ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.