Isolation, Structure-activity Relationship And Toxicity Studies Of Annonaceous Acetogenins

ACGs (Annonaceous acetogenins) isolated from the plants in Annonaceae have strong anti-tumor activity and are the hot interest of the international natural products chemists. However, The antitumor structure-activity relationship of these compounds is obscure, and the in vivo pharmacokinetics and toxicity-activity relationship study have not yet been reported.Objective:To isolate ACGs from Annona squamosa seeds and to investigate the structure-activity relationship of their anti-tumor effects, the in vivo pharmacokinetics, toxicity-activity relationship, and the molecular mechanism of action.Methods:1. Enrich the ACGs parts with95%ethanol extraction and liquid-liquid extraction. This part was isolated and purified by column chromatography and preparative liquid technologies. Using mass spectrometry, a variety of chromatographic spectroscopy was performed to determinate the physical and chemical properties and structures of the isolated compounds. The absolute configuration of isolated new compounds was also determined by the Mosher method. After methyl esterification of fatty oils part, the GC was used to analyze the composition and content with mixed reference substance.2. The isolated ACGs were structurally classified. MTT assay was used to test the inhibitory effect of these compounds against different kinds of human tumor cells. The structure-activity relationship of the anti-tumor effects based on the structures of the compound type was analyzed.3.Healthy mice and solid tumor mouse model were used to observe the toxicity and anti-tumor activity of different structure types of ACGs, respectively, for analysis of the toxicity-activity relationship in vivo.4. Rats were given bullatacin three weeks by tail vein intravenous. The heart, liver and kidney tissues of rat were observed using light microscopy and electron microscopy to analyze the toxicity of bullatacin in rats. The mitochondrial complex I activity, calcium and ROS fluorescence intensity, ATP content, Bax, Bcl-2and case-3expression, mitochondrial DNA damage, mitochondrial ND1and ND2gene and protein expression were detected to analyze the molecular mechanisms of toxic effects of bullatacin in rats.5. Process the plasma samples of bullatacin by liquid-liquid extraction, and analyze the pharmacokinetics of bullatacin in rats by tail vein intravenous using HPLC-MS.Results:1. Thirty compounds were isolated from seeds of Annona squamosa. The structures of22ACGs were identified using IR, UV, MS and NMR. Twelve were new compounds and their absolute configuration was determined using Mosher method. The main components of the fatty oil were fatty acids (80.9%), of which the contents of unsaturated fatty acids reached51.1%.2. Isolated ACGs were structurally divided into three types:mono-THF (tetrahydrofuran), adjacent bis-THF and nonadjacent bis-THF ACGs. MTT assay results showed that different structure types of ACGs have a strong inhibitory activity against tumor cell growth. Structure-activity relationship analysis showed that among mono-THF and adjacent bis-THF ACGs, compounds with less carbon numbers between THF ring and lactone ring exhibited better activity. ACGs bearing three substituted hydroxyl (total carbon number is37, molecular weight is622) exhibited strongest activity among adjacent bis-THF and nonadjacent bis-THF ACGs. There is no definite relationship between the position of substituted4-OH and the activity of adjacent bis-THF and nonadjacent bis-THF ACGs.3. Toxicity-activity relationship of three types of ACGs in vivo showed that adjacent bis-THF ACG had higher anti-tumor activity and toxicity than mono-THF and nonadjacent bis-THF ACGs.4. Rats were treated with bullatacin by tail vein intravenous. Results showed that ALT, AST, BUN and CR activity of300μg/kg dose group was increased compared with control group. Liver and kidney tissues of300μg/kg dose group presented lesions through light microscope and electron microscope detection. Compared with control group, liver mitochondrial complex I activity of all bullatacin treated groups were decreased in a dose-dependent manner. Besides, calcium, ROS concentrations of heart, liver and kidney tissues and Bax and case-3expression and Bax/Bcl-2ratio in liver and kidney tissues were increased. ATP contents of heart, liver and kidney tissue of300μg/kg dose treated rat were decreased compared with control group. Mitochondrial DNA PCR amplification of liver and kidney tissues was subjected to agarose gel electrophoresis and amplified a4.7Kb and0.86Kb stripe, another size of approximately1.5Kb bands amplified in rat kidney tissue samples of bullatacin treated group. Compared with control group, ND1and ND2gene expression in rat liver and kidney in bullatacin treated group were increased. ND1and ND2protein expression in liver tissue was decreased, and ND1, ND2protein expression in renal tissue increased.5. Liquid-liquid extraction and determination method by HPLC-MS technique of serum sample with bullatacin treatment were established. The stability, extraction recovery and matrix effect involved in the method were also validated. This method was applied to measure the plasma bullatacin concentrations after a single tail vein intravenous.Conclusions:22ACGs (12new ACGs) including mono-tetrahydrofuran, adjacent bis-and nonadjacent bis-tetrahydrofuran ACGs were isolated from Annona squamosa seeds. The position of tetrahydrofuran ring and its adjacent substituted hydroxy, the number and position of substituted hydroxyl have a certain impact of anti-tumor activity of ACGs in vitro. Compared with mono-THF and nonadjacent bis-THF ACGs, adjacent bis-tetrahydrofuran ACG has better anti-tumor activity in mice, but exhibit higher toxicity.300μg/kg dose of bullatacin for three weeks in rats showed some toxicity effects. Compared with control group, bullatcin caused blood biochemical parameters increased and liver and kidney tissues pathological changes. Bullatacin led to decrease in mitochondrial complex I activity and ATP content, and increase in calcium, ROS concentration, Bax and case-3expression and Bax/Bcl-2ratio, and mitochondrial DNA of rat liver and kidney damage with a certain impact of mitochondrial ND1and ND2gene protein expression in liver and kidney tissues of rats. Liquid-liquid extraction and HPLC-MS techniques can be used to prepare serum samples with bullatacin and determine its blood samples content in rats.