Hypaconitine Research Articles

Determination of aconitum alkaloids in acute poisoning case by electromembrane extraction

In this work, electromembrane extraction (EME) was used for the first time to separate aconitine (AC), mesaconitine (Mes-AC) and hypaconitine (Hyp-AC) from biological samples and Chinese herbal medicines. Efficient EME of polar and high molecular weight aconitine alkaloids from different sample matrices was achieved with the solvent of 1-ethyl-2-nitrobenzene (ENB). Under the optimal EME conditions, EME provided recoveries for all targets in the range of 72%–74 %, 85%–103 % and 92%–94 % for whole blood, urine and aqueous samples. The proposed EME systems combined with LC-MS/MS and HPLC-UV were evaluated using different sample matrices, and the methods displayed satisfactory analytical characteristic including negligible matrix effect. The LOD and LOQ of AC, Mes-AC, and Hyp-AC by EME-LC-MS/MS were in the range of 0.002–0.068 ng/mL and 0.005–0.228 ng/mL respectively. The LOD and LOQ of AC, Mes-AC, and Hyp-AC by EME-HPLC-UV were in the range of 0.06–0.26 μg/mL and 0.20–0.86 μg/mL, respectively. The coefficient of determination, R2-value was ≥0.9926 for all cases, and the accuracy in the linear ranges was in the range of 91%–111 %. Finally, the method was successfully applied for the qualitative and quantitative analysis of AC and Mes-AC in the whole blood and herbal medicine dreg samples from an actual forensic case, and poisoning by aconitum alkaloids was identified as the cause of death. Therefore, we believe that EME could be a powerful tool to identify poisoning, and EME has great potential for efficient separation of polar and high molecular weight substances. These are of great importance in the fields of but not limited to forensic science, Traditional Chinese Medicine and clinics.

The metabolic pathways and products of ten Aconitum alkaloids in Sanwujiao Pills from eight organs of mice by UHPLC-Q-TOF-MS/MS.

Sanwujiao pill (SWJP) is a Chinese herbal preparation widely used in China. It is an essential medicine for treating rheumatism and blood stasis. However, its safety in clinical use has always been the focus of patients because it contains toxic herbs of Aconitum carmichaelii Debx. and A. vilmorinianum Kom. To further reveal the pharmaceutical and toxic effect substances and the action mechanism of SWJPs, the metabolites and their pathways of ten Aconitum alkaloids (AAs) in the preparation at different time points after oral administration in eight organs of mice were investigated. The biosamples were investigated by a four-step strategy of UPLC-Q-TOF-MS /MS technology. Aconitine (AC), mesaconitine (MA), and hypaconitine (HA) were not detected in any organs. The highest concentrations of the other seven AAs occurred at 0.5 h. Yunaconitine (YAC) was not detected in the brain; all seven AAs had the lowest concentration in the brain, and the metabolism was slow in the stomach. Twelve predicted metabolites were identified, the kidney and stomach were their primary distribution locations, and the most metabolites were found at 0.5h. The main metabolic pathways of the ten AAs were demethylation, deethylation, deoxygenation, hydroxylation, and deacetylation. This is the first report about the metabolism of ten AAs in SWJPs in mice. Significantly, the metabolic pathways and products of four hidden toxic AAs were analyzed in vivo for the first time. The results were of great significance for the safety and effectiveness of SWJPs in clinical application.

Glycyrrhiza uralensis promote the metabolism of toxic components of Aconitum carmichaeli by CYP3A and alleviate the development of chronic heart failure.

Aconitum, as "the first drug of choice for invigorating Yang and saving lives", has been widely used for the treatment of heart failure. However, toxic components of Aconitum can easily lead to serious arrhythmia, even death (Y. CT., 2009; Zhang XM., 2018). In this study, a High Performance Liquid Chromatography (HPLC) method for the determination of aconitine (AC), mesaconitine (MA) and hypaconitine (HA) was established; The effect of Glycyrrhiza on CYP3A1 / 2 mRNA expression was detected by RT-PCR; SD rats were given Aconitum and compatibility of Glycyrrhizae and Aconitum by gavage respectively, the blood concentration of toxic components were determined by LC-MS / MS; The CHF rat model was established by intraperitoneal injection of adriamycin (2.5 mg / kg), and were randomly divided into model, Aconitum, the compatibility of Glycyrrhizae and Aconitum and Captopril group, 5 mice/group. After 4 weeks of gavage, the corresponding indexes were detected by ELISA and HPLC. The results showed that Ketoconazole significantly inhibited the metabolites of AC, MA and HA; Glycyrrhiza induced CYP3A gene expression; The level of ALD in the compatibility of Glycyrrhizae and Aconitum group was significantly lower than that in Aconitum group. After intervention with the compatibility of Glycyrrhizae and Aconitum, ATP increased, ADP decreased significantly. In conclusion, we found Glycyrrhiza promoted the metabolism of toxic components of Aconitum by up regulating the expression of CYP3A, and reduced the content of BNP, Ang II and ALD, improved the energy metabolism disorder of myocardium, alleviated the development of CHF.

Investigation into the protective effects of hypaconitine and glycyrrhetinic acid against chronic heart failure of the rats

BackgroundThe present study aimed to determine the protective effects of hypaconitine (HA) and glycyrrhetinic acid (GA) against chronic heart failure (CHF) in the rats and to explore the underlying molecular mechanisms.MethodsThe CHF rat model was established by transverse-aortic constriction (TAC) operation. Transthoracic echocardiography and hematoxylin eosin (HE) staining were used to evaluate the pathophysiological and histopathological changes of CHF model. The total cholesterol (TCHO) and triglyceride (TG) levels were determined by ELISA assay. The protein expression of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) in the rat ventricular tissues was determined by immunohistochemistry. The serum metabolites were determined by LC-MS/MS assay.ResultsAfter applied the HA + GA, the cardiac tissue and structure were obviously improved, and the HA + GA treatment also significantly reduced the plasma levels of TCHO and TG in the CHF rats. The expression of FGF2 and VEGFA protein was up-regulated and the expression of eNOS protein was down-regulated in the ventricular tissues of CHF rats, which was significantly restored after HA + GA treatment. HA + GA treatment down-regulated serum isonicotinic acid, phosphatidylcholine, cardiolipin, estrogen glucuronide, and glycocholic acid, up-regulated serum sphingosine and deoxycholic acid in the CHF rats.ConclusionsIn conclusion, HA + GA showed protective effects on CHF in the rats, and the HA + GA may exert protective effects by reducing lipid levels, up-regulating the expression of FGF2 and VEGFA proteins, attenuating eNOS protein expression, and modulating metabolic pathways. However, the molecular mechanisms underlying HA + GA-mediated effects still require further examination.

The mechanism underlying hypaconitine-mediated alleviation of pancreatitis-associated lung injury through up-regulating aquaporin-1/TNF-α.

Acute pancreatitis-associated lung injury (APALI) is one of the most common and most dangerous form of extra-pancreatic organ damage in severe acute pancreatitis (SAP). The treatment options for SAP were limited thus far; as a result, approximately 60%-80% of patients with SAP would die within a week. Hypaconitine (HC), one of the most important active ingredients in a Mongolian traditional medicine Radix Aconiti Kusnezoffii has an excellent anti-inflammatory effect. To ascertain whether HC has a protective effect against APALI, we investigated the therapeutic effects and the underlying mechanisms in vivo and in vitro and attempted to elucidate the mechanism in detail. In this study, APALI rats and human pulmonary microvascular endothelial cells were treated with therapeutic doses of HC after establishing a model with sodium taurocholate and lipopolysaccharide, respectively. Serum amylase and lipase activity, lung wet/dry weight ratio, lung myeloperoxidase activity, and pancreatic and lung histopathological changes showed that HC alleviated APALI in a dose-dependent way, which can be abolished by an aquaporin-1 (AQP-1) knockdown. The results of the reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemical staining confirmed the expression of AQP-1, a kind of transmembrane protein that mainly distributed in the membranes of pulmonary cells and contributed to maintain water balance in the body by interacting with tumor necrosis factor-alpha (TNF-α), is negatively associated with APALI. On the contrary, HC treatment up-regulated AQP-1 expression and down-regulated the TNF-α expression as a consequence in APALI. These results suggest that HC has a good anti-inflammatory therapeutic effect on APALI with a possible underlying mechanism that affects the AQP-1/TNF-α pathway.

Mdr1a, Bcrp and Mrp2 regulate the efficacy and toxicity of mesaconitine and hypaconitine by altering their tissue accumulation and in vivo residence

Mesaconitine (MA) and hypaconitine (HA) are the main bioactive/toxic alkaloids of Aconitum carmichaelii Debx, and MDR1, BCRP and MRP2 are involved in their efflux in vitro. This study aimed to explore the effects of Mdr1a, Bcrp and Mrp2 on the efficacy/toxicity of MA and HA by using efflux transporter gene knockout mouse models. The analgesic and anti-inflammatory effects, neurotoxicity/cardiotoxicity, and pharmacokinetic profiles of MA and HA were studied. Compared to wild-type mice, the analgesic effects of MA or HA were significantly enhanced in Mdr1a‐−/−, Bcrp1−/− and Mrp2−/− mice, and the anti-inflammatory effects notably increased in Bcrp1−/− and Mrp2−/− mice. Compared to wild-type mice, Mdr1a−/−, Bcrp1−/− and Mrp2−/− mice suffered from severe karyopyknosis and edema in the brain after MA or HA treatment. Meanwhile, significant arrhythmia appeared, and the heart rate and RR-interval were greatly altered in Mdr1a−/−, Bcrp1−/− and Mrp2−/− mice. Additionally, obvious disorder of cardiomyocytes were observed, and the CK and cTnT (indicators of heart injury) levels were greatly enhanced in efflux transporter gene knockout mice. The brain levels of MA and HA were markedly increased in Mdr1a−/−, Bcrp1−/− and Mrp2−/− mice, and the heart levels of MA and HA enhanced greatly in Mdr1a−/− mice. The MRT0-t values of MA and HA were remarkably enhanced in most efflux transporter gene knockout mice. In conclusion, Mdr1a, Bcrp and Mrp2 were all involved in regulating the efficacy/toxicity of MA and HA by altering their tissue accumulation and in vivo residence. Among the three efflux transporters, Mdr1a had a superior regulatory effect.

In vitro and in vivo evaluation of the influences of polysaccharides derived from Glycyrrhiza uralensis on three alkaloids and potential interaction mechanisms

The purpose of this study was to investigate the influences of the polysaccharides derived from Glycyrrhiza uralensis Fisch. (GCPs) on aconitine (AC), hypaconitine (HA), and benzoylmesaconine (BMA) from Aconitum carmichaelii Debx. and to explore potential interaction mechanisms. Biopharmaceutical properties in vitro including stability, aqueous solubility and permeability were determined by UPLC. Pharmacokinetic parameters in vivo were determined using UPLC-MS/MS. Phase solubility analysis, UV–vis spectrophotometry and differential scanning calorimetry (DSC) were used to explore potential interaction mechanisms. The results showed that GCPs could increase the stabilities of three alkaloids and solubilities of AC and HA, significantly decrease permeabilities of three alkaloids. The pharmacokinetic studies demonstrated that, after combination with GCPs, AC exhibited a higher Cmax value, shorter t0.5, higher elimination rate and greater area under the concentration-time curve (AUC) value compared to free AC. GCPs also improved the Cmax, t0.5, AUC(0–tn) and AUC(0–∞) values of HA to play a therapeutic role, and improved the t0.5 and AUC(0–∞) values of BMA to prolong the pharmacological effect and increase bioavailability. In addition, the results for the apparent formation constants and DSC analysis showed that an inclusion complex could be formed between GCPs and AC, GCPs and HA, and GCPs and BMA.

A proton-coupled organic cation antiporter is involved in the blood-brain barrier transport of Aconitum alkaloids

Ethnopharmacological relevanceThe herbs of Aconitum are the essential Traditional Chinese medicine and have played an indispensable role in many Asian countries for thousands of years to treat critical illnesses, and chronic, stubborn diseases. However, Aconitum may induce severe neurotoxicity and even death. So far the mechanism of Aconitum penetrating the blood-brain barrier (BBB) is still unclear.Aim of the study: To determine whether influx transporters contribute to the brain uptake of the highly toxic alkaloids in Aconitum including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). Materials and methodsThe uptake of AC, MA and HA was characterized using in vitro hCMEC/D3 model and in situ mouse brain perfusion. In hCMEC/D3 cells, the effect of incubation temperature, time, initial drug concentration, energy (NaN3), extracellular and intracellular pH (FCCP and NH4Cl), the prototypical substrates/inhibitors of known organic cation transporting carriers and trans-stimulation (pre-incubating with pyrilamine and diphenhydramine) on the cellular uptake were studied. In addition, the effect of silencing OCTN1, OCTN2 and PMAT by specific siRNA was investigated. In mice, the contribution of the proton-coupled antiporter on the brain uptake of Aconitum was investigated by chemical inhibition. ResultsIn hCMEC/D3 cells, AC, MA and HA were each taken up in a temperature-, time- and concentration-dependent manner, which were reduced by NaN3 and FCCP. Regulation of extracellular and intracellular pH as well as trans-stimulation studies showed that AC, MA and HA were transported by a proton-coupled antiporter expressed at the plasma membrane that could also transport pyrilamine and diphenhydramine. Each uptake was markedly inhibited by various cationic drugs, but insensitive to the prototypical substrates/inhibitors of identified organic cation transporting carriers, such as OCTs, PMAT, MATEs and OCTNs. In addition, silence of OCTN1, OCTN2 and PMAT had no significant inhibitory effect on the uptake of AC, MA and HA. In mice, the brain uptake of each alkaloid measured by in situ brain perfusion was suppressed by diphenhydramine when the transport capacity of P-gp/Bcrp at the BBB was chemically inhibited. ConclusionsA novel proton-coupled organic cation antiporter plays a predominant role in the blood to brain influx of AC, MA and HA at the BBB, and thus affect the safety of Aconitum species.

Circadian clock regulates metabolism and toxicity of Fuzi(lateral root of Aconitum carmichaeli Debx) in mice

BackgroundTherapeutic applications of Fuzi (lateral root of Aconitum carmichaeli Debx) are seriously concerned with its toxic effects. Strategies and approaches to reducing toxicity are of great interest. PurposeWe aimed to characterize the diurnal rhythm of Fuzi toxicity, and to determine the role of metabolism and pharmacokinetics in generating toxicity rhythmicity. MethodsToxicity was determined based on assessment of heart injury and animal survival after dosing mice with Fuzi decoction at different circadian time points. Circadian clock control of pharmacokinetics and toxicity was investigated using Bmal1-deficient (Bmal1−/−) mice. ResultsFuzi exhibited a diurnal rhythmicity in cardiotoxicity (reflected by plasma CK-MB and LDH levels). The highest level of toxicity was observed at ZT10 (5 PM), while the lowest level of toxicity occurred at ZT22 (5 AM). Also, a higher mortality rate was observed at ZT10 and lower mortality rates at other times of the day. ZT10 dosing of Fuzi generated higher systemic exposures of three toxic alkaloid ingredients aconitine (AC), hypaconitine (HA) and mesaconitine (MA) compared to ZT22. This was accompanied by reduced the formation of the metabolites (N-deethyl-AC, didemethyl-HA and 2‑hydroxyl‑MA) at ZT10. Bmal1 ablation resulted in an increased level of Fuzi toxicity at ZT22, while having no influences when drug was dosed at ZT10. As a consequence, circadian time-dependent toxicity of Fuzi was lost in Bmal1-deficient mice. In addition, Bmal1 ablation increased the plasma concentrations of AC, HA and MA in mice after oral gavage of Fuzi, and reduced formation of their metabolites (N-deethyl-AC, didemethyl-HA and 2‑hydroxyl‑MA). Moreover, Fuzi metabolism in wild-type liver microsomes was more extensive at ZT22 than at ZT10. Bmal1 ablation abrogated circadian time-dependency of hepatic Fuzi metabolism. ConclusionsFuzi chronotoxicity in mice was attributed to time-varying hepatic metabolism and systemic exposure regulated by circadian clock. The findings may have implications in reducing Fuzi toxicity with a chronotherapeutic approach.

Tissue Accumulations of Toxic Aconitum Alkaloids after Short-Term and Long-Term Oral Administrations of Clinically Used Radix Aconiti Lateralis Preparations in Rats.

Although Radix Aconiti Lateralis (Fuzi) is an extensively used traditional Chinese medicine with promising therapeutic effects and relatively well-reported toxicities, the related toxic aconitum alkaloid concentrations in major organs after its short-term and long-term intake during clinical practice are still not known. To give a comprehensive understanding of Fuzi-induced toxicities, current study is proposed aiming to investigate the biodistribution of the six toxic alkaloids in Fuzi, namely Aconitine (AC), Hypaconitine (HA), Mesaconitine (MA), Benzoylaconine (BAC), Benzoylhypaconine (BHA) and Benzoylmesaconine (BMA), after its oral administrations at clinically relevant dosing regimen. A ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for simultaneous quantification of six toxic alkaloids in plasma, urine and major organs of Sprague Dawley rats after oral administrations of two commonly used Fuzi preparations, namely Heishunpian and Paofupian, at their clinically relevant dose for single and 15-days. Among the studied toxic alkaloids and organs, BMA demonstrated the highest concentrations in all studied organs with liver containing the highest amount of the studied alkaloids, indicating their potential hepatotoxicity. Moreover, tissue accumulation of toxic alkaloids after multiple dose was observed, suggesting the needs for dose adjustment and more attention to the toxicities induced by chronic use of Fuzi in patients.

Cardiotoxicity evaluation and comparison of diterpene alkaloids on zebrafish

Diterpene alkaloids (DAs) have a broad spectrum of pharmacological activities, but exhibiting extremely serious cardiotoxicity to induce arrhythmia, heart arrest, even death. This study aimed to evaluate the cardiotoxicity of three diester diterpene alkaloids (DDAs) including aconitine (AC), mesaconitine (MAC), hypaconitine (HAC) and three monoester diterpene alkaloids (MDAs) including 14-α-benzoylaconine (BAC), 14-α-benzoylmesaconine (BMAC), 14-α-benzoylhypaconine (BHAC) on zebrafish. Firstly, the zebrafish embryos after a 72-hour post fertilization were treated with different doses of AC, MAC, HAC, and BAC, BMAC and BHAC for 2, 10 and 24 h, respectively. The heart rates of the treated embryos were calculated and the morphological images of body, together with heart fluorescence were obtained. Results demonstrated that AC, MAC, and HAC at low doses (15.6 and 31.3 μM) decreased the heart rates and increased them at high doses (62.5, 125, and 250 μM), while BAC, BMAC, and BHAC decreased the heart rates in the dose range of 31.3–250 μM, but the highest dose (500 μM) of BAC and BMAC increased the heart rates. In addition, AC, MAC, and HAC exhibited serious organic and functional toxicities, while BAC, BMAC, and BHAC did not. It could be induced that DDAs expressed stronger cardiotoxicities than MDAs, which might be due to that they were known as the Na+ channel activators and K+ channel inhibitors, respectively. The β-acetate at C-8 position, along with the protonated nitrogen on ring A of their chemical structures contributed more for their different cardiotoxicities. This is the first study on cardiotoxicity comparison of DAs, providing references for the rational and safe application of these compounds and some plant species containing them to reduce side effects while retaining therapeutic efficacy.

Effects of liquorice on pharmacokinetics of aconitine in rats

Aconite alkaloids are the main bioactive ingredients existing in Aconitum, for instance aconitine (AC), which exhibit potent analgesic, antirheumatic and other pharmacological effects. In this study, effects of long-term treatment with liquorice on pharmacokinetics of AC in rats were investigated.Pharmacokinetics of AC after oral administration of AC at 1.5 mg/kg either with pre-treatment of liquorice water extracts at 0.433 or 1.299 g/kg (crude drug), respectively, for one week or not were studied. Additionally, LS-180 cells and human primary hepatocytes were utilized to explore the potential effects of bioactive ingredients of liquorice on P-glycoprotein (P-gp) and Cytochromes P450 (CYPs), respectively.The results revealed that exposure of AC after pre-treatment with liquorice was altered remarkably. Area under the concentration-time curve (AUC) decreased from 161 ± 37.8 to 58.8 ± 8.97 and 44.7 ± 8.20 ng/mL*h, respectively. Similarly, Cmax decreased from 26.2 ± 5.19 to 11.8 ± 1.15 and 6.86 ± 0.600 ng/mL, respectively. In addition, expressions of CYPs of human primary hepatocytes were enhanced to various contents after induction. Moreover, accumulation of AC and hypaconitine (HA), not mesaconitine (MA) inside of LS-180 cells were reduced after pre-treatment by comparison with control.In conclusion, the exposure of AC in vivo declined after pre-treatment with liquorice extract, which may be highly associated with upregulated expression and/or function of CYPs and P-gp.

Simultaneous quantitation of aconitum alkaloids from You-Gui-yin in rat plasma by UPLC–ESI–MS and its application to a pharmacokinetic study

You-Gui-Yin (YGY), a famous traditional Chinese medicine, has been widely used in clinics for the treatment of kidney-yang deficiency, yang deficiency caused by excessive yin, and osteoporosis. A rapid and sensitive ultraperformance liquid chromatography–electrospray ionization–mass spectrometry (UPLC–ESI–MS) method for simultaneous determination of six Aconitum alkaloids including aconitine (AC), hypaconitine (HA), mesaconitine (MA), benzoylaconine (BAC), benzoylhypaconine (BHA), and benzoylmesaconine (BMA) in rat plasma after oral administration of YGY was developed in this study. Chromatographic separation was performed on an ACQUITY UPLC™ BEH C18 column (2.1 × 100 mm, 1.7 μm) using gradient elution with the mobile phase consisting of 2 mmol/L ammonium formate in 0.05% formic acid aqueous solution and 0.05% formic acid methanol solution, at a flow rate of 0.20 mL/min. MS detection was performed in the positive ion mode. The calibration curves were linear in the concentration range of 0.04160–41.60 ng/mL, 0.1070–107.0 ng/mL, 0.07358–73.50 ng/mL, 0.03228–32.28 ng/mL, 0.01809–18.09 ng/mL, and 0.1320–132.0 ng/mL for AC, HA, MA, BAC, BHA, and BMA, respectively. The intra- and inter-day precisions (relative standard deviation [RSD]) were less than 11.6% and 12.6%, respectively. The accuracies Relative Error (RE) ranged from −10.2% to 5.6%, while the recoveries ranged from 70.4% to 99.3%. The method for simultaneous quantitation of Aconitum alkaloids of You-Gui-Yin in rat plasma is accurate and repeatable, and this method was successfully applied to investigate the pharmacokinetics of the six Aconitum alkaloids in rat plasma after oral administration of YGY. For the pharmacokinetic study, the pharmacokinetics of the six Aconitum alkaloids were best described by a two-compartment open model.

Effects of different decocting methods on quality of Mahuang Xixin Fuzi decoction

To compare the quality difference between Mahuang Xixin Fuzi decoction(MXF) prepared by traditional decocting method and that prepared by two commonly used decocting methods, and explore the scientific nature of the traditional decocting method. By taking effect-toxic components in MXF as the research object, this article investigated these three different decocting methods from the quantitative determination of effect-toxic components in MXF. By using multivariate statistical analysis methods, three characteristic constituents were identified as kakoul, mesaconitine (MA) and hypaconitine (HA) respectively. As compared with two commonly used decocting methods, MXF decoction prepared by traditional decocting method had the shortest boiling time, but with the lowest dissolution rates of MA and AC and the higher dissolution rates of mono-ester aconitum alkaloids. In addition, the traditional decocting method increased the dissolution of ephedra alkaloid and accelerated the hydrolysis of diester diterpenoid alkaloids. There were differences in the content of effect-toxic components in MXF decoctions prepared by three different decocting methods, which can provide a reference for use of the classical prescriptions.

Cytotoxic effect of non-steroidal anti-inflammatory drugs towards colon fibroblast of the rat

Epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis and provides novel strategies for cancer therapy. Hypaconitine (HpA), a diester-diterpenoid alkaloid isolated from the root of the Aconitum species, exhibits anti-inflammatory, analgesic, and especially, cardiotoxic activities. Here, we reported the anti-metastatic potentials of HpA in transforming growth factor-β1 (TGF-β1)-induced EMT in lung cancer A549 cells. The cytotoxic effect of HpA was determined by MTT assay. A549 cells were treated with TGF-β1 with or without HpA co-treatment, and the morphological alterations were observed with a microscopy. The expression of E-cadherin, N-cadherin, and NF-κB was determined by both Western blotting and immunofluorescence analyses. The adhesion, migration, and invasion were detected with Matrigel, wound-healing, and transwell assays, respectively. The expression of Snail was determined by Western blotting. The expression of NF-κB p65, IκBα, and p-IκBα in nuclear and cytosolic extracts was assessed by Western blotting. The results showed that low concentration of HpA (<16 μmol·L−1) had no obvious cytotoxicity to A549 cells. Morphologically, TGF-β1 treatment induced spindle-shaped alteration in the cells. The upregulation of N-cadherin, NF-κB, and Snail and the downregulation of E-cadherin were detected after TGF-β1 treatment. The adhesion, migration and invasion abilities were also increased by TGF-β1. Besides, TGF-β1 induced expression of Snail in a time-dependent manner. Furthermore, TGF-β1 induced nuclear translocation of NF-κB p65. All these alterations were dramatically inhibited by HpA co-treatment. In addition, the NF-κB inhibitor PDTC showed similar inhibitory effect. In conclusion, these results showed that HpA inhibited TGF-β1-induced EMT in A549 cells, which was possibly mediated by the inactivation of the NF-κB signaling pathway, providing an evidence for anti-cancer effect of HpA.

Protective mechanisms of hypaconitine and glycyrrhetinic acid compatibility in oxygen and glucose deprivation injury.

This study investigated the protective effect of the compatibility of hypaconitine (HA) and glycyrrhetinic acid (GA) on H9c2 cells under oxygen and glucose deprivation (OGD)-induced injury, and the possible mechanisms. We found that HA+GA significantly improved pathology and morphology of the nucleus and ultrastructure of H9c2 cells under OGD as determined by Hoechst 33342 staining and transmission electron microscopy (TEM) tests. It also reduced the releases of lactate dehydrogenase (LDH), creatine kinase-myocardial band isoenzyme (CK-MB), and aspartate transaminase (AST) from the cultured supernatant of H9c2 cells, which were tested by enzyme-linked immune sorbent assay (ELISA) kits. In addition, it lessened the apoptotic rate as determined by a fluorescein isothiocyanate-annexin V/propidium iodide (FITC-AV/PI) double staining assay. It was also found that HA+GA might regulate the protein expression associated with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Overall, the study demonstrated that HA+GA protected H9c2 cells against OGD-induced injury, and the signaling mechanism might be related to the PI3K/Akt signaling pathway.

Hypaconitine inhibits TGF-β1-induced epithelial–mesenchymal transition and suppresses adhesion, migration, and invasion of lung cancer A549 cells

Epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis and provides novel strategies for cancer therapy. Hypaconitine (HpA), a diester-diterpenoid alkaloid isolated from the root of the Aconitum species, exhibits anti-inflammatory, analgesic, and especially, cardiotoxic activities. Here, we reported the anti-metastatic potentials of HpA in transforming growth factor-β1 (TGF-β1)-induced EMT in lung cancer A549 cells. The cytotoxic effect of HpA was determined by MTT assay. A549 cells were treated with TGF-β1 with or without HpA co-treatment, and the morphological alterations were observed with a microscopy. The expression of E-cadherin, N-cadherin, and NF-κB was determined by both Western blotting and immunofluorescence analyses. The adhesion, migration, and invasion were detected with Matrigel, wound-healing, and transwell assays, respectively. The expression of Snail was determined by Western blotting. The expression of NF-κB p65, IκBα, and p-IκBα in nuclear and cytosolic extracts was assessed by Western blotting. The results showed that low concentration of HpA (<16 μmol·L−1) had no obvious cytotoxicity to A549 cells. Morphologically, TGF-β1 treatment induced spindle-shaped alteration in the cells. The upregulation of N-cadherin, NF-κB, and Snail and the downregulation of E-cadherin were detected after TGF-β1 treatment. The adhesion, migration and invasion abilities were also increased by TGF-β1. Besides, TGF-β1 induced expression of Snail in a time-dependent manner. Furthermore, TGF-β1 induced nuclear translocation of NF-κB p65. All these alterations were dramatically inhibited by HpA co-treatment. In addition, the NF-κB inhibitor PDTC showed similar inhibitory effect. In conclusion, these results showed that HpA inhibited TGF-β1-induced EMT in A549 cells, which was possibly mediated by the inactivation of the NF-κB signaling pathway, providing an evidence for anti-cancer effect of HpA.

Simultaneous quantitation of six aconitum alkaloids and three flavonoids in the herb couple of radix aconiti lateralis-radix glycyrrhizae (Fuzi-Gancao) by UHPLC-ESI-MS/MS.

Background:Fuzi–Gancao herb couple is one of the commonly used herb couples involved in the traditional Chinese medicine formulations, with radix aconiti lateralis (Fuzi in Chinese) and radix glycyrrhizae (Gancao in Chinese) as a ratio of 1:1. Alkaloids and flavonoids were considered as the main active ingredients of Fuzi–Gancao herb couple. However, no analytical methods have been reported to quantitatively analyze these activity ingredients in Fuzi–Gancao herb couple simultaneously.Objective:To develop a simple, rapid, and sensitive UHPLC-MS/MS method for simultaneous quantitation of six alkaloid and three flavonoid compounds, namely, aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconitine (BAC), benzoylmesaconitine (BMA), benzoylhypaconitine (BHA), liquiritin, isoliquiritin, and licochalcone A (LCA) in Fuzi–Gancao herb couple.Materials and Methods:The chromatographic separation was achieved using a reversed phase C18 column and a mobile phase consisted of water (0.1% formic acid in water, v/v) and acetonitrile. The detection was achieved in multiple reaction monitoring modes. The optimal mass transition ion pairs (m/z) for quantitation were 646.3/586.5 for AC, 632.4/572.5 for MA, 616.3/556.4 for HA, 604.5/572.5 for BAC, 590.4/540.4 for BMA, 574.1/542.5 for BHA, 419.4/257.1 for liquiritin, 419.4/257.1 for isoliquiritin, and 339.2/121.1 for LCA.Results:Good linearity was observed in validated concentration range for each analyte (r > 0.9992), and the intra- and inter-day precisions were <3.12% and 3.65%, respectively. The recovery ranged from 85.36% to 110.14%.Conclusions:The results demonstrated that the method developed was reliable, rapid, and specific. Moreover, this method was successfully applied to control the quality of Fuzi–Gancao herb couple.SUMMARY A simple, rapid, and sensitive UHPLC-MS/MS method for simultaneous quantitation of six alkaloid and three flavonoid compounds in Fuzi–Gancao herb couple has been developed. Abbreviations used: UHPLC-ESI-MS/MS: Ultra High Performance Liquid Chromatography-electrospray ionization-mass spectrometry; RSD: Relative standard deviations; LOD: Limit of detection; LOQ: Limit of quantification; MRM: Multiple reaction monitor; TCMs: Traditional Chinese medicines.

The Effects of Rheum palmatum L. on the Pharmacokinetic of Major Diterpene Alkaloids of Aconitum carmichaelii Debx. in Rats.

Aconitum carmichaelii Debx. (Fuzi) is usually compatible with Rheum palmatum L. (Dahuang) in clinic. The study is conducted to investigate the influence of Dahuang on the pharmacokinetics of Fuzi. Twelve rats were randomly divided into two groups. Fuzi group was orally administered a single dose of 38.4mg/kg total alkaloids from Fuzi, and Fuzi-Dahuang group was given 38.4mg/kg total alkaloids from Fuzi and 76.8mg/kg Dahuang anthraquinones at the same time. The plasma concentrations of aconitine (AC), mesaconitine (MC), and hypaconitine (HC), benzoylaconine (BAC), benzoylmesaconine (BMC), benzoylhypaconine (BHC), and aconine (ACN) were determined by ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry method. The pharmacokinetic parameters were calculated including maximum plasma concentration (C max), area under the plasma concentration-time curve in all time-points (AUClast), apparent volume of distribution (V z/F), apparent plasma clearance (CL/F), elimination half-life (T 1/2), and time to achieve maximum concentration (T max). AUClast of diester diterpene alkaloids (DDAs) were 58.20, 169.78, 278.48ng·h/mL for AC, MC, and HC in Fuzi-Dahuang group which were remarkably lower than that in Fuzi group (71.62, 183.13, 410.59ng·h/mL for AC, MC, HC). CL/F was significantly increased from 173.88 to 218.85mL/h for AC, 433.22 to 800.21mL/h for MC, 1150.61 to 1307.30mL/h for HC after combination. However, with the significantly increased C max, AUClast of monoester diterpene alkaloids (MDAs) and amine diterpenoid alkaloids (ADAs) were 152.42, 1238.95, 287.96, 123.33ng·h/mL for BAC, BHC, BMC, ACN in Fuzi-Dahuang group which were remarkably higher than that in Fuzi group (54.47, 1105.48, 200.75, 86.48 ng·h/mL for BAC, BHC, BMC, ACN). At the same time, CL/F was significantly decreased from 1030.15 to 607.09, 3594.06 to 1437.54, 1441.23 to 1310.14, and 391.30 to 239.50mL/h for each one after combination. Fuzi diterpene alkaloids pharmacokinetics was greatly influenced by Dahuang which may account for the compatibility mechanism of effect-enhancing and toxicity-reducing.

Toxic Constituents Index: A Toxicity-Calibrated Quantitative Evaluation Approach for the Precise Toxicity Prediction of the Hypertoxic Phytomedicine-Aconite.

Complex chemical composition is an important reason for restricting herbal quality evaluation. Despite the multi-components determination method significantly promoted the progress of herbal quality evaluation, however, which mainly concerned the total amount of multiple components and ignored the activity variation between each one, and did not accurately reflect the biological activity of botanical medicines. In this manuscript, we proposed a toxicity calibrated contents determination method for hyper toxic aconite, called toxic constituents index (TCI). Initially, we determined the minimum lethal dose value of mesaconitine (MA), aconitine (AC), and hypaconitine (HA), and established the equation TCI = 100 × (0.3387 ×XMA + 0.4778 ×XAC + 0.1835 ×XHA). Then, 10 batches of aconite were selected and their evaluation results of toxic potency (TP), diester diterpenoid alkaloids (DDAs), and TCI were compared. Linear regression analysis result suggested that the relevance between TCI and TP was the highest and the correlation coefficient R was 0.954. Prediction error values study also indicated that the evaluation results of TCI was highly consistent with that of TP. Moreover, TCI and DDAs were both applied to evaluate 14 batches of aconite samples oriented different origins; from the different evaluation results, we found when the proportion of HA was reached 25% in DDAs, the pharmacopeia method could generate false positive results. All these results testified the accuracy and universality of TCI method. We believe that this study method is rather accurate, simple, and easy operation and it will be of great utility in studies of other foods and herbs.