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Objective: To explore the high-efficiency green environmental extraction method of total flavones from Microcos paniculata (MPTF), and investigate its lipid-lowering activity. Methods: The ionic liquid was used to assist the ultrasonic extraction of MPTF, and the extraction process was investigated by single factor experiment and orthogonal test. Wistar rats were randomly divided into normal control group and positive control group (Resuvastatin Calcium Tablets 5.2 mg/kg) by high fat diet, high-fat model group and MPTF low, medium and high dose groups (ig dose MPTF 300, 600, 900 mg/kg), with 10 rats in each group. The total cholesterol (TC), glycerol lipid (TG), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) levels were determinated. Results: The optimum process conditions for ultrasonic-assisted extraction of MPTF by ionic liquid were as follow: ionic liquid was [BMIM]Cl at concentration of 0.30 mol/L; ratio of material to liquid was 1:40, extraction solvent was 60% ethanol aqueous solution with extraction time of 30 min at 50 ℃. The verification test results showed that the extraction rate of total flavonoids obtained from the extraction process was high and the process was stable. The results of lipid-lowering test showed that the levels of TC, TG and LDL-C were decreased in all doses of MPTF groups, and the level of HDL-C was increased (P < 0.05). With the increase of MPTF dose, the indicators showed obvious trends in a dese-dependent manner. Conclusion: The ionic liquid combined with ultrasonic- assisted extraction of MPTF is stable and feasible, which provides reference for ionic liquid synergistic ultrasound-assisted extraction of poorly soluble active ingredients in Chinese materia medica. MPTF extract has better lipid-lowering effect.
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The shrub Microcos paniulata (MPL; Tiliaceae), distributed in south China, south and southeast Asia, yields a phytomedicine used to treat heat stroke, fever, dyspepsia, diarrhea, insect bites and jaundice. Phytochemical investigations on different parts of MPL indicate the presence of flavonoids, alkaloids, triterpenoids and organic acids. The MPL leaves, fruits, barks and roots extracts showed antidiarrheal, antimicrobial and insecticidal, anti-inflammation, hepatoprotective, cardiovascular protective, blood lipids reducing, analgesic, jaundice-relieving and antipyretic activities, etc. The review aims to summary the traditional uses, botany, phytochemistry, pharmacological bioactivity, quality control, toxicology and potential mechanisms of MPL. Additionally, this review will highlight the existing research gaps in knowledge and provide a foundation for further investigations on this plant.
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Objective To obtain the key enzyme gene involved in flavone C-glycosides biosynthesis pathway, a flavanone 2-hydroxylase (F2H) gene was cloned from Microcos paniculata, and its bioinformatics analysis and gene expression pattern were also performed. Methods The specific primers were designed according to Unigene in F2H annotated in the transcriptome data of M. paniculata. The open reading frame (ORF) of MpF2H gene was amplified by PCR. Then the PCR product was purified and ligated to pET30a, and finally a prokaryotic expression vector pET30a-MpF2H was constructed. The bioinformation of F2H gene cDNA sequences was analyzed by some online tools. Using RT-qPCR with suitable primers, the quantitative expression analysis of MpF2H gene in different tissues, namely, buds, leaves, twigs, flowers and fruits was carried out. Results The length of MpF2H gene ORF was 1 557 bp (GenBank accession number KY652921), which encoded a protein with 518 amino acid residues, relative molecular weight of 54 500, theory isoelectric point of 5.49. In which was no transmembrane domain. It was hypothesized that this protein located in chloroplast. MpF2H gene was expressed in different tissues, with the highest expression in leaves and the lowest expression in twigs and flowers. Conclusion The expression of MpF2H gene varied widely in different tissues. The MpF2H gene was cloned from M. paniculata based on pET30a-MpF2H expression vector. This study will provide the fundamental information for the further preparation and functional research of MpF2H protein in flavone C-glycosides biosynthesis pathway.
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Objective To clone the full-length cDNA of MpCHS and analyze its expression pattern in different parts of Microcos paniculata and different growth periods of M. paniculata leaves. Methods Based on the transcriptome data of M. paniculata, we designed specific primers for MpCHS gene. The full-length cDNA of MpCHS was amplified by PCR and the positive clones were then sequenced, analyzed, and constructed prokaryotic expression vector. The bioinformatics analysis of MpCHS was also performed. Meanwhile, the mRNA expression of MpCHS was detected using real-time quantitative PCR. Results The relative molecular mass was 42 700, and its theoretical isoelectric point was 6.11, with three conserved functional active sites (165 C, 304 H, and 337 N) of the CHS family proteins and the tag sequence of RLMMYQQGCFAGGTVLR and GVLFGFGPGL. Phylogenetic tree analysis showed that MpCHS had close relationship with woody plants such as cocoa and upland cotton. We successfully cloned the full-length cDNA of MpCHS (GenBank: KY472608). It had an ORF of 1 176 bp which encoded a protein of 391 amino acid residues. RT-qPCR results showed that MpCHS was expressed in all parts of M. paniculata and its expression in leaves was gradually decreased along with its development. Conclusion MpCHS is cloned from M. paniculate for the first time, and the gene expression pattern of MpCHS in different parts of M. paniculata and different growth periods of M. paniculata leaves was analyzed. This study facilitates the further purification and functional validation of MpCHS protein and provides reference for further analysis of flavonoids biosynthesis pathway in M. paniculata.
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Objective: To investigate the relationship between variations of chloroplast DNA psbA-trnH and nuclear ribosomal DNA ITS2 sequences and the geographical origins of Microcos paniculata, and seek for some evidence for its germplasm resources evaluation and molecular identification. Methods: Total genomic DNA was extracted from M. paniculata and the psbA-trnH and ITS2 sequences were amplified by PCR and directly sequenced. The raw data were dealt with DNAMAN 8.0 and the pairwise distance was calculated based on Kimura 2-parameter model using MEGA 6.0. The phylogenic tree was constructed by Neighbor-Joining method. Results: The ITS2 sequences of M. paniculata from different populations were 462 bp with 14 variable sites. The nucleotide divergence between ITS2 sequences in pairwise comparison was calculated and the result showed that the distances between M. paniculata populations is about 0.000 0 - 0.019 8. The sequences length of psbA-trnH was 387 bp, except for one isolated from Jinghong, Yunnan province which was 379 bp. Conclusion: The psbA-trnH sequences of M. paniculata are more conservative than the ITS2 sequences, which can effectively identify M. paniculata from its adulterants and the closely relate species. The ITS2 sequence is so sensitive with the geographical variation that it can show the different characteristics of different populations in molecular level.
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Objective: To study the chemical constituents from the effective fraction of the leaves of Microcos paniculata, which can protect acute myocardial ischemia injury induced by isoprenaline. Methods: The chemical constituents were isolated and purified by combined using chromatography on silica gel, and Sephadex LH-20 column, and preparative HPLC. The structures of the isolated compounds were elucidated by NMR and MS spectral data. Results: Eleven compounds were isolated from the effectively protective fraction with the protection on acute myocardial ischemia injury and identified as vitexin (1), isovitexin (2), isorhamnetin (3), kaempferol (4), quercetin (5), 5, 6, 7, 8, 4'-pentamethoxyflavone (6), nobiletin (7), vanillic acid (8), caffeic acid (9), ferulic acid (10), and erucicamide (11). Conclusion: Compounds 6, 7, 9 and 11 are isolated from the plants in Microcos Linn. for the first time. Compound 11 is the firstly reported fatty acid amide and compounds 6 and 7 are the firstly reported polymethoxylated flavonoids with the obvious anti-inflammatory activity in this genus.
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Objective: To study the protective effect of the total flavones in Microcos paniculata (TFMP) on the acute myocardial ischemia (AMI) induced by isoprenaline (ISO) and its mechanism. Methods: The rats were ig administered with TFMP (8, 4, and 2 mg/kg) once daily for consecutive 5 d, and the AMI rat model was established by sc injection with ISO (2 mg/kg) 1 h after the last administration. The effects of TFMP on the electrocardiogram (ECG) at different time points and the myocardial tissue pathological histomorphology dying by hematoxylin were observed; The superoxide dismutase (SOD) and malondialdehyde (MDA) levels of myocardial tissue, glutathione peroxidase (GSH-Px), lactate dehydrogenase (LDH), and creatine kinase (CK) activities in serum were detected using biochemical method. Results: Compared with the model group, the high- and mid-doses (8 and 4 mg/kg) TFMP inhibited the J spot downward of ECG during myocardial ischemia (P < 0.05), especially after 10 min of ISO injection. The myocardial injury induced by ISO was aslo improved in these two groups, the levels of LDH and CK in serum (P < 0.05) and the content of MDA in myocardial homogenate (P < 0.01) were decreased, the activities of SOD (P < 0.01) and GSH-Px (P < 0.05, 0.001) in the myocardial homogenate were increased. Conclusion: The TFMP has the apparent protective effect on AMI injury, its mechanism may be related with improving the myocardial anti-oxidative ability and decreasing the oxidative stress reaction.