaneous determination of six coptis alkaloids in urine and feces by LC-MS/MS and its application to excretion kinetics and the compatibility mechanism of Jiao-Tai-Wan in insomniac rats.

Jiao-Tai-Wan (JTW) is a well-known traditional Chinese medicine prescription composed of Rhizoma Coptidis (RC) and Cortex Cinnamon (10:1, g/g). It has been used to treat insomnia in China for centuries. This study investigates the excretion properties of coptis alkaloids from RC and JTW in normal and insomniac rats, and it examines the compatibility mechanism for this prescription. A new liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of six alkaloids - berberine, epiberberine, coptisine, jatrorrhizine, palmatine and magnoflorine - in rat urine and feces. The normal and model rats were orally treated with RC and JTW powder at a dosage containing 3.0 g kg-1 day-1 RC once per day for 7 days. Briefly, the results showed that the cumulative amounts of urinary and fecal excretion of the six alkaloids were significantly different in the pathological condition, as well as in compatibility. In normal rats, the urinary and fecal excretion of coptis alkaloids, especially berberine, coptisine and palmatine, increased significantly in the JTW group compared with the RC group, while the urinary and fecal excretion of six alkaloids decreased in insomniac rats. These data suggested that pathological conditions might have a notable influence on the excretion of alkaloids in rats, and demonstrated that the compatibility could promote better therapeutic effects through the accumulation of alkaloids in the body. These results might explain the compatibility of JTW.

Pharmacokinetic and excretion study of three alkaloids in rats using UPLC-MS/MS after oral administration of menispermi rhizoma capsules.

A sensitive, specific and rapid ultra-performance liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) was developed for simultaneous determination of three main alkaloids (daucicoline, daurisoline, dauricine) in rat plasma, urine and feces after oral administration of menispermi rhizoma capsules. The chromatographic separation was performed on a Waters ACQUITY UPLC® BEH C18 column (50 mm × 2.1 mm, id, 1.7 μm) with a column temperature of 30 °C and a linear gradient elution using a mobile phase consisting of 0.1% formic acid and acetonitrile. The flow rate was set at 0.3 mL min−1, and the total run time was 10 min. The detection was performed, without interference, using positive electrospray ionization with a multiple reaction monitoring mode. A comprehensive validation of the method was performed. The linearity of the analytical response was good over a wide concentration range with correlation coefficients greater than 0.9903 and the lower limits of quantification were 1.5–5.0 ng mL−1 for all matrices. Both accuracy and precision of the assay were satisfactory. The mean extraction recoveries of analytes and internal standard from rat plasma, urine and feces were all more than 78.3%. The validated methods of the three types of substrates were successfully applied to a pharmacokinetic and excretion study of the three alkaloids in rats after oral administration of menispermi rhizoma capsule. The pharmacokinetics and excretion study of these active components in menispermi rhizoma capsule have not been reported. The results provided a meaningful basis for the clinical application of menispermi rhizoma capsule.

Pharmacokinetics, Tissue Distribution, and Elimination of Three Active Alkaloids in Rats after Oral Administration of the Effective Fraction of Alkaloids from Ramulus Mori, an Innovative Hypoglycemic Agent.

In this study, we systematically investigated the plasma pharmacokinetics, tissue distribution, and elimination of three active alkaloids after oral administration of the effective fraction of alkaloids from Ramulus Mori (SZ–A)—an innovative hypoglycemic agent—in rats. Moreover, the influences of other components in SZ–A on dynamic process of alkaloids were explored for the first time. The results showed that 1-deoxynojirimycin (DNJ), fagomine (FGM) and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) exhibited nonlinear pharmacokinetics following oral administration of SZ–A (40–1000 mg/kg). The prolonged t1/2 and greater area under concentration-time curve (AUC) versus time (AUC0–t) of DNJ for SZ–A than for purified DNJ has been observed after both oral and intravenous administration. It was found that other components in SZ–A could enhance the absorption of DNJ through the intestinal barrier. The major distribution tissues of DNJ, FGM, and DAB were the gastrointestinal tract, liver, and kidney. Three alkaloids were mainly excreted into urine and feces, but less into bile. Interestingly, the excess excretion of FGM was revealed to be partly due to the biotransformation of other components in SZ–A via gut microbiota. These information provide a rational basis for the use of SZ–A in clinical practice.

Simultaneous Determination and Comparative Pharmacokinetics of Fuzi Water-Soluble Alkaloids between Normal and Acute Heart Failure Rats by Ultra Performance Liquid Chromatography Method.

In order to compare the pharmacokinetics of Fuzi water-soluble alkaloids between normal and acute heart failure rats, an ultra performance liquid chromatography (UPLC) method for simultaneous determination of it in rat plasma was developed. Plasma samples were treated by protein precipitation method. Seven water-soluble alkaloids were separated on a CAPCELL column and detected under the optimized chromatography condition. The calibration curves of seven targeted components showed good linearity with r2 > 0.9990 with average recoveries from 82.72 to 103.33% and matrix effect ranged from 90.02 to 104.03%. The intra- and inter-batch relative standard deviations were <10.72%, and the relative error of accuracy was within the range of -12.79-4.44%, respectively. After oral administration, the pharmacokinetic characteristics of it were investigated. Compared with the normal group, the Cmax and AUClast (area under the plasma concentration-time curve from the time of dosing to the time of last quantifiable concentration) of seven components except 3-Methoxy-p-tyramine Hydrochloride were obviously increased with remarkable prolonged t1/2 in acute heart failure group. In conclusion, a rapid, simple and sensitive UPLC method for the simultaneous quantification of seven water-soluble alkaloids in rat plasma was developed and validated for the first time. And the study showed that the disease condition had an impact on pharmacokinetics of Fuzi water-soluble alkaloids in rats in vivo.

Pharmacokinetic Effects of Cinnamic Acid, Amygdalin, Glycyrrhizic Acid and Liquiritin on Ephedra Alkaloids in Rats.

Ephedra alkaloids, including ephedrine (EP), pseudoephedrine (PEP) and methylephedrine (MEP), are sympathomimetic compounds with known toxicities but many Ephedra (Ephedrae herba) preparations, such as Ephedra decoction, have been clinically applied for centuries. In order to explore the possible detoxification mechanism of Ephedra alkaloids, four representative compounds in Ephedra decoction (cinnamic acid, amygdalin, glycyrrhizic acid and liquiritin) were studied for their pharmacokinetic effects on Ephedra alkaloids in Sprague-Dawley rats. Animals were randomly divided into six groups, with six rats in each. Rats were treated orally with EP-PEP-MEP (20mg/kg EP+20mg/kg PEP+20mg/kg MEP) and different combinations of cinnamic acid (3.03mg/kg), amygdalin (56.97mg/kg), glycyrrhizic acid (12.42mg/kg), liquiritin (3.79mg/kg) with EP-PEP-MEP, and 20mg/kg EP+20mg/kg PEP+20mg/kg MEP+3.03mg/kg cinnamic acid+56.97mg/kg amygdalin+12.42mg/kg glycyrrhizic acid+3.79mg/kg liquiritin. Blood samples (0.5mL) were taken from the orbital sinus venous plexus into heparinized tubes at 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 300 and 360min(6 rats per time point in each group) following single administration. The concentrations of Ephedra alkaloids in rat plasma were determined using a validated high performance liquid chromatography method. Area under the concentration-time curve from 0 to 360min (AUC0-t ) of EP, PEP and MEP were 666.99, 650.76 and 632.37µg·min/mL, respectively. Maximum plasma concentration (C max) of EP, PEP and MEP were 4.15, 4.08 and 3.59μg/mL, respectively. Mean residence time (MRT) of EP, PEP and MEP were 197.00, 173.97 and 183.87min, respectively, when the rats were treated with EP-PEP-MEP. Cinnamic acid increased the AUC0-t of EP while decreased C max of EP, amygdalin and glycyrrhizic acid increased C max and AUC0-t of EP and PEP, while liquiritin decreased AUC0-t of EP and PEP. The four representative compounds reduced MRT of EP, PEP and MEP, four compounds decreased AUC0-t of MEP. The EP-PEP-MEP+cinnamic acid+amygdalin+glycyrrhizic acid+liquiritin group increased AUC0-t of EP while decreased MRT of EP, increased MRT of PEP while decreased AUC0-t of PEP. The EP-PEP-MEP+cinnamic acid+amygdalin+glycyrrhizic acid+liquiritin group decreased MRT, AUC0-t and C max of MEP. Significant changes in pharmacokinetic parameters of EP, PEP and MEP were observed after oral administration with different combinations. The pharmacokinetic results reported here might provide reference for clinical usage of Ephedra alkaloids.

Characterization of chemical constituents and rats metabolites of an alkaloidal extract of Alstonia scholaris leaves by liquid chromatography coupled with mass spectrometry.

Alstonia scholaris has been used in "Dai" ethnic medicine to treat chronic respiratory diseases for a long history, and the major bioactive constituents are alkaloids. An alkaloidal extract of A. scholaris leaves (AAS) has been developed into an investigational new drug, and has been approved for phase I/II clinical trials by China Food and Drug Administration. However, little is known on the chemical composition and in vivo metabolism of AAS, thus far. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/qTOF-MS) method was established to characterize the chemical constituents of AAS. Samples were separated on an ACQUITY UPLC CSH column (2.1×100mm, 1.7μm) with acetonitrile and water containing 0.3% formic acid as the mobile phase. On the basis of high-accuracy mass spectral analysis, a total of 35 alkaloids were characterized from AAS, including 11 scholaricine-type, 9 vallesamine-type, 12 picrinine-type, and 3 tubotaiwine-type alkaloids. Furthermore, the metabolic pathways of 4 representative alkaloids in rats were studied. They mainly undertook hydroxylation and glucuronidation reactions. Based on the above metabolic pathways, the metabolism of AAS (10mg/kg) in rats after oral administration was studied by LC/MS. A total of 33 compounds in plasma, 40 compounds in urine, and 38 compounds in feces were characterized. The results indicated that scholaricine-type alkaloids could get into circulation more readily than the other types. This is the first systematic study on chemical profiling and metabolites identification of AAS.

Simultaneous quantification of three active alkaloids from a traditional Chinese medicine Ramulus Mori (Sangzhi) in rat plasma using liquid chromatography–tandem mass spectrometry

Fagomine, 1-deoxynojirimycin (DNJ) and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) are the major bioactive constituents in the active fraction of alkaloids from the traditional Chinese medicine mulberry twig (Ramulus Mori, Chinese name Sang Zhi), which has a strong activity on α-glucosidase in vitro and in vivo. A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of DNJ, fagomine and DAB in rat plasma. Plasma samples were prepared using a simple protein precipitation by the addition of 1% volume of Tris and two volumes of methanol–acetonitrile. The analytes and internal standard (IS, miglitol) were chromatographed in an XBridge™ amide column with a gradient mobile phase of acetonitrile–water (0.1% ammonium hydroxide) at a flow rate of 0.7mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) source in positive ion mode by multiple reaction monitoring (MRM) mode. Linear detection responses were obtained for DNJ ranging from 5.00 to 5000.00ng/mL, 10.00 to 2500.00ng/mL for fagomine and DAB. The lower limits of quantification (LLOQs) were 5.00, 10.00, 10.00ng/mL for DNJ, fagomine and DAB, respectively. Intra-day and inter-day precisions (R.S.D.%) were within 10% for three analytes with accuracies (R.E.%) less than 12%. The mean recoveries of analytes were greater than 85%. All analytes were proved to be stable during the sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of the three alkaloids in rats after oral administration of the active fraction of alkaloids from mulberry twig.

Integrated pharmacokinetics of five protoberberine-type alkaloids in normal and insomnic rats after single and multiple oral administration of Jiao-Tai-Wan

Ethnopharmacological relevanceJiao-Tai-Wan (JTW), an important herbal formula consists of Rhizoma coptidis and Cortex cinnamomi powder, is a famous prescription which has been used for centuries to treat insomnia in Traditional Chinese Medicine. The purpose of this study is to compare the pharmacokinetic properties of five protoberberine-type alkaloids (i.e. berberine, palmatine, coptisine, epiberberine and jatrorrhizine), the main bioactive constituents in JTW, between normal and insomnic rats. We also investigate the differences between single-dose and multiple-dose pharmacokinetics of five protoberberine-type alkaloids. Materials and methodsThe insomnic rat models were induced by intraperitoneal injection of one-dose para-chlorophenylalanine acid (PCPA). Quantification of five protoberberine-type alkaloids in rat plasma was achieved by using a rapid LC–MS/MS method. Plasma samples were collected at different time points to construct pharmacokinetic profiles by plotting drug concentration versus time and estimate pharmacokinetic parameters. An unpaired Student׳s t test was used for comparisons with SPSS 17.0. ResultsThe five protoberberine-type alkaloids of single-dose normal groups had slow absorption and low bioavailability, as well as a delay of peak time. In the single-dose oral administration, the Cmax and Tmax of five ingredients in insomnic rats had significant differences compared with those of normal rats. In the multiple-dose oral administration, the pharmacokinetic parameters of five protoberberine-type alkaloids varied greatly in insomnic rats. In the normal rats, there were significant differences (P<0.05) in the principal pharmacokinetic parameters such as Cmax and Tmax between single-dose and multiple-dose oral administration. In the insomnic rats, the five ingredients of multiple-dose groups showed better absorption than the single-dose groups. Particularly, three peaks were observed in multiple-dose model group of plasma–concentration curves. ConclusionsThe pharmacokinetic behavior of five protoberberine-type alkaloids was described in this paper. In both normal groups and model groups, the pharmacokinetic behavior of multiple-dose had significant differences comparing with the single-dose; either single-dose or multiple-dose, the pharmacokinetic behavior of insomnic rats had significant differences comparing the normal rats. Multiple dosing may improve the absorption of JTW in insomnic rats, which will increase the bioavailability and bring into active role in therapeutical effect.

Studies on absorption kinetics characteristics of diterpenoid alkaloids in rats after oral administration of Sini Tang powder

The purpose of this study was to investigate the absorption kinetics of aconitine, mesaconitine and hypaconitine in rats after oral administration of Sini Tang powder. With cannulate portal and jugular veins cannulated (double-cannulate), conscious moving rats were orally administered Sini Tang. Then samples of portal and systemic blood were collected at the designated periods of time and analyzed for aconitine, mesaconitine and hypaconitine by HPLC. Apparent absorption coefficient of aconitine, mesaconitine and hypaconitine was caculated respectively. The results indicated that the apparent absorption coefficient of aconitine, mesaconitine and hypaconitine come from Sini Tang were 0. 336, 0. 090, 0. 176, respectively, which had some differences among them. It was also suggested that double-cannulated rat was useful for estimating the absorption kinetics of aconitine, mesaconitine and hypaconitine after orally administered Sini Tang by determining the AUC values for drugs in portal and systemic blood samples. The three alkaloids could all be detected in blood, but the absorption differences were existed among the three alkaloids.

A sensitive and selective liquid chromatography−tandem mass spectrometry method for simultaneous determination of five isoquinoline alkaloids from Chelidonium majus L. in rat plasma and its application to a pharmacokinetic study

Chelidonium majus L. is one of the most important medicinal plants of the family Papaveraceae. Its pharmacological effects have been primarily attributed to the presence of a number of alkaloids. In the present study, a sensitive and selective liquid chromatography-tandem mass spectrometry method for simultaneous determination of five isoquinoline alkaloids from Chelidonium majus L. was developed and validated. The analytes (protopine, chelidonine, coptisine, sanguinarine and chelerythrine), together with the internal standard (palmatine), were extracted from acidified rat plasma with ethyl acetate-dichloromethane (4:1, v/v). Chromatographic separation was carried out on a Diamonsil C(18) column with an isocratic mobile phase consisting of acetonitrile and water (adjusted to pH 2.3 with formic acid) (30:70, v/v) at a flow rate of 0.4 ml/min. Mass spectrometric detection was performed by selected reaction monitoring mode via electrospray ionization source operating in positive ionization mode. The assay exhibited good linearity (r ≥ 0.9933) for all the analytes. The lower limits of quantification were 0.197-1.27 ng/ml using only 50 µl of plasma sample. The intra- and inter-day precisions were less than 11.9%, and the accuracy was between -6.3% and 9.3%. The method was successfully applied to the pharmacokinetic study of the five alkaloids in rats after intragastric administration of Chelidonium majus L. extract.

Metabolism studies of the Kratom alkaloids mitraciliatine and isopaynantheine, diastereomers of the main alkaloids mitragynine and paynantheine, in rat and human urine using liquid chromatography–linear ion trap-mass spectrometry

Mitragyna speciosa ( Kratom in Thai), native in Southeast Asia, is increasingly misused as a herbal drug of abuse. During metabolism studies on the Kratom alkaloids mitragynine, its diastereomers speciogynine and speciociliatine as well as paynantheine in rats and humans, further isomeric compounds were detected in Kratom users’ urine. The question arose whether these compounds were formed from the low abundant, isomeric alkaloids mitraciliatine (MC) and isopaynantheine (ISO-PAY). Therefore, the aim of the presented study was to identify using liquid chromatography–linear ion trap-mass spectrometry their phase I and II metabolites in rat urine after administration of pure MC or ISO-PAY, to confirm their formation in humans, and finally to confirm whether the above-mentioned isomeric compounds in human urine represent MC and ISO-PAY and/or their metabolites. The metabolic pathways of both alkaloids in rats were found to be comparable to those of their corresponding diastereomers. In the human urines tested, not all metabolites found in rats could be detected because of the much lower amounts of MC and ISO-PAY in Kratom. However, all the above-mentioned so far unknown isomeric compounds could be identified in the human urine samples as MC, ISO-PAY and/or their metabolites. The used LC separation was also suitable for the differentiation of all other Kratom alkaloids and their metabolites in human urine.