Integrating metabolomics and network toxicology to reveal the mechanism of hypoaconitine-induced hepatotoxicity in mice.

Hypoaconitine (HA), a major secondary metabolite of aconite (a plant-derived rodenticide), is a highly toxic di-ester alkaloidal constituent. The toxicity of HA is intense with a low LD50. However, studies on its toxicity mechanism have mainly focused on cardiotoxicity, with few reports on the mechanism of hepatotoxicity. In this study, we combined metabolomics and network toxicology to investigate the effects of HA on the liver and analyzed the mechanisms by which it causes hepatotoxicity. The results of metabolomics studies indicated diethylphosphate, sphingosine-1-phosphate, glycerophosphorylcholine, 2,8-quinolinediol, guanidinosuccinic acid, and D-proline as differential metabolites after HA exposure. These metabolites are involved in eight metabolic pathways including arginine and proline metabolism, ether lipid metabolism, ß-alanine metabolism, sphingolipid metabolism, glutathione metabolism, and glycerophospholipid metabolism. Network toxicology analysis of HA may affect the HIF-1 signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and so on by regulating the targets of ALB, HSP90AA1, MMP9, CASP3, and so on. Integrating the results of metabolomics and network toxicology, it was concluded that HA may induce hepatotoxicity by triggering physiological processes such as oxidative stress, inflammatory response, and inducing apoptosis in hepatocytes.

Anti-rheumatic potential vis-à-vis aconitine and hypaconitine content analysis in different Aconitum spp. from Sikkim Himalayas (India).

Aconitum spp. are important medicinal plants mentioned in Ayurveda as Ativisa or Vatsanabha. The present study aims to evaluate anti-rheumatic potential in seven Aconitum species and correlation with aconitine and hypaconitine content. Anti-rheumatic potential was analyzed through in vitro xanthine oxidase inhibition, anti-inflammatory and ROS scavenging assays; and quantification of aconitine and hypaconitine with RP-HPLC method validated as per ICH guidelines. The findings reveal that A. palmatum possessed the most promising response (IC50 =12.68±0.15 µg/ml) followed by A. ferox (IC50 =12.912±1.87 µg/ml) for xanthin oxidase inhibition. We observed a wide variation in aconitine and hypaconitine content ranging from 0.018 %-1.37 % and 0.0051 %-0.077 % respectively on dry weight basis. Aconitine and hypaconitine showed moderate positive correlation (r=0.68 and 0.59 respectively) with anti-rheumatic potential. The study identifies potential alternative species of Aconitum that can help in sustainable availability of quality raw material.

Comprehensive insight into the pharmacology, pharmacokinetics, toxicity, detoxification and extraction of hypaconitine from Aconitum plants.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypaconitine (HA), a diterpenoid alkaloid, mainly derived from Aconitum plants such as Acoitum carmichaeli Debx. And Aconitum nagarum Stapf., has recently piqued significant interest among the scientific community given its multifaceted attributes including anti-inflammatory, anticancer, analgesic, and cardio-protective properties. AIM OF THE STUDY: This review presents a comprehensive exploration of the research advancements regarding the traditional uses, pharmacology, pharmacokinetics, toxicity, and toxicity reduction of HA. It aims to provide a thorough understanding of HA's multifaceted properties and its potential applications in various fields. MATERIALS AND METHODS: A systematic literature search was conducted using several prominent databases including PubMed, Web of Science, NCBI, and CNKI. The search was performed using specific keywords such as "hypaconitine," "heart failure," "anti-inflammatory," "aconite decoction," "pharmacological," "pharmacokinetics," "toxicity," "detoxification or toxicity reduction," and "extraction and isolation." The inclusion of these keywords ensured a comprehensive exploration of relevant studies and enabled the retrieval of valuable information pertaining to the various aspects of HA. RESULTS: Existing research has firmly established that HA possesses a range of pharmacological effects, encompassing anti-cardiac failure, anti-inflammatory, analgesic, and anti-tumor properties. The therapeutic potential of HA is promising, with potential applications in heart failure, ulcerative colitis, cancer, and other diseases. Pharmacokinetic studies suggest that HA exhibits high absorption rates, broad distribution, and rapid metabolism. However, toxic effects of HA on the nerves, heart, and embryos have also been observed. To mitigate these risks, HA needs attenuation before use, with the most common detoxification methods being processing and combined use with other drugs. Extraction methods for HA most commonly include cold maceration, soxhlet reflux extraction, and ultrasonic-assisted extraction. Despite the potential therapeutic benefits of HA, further research is warranted to elucidate its anti-heart failure effects, particularly in vivo, exploring aspects such as in vivo metabolism, distribution, and metabolites. Additionally, the therapeutic effects of HA monomers on inflammation-induced diseases and tumors should be validated in a more diverse range of experimental models, while the mechanisms underlying the therapeutic effects of HA should be investigated in greater detail. CONCLUSION: This review serves to emphasize the therapeutic potential of HA and highlights the crucial need to address its toxicity concerns before considering clinical application. Further research is required to comprehensively investigate the pharmacological properties of HA, with particular emphasis on its anti-cardiac failure and anti-inflammatory activities. Such research endeavors have the potential to unveil novel treatment avenues for a broad spectrum of diseases.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: After 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. CONCLUSIONS: In 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.

Mechanism of action of a diterpene alkaloid hypaconitine on cytotoxicity and inhibitory effect of BAPTA-AM in HCN-2 neuronal cells.

Hypaconitine, a neuromuscular blocker, is a diterpene alkaloid found in the root of Aconitum carmichaelii. Although hypaconitine was shown to affect various physiological responses in neurological models, the effect of hypaconitine on cell viability and the mechanism of its action of Ca2+ handling is elusive in cortical neurons. This study examined whether hypaconitine altered viability and Ca2+ signalling in HCN-2 neuronal cell lines. Cell viability was measured by the cell proliferation reagent (WST-1). Cytosolic Ca2+ concentrations [Ca2+ ]i was measured by the Ca2+ -sensitive fluorescent dye fura-2. In HCN-2 cells, hypaconitine (10-50 µmol/L) induced cytotoxicity and [Ca2+ ]i rises in a concentration-dependent manner. Removal of extracellular Ca2+ partially reduced the hypaconitine's effect on [Ca2+ ]i rises. Furthermore, chelation of cytosolic Ca2+ with BAPTA-AM reduced hypaconitine's cytotoxicity. In Ca2+ -containing medium, hypaconitine-induced Ca2+ entry was inhibited by modulators (2-APB and SKF96365) of store-operated Ca2+ channels and a protein kinase C (PKC) inhibitor (GF109203X). Hypaconitine induced Mn2+ influx indirectly suggesting that hypaconitine evoked Ca2+ entry. In Ca2+ -free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished hypaconitine-induced [Ca2+ ]i rises. Conversely, treatment with hypaconitine inhibited thapsigargin-induced [Ca2+ ]i rises. However, inhibition of phospholipase C (PLC) with U73122 did not inhibit hypaconitine-induced [Ca2+ ]i rises. Together, hypaconitine caused cytotoxicity that was linked to preceding [Ca2+ ]i rises by Ca2+ influx via store-operated Ca2+ entry involved PKC regulation and evoking PLC-independent Ca2+ release from the endoplasmic reticulum. Because BAPTA-AM loading only partially reversed hypaconitine-induced cell death, it suggests that hypaconitine induced a second Ca2+ -independent cytotoxicity in HCN-2 cells.

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.

Circadian Cyp3a11 metabolism contributes to chronotoxicity of hypaconitine in mice.

Hypaconitine is an active and highly toxic constituent derived from Aconitum species. Here we aimed to determine the chronotoxicity of hypaconitine in mice, and to investigate a potential role of metabolism in hypaconitine chronotoxicity. Cardiac toxicity was assessed by measuring CK (creatine kinase) and LDH (lactate dehydrogenase) levels after hypaconitine administration to wild-type and Bmal1-/- (a clock disrupted model) mice at different times of day. The mRNA and protein levels of Cyp3a11 in mouse livers were determined by qPCR and western blotting, respectively. In vitro metabolism was assessed using liver microsomes. Pharmacokinetic study of hypaconitine was performed with wild-type mice. We observed injection time-dependent toxicity (i.e., a more severe toxicity during the light phase than the dark phase) for hypaconitine in mice. The chronotoxicity was attributed to a difference in systemic exposure of hypaconitine caused by time of day-dependent metabolism. Furthermore, circadian metabolism of hypaconitine was accounted for by the diurnal expression of Cyp3a11, a major enzyme for hypaconitine detoxification in the liver. Moreover, Bmal1 ablation in mice abolished the daily rhythm of Cyp3a11 expression and abrogated the time-dependency of hypaconitine toxicity. In conclusion, circadian Cyp3a11 metabolism contributed to chronotoxicity of hypaconitine in mice. This metabolism-based chronotoxicity would facilitate the formulation of best timing for drug administration.

Dynamic changes of alkaloids in growth cycle in stems and leaves of Aconitum carmichaelii by HPLC-MS/MS / 中草药

Objective: To investigate the content changes of alkaloids in stems and leaves of Aconiti Radix at different growth period. Methods: The Phenomenex C18 column (150 mm × 4.6 mm, 5 μm) was used and 0.1% formic acid aqueous solution-acetonitrile was selected as mobile phase; The mass spectrum was scanned by ESI+ multiple reaction monitoring (MRM) mode. The HPLC-MS/MS method was established for the simultaneous determination of aconitine, mesaconitine, hypaconitine, indaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconitine, aconine, fuziline, neoline, talatisamine, songorine, higenamine, and salsoline in the stems and leaves of Aconite Radix. Combined with principal component analysis (PCA), the transfer rule of various alkaoids was tracked in the growth cycle of Aconiti Radix. Results: Methodological validation results showed that the linear range of the 14 compounds was good (r2 > 0.990 0). The limit of quantification was 2.27-18.27 ng/mL, and the average recovery was 94.73%-104.50%. The results showed that there was considerable amounts of alkaloids in stems and leaves of Aconiti Radix, and the total contents of alkaloids in stems were higher than those in leaves. In May, June, July, and August, the total content of alkaloids in stems was 0.087 1%, 0.182 8%, 0.141 0%, and 0.199 4% respectively, which showed a wave-like upward trend. The total content of alkaloids in leaves was 0.074 7%, 0.075 9%, 0.081 4%, and 0.058 9% respectively, which showed a trend of rising first and then decreasing, and the total content of alkaloids in leaves was highest in July. Conclusion: PCA found that the alkaloids content in stems and leaves showed different variation trend in the different period. The considerable alkaloids in stems reached the peak value at the harvest time. The amount of alkaloids is considerable, which has the potential and development value of becoming new medicinal resources.

Effect of Tannic Acid on Transport of Diaco-Aconitine in Caco-2 Cell Model / 中国药学杂志

OBJECTIVE: To study the effects of three aconitine transporters in Caco-2 cells and tannin (tannic acid) in Terminalia on the transport of three di-ester aconitine (aconitine, meoaconitine and hypaconitine) in Aconitum chinensis. METHODS: The components were detected by UPLC/Q exactive MS in terms of the cumulative transshipment volume of three aconitine and the apparent permeability coefficient Papp as indicators to investigate the two-way transport behaviors of three aconitine in the Caco-2 cell model, as well as the proportion of tannic acid, and the changes of the transport behaviors of three aconitine. RESULTS: There was a positive correlation between the cumulative transshipment volume of aconitine and the incubation time. There was no statistical difference in Papp values of the three aconitine, and the efflux effect was significantly stronger than the absorption, with an external ratio close to 1.5.When the compatibility ratio of three aconitine with tannic acid was 1∶1 and 1∶0.5, the absorption of aconitine was significantly inhibited (P was 0.05), but the transport behavior with effluent was not significantly affected. CONCLUSION: Aconitine, meoaconitine, and hypaconitine in P. aeruginosa are mainly passive transporters, and may involve in efferent proteins, which are a kind of drug with good absorption.When mixed with tannic acid, the absorption and transshipment of three kinds of alkali were significantly reduced, which proves that Terminalia chebula could detoxify aconitum by inhibiting its absorption.

[Content determination of four diester diterpenoid alkaloids in leaves of Aconitum kusnezoffii by HPLC].

This present study is to develop an HPLC method for simultaneous determination of four diester diterpenoid alkaloids, beiwutine, mesaconitine, hypaconitine and aconitine in the leaves of Aconitum kusnezoffii, so as to provide evidence of the quality control of this herb. The four constituents were measured on a Waters XBridge CC18 column(4.6 mmχ250 mm, 5 µm). The mobile phase was acetonitrile-40 mmol·L⁻¹ ammonium acetate solution(adjusted pH to 10.5 with ammonia solution)(33:67) with isocratic elution at a flow rate of 1.0 mL·min⁻¹; the detection wavelength was 235 nm; the column temperature was 30 °C, and the injection volume was 10 µL. Next, this contents of the four diester diterpenoid alkaloids in 12 samples were 0.025 5-0.088 5, 0.039 1-0.071 5, 0.026 6-0.081 0 and 0.008 12-0.031 2 mg·g⁻¹, respectively. Next, this method has been successfully applied to the analysis of A. kusnezoffii folium in different harvest periods. The contents of the four alkaloids decreased primarily, and then increased with the postponing of harvest. The established method is proved to be accurate and sensitive for the determination of alkaloids in A. kusnezoffii folium, and may be useful for the quality improvement of this herbal medicine. Moreover, these results indicated the scientific significance for the toxicity and the suitable harvest time of this herb.

Content determination of four diester diterpenoid alkaloids in leaves of Aconitum kusnezoffii by HPLC / 中国中药杂志

This present study is to develop an HPLC method for simultaneous determination of four diester diterpenoid alkaloids, beiwutine, mesaconitine, hypaconitine and aconitine in the leaves of Aconitum kusnezoffii, so as to provide evidence of the quality control of this herb. The four constituents were measured on a Waters XBridge CC₁₈ column(4.6 mmχ250 mm, 5 μm). The mobile phase was acetonitrile-40 mmol·L⁻¹ ammonium acetate solution(adjusted pH to 10.5 with ammonia solution)(33:67) with isocratic elution at a flow rate of 1.0 mL·min⁻¹; the detection wavelength was 235 nm; the column temperature was 30 °C, and the injection volume was 10 μL. Next, this contents of the four diester diterpenoid alkaloids in 12 samples were 0.025 5-0.088 5, 0.039 1-0.071 5, 0.026 6-0.081 0 and 0.008 12-0.031 2 mg·g⁻¹, respectively. Next, this method has been successfully applied to the analysis of A. kusnezoffii folium in different harvest periods. The contents of the four alkaloids decreased primarily, and then increased with the postponing of harvest. The established method is proved to be accurate and sensitive for the determination of alkaloids in A. kusnezoffii folium, and may be useful for the quality improvement of this herbal medicine. Moreover, these results indicated the scientific significance for the toxicity and the suitable harvest time of this herb.

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-ß1-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-ß1 (TGF-ß1)-induced EMT in lung cancer A549 cells. The cytotoxic effect of HpA was determined by MTT assay. A549 cells were treated with TGF-ß1 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-ß1 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-ß1 treatment. The adhesion, migration and invasion abilities were also increased by TGF-ß1. Besides, TGF-ß1 induced expression of Snail in a time-dependent manner. Furthermore, TGF-ß1 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-ß1-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.

Histone deacetylase-high mobility group box-1 pathway targeted by hypaconitine suppresses the apoptosis of endothelial cells.

Hypaconitine is an active component of Aconitum carmichaelii Debx, a Chinese medicinal herb for the treatment of cardiovascular diseases, but the mechanism underlying its effect remains elusive. In this study, we found that hypaconitine, rather than aconitum alkaloids in A. carmichaelii (e.g. aconitine, mesaconitine and benzoylaconitine), prevented endothelial cells from damage due to oxidized low-density lipoprotein (oxLDL) challenge. Cleaved caspase 3 expression in endothelial cells was up-regulated by oxLDL and markedly attenuated by hypaconitine, suggesting that hypaconitine inhibited the oxLDL-induced cell apoptosis. Microarray analysis revealed that histone deacetylase 3 (HDAC3) was significantly increased by hypaconitine. The cytoplasmic relocation and extracellular release of high-mobility group box 1 (HMGB1, an HDAC3 downstream effector) in endothelial cells were significantly increased by oxLDL and markedly decreased by hypaconitine. The effect of hypaconitine on the oxLDL-induced apoptosis and HMGB1 release in endothelial cells was significantly reduced by the suppression of HDAC3 by siRNA or a specific inhibitor. Thus, this study proves that the histone deacetylase-HMGB1 pathway targeted by hypaconitine suppresses the apoptosis of endothelial cells. Our findings are of therapeutic significance and provide the potential of hypaconitine exploitation. Impact statement First, our study shows the antiapoptosis effect of Aconitum carmichaelii and its active component hypaconitine on endothelial cells. It may provide new strategies for the treatment of diseases involving endothelium damage. Second, this finding indicates the function of hypaconitine in regulating HDAC3-HMGB1 pathway, which suggests a new anti-inflammatory therapy. Third, due to its poisonousness, A. carmichaelii is always used with caution in clinics. Thus, the identification of hypaconitine as an active component of A. carmichaelii could contribute to the development of toxicity-decreasing procedure for A. carmichaelii.

Simultaneous Determination of Fangchinoline,Tetrandrine,Mesaconitine,Aconitine and Hypaconitine in Huoxue Zhentong Plaster by HPLC with Gradient Elution / 中国药师

Objective:To develop an HPLC gradient elution method for the simultaneous determination of fangchinoline,tetrandrine,mesaconitine,aconitine and hypaconitine in Huoxue Zhentong plaster.Methods::A Dikma-C18 (200 mm×4.6 mm,5 μm) chromatographic column was adopted,the mobile phase was methanol-acetonitrile (3∶1)(A)-0.06% diaethylamin solution (B) with gradient elution at a flow rate of 1.0 ml·min-1,the detection wavelength was 280 nm for fangchinoline and tetrandrine,and 235 nm for mesaconitine,aconitine and hypaconitine.The column temperature was set at 30 ℃,and the injection volume was 10 μl.Results::There was a good linear relationship when the content of fangchinoline,tetrandrine,mesaconitine,aconitine and hypaconitine was within the range of 7.490-149.800 μg·ml-1(r=0.999 9),14.610-292.200 μg·ml-1(r=0.999 8),4.150-83.000 μg·ml-1(r=0.999 2),5.250-105.000 μg·ml-1(r=0.999 6) and 5.140-102.800 μg·ml-1(r=0.999 9),respectively.The average recovery and the corresponding RSD were 99.87%(0.49%),97.79%(1.11%),96.97%(1.75%),98.60%(1.50%) and 97.94%(0.98%)(n=6),respectively.Conclusion:An HPLC gradient elution method is successfully established for the simultaneous determination of 5 components in Huoxue Zhentong plaster.The established method is simple,accurate and reliable,which is helpful to the quality control of Huoxue Zhentong plaster.

Influence of compatibility of glycyrrhetinic acid, liquiritin and hypaconitine on apoptosis pathway in rats with chronic heart failure / 中成药

AIM To explore the anti-heart failure effects of compatibility of glycyrrhetinic acid,liquiritin and hypaconitine on apoptotic pathway in rats with chronic heart failure and the possible mechanism of action.METHODS Sixty rats were operated through transverse aortic constriction to create the chronic heart failure models.After four weeks,these rats were randomly divided into seven groups:sham group (n =6),model group (n =7),digoxin group (n =8),hypaconitine group (n =9),glycyrrhetinic acid + hypaconitine group (n =9),liquiritin + hypaconitine group (n =9),glycyrrhetinic acid + liquiritin + hypaconitine group (n =9).Then the rats were given medicines or saline perfusion by oral gavage for one week.On the 7th day,the rats were executed.Before the rats were killed,the blood samples were obtained and echocardiographic were carried on to get the ejection fraction (EF) and fractional shortening (FS) data;and the ELISA test was done for type B natriuretic peptide (BNP) changes;and myocardial histopathological examinations were performed on Bcl-2,Bax and Caspase-3.And the protein expressions of Fas and Fas-L were detected by Western blot.RESULTS Compared with the model group,the expression of Bcl-2 of all the compatibility groups except the hypaconitine group was higher than that of the model group;the expressions of Bax,Caspase-3,Fas and Fas-L,besides with the measurements of EF,FS and BNP were lower than those of the model group,and the glycyrrhetinic acid + liquiritin + hypaconitine group showed the best effect.CONCLUSION The compatibility of hypaconitine,glycyrrhetinic acid and liquiritin could down-regulate the levels of EF,FS and BNP,together with the protein expressions of pro-apoptosis,meanwhile,up-regulate the protein expressions of anti-apoptosis.This might be one of the mechanisms for the anti-apoptosis effects on chronic heart failure by compatibility of Glycyrrhizea radix et rhizoma (Gancao) and Acontti lateralis Radix praparata (Fuzi).

Influence of compatibility of glycyrrhetinic acid, liquiritin and hypaconitine on apoptosis pathway in rats with chronic heart failure / 中成药

AIM To explore the anti-heart failure effects of compatibility of glycyrrhetinic acid,liquiritin and hypaconitine on apoptotic pathway in rats with chronic heart failure and the possible mechanism of action.METHODS Sixty rats were operated through transverse aortic constriction to create the chronic heart failure models.After four weeks,these rats were randomly divided into seven groups:sham group (n =6),model group (n =7),digoxin group (n =8),hypaconitine group (n =9),glycyrrhetinic acid + hypaconitine group (n =9),liquiritin + hypaconitine group (n =9),glycyrrhetinic acid + liquiritin + hypaconitine group (n =9).Then the rats were given medicines or saline perfusion by oral gavage for one week.On the 7th day,the rats were executed.Before the rats were killed,the blood samples were obtained and echocardiographic were carried on to get the ejection fraction (EF) and fractional shortening (FS) data;and the ELISA test was done for type B natriuretic peptide (BNP) changes;and myocardial histopathological examinations were performed on Bcl-2,Bax and Caspase-3.And the protein expressions of Fas and Fas-L were detected by Western blot.RESULTS Compared with the model group,the expression of Bcl-2 of all the compatibility groups except the hypaconitine group was higher than that of the model group;the expressions of Bax,Caspase-3,Fas and Fas-L,besides with the measurements of EF,FS and BNP were lower than those of the model group,and the glycyrrhetinic acid + liquiritin + hypaconitine group showed the best effect.CONCLUSION The compatibility of hypaconitine,glycyrrhetinic acid and liquiritin could down-regulate the levels of EF,FS and BNP,together with the protein expressions of pro-apoptosis,meanwhile,up-regulate the protein expressions of anti-apoptosis.This might be one of the mechanisms for the anti-apoptosis effects on chronic heart failure by compatibility of Glycyrrhizea radix et rhizoma (Gancao) and Acontti lateralis Radix praparata (Fuzi).

Hypaconitine inhibits TGF-β1-induced epithelial-mesenchymal transition and suppresses adhesion, migration, and invasion of lung cancer A549 cells / 中国天然药物

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) 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.

Botanical drugs in Ayurveda and Traditional Chinese Medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: China and India have a long history in the therapeutic application of botanical drugs in traditional medicine. Traditional Chinese Medicine (TCM) and Ayurveda are considered as two of the most ancient systems of medicine, with history of more than two millennia. Medicinal plants are the principal medicinal materials used in both these systems. AIM OF THE REVIEW: This review discusses about the histories of Ayurveda and TCM, the common medicinal plants species, the drug processing strategies used, and the current statuses of these traditional systems of medicine (TSM). Through the views presented in this article, we aim to provide a new perspective to herbal drug researchers for expanding and improving the utilization of botanical drugs and their therapeutic applications. METHODS: A bibliographic investigation of Chinese and Indian pharmacopoeias, monographs and official websites was performed. Furthermore, information was obtained from scientific databases on ethnobotany and ethno medicines. RESULTS: The review of Ayurveda and TCM ethno medicine indicates that both these systems have many medicinal materials in common. The studies carried out by the authors for comparison of plants from same genus from both these TSM's have been discussed to further bring focus to the utilization of "qualitatively" similar species which can be utilized and substituted for endangered or economically valued species. The overview of ancient literature and scientific findings for drugs in both these systems suggests that, the botanical drugs used in common and their processing methods can be explored further for extensive utilization in traditional medicine. CONCLUSION: This review describes the histories, common medicinal plant species, their processing methods and therapeutic applications in Ayurveda and TCM. The insights provided through this article may be used by herbal drug researchers and pharmacologists for further exploration of botanical drugs from these two traditional systems of medicine.

<b>Three Cases with Intractable Pain Treated with a Dose of Daiuzusen, and an Analysis of Aconite Concentrations in Prescriptions and Patientsʼ </b><b>Serum</b> / 日本東洋医学雑誌

<p>In 2013, we prescribed daiuzusen for 3 patients with intractable pain; pain from complex regional pain syndrome, colic pain of unknown origin after an abdominal operation, and colic pain from advanced colon cancer and ileus. A dose of daiuzusen (containing uzu 0.5-2 g) quickly relieved their pain in several minutes. Another common symptom was “cold” in their bowel or extremities when they were feeling pain. Aconite levels in drugs and patients' serum after taking daiuzusen were analyzed by liquid chromatography tandem mass spectrometry. Daiuzusen per 1 g of uzu contained aconitine 1.28 μg, mesaconitine 2.31 μg, and hypaconitine 92.89 μg, while jesaconitine was not detected; this was about 5 to 35 times the level of tsumyakushigyakuto per 1 g of uzu. Serum concentrations of hypaconitine peaked in the study at 1.11 ng/mL after about an hour of taking daiuzusen (1 g of uzu). We posit that the immediate effect after taking daiuzusen was due to transmucosal absorption of uzu components. However serum hypaconitine, which we are now able to monitor, is at least one practical way of indicating the use of uzu or bushi containing prescriptions.</p>