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1.
Article in Chinese | WPRIM | ID: wpr-906512

ABSTRACT

Objective:To explore the effect of Trichosanthis Fructus-Allii Macrostemonis Bulbus medicine on the proliferation and autophagy levels of aortic plaque vascular smooth muscle cells (VSMCs) in ApoE<sup>-/-</sup> mice with atherosclerosis (AS). Method:A total of 40 ApoE<sup>-/-</sup> mice were fed with high-fat diet to replicate AS animal models. They were randomly divided into model group, Trichosanthis Fructus-Allii Macrostemonis Bulbus group, rapamycin group and atorvastatin group, and 10 mice with normal diet C57BL/6J mice were the blank group. The blank group and the model groups were given normal saline by gavage, while Trichosanthis Fructus-Allii Macrostemonis Bulbus group, rapamycin group and atorvastatin group were given corresponding drugs by gavage for 8 weeks. After the experiment, the mice were sacrificed. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were detected by the Microplate reader, the ratio of the aortic plaque area to the total area was observed and measured by staining with aortic gross oil red O. Western blot method was used to detect the proliferation-related protein proliferating cell nuclear antigen (PCNA) and <italic>α</italic>-smooth muscle actin (<italic>α</italic>-SMA) levels of VSMCs in the aortic media. Transmission electron microscopy was used to observe the autophagosomes of VSMCs and detect the expressions of VSMCs autophagy-related proteins Beclin-1, light chain proteinⅡ (LC3Ⅱ) and p62. Result:Compared with the model group, the Trichosanthis Fructus-Allii Macrostemonis Bulbus group showed significant reduction in the aortic lipid accumulation and plaque area of AS mice and the levels of TC, TG, LDL-C (<italic>P</italic><0.01) and increase of HDL-C (<italic>P</italic><0.05). Trichosanthis Fructus-Allii Macrostemonis Bulbus significantly reduced the levels of proliferation-related antigens PCNA and <italic>α</italic>-SMA in aortic VSMCs (<italic>P</italic><0.01), and inhibited the excessive proliferation of VSMCs. Trichosanthis Fructus-Allii Macrostemonis Bulbus significantly up-regulated Beclin-1 and LC3Ⅱ in aortic VSMCs protein expression, decreased p62 accumulation (<italic>P</italic><0.01), increased the expressions of VSMCs autophagosomes, and increased the autophagy level of VSMCs. Conclusion:Trichosanthis Fructus-Allii Macrostemonis Bulbus regulates blood lipid levels in AS mice, and inhibits the excessive proliferation of aortic VSMCs and plaque formation in the aorta of AS mice. The mechanism may be related to the up-regulation of the autophagy activity of VSMCs.

2.
Article in Chinese | WPRIM | ID: wpr-906060

ABSTRACT

Objective:Based on pharmacokinetics, the antitussive and expectorant related quality markers (Q-marker) of Trichosanthis Fructus were screened from diosmetin-7-<italic>O</italic>-glucopyranoside, diosmetin, apigenin, vanillic acid and cinnamic acid, and the candidate Q-marker was evaluated by multivariate statistical method. Method:Six healthy rats were randomly selected and the 70% ethanol extract of Trichosanthis Fructus (dose of 20 g·kg<sup>-1</sup>) was given by intragastric administration. Blood was collected from the orbital vein at different time points, and the plasma concentrations of 5 components (diosmetin-7-<italic>O</italic>-glucopyranoside, diosmetin, apigenin, vanillic acid and cinnamic acid) from Trichosanthis Fructus were detected simultaneously by high performance liquid chromatography-triple quadrupole tandem mass spectrometry (HPLC-QqQ-MS/MS). The main detection conditions were as following:mobile phase of 0.2% formic acid aqueous solution (A)-acetonitrile (B) for gradient elution (0-4 min, 6%-23%B; 4-5 min, 23%-59.5%B; 5-10 min, 59.5%-60%B), flow rate of 0.5 mL·min<sup>-1</sup>, the detection wavelength at 254 nm, electrospray ionization (ESI), positive ion mode detection, multiple reaction monitoring (MRM) mode scanning, scanning range of <italic>m</italic>/<italic>z</italic> 50-1 500. Diosmetin-7-<italic>O</italic>-glucopyranoside, diosmetin, apigenin and vanillic acid with clear pharmacokinetic behaviors were selected as candidate Q-marker about antitussive and expectorant of Trichosanthis Fructus. The contents of these components in 9 batches of medicinal materials were determined and the main detection conditions were the same as the pharmacokinetic study. SPSS 21.0 was used for cluster analysis and principal component analysis (PAC) based on the results of determination. Result:The pharmacokinetic results showed that the area under concentration-time curve (AUC<sub>0-</sub><italic><sub>t</sub></italic>) of 4 components (diosmetin-7-<italic>O</italic>-glucopyranoside, diosmetin, apigenin and vanillic acid) were (111.28±9.94), (27.08±2.76), (1 376.12±101.86), (631.32±64.72) μg·h·L<sup>-1</sup>, respectively. The 9 batches of Trichosanthis Fructus samples were clustered into 3 groups by systematic cluster analysis. The clustering results were related to the variety of Trichosanthis Fructus and also affected by the origin. The PCA results showed that the comprehensive scores of Gaotang Trichosanthis Fructus, Shanxi Trichosanthis Fructus, Hebei Ben Trichosanthis Fructus were 1.919, 1.356 and 0.299, respectively, ranking in the top 3 among all samples. The comprehensive scores of Nongkeyuan No. 1, Hebei Trichosanthis Fructus and Nongkeyuan No. 2 were -0.804, -1.085, -1.120, respectively, which were in the last 3 positions among all samples. Conclusion:The pharmacokinetic characteristics and quality evaluation of diosmetin-7-<italic>O</italic>-glucopyranoside, diosmetin, apigenin and vanillic acid meet the requirements about antitussive and expectorant related Q-marker of Trichosanthis Fructus.

3.
Article in Chinese | WPRIM | ID: wpr-905874

ABSTRACT

Objective:To compare the effects of different drying methods on the chemical constituents of Trichosanthis Fructus. Method:Trichosanthis Fructus was dried by means of air drying, sun drying, hot air drying (40, 60, 80 ℃) and variable temperature drying (50-80, 80-50 ℃). The contents of nucleosides and flavonoids in Trichosanthis Fructus peels and seeds treated by different methods were compared by high performance liquid chromatography (HPLC), mobile phase was acetonitrile-0.2% acetic acid aqueous solution (3∶7) (A)-acetonitrile (B) for gradient elution (0-15 min, 97-95%B; 15-30 min, 95%-90%B; 30-35 min, 90%-87%B; 35-40 min, 87%-86.5%B; 40-48 min, 86.5%-97%B; 48-50 min, 97%B), the detection wavelength was 260 nm, and the flow rate was 0.4 mL·min<sup>-1</sup>. Gas chromatography-ion mobility spectrometry (GC-IMS) was used to compare the changes of volatile components in the samples treated by different treatments. The volatile components were incubated on a SE-54 capillary column (0.32 mm×30 m, 0.25 μm) at 80 ℃ and 500 r·min<sup>-1</sup> for 15 min, the injection temperature was 85 ℃, the injection volume was 400 μL, the analysis time was 35 min, carrier gas was high purity nitrogen, the flow rate of carrier gas was 2.0 mL·min<sup>-1</sup>, the flow rate of drift gas was 150 mL·min<sup>-1</sup>, and the temperature of IMS detector was 45 ℃. Result:The contents of uridine, adenosine and adenine were higher after hot air drying at >50 ℃. Low temperature drying was conducive to maintaining the stability of cytidine, cytosine, rutin, luteolin and 2ʹ-deoxyadenosine. GC-IMS technology could realize the analysis and identification of Trichosanthis Fructus samples after different treatments. There were more volatile components after hot air drying at 80 ℃ and variable temperature drying. Conclusion:Hot air drying at 40 ℃ and 60 ℃ can retain nucleosides and flavonoids, and the volatile components are similar to those in traditional drying methods, which has the advantages of high efficient, controllable and suitable for industrial production.

4.
Article in Chinese | WPRIM | ID: wpr-921679

ABSTRACT

In this study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS)-based liver metabolomics approach was used to explore the mechanism of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in improving atherosclerosis(AS) of mice with apolipoprotein E gene knockout(ApoE~(-/-)). AS mouse model was induced by high-fat diet. The pathological and biochemical indexes such as the histopathological changes, body weight, liver weight, blood lipid level and inflammatory factors in the liver of mice were determined. The metabolic profiling of mice liver samples was performed with UPLC-Q-TOF-MS. Multiple statistical analysis methods including partial least squares discriminant analysis(PLS-DA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were employed to screen and identify biomarkers. The levels of related enzymes including LCAT, sPLA2, EPT1 and ACER1 were detected. The results showed that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" significantly reduced the areas of aortic plaque and fat vacuoles of liver in AS mice and decreased the accumulation of lipid droplets and liver coefficient. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" also regulated the levels of blood lipid and inflammatory injury in the liver. The metabolites of the control group, the model group and the "Trichosanthis Fructus-Allii Macrostemonis Bulbus" group could be distinguished significantly. Fifteen potential biomarkers related to AS were discovered and preliminarily identified, seven of which could be regulated by "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in a trend of returning to normal. Metabolic pathway analysis screened out two major metabolic pathways. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" obviously regulated the levels of LCAT, sPLA2, EPT1 and ACER1. It was inferred that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" could play a major role in AS treatment by regulating glycerophospholipid and sphingolipid metabolism disorders in the liver, with the mechanism probably relating to the intervention of the expression of LCAT, sPLA2, EPT1 and ACER1.


Subject(s)
Animals , Apolipoproteins E/genetics , Atherosclerosis/genetics , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Liver , Metabolomics , Mice
5.
Article in Chinese | WPRIM | ID: wpr-872903

ABSTRACT

Objective:Based on the hyperlipidemia rat model and network pharmacology technology, the mechanism of action of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia was analyzed. Method:The levels of blood lipids and inflammatory factors were measured through prophylactic administration of low, medium and high-dose Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs in hyperlipidemia rats. The active ingredients of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs were screened out through Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) and text mining. The targets of active ingredients screened through the Swiss Target Prediction, Similarity ensemble approach (SEA), DrugBank database. The disease targets were collected through Therapeutic Target Database (TTD), Online Mendelian Inheritance in Man (OMIM), DrugBank, DisGeNET database. The targets of active ingredients and disease target were integrated, and screened through topological parameters to gain the main candidate targets of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and the gene ontology (GO) functional enrichment analysis were conducted through ClueGO and Database for Annotation, Visualization and Integrated Discovery (DAVID), respectively. The traditional Chinese medicine-chemical ingredient-target network model, and the target-pathway network model were constructed through Cytoscape, and their crosstalk target and signal pathway were analyzed. Result:Animal experiments showed that the prophylactic administration of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs significantly reduced the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) in serum of rats with hyperlipidemia, increased high-density lipoprotein (HDL-C) levels, and inhibited the expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). According to the findings, 27 active ingredients, such as mandenol, diosmetin and α-spinasterol, might be the main active ingredients of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs, 16 crosstalk targets and 10 signal pathways might be the main therapeutic targets and pathways, main targeting apolipoprotein A1 (APOA1), apolipoprotein A2 (APOA2), apolipoprotein C3 (APOC3), lipoprotein lipase (LPL), low-density lipoprotein receptor (LDLR) and other crosstalk targets affected cholesterol metabolism, bile secretion, peroxisome proliferator activated receptor (PPAR) signaling pathway in regulating the lipid level, targeting tumor necrosis factor (TNF), IL-6, interleukin-1β (IL-1β), mitogen-activated protein kinase 1 (MAPK1), C-C motif chemokine 2 (CCL2) and other crosstalk targets affected tumor necrosis factor (TNF) signaling pathway, Toll-like receptor signaling pathway, interleukin-17 (IL-17) signaling pathway, and hypoxia inducible factor (HIF) signaling pathway in regulating the inflammatory factor level. The DAVID database for GO enrichment analysis showed that the hyperlipidemia was treated mainly through biological processes, such as inflammation, lipid localization, storage and lipid metabolism. Conclusion:These findings can predict the mechanism of action of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia, and provide a theoretical basis for the material basis and clinical application of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs.

6.
Article in Chinese | WPRIM | ID: wpr-846535

ABSTRACT

Different parts of Trichosanthes kirilowii can all be used as medicines, including the fruits (Trichosanthis Fructus), pericarps (Trichosanthis Pericarpium), seeds (Trichosanthis Semen) and roots (Trichosanthis Radix). Modern research has confirmed that the main active ingredients of Trichosanthis Pericarpium are flavonoids and amino acids; Trichosanthis Semen mainly contains terpenoids and sterols; Trichosanthis Radix mainly contains protein, steroids and polysaccharides. And the pharmacological effects of various medicinal parts are also different. This paper summarizes the traditional efficacy, chemical composition and modern pharmacological effects of different medicinal parts of T. kirilowii, analyzes the relationship between them, so as to analyze and predict the quality marker of T. kirilowii.

7.
Article in Chinese | WPRIM | ID: wpr-850829

ABSTRACT

Trichosanthis Fructus is a traditional Chinese herbal medicine, which has wide distribution and rich medicine source. With the in-depth study of Trichosanthis Fructus, its effective ingredients and application research has attracted much attention, and has great application prospects. Based on a summary of its chemical composition and main pharmacological effects, according to the definition of quality marker (Q-marker), the quality marker components of Trichosanthis Fructus were predicted from the aspects of the biosynthetic approach and component specificity of chemical components and their correlation with the effectiveness of Chinese medicines and the measurability of components, which provides a scientific basis for quality evaluation of Trichosanthis Fructus.

8.
Article in Chinese | WPRIM | ID: wpr-774509

ABSTRACT

To explore the anti-platelet aggregation and anti-thrombotic mechanisms of Trichosanthis Fructus combined with aspirin based on network pharmacology and the validation of arteriovenous by pass model in rats. The databases of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Drug Repositioning and Adverse Drug Reaction Chemical-Protein Interactome(DRAR-CPI),Universal Protein Resource(Uniprot) and the Database for Annotation,Visualization,and Integrated Discovery(DAVID) were used to predict protein targets and analyze biological pathway and signal pathway in the combination of Trichosanthis Fructus with aspirin. The effects of pretreatment with Trichosanthis Fructus pellets,aspirin pellets and their combination on thromboxane B2(TXB2),6-keto prostaglandin F1α(6-keto-PGF1α) and cyclic adenosine monophosphate(c AMP) in rat thrombotic model were studied. Through the study of network pharmacology,12 components of aspirin and Trichosanthis Fructus,including hydroxygenkwanin,quercetin and adenosine,were found to show the anti-platelet aggregation and anti-thrombosis mechanisms through9 common protein targets,such as SRC,RAC1,MAPK14,MAPK1,AKT1,and 14 common signaling pathways,such as VEGF signaling pathway. After the intervention with Trichosanthis Fructus pellets combined with aspirin pellets,the vascular endothslia growth factor(VEGF) signaling pathway can be activated to inhibit platelet aggregation and improve vascular endothelial function,and show the anti-platelet aggregation and anti-thrombosis mechanisms,which verify the results of the network pharmacology,and explain the anti-platelet aggregation and anti-thrombotic mechanisms of the combination of Trichosanthis Fructus pellets with aspirin pellets.


Subject(s)
6-Ketoprostaglandin F1 alpha , Metabolism , Animals , Aspirin , Pharmacology , Cyclic AMP , Metabolism , Drugs, Chinese Herbal , Pharmacology , Fruit , Chemistry , Platelet Aggregation , Platelet Aggregation Inhibitors , Pharmacology , Rats , Signal Transduction , Thrombosis , Drug Therapy , Thromboxane B2 , Metabolism , Trichosanthes , Chemistry
9.
Article in Chinese | WPRIM | ID: wpr-801717

ABSTRACT

Objective: To analysis and identify the chemical components in Trichosanthis Fructus by UPLC-LTQ-Orbitrap-MS. Method: Samples of Trichosanthis Fructus were extracted by ultrasonic with 70% methanol after smashing and sifting by 40 mesh sieve. Thermo ScientificTM DionexTM UltiMateTM 3000 Rapid Separation LC system performed UPLC separations with Waters HSS T3-C18(2.1 mm×100 mm,1.8 μm) column. The mobile phase was 0.1% formic acid water(A)-methanol(B) with a gradient elution. The volume flow was 0.3 mL ·min-1. A Thermo ScientificTM LTQ-Orbitrap mass spectrometer equipped with a ESI probe was employed. The samples were respectively scanned in MS1 and MS2 mode of positive and negative ions. According to the chromatographic peak separation,mass signal intensity,and the number of molecular ions in MS1 model,the extraction condition,chromatogram and mass spectrum parameters were optimized. The chemical compounds were identified by the accurate mass measurement of molecular ions and fragment ion and comparation with reference substance. Result: 91 chemical compositions in Trichosanthis Fructus were totally identified,including 14 amino acids,5 monoterpenoids,5 tetracyclic triterpenoids,1 pentacyclic triterpene,14 flavonoids, 17 organic acids,3 polysaccharides,7 nucleotides,7 alkaloids and nitrogen compounds,2 volatile components,1 phytosterol,5 other compositions. Conclusion: The established UPLC-LTQ-Orbitrap-MS method can be used to quickly analyze and identify the main chemical constituents of Trichosanthis Fructus. The chemical information concerning the constituents in Trichosanthis Fructus could be helpful to the quality control and further studies of Trichosanthis Fructus.

10.
Article in Chinese | WPRIM | ID: wpr-776418

ABSTRACT

To investigate the spectrum-activity relationship of Trichosanthis Fructus and Trichosanthis Fructus strip pieces for rat myocardial ischemia-reperfusion injury. HPLC fingerprints of Trichosanthis Fructus and Trichosanthis Fructus strip pieces were established, and the values of creatinekinase-MB (CK-MB), myoglobin (MYO) and cardiac troponin-T (cTNT) in 3 dose groups (2.25, 13.5, 27.0 g·kg⁻¹, equivalent to the crude herb g·kg⁻¹) of Trichosanthis Fructus and Trichosanthis Fructus strip pieces with myocardial ischemia-reperfusion injury in rats were measured, and the grey relational analysis was used to study the spectrum-activity relationship of Trichosanthis Fructus and Trichosanthis Fructus strip pieces for rat myocardial ischemia-reperfusion injury. With the dosage increase from 2.25 g·kg⁻¹ to 27.0 g·kg⁻¹, the correlation degree of spectrum-activity relationship of Trichosanthis Fructus and Trichosanthis Fructus strip pieces was also enhanced, but the change trend was different between these two groups. According to the frequency of the top 10 peaks in the correlation degree, peak 17, 14, 16, 19, 32, 12, 26, 30, 4, 6 and 2 were the basic effective substances group of Trichosanthis Fructus, peak 6,14,12,32,30,4 and 6 were the basic effective substances group of Trichosanthis Fructus strip pieces. Peak 6, 14, 12, 32, 30, 4 and 26 in fingerprints of Trichosanthis Fructus and Trichosanthis Fructus strip pieces were the main common pharmacodynamic substance base, among them, peak 6 was 5-hydroxymethyl furfural, peak 14 was vanillic acid and the peak 28 was rutin, but the correlation degree with the efficacy was different. The effect of Trichosanthis Fructus and Trichosanthis Fructus strip pieces on rat myocardial ischemia-reperfusion injury was due to the synergistic effect of the effective substance groups related to the dosage. The essential pharmacodynamic substance groups of Trichosanthis Fructus and Trichosanthis Fructus strip pieces were different, but they shared a common active ingredient group.


Subject(s)
Animals , Chromatography, High Pressure Liquid , Creatine Kinase, MB Form , Blood , Cucurbitaceae , Chemistry , Drugs, Chinese Herbal , Pharmacology , Fruit , Chemistry , Myocardial Reperfusion Injury , Drug Therapy , Myoglobin , Blood , Rats , Troponin T , Blood
11.
Chinese Pharmaceutical Journal ; (24): 1962-1966, 2016.
Article in Chinese | WPRIM | ID: wpr-858912

ABSTRACT

OBJECTIVE: To investigate the microbial contamination of trichosanthis fructus decoction pieces, and provide a reference for the test method and standard of the microbial limit. METHODS: Trichosanthis fructus decoction pieces were tested for the total aerobe microbial count, the total combined yeasts and molds count, the thermoduric bacteria count, the bile-tolerant gram-negative bacteria, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella. RESULTS: The total contamination percentages of aerobes, combined yeasts and molds, thermoduric bacteria, bile-tolerant gram-negative bacteria of trichosanthis fructus are 100%, 59%, 91%, and 91%, respectively. Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and salmonella of trichosanthis fructus were not detected. CONCLUSION: To assure the safety of trichosanthis fructus decoction pieces, it is recommended to control the total aerobe microbial count, the total combined yeasts and molds count, the thermoduric bacteria count, the bile-tolerant gram-negative bacteria, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and salmonella.

12.
Article in Chinese | WPRIM | ID: wpr-476864

ABSTRACT

This study was aimed to investigate the seed protein of different forms ofTrichosanthis fructus from Shandong province. UV analyzer was used to analyze the seed protein content. PAGE fingerprint was drawn by Biosens Gel Imaging System software. The Jaccard coefficient was worked out by using NTSYS-pc software. A cluster dendrogram of different samples was established based on unweighted pair-group method with arithmetic means (UPGMA). The results showed that the content of different samples was obviously different. But there were many common bands in the fingerprints of PAGE. It was concluded that the content of seed protein cannot be used as the main index to evaluate the quality ofTrichosanthis fructus. The common bands in fingerprints of PAGE can be used to identifyTrichosanthis fructus.

13.
Article in Chinese | WPRIM | ID: wpr-854649

ABSTRACT

Objective: To investigate the effect of Aconiti Kusnezoffii Radix (AKR) combined with Trichosanthis Fructus (TF) in 13 different ratios on contents of benzoylmesaconitine, benzoylaconine, benzoylhypaconine, mesaconitine, aconitine, and hypaconitine. Methods: The powdered AKR-TF material, mixture, was alkalized by 10 times 75% ethanol and distilled water for 1 h, respectively, and extracted regurgitantly twice (each time for 1 h). The extracts were combined and centrifuged, and the supernatant was taken. The Welch Xtimate-C18 column (250 mm × 4.6 mm, 5 μm) was eluted with a mobile phase consisted of aceto-nitrile and 40 mmol/L ammonium acetate (pH value 10.0, adjusted with aqueous ammonia) and gradient elution at a flow rate of 1 mL/min. The eluent was monitored by a UV detector at 235 nm. Results: After AKR combined with TF, the content of monoester-alkaloids did not change significantly. Liquid pH value gradually reduced and total quality of diester-alkaloids increased with the ratios of TF increasing. Conclusion: From the view of chemical composition, the toxicity is increasing when AKR combined with TF.

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