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Effect of an aqueous extract of dried immature fruit of Poncirus trifoliata (L.) Raf. on intestinal transit in rodents with experimental gastrointestinal motility dysfunctions.

The effect of an aqueous extract of the dried immature fruit of Poncirus trifoliata Raf. (Rutaceae) (PF-W) on gastrointestinal (GI) motor function was investigated by measuring the intestinal transit rate (ITR) of Evans blue in rats and mice with experimental GI motility dysfunctions (GMDs). GMD was induced by appropriate surgery or by the administration of acetic acid, atropine, L-DOPA, or morphine to the animals. The ITR in these GMD animals was significantly retarded compared to normal animals. The retardation, however, was significantly inhibited by the intragastric administration of PF-W (0.1-1g/kg) in a dose dependent manner for all GMD models except for L-DOPA-induced GMD. The above results suggest that PF-W has the potential for development as a prokinetic agent that may prevent or alleviate GMD in human patients.

Animals↗

Anti-inflammatory effect of Poncirus trifoliata fruit through inhibition of NF-kappaB activation in mast cells.

Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 with immune regulatory properties. We investigated the effect of the fruits of Poncirus trifoliata (L.) Raf (Rutaceae) (FPT) on expression of pro-inflammatory cytokines by activated human mast cell line, HMC-1. FPT dose dependently decreased the gene expression and production of TNF-alpha and IL-6 on phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated HMC-1 cells. In addition, FPT attenuated PMA and A23187-induced activation of NF-kappaB indicated by inhibition of degradation of I kappa B alpha, nuclear translocation of NF-kappaB, NF-kappaB/DNA binding, and NF-kappaB-dependent gene reporter assay. Our in vitro studies provide evidence that FPT might contribute to the treatment of mast cell-derived allergic inflammatory diseases.

Anti-Inflammatory Agents↗

Effect of Poncirus fructus on stem cell factor-induced mast cell migration.

Mast cell hyperplasia can be causally related with chronic inflammation. Stem cell factor (SCF), the ligand of the c-kit protooncogene product, is a major regulator and chemoattractant of mast cells. Poncirus fructus (PF) has been used against allergic diseases for generations in South Korea. PF (1 mg ml(-1)) significantly inhibited the SCF-induced migration of rat peritoneal mast cells (RPMCs). RPMCs exposed to SCF (50 ng ml(-1)) resulted in a drastic shape change with a polarized morphology while the cells exposed to PF (1 mg ml(-1)) remained resting, with little or no shape alteration. The drastic morphological alteration and distribution of polymerized actin were blocked by pretreatment with PF. In addition, PF inhibited both TNF-alpha and IL-6 secretion from RPMCs stimulated with SCF. Our findings provide evidence that PF inhibits chemotactic response and inflammatory cytokines secretion to SCF in mast cells.

Actins↗

Passive cutaneous anaphylaxis-inhibitory activity of flavanones from Citrus unshiu and Poncirus trifoliata.

This study examined the passive cutaneous anaphylaxis-inhibitory activity of the flavanones isolated from the pericarp of Citrus unshiu (Family Rutaceae) and the fruit of Poncirus trifoliata (Family Rutaceae). Naringenin, hesperetin and ponciretin potently inhibited IgE-induced beta-hexosaminidase release from RBL-2H3 cells and the PCA reaction. Among the flavanones examined, naringenin was the most potent with an IC50 value for beta-hexosaminidase release from RBL-2H3 cells of 0.029 mM. Intraperitoneally administered naringenin (5 mg/kg) inhibited PCA by 70 +/- 1.7 %, compared with the control group. The inhibitory activity of naringenin was found to be comparable to that of azelastine, which is a commercially available antiallergic drug. However, their glycosides, hesperidin, naringin and poncirin, did not inhibit the in vitro release of beta-hexosaminidase from the RBL-2H3 cells. On the other hand, these flavanones did not improve the oxazolone-induced dermatitis in the mouse ears. When the flavanone glycosides were administered to rats, the aglycones, but not the flavanone glycosides, were excreted in urine. This suggests that the flavanone glycosides can be activated by intestinal bacteria, and may be effective toward IgE-induced atopic allergies.

Animals↗

Inheritance of organelle DNA sequences in a citrus-poncirus intergeneric cross.

Many land plants deviate from the maternal pattern of organelle inheritance. In this study, heterologous mitochondrial and chloroplast probes were used to investigate the inheritance of organelle genomes in the progeny of an intergeneric cross. The seed parent was LB 1-18 (a hybrid of Citrus reticulata Blanco cv. Clementine x C. paradisi Macf. cv. Duncan) and the pollen parent was the cross-compatible species Poncirus trifoliata (L.) Raf. All 26 progeny examined exhibited maternal inheritance of plastid petA and petD loci. However, 17 of the 26 progeny exhibited an apparent biparental inheritance of mitochondrial atpA, cob, coxII, and coxIII restriction fragment length polymorphisms (RFLPs) and maternal inheritance of mitochondrial rrn26 and coxI RFLPs. The remaining nine progeny inherited only maternal mitochondrial DNA (mtDNA) configurations. Investigations of plant mitochondrial genome inheritance are complicated by the multipartite structure of this genome, nuclear gene control over mitochondrial genome organization, and transfer of mitochondrial sequences to the nucleus. In this study, paternal mtDNA configurations were not detected in purified mtDNA of progeny plants, but were present in progeny DNA preparations enriched for nuclear genome sequences. MtDNA sequences in the nuclear genome therefore produced an inheritance pattern that mimics biparental inheritance of mtDNA.

Citrus↗

Sequence analysis of a 282-kilobase region surrounding the citrus Tristeza virus resistance gene (Ctv) locus in Poncirus trifoliata L. Raf.

Citrus tristeza virus (CTV) is the major virus pathogen causing significant economic damage to citrus worldwide, and a single dominant gene, Ctv, provides broad spectrum resistance to CTV in Poncirus trifoliata L. Raf. Ctv was physically mapped to a 282-kb region using a P. trifoliata bacterial artificial chromosome library. This region was completely sequenced to about 8x coverage using a shotgun sequencing strategy and primer walking for gap closure. Sequence analysis predicts 22 putative genes, two mutator-like transposons and eight retrotransposons. This sequence analysis also revealed some interesting features of this region of the P. trifoliata genome: a disease resistance gene cluster with seven members and eight retrotransposons clustered in a 125-kb gene-poor region. Comparative sequence analysis suggests that six genes in the Ctv region have significant sequence similarity with their orthologs in bacterial artificial chromosome clones F7H2 and F21T11 from Arabidopsis chromosome I. However, the analysis of gene colinearity between P. trifoliata and Arabidopsis indicates that Arabidopsis genome sequence information may be of limited use for positional gene cloning in P. trifoliata and citrus. Analysis of candidate genes for Ctv is also discussed.

Amino Acid Sequence↗

[Plant regeneration from Agobacterium-mediated CTV-cp gene transformation of Poncirus trifoliata Raf].

OBJECTIVE: To lay a foundation for the resistant breeding, the anti-virus CTV-cp gene was transformed into the epicotyles mediated by Agrobacterium tumefaciens in Poncirus trifoliata. METHOD: The explants used for the genetic transformation were the epicotyls from P. trifoliata. The Agrobacterum tumefaciens strain was EHA101 containing vector plasmid pGA482GG. The coat protein gene (CTV-cp gene), GUS gene and NPT II gene were introduced into the transformation plasmid. RESULTS: Ceftaxime used as antibiotics was better than carbenicillin. The concentration was 300 mg.L-1; The selection pressure for kanamycin was 50 mg.L-1; 70.0% of the resistant plants were GUS-positive; extra gene was proved to be in P. trifoliata plant by southern blot examination. CONCLUSION: An effective genetic transformation mediated by Agrobacterium tumefacines, which harbours a CTV-cp gene, has been developed in P. trifoliata. Transgenic CTV-cp plants were obtained.

Capsid↗

Non-equilibrium cooling of Poncirus trifoliata (L.) embryonic axes at various water contents.

The present study investigated the rate of temperature change within axes of Poncirus trifoliata during cooling and warming by various methods. Cooling rates ranged between 0.17 and 170 degree (C per second, and warming rates of 1.25 and 600 degree C per second were measured when axes were warmed at room temperature or in water at 40 degree C, respectively. Partial drying increased the cooling rate within axes in direct contact with the cryogen, but did not affect the cooling or warming rates within axes enclosed in a double layer of lightweight aluminium foil. The procedures described illustrate the orders of magnitude that separate extremes of the range of cooling or warming rates attained using methods commonly employed in cryopreservation studies. Quantifying these rates allows the relationship between cooling rate, water content and survival of hydrated embryonic axes to be explored.

Cryopreservation↗

Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress.

The effect of the arbuscular mycorrhizal (AM) fungus, Glomus versiforme, on growth and reactive oxygen metabolism of trifoliate orange (Poncirus trifoliata) seedlings was studied in potted plants under well-watered (WW) and water stressed (WS) conditions. Water stress significantly decreased root colonization. Shoot dry weight, plant height and stem diameter were higher in AM than in non-AM seedlings regardless of the water status. Inoculation with G. versiforme increased root dry weight and leaf number per plant of WW seedlings. There was less malondialdehyde (MDA) concentration in leaves and roots of AM seedlings, as well as lower hydrogen peroxide (H(2)O(2)) and superoxide anion radical (O(2)(-)) concentrations in AM roots under WW and WS conditions. AM inoculation did not affect the H(2)O(2) and O(2)(-) concentrations of WW leaves. Whether WS or not, AM symbiosis notably increased the guaiacol peroxidase (G-POD) activity of leaves, glutathione reductase (GR) activity of leaves and ascorbate peroxidase (APX) activity of roots. AM infection also markedly increased the APX activity of WS leaves. Soluble proteins and glutathione (GSH) in leaves and roots and ascorbate (ASC) in leaves were higher in WW AM than in WW non-AM seedlings. AM infection also enhanced the ASC and GSH contents of leaves and roots in WS seedlings. Cross-tolerance might occur in AM plants and be enhanced by AM symbiosis. Our results suggest that the increased concentrations of antioxidant enzymes and non-enzymatic antioxidants found in AM plants may serve to protect the organism against oxidative damage, enhancing drought tolerance.

Antioxidants↗

Antianaphylactic activity of Poncirus trifoliata fruit extract.

The effect of an aqueous extract of Poncirus trifoliata (L.) Raf. (Rutaceae) fruits (PTFE) on compound 48/80-induced mortality associated with anaphylaxis was studied in rats. PTFE inhibited compound 48/80-induced anaphylaxis 100% with a dose of 1.6 mg/g body weight (BW) 1 h before or 5 min after injection of compound 48/80. PTFE inhibited compound 48/80-induced anaphylaxis almost 100% with doses above 0.4 mg/g BW intraperitoneally administered. PTFE (1-1000 micrograms/ml) also dose-dependently inhibited the histamine release induced by compound 48/80 (5 micrograms/ml) in rat peritoneal mast cells. The level of cAMP in peritoneal mast cells, when PTFE was added, increased transiently, and significantly increased 53-fold at 10 s compared with that of basal cells. Moreover, PTFE inhibited intracellular calcium release induced by compound 48/80. These results suggest that PTFE has antianaphylactic activity by stabilizing the peritoneal mast cell membrane.

Anaphylaxis↗

Inhibition of immunoglobulin E production by Poncirus trifoliata fruit extract.

Immunoglobulin E (IgE) is the principal immunoglobulin involved in immediate hypersensitivities and chronic allergic diseases. The effect of an aqueous extract of Poncirus trifoliata (L) Raf. (Rutaceae) fruits (PTFE) on in vivo and in vitro IgE production was investigated. PTFE dose-dependently inhibited the active systemic anaphylaxis and serum IgE production induced by immunization with ovalbumin, Bordetella pertussis toxin and aluminum hydroxide gel. PTFE strongly inhibited interleukin 4 (IL-4)-dependent IgE production by lipopolysaccharide-stimulated murine whole spleen cells. In the case of U266 human IgE-bearing B cells, PTFE also showed an inhibitory effect on the IgE production. These results suggest that PTFE has an anti-allergic activity by inhibition of IgE production from B cells.

Aluminum Hydroxide↗

De novo arginine biosynthesis in leaves of phosphorus-deficient citrus and poncirus species.

Young, fully expanded leaves from 7-month-old P-deficient citrus rootstock seedlings had levels of nonprotein arginine that were 10- to 50-fold greater than those from P-sufficient control plants. Arginine content of the protein fraction increased 2- to 4-fold in P-deficient leaves. Total arginine content, which averaged 72 +/- 6 micromoles per gram dry weight of P-sufficient leaf tissue (mean +/- se, n = the four rootstocks) was 207, 308, 241, and 178 micromoles in P-deficient leaves from Citrus limon cv rough lemon, Poncirus trifoliata x C. sinensis cv Carrizo citrange and cv Troyer citrange, and P. trifoliata cv Australian trifoliate orange, respectively. For each rootstock, the accumulation of arginine paralleled an increase in the activity of the pathway for the de novo biosynthesis of arginine. The ratio of the nanomoles NaH(14)CO(3) incorporated into the combined pool of arginine plus urea per gram fresh weight intact leaf tissue during a 3-hour labeling period for P-deficient to P-sufficient plants was 91:34, 49:11, 35:11, and 52:41, respectively. When P-deficient plants were supplied with P, incorporation of NaH(14)CO(3) into arginine plus urea was reduced to the level observed for the P-sufficient control plants of the same age and arginine ceased to accumulate. Arginase and arginine decarboxylase activity were either unaffected or slightly increased during phosphorus deficiency. Taken together, these results provide strong evidence that arginine accumulation during phosphorus deficiency is due to increased activity of the de novo arginine biosynthetic pathway.

Journal Article↗

Effects of Poncirus trifoliata on type I hypersensitivity reaction.

A study was carried out to examine the effect of an aqueous extract from immature fruit of Poncirus trifoliata L. (Rutaceae) (PTIFE) on the type I hypersensitivity reaction. Forty-eight hour PCA (passive cutaneous anaphylaxis) in rats was significantly inhibited by the oral administration of PTIFE (200 mg/kg). It also inhibited histamine release from rat peritoneal mast cells (RPMC) induced by mouse anti-dinitrophenyl (DNP)-IgE and dinitrophenyl-human serum albumin (DNP-HSA). These results suggest that PTIFE has anti-allergic action against the type I hypersensitivity reaction.

Administration, Oral↗

Anti-Helicobacter pylori activity of the metabolites of poncirin from Poncirus trifoliata by human intestinal bacteria.

Poncirin was isolated from water extract of the fruits of Poncirus trifoliata and metabolized by human intestinal bacteria. The inhibitory effect of poncirin and its metabolites by these bacteria on the growth of Helicobacter pylori (HP) was investigated. Among them, ponciretin (5,7-dihydroxy-4'-methoxyflavanone), the main metabolite most potently inhibited the growth of HP, with a minimum inhibitory concentration (MIC) of 10-20 microg/ml. However, poncirin and its metabolites except ponciretin did not inhibit the growth of HP, nor did they inhibit HP urease.

Anti-Bacterial Agents↗

Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.

Amino Acid Sequence↗

[Two complementary dominant genes controlling apomixis in genus Citrus and poncirus].

Apomixis is very common in Citrus genus and its related genus. Two monoembryonic tangerine varieties (C. reticulata Blanco) Clementine and Wilking were used as seed parents to cross with four polyembryonic orange varieties [C. sinensis (L.) Osbeck]. Both sexual and apomictic progenies were found in each F1 population with different segregation ratios. In a total of 74 Wilking progenies, 23 were monoembryonic and 51 polyembryonic and the ratio of sexual and apomictic progenies was about 1:2 tested either in each individual cross or in the whole Wilking cross combionations. In Clementine progenies, 84 were monoembryonic and 71 polyembryonic and the ratio was about 1:1 tested either in each individual cross or in the whole Clementine cross combionations. According to the results together with other data published, a possible apomixis controlling mechanism was proposed, which involves two complementary dominant genes named as A1 and A2 that control apomixis in genus Citrus and Poncirus. Trees of genotype A1-A2-, except for homozygous of dominant gene A1 (which is lethal), can produce apomixis seeds. And those of other genotypes will produce sexual seeds. The segregation and recombination of these two genes accorded with Mendel's genetic laws. The proposed mechanism could explain genotypes controlling polyembryony-monoembryony existing both in nature species and artificial hybridization progenies as well as most of the known hybridization results.

Citrus↗

[Inheritance of organelle genomes of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and flying dragon (Poncirus trifoliata)].

Cleaved Amplified Polymorphic Sequence (CAPS) was successfully applied to analyze the organelle composition of three eight-year-old trees of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and Flying Dragon (Poncirus trifoliata). Five chloroplast and five mitochondrial universal primer pairs were used. All chloroplast primer pairs (rbcL-rbcL, rbcL-PSA I, TrnH-Trnk, TrnD-TrnT, TrnK-TrnK) and three (nad 1 exon B-nad 1 exon C, 18S rRNA-5S rRNA, nad 4 exon 1-nad 4 exon 2) of the five mitochondrial primer pairs, were efficiently amplified, but no polymorphism was detected, when the PCR products were digested by eleven restriction endonucleases, including, Hin6 I, Bus RI, Taq I, Msp I, HinfI, AluI, Dra I, EcoR I, Hind III, BamH I and Pst I respectively, three polymorphic cpDNA-CAPS markers (rbcL-rbcL/Hin 6 I, TrnD-TrnT/BusR I, TrnD-TrnT/Taq I) and one mtDNA-CAPS marker (nad 1-nad1/Msp I) were found. The results showed that cpDNA in the somatic hybrid plants came from Flying Dragon, the mesophyll parent, and mtDNA from Cleopatra mandarin, the embryogenic suspension parent uniformly. In order to prove the reliability of CAPS results, and to get more detailed information about the mtDNA inheritance, RFLP analyses was conducted. Genomic DNA of the somatic hybrids and their corresponding parents were digested by five restriction endonucleases (Dra I, EcoR I, Hind III, BamH I and Pst I), and hybridized with five mitochondrial probes (Cob, Pro 2, Pro I, atp 6, 26S rRNA) as well as one chloroplast probe, i.e. the PCR product of Flying Dragon with the primer pair of trnd 1-trnt 1. The results were in line with those of CAPS, and no novel bands were detected, which indicated that no organelle DNA recombination or rearrangement have been detected in the hybrid plants. The research showed that novel pattern of nuclear-mitochondria-chloroplast interaction could be reached via protoplast fusion.

Cell Fusion↗