The Effect of Yangyin Xifeng Tongluo Formula （养阴熄风通络方） and Its Core Herb-Pairs on Prevention of Aconitine-Induced Ventricular Precontraction in Rats / 中医杂志

ObjectiveTo observe the effect of Yangyin Xifeng Tongluo Formula （养阴熄风通络方） and its core herbs combination on prevention of ventricular precontraction （PVC）. MethodsExperiment 1： Forty SD rats were randomly divided into model group， formula group， core herb-pairs medium-dose group and amiodarone group， with 10 rats in each group. Rats in the model group were given 10 ml/（kg·d） of pure water by gavage， rats in the formula group were given 1.125 g/（kg·d） of Yangyin Xifeng Tongluo Granules （养阴熄风通络方颗粒） by gavage， rats in the core herb-pairs medium-dose group were given 0.585 g/（kg·d） of granules of core herb-pairs by gavage， and rats in the amiodarone group were given 18 mg/（kg·d） of amiodarone hydrochloride tablets by gavage. The rats in each group were gavaged once a day for 14 consecutive days， and then a PVC model was established using rat tail vein injection of aconitine 25 μg/kg to compare the mortality and incidence of PVC， ventricular tachycardia （VT） and ventricular fibrillation （VF）， the time of the appearance of PVC， and the duration of PVC in the rats in each group. Experiment 2： Sixty SD rats were randomly divided into model group， the amiodarone group， and the core herb-pairs of low， medium and high dose groups， 12 rats in each group. Rats in the model group were gavaged with 10 ml/（kg·d） of purified water， rats in the low， medium， and high dose groups were gavaged with 0.2925， 0.585， and 1.17 g/（kg·d） of the core herb-pairs granules respectively， and rats in the amiodarone group were gavaged with 18 mg/（kg·d） of amiodarone hydrochloride tablets. All of them were gavaged once a day. After 14 days， rats in each group were injected intravenously into the tail vein with aconitine 25 μg/kg， and then the rats in each group were observed to show the mortality and incidence of PVC， VT and VF， and the time of appearance and duration of PVC. ResultsExperiment 1： Compared with the model group， The difference in the incidence of PVC in rats of all groups was not statistically signi-ficant （P＞0.05）， and the mortality and incidence of VT and VF in rats in the formula group， the core herb-pairs medium-dose group， and the amiodarone group significantly reduced， with delayed time of the occurrence of PVC and shorter duration of the PVC （P＜0.05 or P＜0.01） as compared with the model group， whereas the difference between each group with medication intervention was not statistically significant （P＞0.05）. Experiment 2： There was no statistically significant difference in the incidence of PVC in all groups （P＞0.05）， and the mortality and incidence of VT and VF reduced in the core herb-pairs of low-， medium-， and high-dose groups and in the amiodarone group， and the appearance of PVC delayed and its duration shortened （P＜0.05 or P＜0.01） as compared with the control group， whereas the differences were not statistically significant in the comparison among groups with medication （P＞0.05）. ConclusionBoth Yangyin Xifeng Tongluo Formula and its core herb-pairs could delay the time of occurrence and shorten the duration of PVC.

Chemical reaction mechanism of decoction of traditional Chinese medicines: a review / 中国中药杂志

Complicated chemical reactions occur in the decoction of traditional Chinese medicines(TCMs) which features complex components, influencing the safety, efficacy, and quality controllability of TCMs. Therefore, it is particularly important to clarify the chemical reaction mechanism of TCMs in the decoction. This study summarized eight typical chemical reactions in the decoction of TCMs, such as substitution reaction, redox reaction, isomerization/stereoselective reaction, complexation, and supramolecular reaction. With the "toxicity attenuation and efficiency enhancement" of aconitines and other examples, this study reviewed the reactions in decoction of TCMs, which was expected to clarify the variation mechanisms of key chemical components in this process and to help guide medicine preparation and safe and rational use of medicine in clinical settings. The current main research methods for chemical reaction mechanisms of decoction of TCMs were also summed up and compared. The novel real-time analysis device of decoction system for TCMs was found to be efficient and simple without the pre-treatment of samples. This device provides a promising solution, which has great potential in quantity evaluation and control of TCMs. Moreover, it is expected to become a foundational and exemplary research tool, which can advance the research in this field.

Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry / 药物分析学报

Aconitine,a common and main toxic component of Aconitum,is toxic to the central nervous system.However,the mechanism of aconitine neurotoxicity is not yet clear.In this work,we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine.HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system.Meanwhile,to confirm the metabolic mechanism of aconitine toxicity on HT22 cells,the levels of lactate dehydrogenase,intracellular Ca2+,reactive oxygen species,glutathione and superoxide dismutase,and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology.Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid,which was followed by the accumulation of lactic acid and reduction of glucose.The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca2+overload and oxidative stress,and eventually result in cell apoptosis.In general,we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.

Ginsenoside Rg1 Reduces Cardiotoxicity While Increases Cardiotonic Effect of Aconitine in vitro / 中国结合医学杂志

OBJECTIVE@#To explore the synergic mechanism of ginsenoside Rg1 (Rg1) and aconitine (AC) by acting on normal neonatal rat cardiomyocytes (NRCMs) and pentobarbital sodium (PS)-induced damaged NRCMs.@*METHODS@#The toxic, non-toxic, and effective doses of AC and the most suitable compatibility concentration of Rg1 for both normal and damaged NRCMs exposed for 1 h were filtered out by 3- (4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide, respectively. Then, normal NRCMs or impaired NRCMs were treated with chosen concentrations of AC alone or in combination with Rg1 for 1 h, and the cellular activity, cellular ultrastructure, apoptosis, leakage of acid phosphatase (ACP) and lactate dehydrogenase (LDH), intracellular sodium ions [Na+], potassium ions [K+] and calcium ions [Ca2+] levels, and Nav1.5, Kv4.2, and RyR2 genes expressions in each group were examined.@*RESULTS@#For normal NRCMs, 3000 µ mol/L AC significantly inhibited cell viability (P<0.01), promoted cell apoptosis, and damaged cell structures (P<0.05), while other doses of AC lower than 3000 µ mol/L and the combinations of AC and Rg1 had little toxicity on NRCMs. Compared with AC acting on NRCMs alone, the co-treatment of 3000 and 10 µ mol/L AC with 1 µ mol/L Rg1 significantly decreased the level of intracellular Ca2+ (P<0.01 or P<0.05), and the co-treatment of 3000 µ mol/L AC with 1 µ mol/L Rg1 significantly decreased the level of intracellular Ca2+ via regulating Nav1.5, RyR2 expression (P<0.01). For damaged NRCMs, 1500 µ mol/L AC aggravated cell damage (P<0.01), and 0.1 and 0.001 µ mol/L AC showed moderate protective effect. Compared with AC used alone, the co-treatment of Rg1 with AC reduced the cell damage, 0.1 µ mol/L AC with 1 µ mol/L Rg1 significantly inhibited the level of intracellular Na+ (P<0.05), 1500 µ mol/L AC with 1 µ mol/L Rg1 significantly inhibited the level of intracellular K+ (P<0.01) via regulating Nav1.5, Kv4.2, RyR2 expressions in impaired NRCMs.@*CONCLUSION@#Rg1 inhibited the cardiotoxicity and enhanced the cardiotonic effect of AC via regulating the ion channels pathway of [Na+], [K+], and [Ca2+].

Preliminary study on mechanism of electroacupuncture with the involvement of SERCA2a/PLB on the synergistic and attenuated effect of aconitine in treatment of heart failure / 中国针灸

OBJECTIVE@#To investigate the mechanism of electroacupuncture (EA) with the involvement of sarcoplasmic reticulum Ca@*METHODS@#Thirty SPF-ranked SD rats were randomly divided into a control group, a model group, an EA group, an aconitine group and an EA plus aconitine group, with 6 rats in each group. The rat model of acute heart failure was established by infusion of high-dose propranolol hydrochloride solution into the right femoral vein. After stabilized for 10 min in the modeled rats, EA was exerted at "Neiguan" (PC 6), with disperse-dense wave, 2 Hz/15 Hz in frequency, 3 mA in intensity, for 30 min in the EA group and the EA plus aconitine group; aconitine solution (10 μg/kg) was injected from the left femoral veins in the rats in the aconitine group and the EA plus aconitine group. Hemodynamic indexes such as the left ventricular systolic pressure (LVSP) and the maximum rate of increase/decrease of left ventricular pressure (±dp/dt@*RESULTS@#Compared with the control group, LVSP and ±dp/dt@*CONCLUSION@#The intervention with electroacupuncture achieves the synergism/ attenuation effect of aconitine for the improvements in heart failure probably by up-regulating the expression of SERCA2a and down-regulating the expression of PLB in myocardial tissue.

The Significances of Water Volume to Decoct Formulas Described in Classical Chinese Medicine / 日本東洋医学雑誌

Although the descriptions of shigyakukachotanto in “Waitaimiyaofang” and tsumyakushigyakukachotanjuto in “Songban Shanghanlun” are quite similar to each other, the specifications of the dosages of crude drugs and the water volume in the books were considerably different. Focused on the specified water volume to decoct these formulas, each reasonable decocting period was estimated, then the decoctions were prepared using hard water that was common in mainland China. The dosages of aconite root were 2­-fold different between these two formulas, but the contents of aconitine-­type diester alkaloids (ADA) in both decoctions were found in the range of 1.2—1.4­-fold. It was suggested that in order to control the efficacy and the safety of aconite, the decocting period was well regulated by the specification of water volume for decocting at this ancient era. Moreover, the dosages of aconite root and glycyrrhiza in bukuryoshigyakuto (BSGT) formula of “Songban Shanghanlun” are equal to those of shigyakuto (SGT) but the specified water volume to begin decocting is as about twice as that of SGT. When prepared using hard water, BSGT resulted to make the contents of ADA lower and those of non-­ester alkaloids higher compared with those of SGT decoction. It was suggested the spe­cific water volume for each formula prescribed in classical Chinese medicine had considerable significance to determine the dosages of chemical ingredients in the decoctions especially in the circumstances using hard water to prepare them.

Repeated administration of low-dose aconitine remodels mitochondrial energy metabolism pattern of human iPSCs-CM / 中国药理学通报

Aim To study the effect of low dose aconitine on the metabolism of hiPSCs-CM. Methods After 100 nmol · L

Construction of a Stable Expression Cell Line of Human Phospholamban / 法医学杂志

OBJECTIVES@#To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism.@*METHODS@#FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-InTM T-RExTM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation.@*RESULTS@#After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine.@*CONCLUSIONS@#This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.

Limitation standard of toxic aconitines in proprietary Chinese medicines using on-line extraction electrospray ionization mass spectrometry

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 proprietary Chinese medicines (APCMs). Multi-type dosage forms (, tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 μL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography-mass spectrometry (LC-MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.

Real-time toxicity prediction of stewing system using extractive electrospray ionization mass spectrometry

Due to numerous obstacles such as complex matrices, real-time monitoring of complex reaction systems (, medicinal herb stewing system) has always been a challenge though great values for safe and rational use of drugs. Herein, facilitated by the potential ability on the tolerance of complex matrices of extractive electrospray ionization mass spectrometry, a device was established to realize continuous sampling and real-time quantitative analysis of herb stewing system for the first time. A complete analytical strategy, including data acquisition, data mining, and data evaluation was proposed and implemented with overcoming the usual difficulties in real-time mass spectrometry quantification. The complex Fuzi (the lateral root of )-meat stewing systems were real-timely monitored in 150 min by qualitative and quantitative analysis of the nine key alkaloids accurately. The results showed that the strategy worked perfectly and the toxicity of the systems were evaluated and predicated accordingly. Stewing with trotters effectively accelerated the detoxification of Fuzi soup and reduced the overall toxicity to 68%, which was recommended to be used practically for treating rheumatic arthritis and enhancing immunity. The established strategy was versatile, simple, and accurate, which would have a wide application prospect in real-time analysis and evaluation of various complex reaction systems.

Changes of processing process of Aconiti Radix Cocta based on bionic technology / 中草药

Objective: To study the characteristics of gas and taste changes in different stages during the processing of Aconiti Radix Cocta (ARC), and characterize the relationship between the changes of gas and taste and the internal active components. Methods: The changes of gas and taste of three batches of ARC samples with different processing time were detected by using electronic nose and electronic tongue system, the obtained data from processing were analyzed through the radar map, linear discriminant analysis (LDA), principal component analysis (PCA), and the correlation analysis was made with alkaloids from ARC processing. Results: Electronic nose and electronic tongue could distinguish the samples of different processing degree of ARC. Pearson correlation analysis showed that electronic nose FAC1 was significantly correlated with aconitine, neoaconitine, benzoylaconitine, benzoylneoaconitine and monoester alkaloids (P < 0.01). Electronic tongue FAC2 was significantly correlated with the total amount of benzoylaconitine and monoester alkaloids (P < 0.01), and correlated with the content of benzoylaconitine (P < 0.05). Conclusion: The bionic technology of electronic nose and electronic tongue could be used to study the processing quality of ARC. This method provides a new way of thinking for the Chinese medicine processing study.

Study on influence factors of Aconiti Lateralis Radix Praeparata by microwave processing technology / 中草药

Objective: To explore the influence factors of processing Aconiti Lateralis Radix Praeparata by microwave-drying, so as to lay a foundation for its industrial development. Methods: The drying process of Aconiti Lateralis Radix Praeparata was investigated by water ratio, drying rate and energy consumption analysis. The aconite morphology, contents of diester alkaloids, monoester alkaloids and alkanolamine alkaloids were used as indexes to study the effects of slice thickness, water content, microwave power and processing time on aconite processing. Results : The thickness, rehydration rate, microwave power and processing time had influence on the processing process and alkaloid content of aconite. When the thickness was 5 mm, the drying process had shorter drying time, faster drying rate and lower the energy consumption, but it had a higher content of diester alkaloids and lower content of the total alkaloids. When the rehydration rate of raw aconite slices was 60%, the drying time was the shortest. The order of average drying rate was 100% rehydration rate > 60% rehydration rate > 80% rehydration rate > 40% rehydration rate. The order of content of diester alkaloids was 60% rehydration rate > 100% rehydration rate > 80% rehydration rate > 40% rehydration rate. The order of total alkaloids content was 60% rehydration rate > 80% rehydration rate = 100% rehydration rate > 40% rehydration rate. The influence of rehydration rate on energy consumption was not obvious. When the processing time was the same, the drying process under the microwave power of 550 W had the shortest drying time, the fastest drying rate, the lowest content of diester aconitine and the highest content of total alkaloid. However, with the increase of processing time, the aconite was easier to be burnt under a high microwave power. Conclusion: The appropriate thickness, moisture content, microwave power and processing time in aconite processing process are helpful to reduce the toxicity of aconite and improve the quality of aconite. Therefore, the thickness of the slices should choose 3 mm, the moisture content should be 60%-80%, and the processing time should be reduced according to the increase of microwave power.

Study on multi-index components of Tibetan medicine Bangna of Aconitum genus according to fingerprints and chemometrics / 中草药

Objective: To establish the chemical fingerprint and multi-index components determination of Tibetan medicine Bangna of Aconitum genus, and provide references for the formulation of quality standards of multi-base original medicinal materials and clinically safe medication. Methods: HPLC fingerprint of Bangna was established and evaluated by the similarity evaluation system of TCM. In addition, the content of the seven components of Bangna from 30 batches and the difference of chemical information between the two species of Bangna was investigated by principal component analysis and orthogonal partial least squares discrimination analysis (OPLS-DA) respectively. Results: A total of 17 common peaks were identified in the fingerprint that was established by the determination of 30 batches of Bangna, and seven components of which were identified with 12-epi-napelline, songorine, benzoylmesaconitine, benzoylaconine, mesaconitine, aconitine, 3-acetylaconitine. Based on similarity results, the fingerprint had good consistency between the same origin and minor diversity between the different sources. The results of principal component analysis and OPLS-DA showed that there were some differences in the content of seven components between the two species. Based on the results of OPLS-DA and t test, it could be determined that the contents of 12-epi-napelline and aconitine of Aconitum flavum were significantly higher than those in Aconitum pendulum (P < 0.01). Conclusion: The fingerprint and multi-component quantitative analysis methods were used for the quality and clinically safe medication control of Bangna in this paper is simple, easy to operate, and informative. Moreover, it is necessary to establish and improve the limit determination of diester alkaloids.

Research Progress on the Molecular Mechanisms of Toxicology of Ethanol-Aconitine Induced Arrhythmia / 法医学杂志

Aconitum is one of the most widely used Chinese herbal medicines, and aconitine is the major toxic component in it. Aconitine can induce a variety of arrhythmias, resulting in death. Acute ethanol consumption causes arrhythmia as well. Poisoning cases caused by aconitum medicinal liquor are frequently encountered in the practice of forensic medicine. The molecular mechanisms of myocardial toxicity of these two drugs have much in common, and both of them affect the sodium channel, calcium channel and potassium channel of myocardial cell membrane and so on. This paper analyzes and discusses the possible co-effects of ethanol-aconitine on cardiomyocyte channel proteins, by reviewing researches on the mechanism of cardiotoxicity of ethanol and aconitine in recent years, in order to provide ideas and references for the research on the molecular mechanism of arrhythmia caused by combined poisoning.

In vitro evaluation of aconitine-induced arrhythmia based on RTCA Cardio technology / 中国药理学通报

Aim To establish an in vitro arrhythmia detection technique based on real-time cell analysis (RTCA) Cardio sys-tem with aconitine as a tool drug, and to provide a reliable method for the development of antiarrhythmic drugs. Methods The effects of aconitine on rat cardiac rhythm were detected by eight-channel physiological recorder at the level of whole animal. In vitro cultured cardiac myocytes, the inoculation density of cardiac myocytes was investigated by HTCA method, the effect of aconitine on cardiac beating was monitored by RTCA Cardio system, and the CI value, beating rate, amplitude and irregular rhythm of cardiac myocytes were analyzed. Results Eight-channel physiological recorder was used to detect the effects of aconitine on whole animals. The results showed that aconitine(50 mg • g"1) could induce arrhythmias such as ventricular tachycardia, ventricular fibrillation, shortened RR interval and increased heart rate. RTCA Cardio system showed that aconitine (2-8 p,M) could induce arrhythmias such as increased cardiac cell beating frequency, decreased beating amplitude and abnormal beating state in a dose-dependent manner. Conclusions RTCA Cardio system can rapidly, sensitively and accurately detect the arrhythmia induced by aconitine in cardiac myocytes, which provides methodological reference for the development of antiarrhythmic drugs.

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.

The Decocting Time and the Contents of the Aconitine-type Diester Alkaloids in the Decoctions of the Formula Containing Unprocessed Aconite Root in “Songban Shanghanlun” / 日本東洋医学雑誌

We investigated the decocting time to prepare the formulas containing unprocessed aconite root, such as shigyakuto, tsumyaku shigyakuto, and kankyobushito, which had been registered in “Shanghanlun” edited in Song Dynasty, using the weights and measures in Houhan Dynasty when the original “Shanghanlun” was regarded to have been established. Also the contents of aconitine-type diester alkaloids (ADA) eluted from unprocessed aconite root in the decoction were analyzed in time-dependent manners. As regards the modified formula for the “physically strong patients” in the texts of tsumyakushigyakuto in “Shanghanlun”, adding dried ginger was found to lead the decocting time to be shorter and the sum of ADA content in the decoction of the modified formula to increase about 20%. It was also found that the compositions of diterpene alkaloids derived from aconite root in kankyobushito decoction were highly different from those in shigyakuto decoction, containing less ADA and more aconine and hypaconine, due to the high pH of the decoction, which was the consequence of lacking glycyrrhiza in kankyobushito formula. It is suggested that the doctors in the era of “Shanghanlun” establishment may have carefully adjusted the contents of ADA in the decoctions using unprocessed aconite root by choosing co-decocted crude drugs.

Effects of Benzoyl Aconitine on Autophagy and Apoptosis of Human Lung Cancer Cell Line A 549 / 中国药房

OBJECTIVE： To study the effects of benzoyl aconitine （BAC） on autophagy and apoptosis of human lung cancer A549 cells， and to investigate its mechanism in anti-non-small cell lung cancer. METHODS： A549 cells were treated with different doses of BAC （10， 50， 100， 200， 400 μmol/L）， and then cell morphology was obtained； the proliferation inhibition rate of the cell was determined by CCK-8 assay. The cells were divided into control group （without drug）， BAC low-dose and high-dose groups （200， 400 μmol/L）. After treated with relevant drugs， the apoptosis rate of cells was determined by flow cytometry. The gene and protein expression of apoptosis-related factors Bcl-2， Bax， Caspase-3 as well as autophagy-related factors Beclin1， LC3， P62 were determined by RT-PCR and Western blotting assay. RESULTS： After treated with different doses of BAC， the cells were shrunken and sparsely arranged； inhibitory rate of cell proliferation was increased significantly in BAC 100， 200， 400 μmol/L groups （P＜0.05 or P＜0.01）. Results of flow cytometry showed that the apoptotic rates of cells were increased to different extents in BAC low-dose and high-dose groups after treated for 24 and 48 h， in a concentration and time-dependent manner. Compared with control group， mRNA and protein expression of Bcl-2 and P62 were decreased to different extents in BAC groups； mRNA expression of Bax， Caspase-3， Beclin1 and LC3 as well as protein expression of Bax， Active caspase-3， P62， Beclin1， LC3Ⅱ/Ⅰ were increased to different extent； there was statistical significance in mRNA expression of Caspase-3， and protein expression of Bcl-2， Active Caspase-3， Beclin1， LC3Ⅱ/Ⅰ and P62 in BAC low-dose group as well as all target mRNA and protein expression in BAC high-dose group （P＜0.05 or P＜0.01）， in dose-dependent manner. CONCLUSIONS： BAC can inhibit the proliferation and promote the apoptosis of A549 cells， promote Beclin1， LC3（LC3Ⅱ/Ⅰ），Bax and Caspase-3 （Active Caspase-3） gene and their protein expression， but inhibit P62 and Bcl-2 gene and their protein expression. The mechanism may be related to BAC inducing apoptosis by promoting excessive autophagy of cells.

Recovery and Utilization of Alkaloids in Processing Wastewater of Aconiti Radix / 中国实验方剂学杂志

Objective: To optimize the recovery technology for 6 kinds of toxic alkaloids in the toxic wastewater from processing of Aconiti Radix with macroporous resin. Method:With the rates of adsorption and elution of benzoylmesaconine,benzoylaconine,benzoylhypaconine,mesaconitine,hypaconitine and aconitine as indexes,static and dynamic adsorption-elution tests were used to select the best one from 15 kinds of macroporous resin,and the recovery technology parameters of six toxic alkaloids in the wastewater from processing of Aconiti Radix were optimized. Result:D101 macroporous resin had a good adsorption and elution effect on 6 kinds of toxic alkaloids in the wastewater from processing of Aconiti Radix,its optimum technology conditions were as follows:each gram of macroporous resin could be used to treat processing wastewater from 4.3 g of Aconiti Radix,the sample loading speed was not higher than 3.0 mL·min-1,the resin column was eluted with 6 BV of 70% ethanol after removing impurities with 2 BV of water.The recoveries of benzoylmesaconine,benzoylaconine,benzoylhypaconine,mesaconitine, hypaconitine and aconitine were 98.03%,94.09%,96.53%,78.15%,85.40% and 70.57%,respectively. Conclusion:D101 macroporous resin can be used for detoxification treatment of processing wastewater of Aconiti Radix,at the same time,6 kinds of alkaloids are effectively recovered,which can solve the environmental problems and create certain economic benefits,and the optimized process conditions are stable and feasible.

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.