Citrus Hassaku Research Articles

Auraptene Enhances AMP-Activated Protein Kinase Phosphorylation and Thereby Inhibits the Proliferation, Migration and Expression of Androgen Receptors and Prostate-Specific Antigens in Prostate Cancer Cells.

Citrus hassaku extract reportedly activates AMPK. Because this extract contains an abundance of auraptene, we investigated whether pure auraptene activates AMPK and inhibits proliferation using prostate cancer cell lines. Indeed, auraptene inhibited the proliferation and migration of LNCaP cells and induced phosphorylation of AMPK or its downstream ACC in LNCaP, PC3, and HEK-293 cells, but not in DU145 cells not expressing LKB1. In addition, the mTOR-S6K pathway, located downstream from activated AMPK, was also markedly suppressed by auraptene treatment. Importantly, it was shown that auraptene reduced androgen receptor (AR) and prostate-specific antigen (PSA) expressions at both the protein and the mRNA level. This auraptene-induced downregulation of PSA was partially but significantly reversed by treatment with AMPK siRNA or the AMPK inhibitor compound C, suggesting AMPK activation to, at least partially, be causative. Finally, in DU145 cells lacking the LKB1 gene, exogenously induced LKB1 expression restored AMPK phosphorylation by auraptene, indicating the essential role of LKB1. In summary, auraptene is a potent AMPK activator that acts by elevating the AMP/ATP ratio, thereby potentially suppressing prostate cancer progression, via at least three molecular mechanisms, including suppression of the mTOR-S6K pathway, reduced lipid synthesis, and AR downregulation caused by AMPK activation.

Auraptene, a citrus peel-derived natural product, prevents myocardial infarction-induced heart failure by activating PPARα in rats

BackgroundAuraptene derived from the peel of Citrus hassaku possesses anti-tumor, anti-inflammatory, and neuroprotective activities. Thus, it could be a valuable pharmacological alternative to treat some diseases. However, the therapeutic value of auraptene for heart failure (HF) is unknown. Study design/methodsIn cultured cardiomyocytes from neonatal rats, the effect of auraptene on phenylephrine-induced hypertrophic responses and peroxisome proliferator-activated receptor-alpha (PPARα)-dependent gene transcriptions. To investigate whether auraptene prevents the development of heart failure after myocardial infarction (MI) in vivo, Sprague–Dawley rats with moderate MI (fractional shortening < 40%) were randomly assigned for treatment with low- or high-dose auraptene (5 or 50 mg/kg/day, respectively) or vehicle for 6 weeks. The effects of auraptene were evaluated by echocardiography, histological analysis, and the measurement of mRNA levels of hypertrophy, fibrosis, and PPARα-associated genes. ResultsIn cultured cardiomyocytes, auraptene repressed phenylephrine-induced hypertrophic responses, such as increases in cell size and activities of atrial natriuretic factor and endothelin-1 promoters. Auraptene induced PPARα-dependent gene activation by enhancing cardiomyocyte peroxisome proliferator-responsive element reporter activity. The inhibition of PPARα abrogated the protective effect of auraptene on phenylephrine-induced hypertrophic responses. In rats with MI, auraptene significantly improved MI-induced systolic dysfunction and increased posterior wall thickness compared to the vehicle. Auraptene treatment also suppressed MI-induced increases in myocardial cell diameter, perivascular fibrosis, and expression of hypertrophy and fibrosis response markers at the mRNA level compared with vehicle treatment. MI-induced decreases in the expression of PPARα-dependent genes were improved by auraptene treatment. ConclusionsAuraptene has beneficial effects on MI-induced cardiac hypertrophy and left ventricular systolic dysfunction in rats, at least partly due to PPARα activation. Further clinical studies are required to evaluate the efficacy of auraptene in patients with HF.

Citrus hassaku Extract Powder Increases Mitochondrial Content and Oxidative Muscle Fibers by Upregulation of PGC-1α in Skeletal Muscle.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is expressed in skeletal muscles and regulates systemic metabolism. Thus, nutraceuticals targeting skeletal muscle PGC-1α have attracted attention to modulate systemic metabolism. As auraptene contained in citrus fruits promotes lipid metabolism and improves mitochondrial respiration, it could increase mitochondrial function through PGC-1α. Therefore, we hypothesized that PGC-1α is activated by auraptene and investigated its effect using Citrus hassaku extract powder (CHEP) containing >80% of auraptene. C2C12 myotubes were incubated with vehicle or CHEP for 24 h; C57BL/6J mice were fed a control diet or a 0.25% (w/w) CHEP-containing diet for 5 weeks. PGC-1α protein level and mitochondrial content increased following CHEP treatment in cultured myotubes and skeletal muscles. In addition, the number of oxidative fibers increased in CHEP-fed mice. These findings suggest that CHEP-mediated PGC-1α upregulation induced mitochondrial biogenesis and fiber transformation to oxidative fibers. Furthermore, as CHEP increased the expression of the protein sirtuin 3 and of phosphorylated AMP-activated protein kinase (AMPK) and the transcriptional activity of PGC-1α, these molecules might be involved in CHEP-induced effects in skeletal muscles. Collectively, our findings indicate that CHEP mediates PGC-1α expression in skeletal muscles and may serve as a dietary supplement to prevent metabolic disorders.

Metabolomic analysis of primary metabolites in citrus leaf during defense responses

Mechanical damage is one of the unavoidable environmental stresses to plant growth and development. Plants induce a variety of reactions which defend against natural enemies and/or heal the wounded sites. Jasmonic acid (JA) and salicylic acid (SA), defense-related plant hormones, are well known to be involved in induction of defense reactions and play important roles as signal molecules. However, defense related metabolites are so numerous and diverse that roles of individual compounds are still to be elucidated. In this report, we carried out a comprehensive analysis of metabolic changes during wound response in citrus plants which are one of the most commercially important fruit tree families. Changes in amino acid, sugar, and organic acid profiles in leaves were surveyed after wounding, JA and SA treatments using gas chromatography-mass spectrometry (GC/MS) in seven citrus species, Citrus sinensis, Citrus limon, Citrus paradisi, Citrus unshiu, Citrus kinokuni, Citrus grandis, and Citrus hassaku. GC/MS data were applied to multivariate analyses including hierarchical cluster analysis (HCA), primary component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) to extract stress-related compounds. HCA showed the amino acid cluster including phenylalanine and tryptophan, suggesting that amino acids in this cluster are concertedly regulated during responses against treatments. OPLS-DA exhibited that tryptophan was accumulated after wounding and JA treatments in all species tested, while serine was down regulated. Our results suggest that tryptophan and serine are common biomarker candidates in citrus plants for wound stress.

Isolation and characterization of wound-induced compounds from the leaves of Citrus hassaku

Citrus plants are world widely cultivated as horticultural tree crops, and nowadays their pharmacological activities have been well studied. Since research of defense responses in citrus plants have been mainly focused on the post-harvested fruits because of their commercial importance, defense mechanisms during their developmental stages have not been well understood. In the present study, two wound-induced compounds were isolated from leaves of Citrus hassaku, and their structures were elucidated by high-resolution electron spray ionization mass spectra (HRESIMS) and nuclear magnetic resonance (NMR) analyses. One of these compounds was identified as a known flavanone, hesperetin. The other was characterized as a novel furofuran lignan, and was named 'biscitrusnin-A'. Their antimicrobial activities were also evaluated.

미성숙 감귤 과피 초임계 추출물의 성분 분석과 자궁암세포 성장억제효능

본 연구의 목적은 제주도 재래감귤인 팔삭과 이예감 초임계 추출물에 대한 인간 자궁암 세포인 HeLa 세포에서의 성장억제 효능을 탐색하고, 각 추출물에서의 유효 활성 성분을 분석하는데 있다. HeLa 세포에서의 성장억제 및 세포사멸 효능을 탐색하기 위해 MTT assay와 Hoechst 33342 염색을 수행하였으며, 성분분석은 가스크로마토그래피 질량분석기(GC/MS)를 이용하였다. 두 종류의 미성숙 감귤 과육, 과피 초임계 추출물에 대한 HeLa 세포의 성장 억제 효능을 비교해 본 결과, 과피 추출물은 팔삭과 이예감 모두 비슷한 세포사멸 효능을 나타냈다. 과피 추출물을 처리한 세포에서는 농도 의존적으로 성장률이 억제되었으며, 처리 농도 <TEX>$400{\mu}g/mL$</TEX>에서 팔삭과 이예감 추출물이 각각 87.16%와 92.95%의 세포사멸 효능을 나타냈으나, 과육 추출물의 세포 성장 억제 효능은 처리 농도 <TEX>$400{\mu}g/mL$</TEX>까지 관찰되지 않았다. Hoechst 33342 염색을 통해 apoptotic body 형성을 현미경으로 관찰한 결과, 100, <TEX>$200{\mu}g/mL$</TEX> 과육 초임계 추출물을 처리한 세포에서는 apoptpotic body를 관찰할 수 없었으나, 동일한 농도의 과피 추출물을 처리한 세포의 경우에서는 apoptotic body가 현저하게 증가하는 것을 확인 할 수 있었다. GC/MS 분석을 통해 미성숙 팔삭 과육과 과피 초임계 추출물에서 각각 27개와 31개의 화합물을 검출하였으며, 미성숙 이예감 과육과 과피 초임계 추출물에서는 각각 27개와 29개의 화합물을 검출하였다. 팔삭 과피 초임계 추출물에는 1,1,4,4-Tetramethyl-2-tetralone(20.86%), alloimperatorin(8.15%), limonene (11.23%), auraptene(7.29%) 등이 주로 함유되어 있었으며, 이예감 과피 초임계 추출물에는 limonene(22.19%), linalool(11.23%), <TEX>${\gamma}$</TEX>-sitosterol(9.12%) 등이 주로 함유되어 있었다. This study was performed to investigate the antiproliferative activities of supercritical extracts from phalsak(Citrus hassaku Hort ex Tanaka) and yeagam(Citrus iyo Hort. ex Tanaka) against human cervical carcinoma HeLa cells and the chemical compositions of the extracts. The anticancer properties of supercritical extracts were demonstrated using the MTT assay and Hoechst 33342 staining and the compositional analyses were conducted by using gas chromatography-mass spectrometry(GC-MS). The peel extracts of both species exhibited similar antiproliferative effect. The antiproliferative activity of the flesh extracts was not detected up to <TEX>$400{\mu}g/mL$</TEX>, whereas peel extracts of phalsak and yeagam reduced cell viability with 87.16% and 92.95% at <TEX>$400{\mu}g/mL$</TEX>, respectively. There was a dramatic increase of the apoptotic body formation in the cell treated with peel extracts while no apoptotic body formation detected in the cell treated with flesh extracts at 100, <TEX>$200{\mu}g/mL$</TEX>. By GC-MS analysis, 27 and 31 kinds of compounds identified in flesh and peel of phalsak, while 27 and 29 kinds of compounds were identified in flesh and peel of yeagam, respectively. 1,1,4,4-Tetramethyl-2-tetralone(20.86%), alloimperatorin(8.15%), limonene(11.23%), and auraptene(7.29%) were major in peel of phalsak, whereas limonene(22.19%), linalool(11.23%), and <TEX>${\gamma}$</TEX>-sitosterol(9.12%) were major in peel of yeagam.

Mixture of guava leaves extract and Citrus hassaku pericarp extract inhibits in vitro inflammatory biomarkers by blocking ERK, JNK, and p38 MAPK signaling and protects mice from lethal endotoxemia

Although anti-inflammatory activity of guava leaves (GLE) and fruits of Citrus hassaku Hort ex Tanaka (phalsak, PSE) has been demonstrated in cell culture system, the combination therapy of GLE and PSE with better anti-inflammatory activity and the exact mechanism remains unclear. In the present study, we set out to determine whether the anti-inflammatory effects of optimal mixture (OM) are mediated to suppress mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB), and activated protein-1 (AP-1) activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. We found that OM significantly suppressed LPS-stimulated NO production without a cytotoxic effect on RAW 264.7 cells. OM inhibited the expression of LPS-induced iNOS and COX-2 protein and their mRNA expression. Also, TNF-α, IL-6, and PGE2 secretion were decreased by OM in LPS-stimulated macrophages. As a result, OM inhibited pro-inflammatory markers such as TNF-α, IL-6, and PGE2, which is hypothesized as being due to the suppression of LPS-induced ERK, p38, and JNK signaling pathway, but not NF-κB and AP-1. Moreover, OM improved the survival rate during lethal endotoxemia by inhibiting the production of TNF-α and IL-6 in an animal model.

Abstract 11983: Auraptene, a Coumaric Compounds Analogous, Improved the Worsening of Systolic Function by Activating Mitochondrial Function After Myocardial Infarction in Rats

Introduction: Heart failure is associated with pathological growth and mitochondrial dysfunction of constituent cardiomyocytes. To achieve effective oral pharmacological therapy for heart failure, we screened compounds isolated from natural products and found that auraptene derived from the peel of Citrus Hassaku may be applicable to pharmacological therapy for heart failure. Hypothesis: We assessed the hypothesis that auraptene could improve the deterioration of mitochondrial function and the development of heart failure in rats with myocardial infarction (MI). Methods and Results: In cultured cardiomyocytes, auraptene (2.5-10 μM) dose-dependently repressed phenylephrine-induced hypertrophic responses such as increase in cell size and ANF and ET-1 promoter activations. Auraptene also activated mitochondrial- and lipid metabolism-related gene transcriptions, such as PGC1α, PPARα/γ, mCPT1, UCP3, and PDK4. One week after operation, 22 rats with a moderate size of MI (Fractional shortening (FS) &lt; 40%) were then randomly assigned to vehicle (n=8), auraptene low-dose (5 mg/kg/day, n=7), or high-dose (50 mg/kg/day, n=7). Oral daily treatments with these agents were continued for 6 weeks. There were no differences in left ventricle (LV) geometric and functional data among the 3 MI groups before treatment. After treatment, LVFS was significantly higher in the auraptene low-dose (21%, p &lt; 0.0001) and high-dose (26%, p &lt; 0.0001) groups than the vehicle group (16%). LV wall thickness in the remote non-infarct area was significantly thinner in the auraptene low-dose (1.4 mm, p &lt; 0.01) and high-dose (1.2 mm, p &lt; 0.0001) groups than the vehicle group (2.5 mm). Histological analysis demonstrated that auraptene treatment significantly suppressed MI-induced increases in myocardial cell diameter and perivascular fibrosis compared with vehicle treatment. Moreover, auraptene also prevented the activations of ANF and MCP-1 mRNA levels and up-regulated mitochondrial- and lipid metabolism-related gene transcriptions in LV. Conclusions: Auraptene treatment prevents the worsening of LV systolic function and represses hypertrophy after MI in adult rats. A natural compound, auraptene is expected as a novel useful agent for heart failure therapy in humans.

Construction of genetic linkage map and graphical genotyping of pseudo-backcrossed F2 (BC’2) progeny to introduce a CTV resistance from Poncirus trifoliata (L.) Raf. into Citrus by introgression breeding

Most citrus tristeza virus (CTV) strains infect almost all Citrus species and have caused serious damage on citrus production in many countries. Poncirus trifoliata (trifoliate orange) has a single dominant resistance gene effective against a broad range of CTV strains. P. trifoliata is sexually compatible with Citrus species, and introgression breeding programs have been initiated to introduce the CTV resistance into Citrus. “Kankitsu Chukanbohon Nou 8 Gou” (Nou-8) was developed by crossing Citrus sp. “Kiyomi” with an F1 hybrid derived from Citrus hassaku × P. trifoliata, but it still had undesirable eating traits. In aim to progress marker-assisted introgression breeding for CTV resistance efficiently, we selected 189 DNA markers which could be applied to genotyping for Citrus and P. trifoliata and constructed a genetic linkage map of Nou-8. The map included 189 DNA markers on 10 linkage groups and extended about 722.8 cM in total length. CTV resistance locus was located at 2.4 cM position in linkage group 02_(1), with the same location found when progeny were scored for resistance using an ELISA assay and for DNA marker for CTV resistant (CTV103). Graphical genotypes of Nou-8 and its progeny suggested that (1) 36 % of the chromosome in the BC’2 population were inherited from Nou-8 without any recombination sites, (2) Nou-8 had been already completely substituted by Citrus genotype in two linkage groups, and (3) four of 93 progeny of Nou-8 had recombination sites in the flanking region of the CTV resistance gene locus.

Induction of apoptosis in anoikis-resistant breast cancer stem cells by supercritical CO2 extracts from Citrus hassaku Hort ex Tanaka

Anoikis (or cell-detachment-induced apoptosis) is a special form of apoptosis in which cells loose contact with other cells or the extracellular matrix. In the present study, we have found that the breast cancer stem cell line MCF-7-SC, isolated from MCF-7 breast cancer cells, shows characteristics of anoikis resistance. Treatment of supercritical CO2 extract from Citrus hassaku Hort ex Tanaka, locally known as Phalsak, reduced the expression of ATP-binding cassette sub-family G member 2 mRNA and induced apoptosis in MCF-7-SCs, as evidenced by an increase of apoptotic body formation, increased cell population in the sub-G1 phase, increase in the Bax/Bcl-2 ratio, proteolytic activation of caspase-9 and caspase-3, and the degradation of poly(ADP-ribose) polymerase (PARP) protein. The effects of supercritical CO2 extract from Phalsak on anoikis-resistant breast cancer stem cells suggest the use of this extract as a potential therapeutic agent for breast cancer stem cell treatment.

Construction of a citrus framework genetic map anchored by 708 gene-based markers

We developed 708 gene-based markers for citrus genome analysis. Sequence-tagged site (STS) primers were designed that were located in conserved exon regions and whose PCR products spanned genomic introns. Of these, 79.7 % comprised cleaved amplified polymorphic sequence markers. The gene-based markers and their annotation and position on Clementine scaffolds ver. 1.0 permitted comparison of the genetic map and the Clementine genome sequence. The 708 gene-based markers were used to construct a genetic map using the 87 progenies (AG population) from the cross between ‘Okitsu 46 gou’ (‘Sweet Spring’ (‘Ueda unshiu’ (Citrus unshiu) × Hassaku (Citrus hassaku Hort. ex Tanaka)) × ‘Trovita’ orange) × ‘Kankitsu Chukanbohon Nou 5 gou’ (‘Lee’ (Citrus clementina × tangelo) × Citrus kinokuni). The markers were integrated using common STSs on different phase maps in cross-pollination mode. The integrated map (AGI map) comprised 706 loci, including two morphological traits, and spanned 990.9 centimorgans (cM) with an average marker distance of 1.40 cM. These markers formed nine linkage groups (LGs) (corresponding to citrus physical chromosomes): LG-01 to LG-09 corresponded to Scaffold_01, Scaffold_07, Scaffold_09, Scaffold_06, Scaffold_03, Scaffold_02, Scaffold_04, Scaffold_08, and Scaffold_05, respectively. LG-08 and LG-09 contained morphological traits controlling embryo color and seedlessness. Eighty-eight loci comprised three or more alleles on the AGI map; 36.4 % of them were related to transcription factors and DNA-binding proteins. The 708 gene-based markers and the AGI map are valuable for integrating various citrus genetic maps, alignment of genomic sequences, chromosome assignment, and understanding the diversity of citrus germplasms.

Anti‐Metastatic Effect of Supercritical Extracts from the Citrus hassaku Pericarp via Inhibition of C‐X‐C Chemokine Receptor Type 4 (CXCR4) and Matrix Metalloproteinase‐9 (MMP‐9)

The fruit of hassaku (Citrus hassaku Hort. ex Tanaka) is locally known as phalsak in Korea. Recently, the fruit extract has been known to exhibit in vivo preventive effects against UVB-induced pigmentation, antiallergic activity, and enhancement of blood fluidity. However, the exact mechanisms of how supercritical extracts of phalsak peel (SEPS) inhibits tumor metastasis and invasion are still not fully understood. We found that SEPS could downregulate the constitutive expression of both CXCR4 and HER2 in human breast cancer MDA-MB-231 cells as compared with other cells. SEPS also suppressed matrix metalloproteinase-9 (MMP-9) expression and its enzymatic activity under non-cytotoxic concentrations. Neither proteasome inhibition nor lysosomal stabilization had any effect on the SEPS-induced decrease in CXCR4 expression. A detailed study of the underlying molecular mechanisms revealed that the regulation of the downregulation of CXCR4 was at the transcriptional level, as indicated by downregulation of mRNA expression, suppression of NF-κB activity, and inhibition of chromatin immunoprecipitation activity. Suppression of CXCR4 expression by SEPS correlated with the inhibition of CXCL12-stimulated invasion of MDA-MB-231 cells. Overall, our results indicate, for the first time, that SEPS can suppress CXCR4 and MMP-9 expressions through blockade of NF-κB activation and thus has the potential to suppress metastasis of breast cancer.

Henderson Altıntopu ve Bazı Şadok Çeşitlerinin Adana Ekolojik Koşullarında Verim ve Meyve Kalite Özelliklerinin Belirlenmesi

Pomelo [Citrus grandis (L.) Osbeck] has the largest fruit size among citrus species. There are several cultivars for pomelo species which is sensitive to low temperatures thus its cultivation is possible in relatively preserved areas in subtropical regions. The interest to pomelo production has increased due to several reasons such as, its has a little or no bitterness in fruit flesh conversaly to grapefruit, avaibility of pigmented varieties, high concentration of vitamin C, total phenolics and carotenoids in fruit flesh, as well as its efficiency in destroying free radicals such as superoxide anion and hydrogen peroxide. Several pomelo varieties have been introduced to Turkey and adaptation studies have been established. In the present study, Reinking, Pomelo WN, Kao Panne and Citrus hassaku which is a spontaneous hybrid between pomelo and mandarin were investigated in terms of fruit yield and pomological characteristics. In addition, Henderson grapefruit was included in this study in order to obtain a comparasion between pomelo and grapefruit. A large variation was observed in fruit yield of cultivars established in 2011 and 2012. In 2011, the highest yield was determined in Henderson whereas it was obtained from Citrus hassaku in 2012. Pomelo varieties yielded between 5-60 kg fruit per tree and 8.6-50 kg fruit per tree in 2011 and 2012, respectively. In terms of pomological traits, Citrus hassaku had the highest fruit weight; Pink, Kao Panne and Reinking pomelos had the highest peel thickness and Pomelo WN yielded the highest TSS/TA ratio.

ブラッドオレンジとハッサクのす上がり果判別における音響振動法の適用

カンキツのす上がり果の判別に音響振動法の適用を検討した．ブラッドオレンジの凍結によるす上がり果は弾性指標が健全果より有意に低かった．す上がり程度との関係は認められなかった．す上がり程度が高いほど果実比重は有意に低下した．果実比重と弾性指標との間に，比較的高い正の相関が認められた．す上がりが見られない弾性指標の最小値（閾値）を80 × 105とすると，す上がり果の判別率は約70％であった．比重法によるす上がり果の判別率は，閾値を0.88とすると約78％であり，弾性指標より若干高かった．ハッサクでは，初期の生理的す上がり果は健全果よりも弾性指標が有意に高かった．果実比重はす上がり程度が高いほど有意に低下した．ブラッドオレンジでは，可溶性固形物含量は，す上がり程度による差がなかったが，クエン酸含量はす上がり程度の高い果実が健全果より低かった．ハッサクでは，可溶性固形物含量は，健全果がす上がり程度の高い果実よりわずかに高かったが，クエン酸含量はす上がり程度による有意差が認められなかった．以上の結果，音響振動法は改良によって凍結によるす上がり果および生理的す上がり果の判別に適用できる可能性があることが示された．

Inhibitory effect of a formulated extract from multiple citrus peels on LPS-induced inflammation in RAW 246.7 macrophages

Background: Formulated Citrus Peel Extract (GL) made from the peels of six citrus fruits available in Japan, namely navel oranges, citrus hassaku, citrus limon, citrus natsudaidai, citrus miyauchi and satsuma, was initially developed as a cosmetic product to protect skin from UV irradiation. Anecdotal evidences of anti-cancer property of GL have been reported by consumers based on the cases such as topical application for melanoma, and oral ingestion for prostate, lung and liver cancers. Those anecdotal reports stimulated us to investigate anti-tumorigenesis activity of GL. In the previous study, we reported that the topical application of GL inhibited DMBA/TPA-induced skin tumor formation by decreasing inflammatory gene parameters.Objective: In this study, we mainly investigated the effect of GL on translocation of NF-kB together with production of nitric-oxide and TNF-α induced by LPS in RAW 264.7 cells.Results: This investigation showed that GL decreased the release of TNF-α and nitric oxide from macrophage RAW264.7 cells stimulated by LPS in a dose-dependent manner. In addition, GL suppressed the expression of iNOS and nuclear translocation of NF-kB in RAW264.7 cells, inhibited the degradation of IκB-α, and scavenged hydroxyl radicals (DMPO/OH adduct) in vitro.Conclusions: Our findings suggest that GL suppresses the inflammation in vitro, and exerts chemopreventive activity through the inhibition of production of TNF-α and iNOS proteins due to the inhibition of nuclear translocation of NF-kB and oxidative stress. GL appears to be a novel functional natural product capable of preventing inflammation and inflammation-associated tumorigenesis. Keywords: GL, Citrus peel extract, anti-inflammation, Nitric oxide, iNOS, NF-kB, TNF-α

Distinguishing grapefruit and pummelo accessions using ISSR markers

Grapefruit is the fourth economically most important citrus fruit in the world. In this research Inter-Simple Sequence Repeat (ISSR) markers were used to distinguish twenty-nine grapefruit (Citrus paradisi Macf.), five pummelo (Citrus maxima (Burm.) Merr.) and one Citrus hassaku Hort. Ex Tanaka accessions. Twelve ISSR primers produced a total of 100 fragments and 62 of them were polymorphic. The number of average polymorphic fragments per primer was 5.2. The mean polymorphism information content (PIC) was 0.37. The unweighted pair group method arithmetic average (UPGMA) analysis demonstrated that the accessions had a similarity range from 0.79 to 1.00. The accessions were separated into two main clusters; group A with five pummelos and group B with grapefruits. In the pummelo cluster, all pummelos were distinguished whereas in the grapefruit cluster some accessions were not clearly separated. There was a low level of variation in the grapefruits due to their mutation origin.

Effects of Unripe Citrus hassaku Fruits Extract and Its Flavanone Glycosides on Blood Fluidity

The enhancement of blood fluidity may lead to improvements in skin problems resulting from unsmooth circulation or blood stagnation. Since a 50% ethanolic extract (CH-ext) obtained from unripe Citrus hassaku fruits may be a useful ingredient in skin-whitening cosmetics, the present study was designed to examine the effect of CH-ext on blood fluidity. CH-ext concentration-dependently inhibited in vitro collagen-induced rabbit platelet aggregation and in vitro polybrene-induced rat erythrocyte aggregation. The CH-ext showed in vitro fibrinolysis activity in fibrin plate assay. Activity-guided fractionation of the CH-ext using antiplatelet activity, inhibitory activity of erythrocyte aggregation, and fibrinolysis activity revealed that these activities of CH-ext were attributable to naringenin-7-glycoside (prunin). Successive oral administration of CH-ext to rats inhibited the lipopolysaccharide (LPS)-induced decrease of blood platelets and fibrinogen, and LPS-induced increase of fibrin degradation products (FDP) in LPS-induced disseminated intravascular coagulation (DIC) model rats. Effects of CH-ext on blood fluidity were analyzed by a micro channel array flow analyzer (MC-FAN). Preventive oral administration of CH-ext to rats showed dose-dependent reduction of the passage time of whole blood flow of the DIC model rats in comparison with that of the vehicle control rats. These results imply that CH-ext may have effects which improve effects on blood fluidity.

Antiallergic activity of unripe Citrus hassaku fruits extract and its flavanone glycosides on chemical substance-induced dermatitis in mice

Oral administration of a 50% ethanolic extract (CH-ext) obtained from unripe Citrus hassaku fruits collected in July exhibited a potent dose-dependent inhibition of IgE (immunoglobulin E)-mediated triphasic cutaneous reaction at 1 h [immediate phase response (IPR)], 24 h [late phase response (LPR)] and 8 days [very late phase response (vLPR)] after dinitrofluorobenzene challenge in mice. Naringin, a major flavanone glycoside component of CH-ext, showed a potent dose-dependent inhibition against IPR, LPR and vLPR. Neohesperidin, another major glycoside component of CH-ext, showed an inhibition against vLPR. The effect of CH-ext on type IV allergic reaction was examined by determining inhibitory activity against ear swelling in mice by using the picryl chloride-induced contact dermatitis (PC-CD) model. Oral administration (p.o.) of CH-ext and subcutaneous administration (s.c.) of prednisolone inhibited ear swelling during the induction phase of PC-CD. The inhibitory activities of combinations of CH-ext (p.o.) and prednisolone (s.c.) against PC-CD in mice were more potent than those of CH-ext alone and prednisolone alone, without enhancing the adverse effects. Other combinations of prednisolone (s.c.) and flavanone glycoside (p.o.) components of CH-ext, i.e. naringin and neohesperidin, exerted similar synergistic effects.

Inhibitory Effects of Citrus hassaku Extract and Its Flavanone Glycosides on Melanogenesis

The 50% ethanolic extract (CH-ext) obtained from the unripe fruit of Citrus hassaku exhibited significant tyrosinase inhibitory activity. The CH-ext showed antioxidant activity, such as superoxide dismutase (SOD)-like activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. Activity-guided fractionation of the CH-ext indicated that flavanone glycoside-rich fractions showed potent tyrosinase inhibitory activity. Further examination revealed that the tyrosinase inhibitory activity and antioxidant activity of the CH-ext were attributable to naringin and neohesperidin, respectively. The CH-ext showed inhibition of melanogenesis without any effects on cell proliferation in cultured murine B16 melanoma cells after glucosamine exposure. The topical application of the CH-ext to the dorsal skin of brownish guinea pigs showed in vivo preventive effects against UVB-induced pigmentation.

Rapid and convenient method for preparing aurapten-enriched product from hassaku peel oil: implications for cancer-preventive food additives.

Aurapten (7-geranyloxycoumarin) has been reported to be an effective inhibitor of chemical carcinogenesis in some rodent models. In the present study, a method for preparing an aurapten-enriched agricultural product has been established. Out of 17 Rutaceae varieties, the aurapten content in hassaku (Citrus hassaku Hort ex Y. Tanaka) fruit peel was marked, as well as that in natsumikan (C. natsudaidai) and grapefruit (C. paradisi). The aurapten content in hassaku peel was most abundant in April. Hassaku fruit peel oil, which was dissolved by heating precipitates including aurapten which had formed after freezing the peel oil at -20 degrees C, was used. After adsorbing aurapten from peel oil onto synthetic adsorbent SP70, the adsorbent was washed with 40% (v/v) ethanol in water to remove essential oils and pigments remaining on the adsorbent. Aurapten was then eluted with 80% (v/v) ethanol. In a laboratory-scale test, the recovery rates of aurapten and total carotenoids from the eluates were 74.3 and 4.6%, respectively. In a pilot-scale test, the recovery rate of aurapten in the aurapten-enriched preparation from dissolved hassaku oil was 91.0%, and its concentration was 64.1% (w/w). When stored for 180 days under sunlight, aurapten in powder form remained at 88.0-89.0% of the initial level, but only 31.3-43.8% in ethanol. The stability of aurapten in the aurapten-enriched preparation was higher than that of purified aurapten. These results suggest that aurapten is readily recovered from hassaku peel oil using SP70, and thus may be used as a food additive.