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Objective: To study the glycosides from the 70% ethanol extract of Lonicera macranthoides. Methods: The compounds were isolated and purified by column chromatography of HP-20 macroporous resin, silica gel, ODS, Sephadex LH-20, and semi-preparative RP-HPLC. Their structures were elucidated by physicochemical properties and spectral analyses. Results: Eight compounds were isolated and identified as 7,3’,4’-trimethoxylquercetin-3-O-α-L-arabinadosyl-(1→6)-O-β-D-glucopyranoside (1), 7,3’,4’-trimethoxylquercetin-3-O-rutoside (2), quercetin-3-O-β-D-glucopyranoside (3), (2E,6S)-8-[α-L-arabinopyranosyl-(1″→6’)- β-D-glucopyranosyl]-2,6-dimethyloct-2-eno-1,2″-lactone (4), kankanoside E (5), betulalbuside A (6), shomaside F (7), and amarantholidoside V (8), respectively. Conclusion: Compound 1 is a new compound named methoxylquercetinside, while compounds 5-8 are isolated from the genus of Lonicera for the first time.
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Objective To clone the full length of LmPAL1 gene and analyze bioinformatics and expression patterns from Lonicera macranthoides. Methods The total RNA of L. macranthoides was extracted. The full-length cDNA sequence of LmPAL1 gene was cloned by RT-PCR and RACE technique; The genome sequence in bioinformatics was analyzed by using the relevant software; The relative expression of the gene in stem, leaf, and different flower period was determined by using real-time PCR. Results The cloned LmPAL1 gene open reading frame (ORF) was 2 145 bp, encoding 714 amino acids. It was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts, containing PAL shielding structure domain (527-641 aa). This gene contained PAL/HAL active center sequence GTITASGDLVPLSYIAG (196-212 aa), which was highly similar to other phenylalanine ammonia-lyase. Real-time PCR results showed that the relative expression level of golden yellow flowering flower was higher in seven florescence periods. When comparing the stem, leaf, and white flower bud period, the relative expression of flower was the highest and the leaf was the lowest. Conclusion In this study, PAL1 gene of L. macranthoides was cloned successfully, laying a foundation for further study of the function of this gene and genetic improvement of L. macranthoides quality and providing the research basis for exploring the biosynthesis and regulation of chlorogenic acid in L. macranthoides.
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Objective To clone ACS3 gene from wild type and ‘Xianglei’ cultivar of Lonicera macranthoides, respectively, followed by bioinformatics analysis and detection of spatio-temporal expression pattern. Methods Unigene sequence which is highly homologous with ACS protein from the transcriptome database of L. macranthoides was screened, the primers were designed based on it to amplify the full length of the Unigene by qRT-PCR and RACE techniques; Bioinformatics tools were used to analyze and identify the physicochemical property, conserved domain and gene homology of ACS3 proteins; Finally, qRT-PCR technique was used to detect the gene expression patterns of different species of L. macranthoides. Results Lm-ACS3 (GenBank: MH724196) and Lm-XL-ACS3 (GenBank: MH724197) were isolated from wild type and ‘Xianglei’ cultivar of L. macranthoides, respectively. The length of open reading frame (ORF) were all 1 452 bp, encoding 483 amino acids, containing the conserved Aminotran_1_2 structural domain, which were highly similar to the ACC synthase of other plants; And qRT-PCR results showsed that the expression quantity of ACS3 gene in wild L. macranthoides changed significantly at different blossoming stages, the overall trend was upward from flowering stage 3, while the expression difference between the flowering stages was relatively small in “Xianglei” cultivar. Conclusion Lm-ACS3 and Lm-XL-ACS3 gene were separately obtained from L. macranthoides and L. macranthoides ‘Xianglei’ cultivar, the expression patterns of ACS3 in this two varieties were different; It’s speculated that ACS3 gene might be a possible functional gene that causing different phenotypes of two strains of L. macranthoides, it provides theoretical basis for further verifying the biological function of ACS3 gene in regulating flower’s bud duration and phenotype.
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Objective To investigate the effects of different processing methods on the appearance, quality components and processing efficiency of Lonicera macranthoides that produced in Guizhou Province, in order to provide basis for the optimization of processing methods. Methods Eleven processing methods were used such as the traditional method group (sun drying, sun drying after steaming, drying in shade, and drying in shade after steaming), constant temperature drying group (50, 60, 70, and 80 ℃) and variable temperature drying group (50→60 ℃, 50→70 ℃, 50→80 ℃). Then the indicators such as color, aroma, moisture, total ash, acid insoluble ash, chlorogenic acid, total saponins, processing time, and drying rate were measured. Results According to all the indicators, the comprehensive correlation degree of the 50 ℃ and the variable temperature drying method was between 0.94 and 0.96, which was much higher than other methods, and the comprehensive correlation degree of 50→60 ℃ degrees was the highest, reaching 0.959 8. Conclusion In conclusion variable temperature drying method is a better processing method for L. macranthoides. This method can be used as an important technical means for large-scale processing.
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Objective: To clone the AP1 (Lm-XL-AP1) gene from a Special Variant Varieties of Lonicera macranthoides "Xianglei", and to analyze its bioinformatics and spatio-temporal expression. Methods: Amplifing the full length of Lm-XL-AP1 gene by RACE technique, using bioinformatics method to analyze homology and similarity of the gene, predicting the coding protein and analyzing the various physical and chemical properties. The expression of the gene in different parts of Lonicera macranthoides Special Variant Varieties was detected by fluorescence quantitative PCR (qRT-PCR). Results: The AP1 gene, containing a 729 bp ORF that encoding 242 amino acids, was cloned. And the similarity of the gene compared with the AP1 gene from the MADS-box gene family of Chrysanthemum lavandulifolium up to 80% (Containing a conserved sequence of MADS and K-box). Without transmembrane domain, AP1 was located in cell nucleus. It is expressed in various organs of Lonicera macranthoides Special Variant Varieties. Conclusion: For the first time, the AP1 gene which may be involved in the control of the expression of floral organ was cloned from the total RNA of Lonicera macranthoides Special Variant Varieties.
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Objective To study the chemical constituents from the flower buds of Lonicera macranthoides and their antitumor activities. Methods The constituents were separated by chromatography of silica gel, ODS, Sephadex LH20, and semi-pre HPLC. Their structures were elucidated by spectral means. The in vitro cytotoxic activities of the isolated compounds were studied by MTT method. Results Seven compounds were isolated and identified as 3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranosyl- hederagenin 28-O-β-D-glucopyranosyl ester (1), 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-oleanolic acid 28-O-α- L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (2), 3-O-α-L-rhamnopyranosyl-(1→2)-α-L- arabinopyranosyl-hederagenin 28-O-β-D-glucopyranosyl ester (3), 3-O-α-L-rhamnnopyranosyl-(1→2)-α-L-arabinopyranosyl- hederagenin 28-O-α-L-rhamnopyransyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4), 3-O-α-L-arabinopyranosyl- hederagenin 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (5), 3-O-β-D-glucopyra- nosyl-(1→4)-α-L-arabinopyranosyl-hederagenin 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside ester (6), and 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-hederagenin 28-O-α-L-rhamno- pyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (7). Conclusion Compound 1 is a new compound named macranthoidin C, and compounds 2-7 are isolated from L. macranthoides for the first time. Compounds 1, 4, and 5 show cytotoxicities against HeLa cells with IC50 of 54.3, 43.9 and 61.2 μmol/L, respectively.
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Objective: To clone the MADS-box gene denoted AGL15 from total RNA of Lonicera macranthoides based on previous transcriptome sequencing data and analyze the bioinformatics and expression of the gene. Methods: The gene containing intact open reading frame (ORF) was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The similarity comparison and homology analysis on the sequence were carried out using bioinformatic method, the coding protein was predicted and the physicochemical properties were analyzed. The expression of the gene in different locations of L. macranthoides was determined by semiquantitative PCR using gene-specific primers. Results: The Lm-AGL15 gene, containing a 795 bp ORF that encoded 264 amino acids, was cloned. The deduced protein sequence had the most similarity to the AGL15 in Vitis vinifera and exhibited two conserved motifs (MADS and K-BOX). Without transmembrane domain, Lm-AGL15 was located in cytoplasm and expressed only in each part. Conclusion: For the first time from L. macranthoides cloning could prolong the period of bud of gene, analysis of the gene expression in different parts of L. macranthoides which would provide a reference for the study of prolonging L. macranthoides bud stage.
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Objective: To screen the reference genes of Lonicera macranthoides for gene expression analysis and to study the spatio-temporal expression characteristics of LmAGL15 which was a member of Mads-Box family. Methods: In this study, 18 S rRNA, Ubiquilin, Actin and Efl-β of L. macranthoides were cloned and the stabilities of the four housekeeping genes were evaluated in different positions (leaves, stems, and buds) and different periods of bud development. In addition, the spatio-temporal expression of LmAGL15 gene was analyzed. Results: 18 S rRNA was the most suitable reference gene for spatio-temporal expression analysis in L. macranthoides; The relative expression of LmAGL15 was low in leaves and stems, and that in buds was higher. Conclusion: 18 S rRNA is the most suitable reference gene in L. macranthoides. The relative expression of LmAGL15 changes significantly in leaves, stems, and buds.
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OBJECTIVE: To study the immunomodulatory and the protection of experimental liver injury of Lonicera macranthoides Hand: Mazz (LHM) which is the main source of Lonicera confusa DC. METHODS: Immunocompromised mice model induced by intraperitoneal injection of cyclophosphamide and in vitro proliferation experiment of its peritoneal macrophages and spleen lymphocytes were used to evaluate the immunomdulatory effect of LHM. The inhibition of LHM on hepatic injury induced by CCl4 or alcohol on mice was measured. RESULTS: The doses of 1.6, 3.2 and 6.4 g · kg-1 of LHM could significantly increased the thymus index, the spleen index, the carbon dissection index and the macrophage phagocytic index of mice (P<0.01), and obviously raised the proliferation ability of peritoneal macrophages and transformation ability of splenic lymphocytes (P<0.01): The doses of 3.2 and 6.4g · kg-1 of LHM could significantly lower the incremental ALT, AST and MDA level (P<0.01), raise the low SOD level in liver (P<0.01) of hepatic injury mice induced by CCl4 or alcohol, and obviously lighten the pathological damage of hepatic tissue. CONCLUSION: The LHM could significantly promote the proliferation and phagocytosis function of immunocytes on immunocompromised mice. Moreover, it has good protection effect on hepatic injury of mice which is induced by CCl4 or alcohol.