Citrus Unshiu Fruits Research Articles

Dyeing properties and antimicrobial activity of silk fabric with extract of unripe Citrus Unshiu fruits

This study was carried out to optimize dyeing conditions of unripe Citrus Unshiu extract on silk fabric and to evaluate antimicrobial activity of the dyed fabric for its potential use as a functional natural dye. Unripe fruits of Citrus Unshiu in Jeju Island, Korea, extracted in 80 % Ethanol solution to final solid dye powder were dyed on silk fabric under a variety of conditions such as dye bath concentrations, temperature, and dyeing duration together with mordanting. Dyeing fastness properties to washing, rubbing, perspiration, and light were tested and the antimicrobial activities of the dyed fabric against Staphylococcus aureus and Klebsiella pneumoniae were investigated quantitatively. As results, the fabric showed the maximum dye uptake (K/S) under the conditions of 80 °C for temperature, 30 min for duration, and 600 % (o.w.f.) for dye concentration. Both pre- and post-mordanting seemed not to be effective on increasing K/S values of the dyed silk fabrics under the optimum dyeing conditions. Fastness ratings to washing, rubbing, and perspiration were all very good (4–5 grades) for both 300 and 600 % dyed silk fabrics. Excellent antimicrobial activities over 99 % reduction rate against two both bacteria were exhibited for all of dyed fabrics undergone more than 300 % of dye concentration. From these results, it was concluded that the dye concentration of 300 % of unripe Citrus Unshiu could be employed to produce antimicrobial silk fabric. Furthermore, to get more saturated shades on the fabric by the citrus, higher dye concentration such as 600 % was available as well.

Isolation of hesperidin from peels of thinned Citrus unshiu fruits by microwave-assisted extraction

Simultaneous extraction by microwave-irradiation and crystallisation were performed in the same pot of solvent of 70% (v/v) aqueous ethanol for isolation of hesperidin from thinned immature fruit peels of Citrus unshiu as refining of Citrus waste biomass. The hesperidin content in immature fruits peels was about 3.2-fold higher than that of mature fruit. After microwave-assisted extraction (MAE), the yield of hesperidin reached 58.6 mg/g, which was comparable to the amount obtained after extraction using DMSO:methanol (1:1, v/v) as a solvent for 30 min at room temperature. Heating temperature and time for isolation of hesperidin crystallites were optimised as 140 °C and 8 min by using response surface methodology. Under this optimal condition, 86.8% (47.7 mg/g) of total hesperidin was isolable by MAE and low-temperature storage (5 °C, 24 h).

Peucedanum japonicum andCitrus unshiu essential oils inhibit the growth of antibiotic-resistant skin pathogens

In this study, the chemical compositions and the anti-bacterial activities of hydrodistilled essential oils of the whole parts ofPeucedanum japonicum and the immature fruits ofCitrus unshiu were investigated. The chemical constituents of theP. Japonicum (PJE) andCitrus unshiu (CuE) essential oils were further analyzed by GC-MS and their major components were β-pinene (66.07%) and limonene (77.93%), respectively. The antibacterial activities of PJE and CuE against drug-susceptible and -resistant skin pathogens were also examined. Anti-bacterial tests using the disk diffusion method and the minimum inhibitory concentration (MIC) values indicated that PJE and CuE have excellent activities. The MIC of PJE against drug-susceptible and -resistant skin pathogens ranged from 0.13 to 5.0 μL/mL, whereas that of CuE ranged from 0.08 to 1.25 μL/mL. In addition, CuE reduced the lipopolysaccharide (LPS)-induced secretion of nitric oxide (NO) in RAW 264.7 cells, indicating that it has anti-inflammatory effects. These findings demonstrate that PJE and CuE have great potential for use in promoting human skin health.

Comparative Evaluation of 12 Immature Citrus Fruit Extracts for the Inhibition of Cytochrome P450 Isoform Activities

In a previous study we found that 50% ethanol extracts of immature fruits of Citrus unshiu (satsuma mandarin) have anti-allergic effects against the Type I, II and IV allergic reactions. However, many adverse interactions between citrus fruit, especially grapefruit juice, and drugs have been reported due to the inhibition of cytochrome P450 (CYP) activities. The purpose of this study was to examine the competitive inhibitory effects of extracts from immature citrus fruit on CYP activity. Extracts were prepared from 12 citrus species or cultivars, and were tested against three kinds of major CYPs, CYP2C9, CYP2D6 and CYP3A4, in human liver microsomes. We also estimated the amounts of flavonoids (narirutin, hesperidin, naringin and neohesperidin) and furanocoumarins (bergapten, 6',7'-dihydroxybergamottin and bergamottin) in each extract using HPLC. Citrus paradisi (grapefruit) showed the greatest inhibition of CYP activities, while Citrus unshiu which has an antiallergic effect, showed relatively weak inhibitory effects. Extracts having relatively strong inhibitory effects for CYP3A4 tended to contain higher amounts of naringin, bergamottin and 6',7'-dihydroxybergamottin. These results, providing comparative information on the inhibitory effects of citrus extracts on CYP isoforms, suggest that citrus extracts containing high levels of narirutin and hesperidin and lower levels of furanocoumarins such as C. unshiu are favorable as antiallergic functional ingredients.

Anti-allergic effect of a combination of Citrus unshiu unripe fruits extract and prednisolone on picryl chloride-induced contact dermatitis in mice

Effect of 50% ethanolic extract of unripe fruits of Citrus unshiu (CU-ext) on type IV allergic reaction was examined by inhibitory activity of ear swelling of picryl chloride-induced contact dermatitis (PC-CD) in mice. Oral administration of CU-ext and subcutaneous administration of prednisolone showed inhibition of ear swelling during both induction and effector phases of PC-CD. The inhibitory activities of combinations of CU-ext (p.o.) and prednisolone (s.c.) during induction phase of PC-CD were more potent than those of CU-ext alone and prednisolone alone. Successive oral administration of hesperidin, a major flavanone glycoside of CU-ext, inhibited ear swelling during induction phase of PC-CD. The inhibitory activities of combinations of hesperidin (p.o.) and prednisolone (s.c.) were more potent than those of hesperidin alone and prednisolone alone. These results indicated that the combinations of prednisolone and CU-ext or hesperidin exerted a synergistic effect.

Water Soluble Fluorescent Substances in Citrus unshiu Fruit and their Possible Participation in Chlorophyll Catabolism.

Changes in levels of fluorescence substances (FS) in aqueous extracts of Citrus unshiu (C. unshiu) peels were measured by the 3-dimensional (3-D) fluorescence spectrophotometer. FS, extracted from C. unshiu peels, have characteristic fluorescence at excitation (Ex) 320-330 nm and emission (Em) 420-440 nm. Ex and Em of FS are very similar to those of lipofucsin-like-compounds known to accumulate in tissues during aging and fluorescent-chlorophyll-catabolites (FCC) appeared to be a product of oxidative cleavage of chlorophyll (Chl)-porphyrin. The amount of FS increased in yellow-green C. unshiu peels and decreased in yellow C. unshiu peels. In ethylene-treated C. unshiu peels, the amount of FS increased at the time of rapid disappearance of Chl, and decreased after that, whereas FS of non-treated C. unshiu peels did not change during incubation. A possible participation of FS in Chl catabolism is discussed.

ウンシュウミカン果実のエチレン誘導クロロフィル代謝に及ぼす２，２′‐ビピリジルの影響について

エチレン処理により促進されるウンシュウミカン果実の脱緑に対する2, 2'-ビピリジルの影響を調査した.2, 2'-ビピリジル処理はエチレン処理したウンシュウミカン果実の脱緑を抑制した.エチレン処理した果実で, 2, 2'-ビピリジル処理は脱フィチルした色素(クロロフィリッドaおよびフェオフォルビドa)の蓄積を起こさず, 脱フィチルしていない色素(クロロフィルa, クロロフィルbおよびフェオフィチンa)を蓄積した.2, 2'-ビピリジル処理は, エチレン処理により誘導されるクロロフィラーゼ活性を抑制したが, in vitroにおける反応液中への2, 2'-ビピリジルの添加はクロロフィラーゼ活性には影響しなかった.これらの結果は, 2, 2'-ビピリジルが, クロロフィル代謝の初期段階の鍵酵素であるクロロフィラーゼの誘導を抑制し, エチレン処理により促進されるウンシュウミカン果実の脱緑を抑制すると推察された.

ウンシュウミカン（Ｃｉｔｒｕｓ ｕｎｓｈｉｕ）果皮のピロフェオホルビドａ分解酵素

Acetone powder extracts (APE), prepared from Citrus unshiu fruit, contained a pyropheophorbide a degrading enzyme. The enzyme catalyses the degradation of pyropheophorbide a in the presence of H2O2 and 2, 4-dichlorophenol (DCP). The optimum pH values were 6.2 with potassium phosphate buffer and 4.2 with acetate buffer. Linearity between the rate of the degradation and protein concentration existed over the range 0.0a250μg protein per 3.0ml of the reaction mixture. The Km for the pyropheophorbide a, DCP and H2O2 was ca. 140μM, ca. 1.3mM, and ca. 45μM, respectively. The enzymatic degradation of pyropheophorbide a was inhibited by ascorbate, hydroquinone, n-propyl gallate. We confirmed that tiron and potassium cyanide (KCN) are involved with free radicals. Pyropheophorbide a degradation by APE resulted in the opening of the chlorophyll-ring and a decrease in the red and Soret bands of the UV/VIS differential spectrum of the reaction mixture. Moreover, pyropheophorbide a and pheophorbide a degradation by horseradish peroxidase (HRP) followed a similar reaction pattern. The velocity of pheophorbide a degradation by APE was slower than that of pyropheophorbide a under the same reacting condition. These results suggest that pyropheophorbide a-degrading enzyme from Citrus unshiu fruit is a peroxidase isozyme.

Ethylene-enhanced Chlorophyll Catabolism in Citrus unshiu Fruit: Effects of Inhibitors on the Activity of Enzymes Involved in Type I Reactions.

Ethylene-enhanced Chlorophyll Catabolism in Citrus unshiu Fruit: Effects of Inhibitors on the Activity of Enzymes Involved in Type I Reactions.

A Novel Pathway of Chlorophyll Catabolism in Citrus unshiu Fruit: Enzymatic Conversion of Chlorophyllide a to Mg-C132-carboxyl-pyropheophorbide.

A Novel Pathway of Chlorophyll Catabolism in Citrus unshiu Fruit: Enzymatic Conversion of Chlorophyllide a to Mg-C132-carboxyl-pyropheophorbide.

ウンシュウミカン果皮（Ｃｉｔｒｕｓ ｕｎｓｈｉｕ Ｍａｒｃ．）におけるエチレン誘導可溶性クロロフィラーゼ

To elucidate the mechanism of ethylene-enhanced degreening in satsuma mandarin (Citrus unshiu Marc. c.v. Nichinan No. 1) fruits, a water-soluble chlorophyllase (chlorophyll-chlorophyllide-hydrolase, E.C. 3.1.1.14) was prepared from the peel of ethylene-treated fruits. The maximum catabolic rate of chlorophyll a (Chl a) to chlorophyllide a occurred in the presence of 30a40% concentration of acetone at 30°C. Optimum concentrations of substrate and protein for the reaction were 12.4 μM and 2.6 mg/4.5ml, respectively. The optimum pH value was 7.6 with a phosphate buffer, 7.86 with a borate buffer, and 7.5 with an acetate buffer. At pH 7.6 (phosphate buffer) and 25°C in the presence of 33% acetone, the tentative Km value for Chl a hydrolysis activity was approximately 6.5 μM. Moreover, when the reaction mechanism of Chl a degradation was investigated using the 3-dimensional fluorescence spectral measurements, fluorescent Chl catabolites (FCC, Ex/Em : 440/670 nm) were detected but the fluorescent Chl-ring cleavage catabolites (Ex/Em : around 320a360/440a460 nm) were not. The FCC formation was inhibited by p-chloromercuribenzoate (PCMB), an inhibitor of chlorophyllase, but the inhibition was reversible by the addition of reduced glutathione. These results suggest that Chl a degradation occurred through chlorophyllide a formation catalyzed by a water-soluble chlorophyllase from ethylene-treated Citrus unshiu fruits. A possible participation of the enzyme in ethylene-enhanced degreening of the fruit peel is briefly discussed.

Chlorophyll Catabolism in Ethylene-treated Citrus unshiu Fruits.

Changes in the levels of chlorophylls (Chls) and their derivatives were determined to clarify the Chl metabolic pathway in ethylene-treated satsuma mandarin (Citrus unshiu Marc. cv. Nichinan No.1) fruits held in the dark. The fruits were treated with or without 200 ppm ethylene for 16 hr in a 20-1 desiccator at 25°C in darkness. After the treatment, the fruits were further stored in fresh air at 25°C in the dark for 72 hr. Chl pigments in the peel monitored with HPLC revealed that they remained unchanged during ethylene treatment. After 40 hours of treatment, a decrease occurred in the levels of Chl a, Chl b, and pheophytin a. With or without ethylene, low levels of chlorophyllide a (Chlide a), 132-Hydroxy (HO) Chl a, pheophorbide a (Phed a), and pyropheophorbide a (Pyrophed a) were eluted as Chl a derivatives. Chlide b and its derivatives could not be detected. The ratio of Chl a to b in the ethylene-treated fruits increased significantly with the advance of degreening. The ethylene-induced Chl metabolic process in Citrus unshiu fruits is discussed.

A Possible Participation of Pyropheophorbide a in Chlorophyll Catabolism during Ripening of Citrus unshiu Fruits.

Chlorophyll (chl) derivatives in the peel of green satsuma mandarin (Citrus unshiu Marc. cv Nichinan No.1) fruits were studied to clarify the mechanism of chl degradation. High performance liquid chromatography (HPLC) and high performance thin-layer chromatography (HPTLC) were used to separate chl derivatives in acetone extracts from green satsuma mandarin fruits. Pyropheophorbide (pyrophed) a was present in the green skin but it gradually disappeared with ripening ; it was undetectable in the last harvest. A possible participation of pyrophed a in chl catabolism is discussed.

Flavonoids of the peel of the fruit ofCitrus unshiu

Flavonoids of the peel of the fruit ofCitrus unshiu

ウンシュウミカン果実におけるエチレン誘導クロロフィル分解のアリルイソチオシアネートによる抑制

ウンシュウミカン果実におけるエチレン誘導クロロフィル分解のアリルイソチオシアネートによる抑制

ウンシュウミカン (<I>Citrus unshiu</I>) 果実におけるエチレン処理後のクロロフィル分解の経路について

Two enzymatic metabolites from chlorophyll a were detected in crude enzyme extracts preparated from ethylene-treated fruits of Citrus unshiu. They were identified as pheophorbide a and pyropheophorbide a using high performance thin layer and liquid chromatographic techniques and fluoresence spectroscopy.Sodium ascorbate enhanced conversion of chlorophyll a to pheophorbide pigments, whereas p-chloromercuribenzoate strongly inhibited the formation of pyropheophorbide a. These results suggest that the crude enzyme extracts contain the two enzymes, 'Mg-de-chelatase' and 'Decarbomethoxylase'

ウンシュウミカン (<I>Citrus unshiu</I> Marc.) 果実のエチレン処理後のクロロフィル分解におけるpheophorbide aの代謝

Two enzymatic reaction metabolites were isolated from a crude enzyme extract of degreening Citrus unshiu fruits when pheophorbide a was used as a substrate. One was identified as pyropheophorbide a but the other was unidentified.A time course study of pheophorbide a degradation and metabolite formation disclosed that pheophrbide a is firstly metabolized to an unidentified intermediate X, which is then degraded top pyropheophorbide a.Optimum pH for X formation from pheophorbide a was 7.0 with potassium phosphate buffer. Linearity between the rate of the conversion and protein concentration was obtained over the range of 0.250.50 mg protein per 10 ml of the reacetion mixture. Tentative Km for pheophorbide a is 4.0 mM. The reaction was strongly inhibited by p-chloromercuribenzoate and AgNO3.

Fruit set, young fruit and leaf growth of Citrus unshiu in relation to assimilate supply

Fruit set, fruitlet and young leaf growth at an early stage of fruit ontogeny were promoted significantly by girdling at the base of autumn-shoots of the preceding year on Day 10 pre-full bloom. In contrast, defoliation or darkening of the old leaves on the autumn-shoots aggravated fruit drop and inhibited fruitlet and young leaf growth. During this period the net photosynthetic rate (Pn) of old leaves rose sharply. These observations clearly indicate that assimilate supply to fruitlets and young leaves of Citrus unshiu during this period is mainly affected by the current photosynthates from the old leaves instead of from the remobilization of stored reserves within the trunks. On Day 20 after full bloom the young leaves reached 48% of their final area and increased their Pn rapidly. Subsequently, the Pn of old leaves began to decrease dramatically. The impeding effect of darkening of old leaves on fruit growth from Day 20 after full bloom was absent. The results show that the young leaves gradually substitute for the old leaves to supply photosynthates to the developing fruits from about Day 20 after full bloom.

ウンシュウミカン果実のクロロフィリドａ 分解酵素（Ｈ２Ｏ２‐ＤＣＰ要求性）

A chlorophyllide a-breaching enzyme from ethylene-treated fruits of Citrus unshiu was ob-tained. The enzyme catalyses the breaching of chlorophyllide a in the presence of H2O2 and 2, 4-dichlorophenol. Its pH optimum was 6.2 with potassium phosphate buffer, 6.2 with borate buffer, and 5.6 with acetate buffer. The reaction was inhibited by potassium cyanide, Tiron, and hydroquinone. 2, 5-Dimethylfuran caused no inhibition. Breaching increased for 3 min and reached a plateau after 5 min. Linearity between the rate of the breaching activity and pro-tein concentration was obtained over the range 0.4 2.8 mg protein per 3.3 ml of the reac-tion mixture. The Km for chlorophyllide a is approximately 20 AM.Cycloheximide, a protein synthesis inhibitor, inhibited the ethylene-enhanced chlorophyll degradation and repressed the enhancement of chlorophyllase activity, whereas it had no effect on the chlorophyllide a-breaching activity. A high correlation coefficient (r=0.8701) was obtained between chlorophyll contents and chlorophyllase activity.Visible-adsorption spectrum of the reaction mixture during chlorophyll degradation indicates occurrences of the shift of the red band around 670 nm to a longer wavelength, a shift to Soret band around 420 nm to shorter wavelength, and a shift around 470 nm to a longer wavelength.

ウンシュウミカン果実のクロロフィリドａ分解酵素（クロロフィリドａペルオキシダーゼ）の精製と酵素化学的性質について

A chlorophyllide a breaching enzyme extracted from degreening Citrus unshiu fruits was purified some 1, 000-fold and characterized.1. The enzyme catalyses chlorophyllide a degradation in the presence of H2O2 and the phenols, 2, 4-dichlorophenol or p-coumaric acid. It behaves as a protein with a MW of 42, 000 on Sephacryl S-200 gel filtration but it does not give a single band on polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, and upon staining with guaiacol in the presence of H2O2. The Michaelis constant, Km, for chlorophyllide a was 13.2 μM.2. The enzyme exhibited IAA oxidase activity in the presence of Mn++; it was inhibited by hydroquinone, Tiron, potassium cyanide (KCN), diethyldithiocarbamate (DIECA) but not by iodoacetate, p-chloromercuribenzoate (PCMB), N-ethylmaleimide, histidine, D-mannose, dimethylfuran, triethylenediamine, ethylenediaminetetraacetic acid (EDTA), NaN3 and thiourea.The possible in vivo participation of this in vitro enzyme system as a H2O2-scavenging system in the dark is discussed.