In order to identify the source of Citrus grandis and evaluate its quality originate from two areas comprehensively,DNA barcode was used to identify 26 samples of C. grandis. The content of naringin,rhoifolin,naringenin and apigenin was determined by UPLC method,and the color difference was numerically studied by color difference analyzer,which was related to the effective components of C. grandis. The results showed that samples was the source of C. grandis in both regions. The ITS2 sequence length was about400-500 bp,and the sequence similarity reached 99. 82%. There was only one base deletion in the two groups. There was one base A in some medicinal materials of Guangdong at 330 bp,but no base in Chongqing. The contents of naringin and rhoifolin in Chongqing samples were higher than those in Guangdong samples,and there were statistical differences between naringenin and apigenin. The chroma value showed that L*value of Guangdong was larger,a*value was smaller,L*value of Chongqing was smaller,and a*value was larger,while the b*value of both was not significantly different; The results of correlation analysis showed that naringin,rhoifolin,naringenin were positively correlated with L*,b*value,negatively correlated with a*value,and apigenin had no correlation with L*,a*,b*value. In this study,the scientific identification and evaluation of C. grandis was carried out to provide a new idea for the further study of the rapid identification and evaluation of C. grandis.
Apigenin ; Citrus/genetics* ; DNA Barcoding, Taxonomic ; Drugs, Chinese Herbal

The present study established the spectrum-effect relationship model of flavonoids in Citri Reticulatae Pericarpium(CRP) from 15 batches of Liujunzi Decoction and statistically analyzed the correlation between chemical peaks and efficacy to identify the main effective components. HPLC fingerprints of flavonoids in CRP from 15 batches of Liujunzi Decoction were established. HPLC analysis was carried out on the Venusil XBP C_(18)(L) column(4.6 mm×250 mm, 5 μm) at 30 ℃ with acetonitrile-water(containing 0.1% formic acid) as mobile phase for gradient elution, a flow rate of 1.0 mL·min~(-1), and detection wavelength of 300 nm to obtain chemical fingerprints. Additionally, the effects of flavonoids from CRP in 15 batches of Liujunzi Decoction on the content of GAS, MTL, and VIP, TFF3 mRNA expression, and percentage of CD3~+ T-cells of model rats with spleen deficiency were determined. The spectrum-effect relationship model was established by gray correlation analysis. The results showed that the main characteristic peaks with great contribution to the regulation of gastrointestinal tract were peak 16(vicenin-2), peak 63(sinensetin), peak 64(isosinensetin), peak 65(nobiletin), peak 67(3,5,6,7,8,3',4'-heptemthoxyflavone), peak 68(tangeretin), and peak 69(5-desmethylnobiletin). Therefore, there was a linear correlation between flavonoids from CRP in Liujunzi Decoction and the efficacy, and the medicinal effect was achieved by multi-component action. This study is expected to provide a new idea for exploring the material basis of the effect, i.e., regulating qi prior to replenishing qi, of CRP in Liujunzi Decoction.
Animals ; Citrus/chemistry* ; Drugs, Chinese Herbal ; Flavonoids/pharmacology* ; Hormones ; RNA, Messenger/genetics* ; Rats ; Spleen

In order to study the transcriptional differences of Citrus medica var. sarcodactylis at different developmental stages, we explored the genes regulating the biosynthesis of the effective components. In this study, Illumina Hiseq 4 000 high-throughput sequencing technology was used to sequence the transcriptome of C. medica var. sarcodactylis at different developmental stages, 121 235 unigenes were obtained with an average length of 2 434 bp, 3 379 different genes were obtained using DESeq screening, which mainly connected to biological processes such as signal transmission, biological regulation, and metabolic processes, and enriched in metabolic pathways such as starch, sucrose metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. Further dynamic comparison of biosynthesis related genes of active ingredients: the expression levels of PAL, CHI, CYP75B1, ZDS, 4CL and FLS gradually increased as the fruit turned from green to yellow; the expressions of COMT, F3H and CYP73A increased at first and then decreased; CCR, HCT and HRP were down-regulated whereas up-regulated. This study provides references for further excavation of key genes in the biosynthesis of active components, as well as biopathway analysis of active components for C. medica var. sarcodactylis.
Citrus/genetics* ; Computational Biology ; Fruit/genetics* ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; High-Throughput Nucleotide Sequencing ; Transcriptome

OBJECTIVETo screen out active substances on Neuromedin U2 receptor (NMU2R) by using stable NMU2R cell lines and negative cell lines and analyzing siRNA interference.

METHODNMU2R cells were used to observe the activating effect of nine nine citrus flavonoids on NMU2R cell. Afterwards, false-positive interference of citrus flavonoids that showed higher activating effect was eliminated by using negative cells and analyzing the efficiency of siRNA interference.

RESULTHesperidin and nobiletin contained in citrus flavonoids were found to effectively activate NMU2R. The efficacy, EC50 and potency values of hesperidin were 4.688, 318.970 micromol x L(-1) and 200.933 micromol x L(-1), while the efficacy, EC50 and potency values of nobiletin were 4.758, 5.832 micromol x L(-1) and 3.124 micromol x L(-).

CONCLUSIONHesperidin and nobiletin contained in citrus flavonoids can activate NMU2R. Nobiletin shows such a low EC50 that it has medicinal value.

Cell Line ; Citrus ; chemistry ; Flavonoids ; pharmacology ; Gene Expression ; drug effects ; Humans ; Plant Extracts ; pharmacology ; RNA Interference ; drug effects ; RNA, Small Interfering ; genetics ; metabolism ; Receptors, Neurotransmitter ; genetics ; metabolism

OBJECTIVEA total of 27 samples belonging to 5 cultivars of Fructus Aurantii (Citrus aurantium), i. e. cv. Xiucheng, cv. Xiangcheng, cv. Lecheng, cv. Jizicheng, and cv. Youzicheng, collected at Changfu and Huanggang, Zhangshu City, Jiangxi Province, were assayed to reveal the genetic relationship among the cultivars and the accordance between morphological and molecular markers.

METHODCultivar identification was based on morphology and cultivar relationship was based on Inter-simple sequence repeats (ISSR).

RESULTTwenty out of 40 ISSR primers screened generated 392 loci across all 27 samples with 315 informative loci. The UPGMA dendrogram showed that samples within cv. Xiucheng and cv. Xiangcheng from Changfu were closely related. However, samples of cv. Lecheng, cv. Jizicheng and cv. Youzicheng from Huanggang, or cv. Xiucheng and cv. Xiangcheng from both Changfu and Huanggang did not exhibited close relationships within each cultivars.

CONCLUSIONBased on morphology the same cultivar grown in different plantations, or even within a single plantation sometimes do not show close genetic relationship, indicating diverse origin of the cultivars. Synonyms or homonyms are believed to common phenomenon in Fructus Aurantii production. To solve the problem ISSR markers can serve a kind of molecular markers which are preferable to partition genetic variations within and between cultivars and to establish genetic relationships among them.

Citrus ; anatomy & histology ; classification ; genetics ; DNA Primers ; DNA, Plant ; genetics ; Fruit ; anatomy & histology ; genetics ; Genetic Markers ; Genetic Variation ; Phylogeny ; Plants, Medicinal ; anatomy & histology ; classification ; genetics ; Repetitive Sequences, Nucleic Acid

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 10(6) conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.
Agrobacterium tumefaciens ; genetics ; Base Sequence ; Citrus ; microbiology ; DNA Primers ; genetics ; DNA, Bacterial ; genetics ; DNA, Fungal ; genetics ; DNA, Recombinant ; genetics ; Drug Resistance, Fungal ; genetics ; Hygromycin B ; pharmacology ; Molecular Sequence Data ; Mutagenesis, Insertional ; Penicillium ; drug effects ; genetics ; pathogenicity ; Plant Diseases ; microbiology ; Plasmids ; genetics ; Transformation, Genetic

OBJECTIVETo observe the effect of pure total flavonoids from Citrus (PTFC) on the hepatic fatty degeneration, inflammation, oxidative stress and SIRT1/PGC-1alpha expressions in mice with non-alcohol steatohepatitis (NASH), and discuss the action mechanism of PTFC on NASH.

METHODMice were given high-fat diet for 16 weeks to induce the NASH model. Since the seventh week after the model establishment, the mice were intervened with 100, 50 and 25 mg x kg(-1) x d(-1) PTFC for 10 weeks. The pathologic changes in hepatic tissues were observed with HE staining. The contents of TG, CHOL in hepatic tissue, as well as the levels of AST, ALT in serum were detected by using the biochemical process. The expression of SIRT1, PGC-1alpha and MnSOD mRNA in hepatic tissues were detected with Real-time PCR assay. SIRT1, PGC-1alpha protein and 8-OHdG expressions were determined with the immunohistochemical method. The SOD level in hepatic tissues was tested by the xanthine oxidase method. The MDA content in hepatic tissues was examined by the thiobarbituric acid method.

RESULTThe contents of TG, CHOL, NAFLD activity scores and ALT level in serum in hepatic tissues of mice in the model induced by fat-rich diet were obviously higher than that of the normal group (P < 0.010. The SIRT1, PGC-1alpha, MnSOD mRNA and protein expression in hepatic tissues were significantly lower than that of the normal group (P < 0.01). The expression of 8-OHdG and the content of MDA in hepatic tissues were obviously higher than that of the normal group (P < 0.01). After the intervention with different doses of PTFC, the NAFLD activity scores, the content of TG and the level of AST in serum were notably lower than that of the normal group (P < 0.01, P < 0.05); whereas the SIRT1, PGC-1alpha, MnSOD mRNA and protein expression were obviously higher than that of the normal group (P < 0.01, P < 0.05), with the significant decrease in the expression of 8-OHdG and the content of MDA (P < 0.01).

CONCLUSIONOxidative stress/lipid peroxidation enhancement in in NASH mice induced by high-fat diet may be related to the changes in SIRT1/PGC-1alpha signal transduction pathway. PTFC could enhance the anti-oxidant capacity in liver, relieve the damage of reactive oxygen during the fatty acid metabolic process, and prevent NASH from the occurrence and development by regulating the SIRT1/PGC-1alpha signal pathway.

Animals ; Citrus ; chemistry ; Fatty Liver ; drug therapy ; genetics ; metabolism ; Flavonoids ; chemistry ; pharmacology ; Inflammation ; drug therapy ; genetics ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease ; Oxidative Stress ; drug effects ; genetics ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Sirtuin 1 ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism

OBJECTIVETo observe the inhibitory effect of citrus extract nobiletin on K562 cells xenograft in nude mice and discuss its anticancer activity and mechanism.

METHODThe model of K562 cells xenograft was established in nude mice. Twenty-five nude mice were divided to five groups. After 24 hrs of inoculation with K562 tumor cells subcutaneously, 1% CMC-Na in the nude mice of model control group, nobiletin (12.5, 25, 50 mg x kg(-1)) in the nude mice of nobiletin groups and CTX (20 mg x kg(-1)) in positive control group were administered once every day. The nude mice were killed at 18th day-point of administration. The inhibitory rate of nobiletin on tumor was calculated according to the measured tumor weight. Immunohistochemistry assay was used to determine the effect of nobiletin on VEGF expression and MVD, and CAM assay was used to detect the effect of nobiletin on vessel regeneration.

RESULTNobiletin have notable inhibition on K562 cells xenograft in nude mice comparing with model control group (P < 0.01), the inhibitory rate of nobiletin groups were 36% -58%. The results of immunohistochemical technology showed that the expression of VEGF in nobiletin groups decreased significantly comparing with the model control group (P < 0.05, P < 0.01, P < 0.01). Nobiletin could remarkably decrease the angiogenesis within tumor tissues. The expression of CD34 in nobiletin low dose group and high dose group was lower than that in model control group (P < 0.05, P < 0.01). The result of CAM indicated that 4 microg and 2 microg nobiletin could inhibit the new blood vessels of CAM (P < 0.01).

CONCLUSIONNobiletin inhibited the tumor growth and angiogenesis by reducing the VEGF expression of K562 cells xenograft in nude mice.

Angiogenesis Inhibitors ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; Citrus ; chemistry ; Disease Models, Animal ; Flavones ; pharmacology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; K562 Cells ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neoplasms ; drug therapy ; genetics ; metabolism ; Transplantation, Heterologous ; Vascular Endothelial Growth Factors ; genetics ; metabolism