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.

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.

Synergetic effects of aqueous extracts of Fuzi(Radix Aconiti Lateralis Preparata) and Tubeimu(Rhizoma Bolbostemmatis) on MDA-MB-231 and SKBR3 cells

OBJECTIVE： To test the synergistic effects of the aqueous extract of Tubeimu （Rhizoma Bolbosternmatis） and Fuzi （Radix Aconiti Lateralis Preparata） on MDA-MB-231 and SKBR3 breast cancer cells.METHODS： A combined index was created for the effects of Tubeimu （Rhizoma Bolbostemmatis） and Fuzi （Radix Aconiti Lateralis Preparata） extracts. Cell proliferation was performed by trypan blue exclusion and 3-（4,5-dimethylthiazol-2-yl）-5-（3- carboxy- methoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） assays. Flow cytometry was used to assess cell cycle distribution and apoptosis. Cell migration was determined by wound-healing and transwell assays. Confocal microscopy was used to detect E-cadherin and actin filaments. RESULTS： The aqueous extract from Tubeimu （Rhizoma Bolbostemmatis）and Fuzi （Radix Aconiti lateralis Preparata） exerted synergetic effects on the growth of MDA-MB-231 cells and G1 phase arrest. When exposed to extracts at concentrations of 62.5 ：62.5 and 62.5：31.3 μg/mL, the combination index was 0.83 and 0.74, respectively. Interestingly, 62.5： 31.3 μg/mL of combined drugs enhanced the inhibitory effect of Tubeimu （Rhizoma Bolbostemmatis） on the migration of SKBR3 cells and reduced the stimulative effect of Fuzi （Radix Aconiti Lateralis Preparata） （P 〈 0.01）, in which cells showed an increased expression of E-cadherin and reorganization of actin filaments （P 〈 0.001）. 62.5：62.5 μg/mL extract also synergistically induced apoptosis of MDA-MB-231 cells （P 〈 0.05 and P 〈 0.001）. Acting as the main active ingredients in the extract, tubeimoside I and acetylbenzoylaconine at 10：10μg/ mL and 5：2.5 μg/mL also produced inhibitory effects on the proliferation and migration of cells （P 〈 0.01）. CONCLUSION： Tubeimu （Rhizoma Bolbostemmatis） and Fuzi （Radix Aconiti Lateralis Preparata） extracts had synergic effects on MDA-MB-231 and SKBR3 cells.

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.

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.