Optimization of the Ethanol Extraction Method of Monoester Alkaloids from Radix Aconiti Preparata Based on HPLC-MS and Orthogonal Test

[Objectives] This study was conducted to optimize the ethanol extraction technology of monoester alkaloids from Radix Aconiti Preparata. [Methods]On the basis of defined extraction times,ethanol concentration,ethanol times and extraction time were investigated by HPLC-MS combined with orthogonal test to optimize extraction process using the content of monoester alkaloids( the sum of benzoyl neoaconitine,benzoyl hypoaconitine and benzoyl aconitine) as an index.[Results]The optimum ethanol extraction technology was as follows: 75% ethanol,ethanol amount 25 times of the medicinal material,and each extraction for 1. 5 h.[Conclusions] The optimal extraction technology is simple,feasible,stable and reliable. It can provide reference for the industrial production and quality control of monoester alkaloids from Radix Aconiti Preparata.

Research Progress in Toxicity and Toxicity-reducing and Efficacy-enhancing Compatibility of Radix Aconiti Lateralis Praeparata

Radix Aconiti Lateralis Praeparata contains alkaloids,flavonoids,polysaccharides,organic acids and organic alkalis,among which diester alkaloids are important effective ingredients,which can be used for treating heart failure,rheumatic heart disease,coronary heart disease,hypotension,shock and other diseases.However,it also contains the main ingredients that produce toxic effects and are highly toxic to the cardiovascular system,nervous system,digestive system,reproductive system,embryos,and livers and kidneys.In this study,we analyzed the toxicity-reducing and efficacy-enhancing effects of the compatibility of Radix Aconiti Lateralis Praeparata with with single herbs Glycyrrhiza uralensis Fisch.,Panax ginseng C.A.Meyer,Zingiber ojjicinale Rosc.and Rheum palmatum L.,aiming to provide references for further exploring the appropriate compatibility conditions of traditional Chinese medicine in the future and improving the safety of the clinical application of Radix Aconiti Lateralis Praeparata.

Advances in Research of Anti-tumor Effect of Aconiti Radix

In this paper,the anti-tumor effects of Aconiti Radix were reviewed and summarized,and the clinical feasibility of Aconiti Radix as a potential anti-tumor drug was analyzed,in order to provide a useful reference for the future research and development of new anti-cancer drugs of Aconiti Radix.

Effects of Radix Aconiti Lateralis Preparata and Rhizoma Zingiberis on Energy Metabolism and Expression of the Genes Related to Metabolism in Rats

Objective： To study the influence of Radix aconiti lateralis preparata and Rhizoma zingiberis, two species of Chinese medicinal herbs with hot property, on energy metabolism and gene expression spectrum, and to analyze the possible mechanism of their effects. Methods： Forty-eight specific pathogen free Wistar rats were randomly divided into a Radix aconiti lateralis preparata group, a Rhizoma zingiberis group, and a control group. They were intragastrically treated with concentrated decoction of Radix aconiti lateralis preparata, Rhizoma zingiberis and normal saline respectively for 20 days. Toe temperature （TT）, energy intake （El）, digestible energy （DE）, and metabolizable energy （ME） were measured. The content of adenosine triphosphate （ATP） and energy charge （EC） in hepatic tissue were measured with high performance liquid chromatography （HPLC）. The activity of ATPase and succinate dehydrogenase （SDH） in the liver were detected with chemical colorimetry. The gene expression in the liver was detected with Illumina＇s rat Ref-12 gene array. The differential expression genes were selected, annotated and classified based on Gene Ontology （GO）. Real-time quantitative reverse-transcriptase PCR （Q-RT-PCR） was used to test the accuracy of results. Results： Compared with the control group, the -I-r on the 10^th day after the beginning of administration and ATP in the Radix aconiti lateralis preparata and Rhizoma zingiberis groups increased significantly （P〈0.05）. El/body mass （BM）, DE/BM, ME/BM, the hepatic EC and the activity of Na^＋-K^＋-ATPase, Ca^2＋-Mg^2＋-ATPase and SDH of liver increased significantly only in the Radix aconiti lateralis preparata group （P〈0.05）. There were 592 differential expression genes in the Radix aconiti/ateralis preparata group and 1 159 in the Rhizoma zingiberis group compared with the control group. Among the differential expression genes, genes related to metabolic processes were the most significant based on GO analysis. There were 337 strips of gene differential expression in common in both Radix aconiti lateralis preparata and Rhizoma zingiberis groups compared with the control group. Conclusions： Herbs with hot property such as Radix aconiti lateralis preparata and Rhizoma zingiberis could improve the energy metabolism in rats, through influencing the metabolic process of sugar, lipid, and amino acid. It could also promote the production, storage, and utilization of energy by regulating the gene expression related to metabolism, which may be the main molecular mechanism of warming yang and dispelling cold for the treatment of the cold syndrome according to Chinese medicine theory.

Study on the Metabolic Characteristics of Aconite Alkaloids in the Extract of Radix aconiti under Intestinal Bacteria of Rat by UPLC/MSn Technique

The extract of Radix aconiti was incubated with rat intestinal bacteria in vitro. Further, aconitine and hypaconitine standard were incubated at the same condition as the extract of Radix aconiti, respectively. Ultra Performance Liquid Chromatography/Multi-Mass Spectrometry （UPLC/MSn） was used for detecting and identifying all the aco- nite alkaloids. Results showed that there were four metabolites which were identified as 8-butyryl-14-benzoylmesa- conine （m/z 660）, 8-propionyl-14-benzoylaconine （m/z 660）, 8-butyryl-14-benzoylaconine （m/z 674） and 8-valeryl-14-benzoylmesaconine （m/z 674） in the metabolized sample of the extract of Radix aconiti. The relative area ratio of them presented increasing trend during 6 d. On the basis of all results, we could concluded that substi- tution at N atom mainly influenced the metabolizing rate of diester-diterpenoid alkaloids （DDAs）, C-8 substitute was active metabolized site, intestinal bacterial metabolites of the aconite alkaloids in the extract of Radix aconiti were mainly the substitute with propionyl group, butyl group or valeryl group at C-8. This paper illustrated holistic metabolizing profile of the extract of Radix aconiti in vitro and possible metabolizing reaction type of main DDAs, which could provide reference for finding out potential bioactive components in the extract and the prescription of Chinese Medicine.

Efficacy of Renshen(Radix Ginseng) plus Fuzi(Radix Aconiti Lateralis Preparata) on myocardial infarction by enhancing autophagy in rats

OBJECTIVE:To elucidate the protective effects of Renshen(Radix Ginseng)and Fuzi(Radix Aconiti Lateralis Preparata)on myocardial infarction(MI)through regulating myocardial autophagy.METHODS:Thirty-one male Sprague-Dawley rats were randomized into five groups(n=6 or 7 for each).After treatment for 3 weeks,electrocardiogram(ECG)and cardiac function were recorded.Hematoxylin and eosin(HE)staining was used to detect pathological changes in the heart.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum B-type brain natriuretic peptide(BNP),cardiac troponin T(c Tn T),tumor necrosis factor-α(TNF-α),and serum inflammatory cytokines.Metabolomic analysis was used to identify differential biomarkers of MI in rats.Immunohistochemistry and western blotting were used to detect BNP,cTnT,TNF-α,LC3B,Beclin-1,p62,and adenosine monophosphate activated protein kinase(AMPK)expression in cardiac tissue.RESULTS:Fuzi(Radix Aconiti Lateralis Preparata)alone or Renshen(Radix Ginseng)plus Fuzi(Radix Aconiti Lateralis Preparata)markedly ameliorated cardiac dysfunction and abnormal ECGs,demonstrated by decreases in the heart weight/body weight ratio,BNP,and c Tn T.Pro-inflammation cytokine interleukin(IL)-1αsignificantly decreased and anti-inflammatory cytokine IL-10 significantly increased in Renshen(Radix Ginseng)single or Renshen(Radix Ginseng)plus Fuzi(Radix Aconiti Lateralis Preparata)groups compared with the control group.HE results suggested that co-treatment produced a greater reduction in cardiomyocyte cross-sectional area than Renshen(Radix Ginseng)or Fuzi(Radix Aconiti Lateralis Preparata)alone.Renshen(Radix Ginseng)plus Fuzi(Radix Aconiti Lateralis Preparata)reversed these changes to different degrees in MI rats.Furthermore,Renshen(Radix Ginseng)plus Fuzi(Radix Aconiti Lateralis Preparata)down-regulated LC3 B,Beclin-1,and AMPK expression in cardiac tissue and upregulated p62 expression.CONCLUSIONS:Renshen(Radix Ginseng)plus Fuzi(Radix Aconiti Lateralis Preparata)may have a greater effect on heart injury induced by MI in rats than Fuzi(Radix Aconiti Lateralis Preparata)treatment alone,and the underlying mechanism may be associated with the regulation of myocardial autophagy and anti-inflammation effects.These results provide fresh insight into the mechanism of co-treatment with Renshen(Radix Ginseng)and Fuzi(Radix Aconiti Lateralis Preparata)for MI.

Relationship between cardiotonic activity of Fuzi(Radix Aconiti Lateralis Preparata)and its fingerprint determined by liquid chromatography-mass spectrometry

OBJECTIVE:To investigate the relationship between the cardiotonic activity of Fuzi(Radix Aconiti Lateralis Preparata,RALP)and its fingerprint determined by liquid chromatography-mass spectrometry(LC-MS).METHODS:First,the fingerprints of six processed products of RALP were established by high performance liquid chromatography quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS)followed by analysis of the principal component of the relative peak area of its common peaks.Next,the scores of the first five principal components were used as input for an artificial neural network(ANN).Additionally,the therapeutic effect of RALP was assessed by measuring the hemodynamic indexes of heart failure model rats.Subsequently,fluorescence semi-quantitative polymerase chain reaction and an enzyme-linked immunosorbent assay kit were used to determine the effects of RALP-processed products on the serum levels of noradrenaline(NA),angiotensin-Ⅰ(Ang-Ⅰ),and the expression ofβ-norepinephrine receptor m RNA(β-NRm)in the rat cardiac tissues.P<0.05 was used as the output of the ANN.Finally,a network was constructed to display the relationship between the LC-MS fingerprints and the cardiotonic activity of the RALP-processed products.RESULTS:Several types of RALPs can improve diastolic function,systolic function and heart rate.On the basis of the findings from the principal component analysis(PCA)of 16 common peaks of fingerprints of six RALP-processed products,it was revealed that the first five principal components may include 100%of the information of the original data.As observed from the multilayer perceptron neural network analysis,principal component 4 presented with the strongest effects on serum levels of NA and Ang-Ⅰin rats,while principal component1 exerted the greatest effect onβ-NRm expression in cardiac tissue.CONCLUSION:The key findings obtained from this study indicated that the network constructed by the PCA-ANN may predict pharmacodynamic effects of the main ingredients of Traditional Chinese Medicine(TCM).This method may serve as a new approach to identify the relationship between LC-MS fingerprints and the pharmacodynamic effects of TCM ingredients.

Identification of Xunxi Shujin Decoction by TLC

[Objectives]To establish a TLC qualitative method for the determination of Radix Aconiti,Radix Aconiti Kusnezoffii,Caesalpinia sappan L.and Radix zanthoxyli.[Methods]TLC was used to study the quality of Radix Aconiti,Radix Aconiti Kusnezoffii and C.sappan L.in Xunxi Shujin Decoction,and the influencing factors were investigated to optimize the TLC conditions.[Results]In the TLC identification test of Radix Aconiti,Radix Aconiti Kusnezoffii,C.sappan L.and R.zanthoxyli in Xunxi Shujin Decoction,the spots were clear,the resolution was good,the specific value was moderate,the negative control was not disturbed,and the specificity was strong.[Conclusions]The established TLC method was simple,specific and reproducible,and could be used for the quality control of Xunxi Shujin Decoction.

Study on Processing Technology for Aconiti Kusnezoffii Radix by Bean Curd

[Objectives] The aim was to explore the best parameters for processing Aconiti Kusnezoffii Radix by bean curd. [Methods] Orthogonal test( L_(16)(4~3)) was designed to investigate the optimal soaking time,decocting time and bean curd proportion for processing Aconiti Kusnezoffii Radix by bean curd. The contents of monoester alkaloids and diester-alkaloids were measured as the evaluation indexes. [Results]The contents of monoester alkaloids were 0. 11%,0. 07%,0. 062% and 0. 048% respectively under the four levels of soaking time,were0. 081%,0. 066%,0. 074% and 0. 067% respectively under the four levels of decocting time,and were 0. 070%,0. 072%,0. 080% and0. 073% respectively under the four levels of bean curd proportion. The content of diester-alkaloids was all below 0. 034%. The influence intensity of the three factors on the content of monoester alkaloids ranked as soaking time > decocting time > bean curd proportion. Under any of the above conditions,the content of diester-alkaloids was below the limit of Chinese Pharmacopoeia. The content of diester-alkaloids was reduced obviously with the increased use of bean curd. [Conclusions] The optimum processing conditions for Aconiti Kusnezoffii Radix were A_3B_3C_2,i. e.,soaking time of 6 d,decocting time of 5 h and bean curd proportion of 25%.

Comparative study of anti-inflammatory effects of different processed products through the COX-2/PGE2 signaling pathway: based on network pharmacology and molecular docking

Background:Radix Aconiti Lateralis Preparata(Fu-zi)is a traditional Chinese medicinal herb,which has been widely used in the clinic and has potent anti-inflammatory activities.we aimed to explore the mechanisms of extract containing alkaloids from different Fu-zi Processed Products(FPP)in treating inflammation,especially rheumatoid arthritis(RA).Methods:Firstly,using network pharmacology technology,the ingredients,and targets of Fu-zi were obtained by searching and screening,the targets involving RA were acquired,the intersection targets were constructed a"component-target-pathway"network.A comprehensive investigation was conducted on the anti-rheumatoid arthritis mechanisms of 5 FPPs in lipopolysaccharide(LPS)induced RAW264.7 cells,which serve as a model for RA.The production of NO and inflammatory cytokines were measured by ELISA kit.Quantitative Real-time PCR(qRT-PCR)was utilized to measure the mRNA levels.COX-2/PGE2 signaling pathway-associated proteins were determined by western blot.Results:According to a network pharmacological study,16 chemical components and 43 common targets were found in Fu-zi and 6 key targets including PTGS2 were closely related to the mechanism of Fu-zi in treating RA.The in vitro study revealed that the levels of NO,TNF-α,and IL-1βwere substantially decreased by the 5 FPPs.The 5 FPPs significantly suppressed the expression of proteins COX-2,iNOS,and NF-κB,with particularly notable effects observed for PFZ and XFZ.Conclusion:Altogether,these results demonstrated that the 5 PPS containing alkaloids have a good anti-RA-related inflammatory effect,and the mechanism may be related to COX-2/PGE2 signaling pathway,particularly,Fu-zi prepared utilizing a traditional Chinese technique.

Metabolomics of Fuzi-Gancao in CCl4 induced acute liver injury and its regulatory effect on bile acid profile in rats

BACKGROUND Fuzi(Radix aconiti lateralis)-Gancao(Radix glycyrrhizae)is one of the most classical drug pairs of traditional Chinese medicine.In clinical practice,decoctions containing Fuzi-Gancao(F-G)are often used in the treatment of liver diseases such as hepatitis and liver failure.AIM To investigate the metabolomics of F-G in CCl4 induced acute liver injury in rats and its regulatory effect on the bile acid profile.METHODS The pharmacodynamic effect of F-G on CCl4 induced acute liver injury in rats was evaluated,and an ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the simultaneous determination of 92 metabolites from multiple pathways was established to explore the protective metabolic mechanism of F-G in serum on the liver.RESULTS Twenty-four differential metabolites were identified in serum samples.The primary bile acid biosynthetic metabolic pathway was the major common pathway in the model group and F-G group.Subsequently,a UPLC-MS/MS method for simultaneous determination of 11 bile acids,including cholic acid,ursodeoxycholic acid,glycochenodeoxycholic acid,glycochenodeoxycholic acid,taurocholic acid,glycocholic acid,chenodeoxycholic acid,deoxycholic acid,taurochenodeoxycholic acid,taurocholic acid,and glycinic acid,was established to analyze the regulatory mechanism of F-G in serum.F-G decreased the contents of these 11 bile acids in serum in a dose-dependent manner compared with those in the model control group.CONCLUSION F-G could protect hepatocytes by promoting the binding of free bile acids to glycine and taurine,and reducing the accumulation of free bile acids in the liver.F-G could also regulate the compensatory degree of taurine,decreasing the content of taurine-conjugated bile acids to protect hepatocytes.

A feasible,economical,and accurate analytical method for simultaneous determination of six alkaloid markers in Aconiti Lateralis Radix Praeparata from different manufacturing sources and processing ways

Aconiti Lateralis Radix Praeparata(Fuzi) is a commonly used traditional Chinese medicine in clinic for its potency in restoring yang and rescuing from collapse. Aconiti alkaloids, mainly including monoester-diterpenoidaconitines(MDAs) and diester-diterpenoidaconitines(DDAs), are considered to act as both bioactive and toxic constituents. In the present study, a feasible, economical, and accurate HPLC method for simultaneous determination of six alkaloid markers using the Single Standard for Determination of Multi-Components(SSDMC) method was developed and fully validated. Benzoylmesaconine was used as the unique reference standard. This method was proven as accurate(recovery varying between 97.5%-101.8%, RSD < 3%), precise(RSD 0.63%-2.05%), and linear(R > 0.999 9) over the concentration ranges, and subsequently applied to quantitative evaluation of 62 batches of samples, among which 45 batches were from good manufacturing practice(GMP) facilities and 17 batches from the drug market. The contents were then analyzed by principal component analysis(PCA) and homogeneity test. The present study provided valuable information for improving the quality standard of Aconiti Lateralis Radix Praeparata. The developed method also has the potential in analysis of other Aconitum species, such as Aconitum carmichaelii(prepared parent root) and Aconitum kusnezoffii(prepared root).

Metabolic regulation mechanism of Aconiti Radix Cocta extract in rats based on ^(1)H-NMR metabonomics

Objective:To establish a metabonomics research technique based on the combination of^(1)H-NMR and multivariate statistical analysis,so as to explore the metabolic regulation mechanism of Aconiti Radix Cocta extract(ARCE)in rat tissues and serum.Methods:SD rats were randomly divided into blank group,female group and male group.The^(1)H-NMR technique was used to collect the information of rat tissues and serum samples in each group.The principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA)and other methods were used for data pattern recognition,so as to screen out potential differential metabolites and metabolic pathways,and then network analysis and KEGG database were used to analyze the relationship between metabolites,metabolic pathways and diseases.Results:The external features and^(1)H-NMR analysis showed that the sex of rats had no obvious effect on the drug action.A total of 15 potential differential metabolites and six metabolic pathways were screened out through data pattern recognition.Through network analysis and KEGG pathway analysis,three target diseases closely related to differential metabolites were found,and the metabolic pathway related to lung cancer was the central carbon metabolism of cancer.Conclusion:This study shows that Aconiti Radix Cocta(ARC)may regulate the energy metabolism of the body by influencing arginine synthesis,so as to play the roles of anti-inflammation,analgesia,anti-tumor and immune regulation.

Rapid Differentiation of Aconiti Kusnezoffii Radix from Different Geographic Origins Using Ultra-Performance Liquid Chromatography Coupled with Time-of-Flight Mass Spectrometry

Objective:There are different geographic origins of Aconiti Kusnezoffii Radixs(AKRs)sold in the market with different quality.This study aims to establish a rapid analysis method to distinguish the different geographic origins of AKRs and to realize the rapid evaluation of their quality.Methods:An ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry(UPLC-Q-TOF MS)method was utilized to acquire the constituents'information of AKRs from different geographic origins.MSE data and Progenesis QI software were employed to identify the chemical constitutes.Principal component analysis(PCA)was applied to comparing MS data to find the chemical markers of AKRs from different geographic origins.Results:Twenty-three components were detected and 17 out of them were identified,including diester-diterpenoid alkaloids,monoester-diterpenoid alkaloids,and amine-diterpenoid alkaloids.Three pairs of isomers were detected and two of them were distinguished by the retention time of standard samples.Thirteen chemical markers were screened out through PCA and orthogonal partial least square discriminant analysis.Through detecting Napelline or isomer of Napelline(m/z 360.2530)and Aconifine(m/z 662.3170),AKRs from inner Mongolia autonomous could be screened.According to the existence of benzoylaconine(m/z 604.3108)and Indaconitine(m/z 630.3159),it could be confirmed that the AKRs are from Xinjiang Uygur autonomous.AKRs that cannot detect compounds above-mentioned could be from Liaoning or Shanxi Province.Conclusions:The chemical profile could be used not only to distinguish the AKRs from different geographic origins but also to identify the true and false of AKRs.This study lays a foundation for the study of efficacy and toxic of AKRs.

Study on Hot Property of Aconiti Lateralis Radix Praeparata and Its Compatibility with Zingiberis Rhizoma Based on Animal Temperature Tropism

Objective To establish an objective method for evaluating the intrinsic characteristics between hot and cold properties of herbal drugs and to study the hot property of Aconiti Lateralis Radix Praeparata(ALRP) and its compatibility with Zingiberis Rhizoma(ZR) on animal temperature tropism.Methods The equipment with cold/hot pads was used to investigate the variety of temperature tropism among mice treated by ALRP and its compatibility with ZR.Meanwhile,the activities of adenosine triphosphatase(ATPase),total anti-oxidant capability(T-AOC),and total superoxide dismutase(T-SOD) were measured.Results Compared with the homologous ALRP group,the compatibility between ALRP and ZR(AZ) had a stronger action against the decreasing of remaining rate on hot pad(P < 0.05),suggesting an enhancement of cold tropism.Meanwhile,the internal indexes of Na+-K+-ATPase activity,Ca2+-Mg2+-ATPase activity,and oxygen consuming volume increased significantly(P < 0.05),suggesting an enhancement of energy metabolism.The changes of T-AOC and T-SOD activity suggested that AZ reinforced the anti-oxidative capability of mice.In addition,it could be inferred from cluster analysis that the activity of AZ was concretely stronger than that of homologous ALRP.Conclusion ALRP and ZR are both herbs with hot property,but AZ has an enhanced trend of hot property.This external behavior of cold tropism and the internal activity of energy metabolism and anti-oxidant might reflect the internal hot property in an intuitive and objective way.The changes of ATPase activity of liver tissue might be the mechanism of drug action.

附子黄芩配伍减毒存效研究

目的:观察附子配伍不同比例的黄芩对附子毒性和药效的影响。方法:ICR小鼠,0.4m L/10g体质量灌胃给药,平行比较附子和附子配伍不同比例黄芩的急性毒性,测定其半数致死量(LD50);SD大鼠1m L/100g体质量灌胃给药,平行比较附子和附子配伍黄芩的心脏毒性;采用高效液相色谱法测定附子配伍前后乌头类生物碱成分的含量;通过小鼠耳肿胀试验、醋酸扭体试验和回阳救逆试验考察附子配伍黄芩对附子药效的影响。结果:随着黄芩配伍比例的增高,附子急性毒性LD50和附子心脏心律失常抑制率均有增大趋势,附子配伍黄芩后主要乌头类生物碱成分含量降低,但附子配伍黄芩后对附子抗炎、镇痛和回阳救逆药效没有明显影响。结论:黄芩对附子毒性具有剂量依赖型拮抗作用,黄芩配伍附子在减轻附子毒性的同时不影响附子的药效,附子配伍黄芩减毒时应该考虑黄芩的配伍比例。

附子中3种双酯型生物碱在体外血浆中的水解规律研究

目的:建立体外血浆温孵法测定乌头碱、中乌头碱和次乌头碱水解规律的方法。方法:空白兔血浆加入不同浓度的3种生物碱混合对照品溶液,通过对温孵不同时间血浆样品的预处理(沉淀蛋白、除杂)、分离和检测等过程实现复杂生物样品中待测成分的分离分析。结果:乌头碱、中乌头碱和次乌头碱浓度在血浆中均随温孵时间的延长逐渐降低,水解速率中乌头碱>乌头碱>次乌头碱,与附子饮片在炮制过程中3种双酯型生物碱的水解规律一致。结论:所建立的体外水解方法简单、稳定、可控,对上述3种成分的体内药动学研究具有一定的参考意义。