• Journal of Veterinary Science
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• v.23 no.1
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• pp.4.1-4.15
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• 2022

Background: Flavonoids are natural polyphenols found widely in citrus fruit and peel that possess anti-adipogenic effects. On the other hand, the detailed mechanisms for the antiadipogenic effects of flavonoids are unclear. Objectives: The present study observed the anti-adipogenic effects of five major citrus flavonoids, including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), on AMP-activated protein kinase (AMPK) activation in palmitate (PA)-treated HepG2 cells. Methods: The intracellular lipid accumulation and triglyceride (TG) contents were quantified by Oil-red O staining and TG assay, respectively. The glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The levels of AMPK, acetyl-CoA carboxylase (ACC), and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and levels of sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression were analyzed by Western blot analysis. The potential interaction between the flavonoids and the γ-subunit of AMPK was investigated by molecular docking analysis. Results: The flavonoid treatment reduced both intracellular lipid accumulation and TG content in PA-treated HepG2 cells significantly. In addition, the flavonoids showed increased 2-NBDG uptake in an insulin-independent manner in PA-treated HepG2 cells. The flavonoids increased the AMPK, ACC, and GSK3β phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. Molecular docking analysis showed that the flavonoids bind to the CBS domains in the regulatory γ-subunit of AMPK with high binding affinities and could serve as potential AMPK activators. Conclusion: The overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK by flavonoids.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.11
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• pp.1688-1694
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• 2014

Jeju Hallabong Tangor (Citrus kiyomi${\times}$ponkan) is a Citrus species with a variety of physiological properties such as anti-oxidant, anti-inflammation, anti-cancer, and anti-obesity. We investigated the anti-obesity effects of Hallabong Tangor peel extracts before (HLB) and after (HLB-C) bioconversion with cytolase based on modulation of adipocyte differentiation and lipid metabolism in 3T3-L1 adipocytes. Treatment with cytolase decreased flavanone rutinoside forms (narirutin and hesperidin) and increased flavanone aglycone forms (naringenin and hesperetin). During adipocyte differentiation, 3T3-L1 cells were treated with 0.5 mg/mL of Sinetrol (a positive control), HLB or HLB-C. Adipocyte differentiation was inhibited in both citrus groups, but not in control and Sinetriol groups. HLB and HLB-C tended to reduce insulin-induced mRNA levels of CCAAT/enhancer-binding protein ${\alpha}$ ($C/EBP{\alpha}$) and sterol regulatory element-binding protein 1c (SREBP1c). Compared to the control and Sinetrol groups, HLB and HLB-C markedly suppressed insulin-induced protein expression of $C/EBP{\alpha}$ and peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$). The HLB and Sinetrol groups, but not HLB-C group, significantly increased adipolytic activity with higher release of free glycerol compared to the control group in differentiated 3T3-L1 adipocytes. These results suggest that bio-conversion of Hallabong Tangor peel extracts with cytolase increases aglycone flavonoids. Irrespective of bioconversion, both Hallabong Tangor peel extracts exert anti-obesity effects that may contribute to prevention of obesity through inhibition of adipocyte differentiation or induction of adipolytic activity.

• Journal of Nutrition and Health
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• v.42 no.3
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• pp.278-290
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• 2009

This study aims to evaluate the changes of flavonoid contents and antioxidants activity of Jeju native citrus fruits juice according to the harvest date. Flavonoids such as quercatagetin, narirutin, hesperidin and neohesperidin were contained most plentifully in the juice of Jigak (Citrus aur-antium) by 573.73 mg/100 mL, Sadoogam (C. pseudogulgul) by 393.99 mg /100 mL, Soyooja by 29.63 mg/100 mL and Jigak (C. aurantium) by 201.23 mg/100 mL in the late August, respectively. The highest contents of nob-iletin, sinensetin and tangeretin among polymethoxyflavones were found in the juice of Hongkyool (C. tachibana) by 7.39 mg/100 mL, 2.24 mg/100 mL, 0.63 mg/100 mL in the late August, respectively. 3,5,6,7,8,3',4'- Heptamet- hoxyflavone recorded the highest amount in Punkyool (C. tangerina) by 0.27 mg/100 mL in the late August, but the other polymethoxyflavones including 3',4',7,8-tetramethoxyflavone, 3',4'-dimethoxyflavone, 4'-methoxyflavone, 5,6,7,3',4',5'-hexamethoxyflavone, scutellarein tetramethylether were observed only trace amount in all the citrus fruits. Flavonoid contents in the citrus fruit juices were the highest during early maturation and decreased rapidly while ripening. Total polyphenol contents were the highest in the late August and decreased with ripening. However from the late December, the contents were increased again. Antioxidant activities of the fruits were evaluated as electron donating ability and were the lowest in the late September and increased with the fruit ripening. These results suggest that quercetagetin among all the flavonoids was most plentiful in Jigak and Dangyooja (C. grandis), so that the fruits could be used for industrial material of flavonoids and antioxidant agents.

• Food Science and Preservation
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• v.13 no.2
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• pp.144-153
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• 2006

The effects on deacidification of citrus fruits produced at early harvesting season for juice products were examined by heating treatment of raw fruits and electrodialysis of juice. Weight and total acidities were decreased by heating treatment for 40 hr at $25^{\circ}C,\;30^{\circ}C,\;35^{\circ}C\;and\;40^{\circ}C$, but $^{\circ}Brix$ and pH increased Sugar to acid ratio also increased. Total acidities were decreased from 1.25 (Oct30, 2004), 1.24 (Nov.5, 2004), 0.99 (Nov.13, 2004) and 0.98% (Nov.19, 2004) to 0.48 (Oct30, 2004), 0.51 (Nov.05, 2004), 0.37 (Nov.13, 2004) and 0.42% (Nov.19, 2004) by electrodialysis for 100 min respectively, and $^{\circ}Brix$ also slightly decreased, but solid to acid ratio was increased as a result. However pH and color remained almost unchanged by electodialysis. Also, free sugar contents of citrus juice little were changed, but organic acid content were decreased fairly. Narirutin and hesperidin content among flavonoids were slightly decreased by electrodialysis, but they were not significantly different. $K^+,\;PO_4^{2-},\;SO_4^{2-}\;and\;Cl^-$ content were decreased by electrodialysis, and $K^+$ contents decreased by more than 80% However, $Na^+$ consent was increased by about 2 times. Total polyphenol contents and electron donating abilities were decreased a little by electrodialysis but nitrite scavenging abilities were little changed. By acceptability test citrus juice prepared by electrodialysis for 100 min was superior to original citrus juice.

• Nutrition Research and Practice
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• v.10 no.2
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• pp.131-138
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• 2016

BACKGROUND/OBJECTIVES: Citrus and its peels have been used in Asian folk medicine due to abundant flavonoids and usage of citrus peels, which are byproducts from juice and/or jam processing, may be a good strategy. Therefore, the aim of this study was to examine antioxidant and anti-inflammatory effects of bioconversion of Jeju Hallabong tangor (Citrus kiyomi ${\times}$ ponkan; CKP) peels with cytolase (CKP-C) in RAW 264.7 cells. MATERIALS/METHODS: Glycosides of CKP were converted into aglycosides with cytolase treatment. RAW 264.7 cells were pre-treated with 0, 100, or $200{\mu}g/ml$ of citrus peel extracts for 4 h, followed by stimulation with $1{\mu}g/ml$ lipopolysaccharide (LPS) for 8 h. Cell viability, DPPH radical scavenging activity, nitric oxide (NO), and prostagladin $E_2$ ($PGE_2$) production were examined. Real time-PCR and western immunoblotting assay were performed for detection of mRNA and/or protein expression of pro-inflammatory mediators and cytokines, respectively. RESULTS: HPLC analysis showed that treatment of CKP with cytolase resulted in decreased flavanone rutinoside forms (narirutin and hesperidin) and increased flavanone aglycoside forms (naringenin and hesperetin). DPPH scavenging activities were observed in a dose-dependent manner for all of the citrus peel extracts and CKP-C was more potent than intact CKP. All of the citrus peel extracts decreased NO production by inducible nitric oxide synthase (iNOS) activity and $PGE_2$ production by COX-2. Higher dose of CKP and all CKP-C groups significantly decreased mRNA and protein expression of LPS-stimulated iNOS. Only $200{\mu}g/ml$ of CKP-C markedly decreased mRNA and protein expression of cyclooxygenase-2 in LPS-stimulated RAW 264.7 cells. Both 100 and $200{\mu}g/ml$ of CKP-C notably inhibited mRNA levels of $interleukin-1{\beta}$ ($IL-1{\beta}$) and IL-6, whereas $200{\mu}g/ml$ CKP-C significantly inhibited mRNA levels of $TNF-{\alpha}$. CONCLUSIONS: This result suggests that bioconversion of citrus peels with cytolase may enrich aglycoside flavanones of citrus peels and provide more potent functional food materials for prevention of chronic diseases attributable to oxidation and inflammation by increasing radical scavenging activity and suppressing pro-inflammatory mediators and cytokines.

• Nutrition Research and Practice
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• v.9 no.6
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• pp.599-605
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• 2015

BACKGROUND/OBJECTIVES: Citrus flavonoids have a variety of physiological properties such as anti-oxidant, anti-inflammation, anti-cancer, and anti-obesity. We investigated whether bioconversion of Citrus unshiu with cytolase (CU-C) ameliorates the anti-adipogenic effects by modulation of adipocyte differentiation and lipid metabolism in 3T3-L1 cells. MATERIALS/METHODS: Glycoside forms of Citrus unshiu (CU) were converted into aglycoside forms with cytolase treatment. Cell viability of CU and CU-C was measured at various concentrations in 3T3L-1 cells. The anti-adipogenic and lipolytic effects were examined using Oil red O staining and free glycerol assay, respectively. We performed real time-polymerase chain reaction and western immunoblotting assay to detect mRNA and protein expression of adipogenic transcription factors, respectively. RESULTS: Treatment with cytolase decreased flavanone rutinoside forms (narirutin and hesperidin) and instead, increased flavanone aglycoside forms (naringenin and hesperetin). During adipocyte differentiation, 3T3-L1 cells were treated with CU or CU-C at a dose of 0.5 mg/ml. Adipocyte differentiation was inhibited in CU-C group, but not in CU group. CU-C markedly suppressed the insulin-induced protein expression of CCAAT/enhancer-binding protein ${\alpha}$ ($C/EBP{\alpha}$) and peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) as well as the mRNA levels of $CEBP{\alpha}$, $PPAR{\gamma}$, and sterol regulatory element binding protein 1c (SREBP1c). Both CU and CU-C groups significantly increased the adipolytic activity with the higher release of free glycerol than those of control group in differentiated 3T3-L1 adipocytes. CU-C is particularly superior in suppression of adipogenesis, whereas CU-C has similar effect to CU on stimulation of lipolysis. CONCLUSIONS: These results suggest that bioconversion of Citrus unshiu peel extracts with cytolase enhances aglycoside flavonoids and improves the anti-adipogenic metabolism via both inhibition of key adipogenic transcription factors and induction of adipolytic activity.

• Korean Journal of Food Science and Technology
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• v.37 no.5
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• pp.783-788
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• 2005

Major components and antioxidative properties of whole fruit powders and juices prepared from premature mandarin, Citrus unshu, were investigated. Dietary fiber, flavonoid, and organic acid contents greatly increased in samples with lower degree of fruit maturity (August) than higher ones (October), and in whole fruit powders than extracted juices, whereas free sugar contents, especially sucrose content, increased in fruits with higher degree of maturity than lower ones. Antioxidative properties were not affected by degree of fruit maturity, whereas total polyphenol content and electron-donating abilities were higher in extracted juices than whole fruit powders. Nitrite-scavenging abilities of all samples were similar except at pH 6.0.

• Food Science and Preservation
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• v.18 no.4
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• pp.567-574
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• 2011

In this work, the major flavonoid content of citrus peel ethanol extract and the changes in the serum lipid and body fat of high-fat-diet-fed rats were examined. The subjects were divided into four groups: the normal, control, O1 (0.1% citrus peel ethanol extract), and O5 (0.5% citrus peel ethanol extract) groups. The experimental groups (O1 and O5) were fed a high-fat diet mixed with 0.1 and 0.5% citrus peel ethanol extract, respectively, for 10 weeks. The results of the analysis showed that the main ingredients of the flavonoid in the citrus peel ethanol extract (60 brix) were $38.21{\pm}1.37$ mg/g hesperidin and $15.02{\pm}0.48$ mg/g narirutin. With regard to the changes in the serum total-cholesterol level, compared with the control group's $79.1{\pm}4.6$ mg/dL, the serum total-cholesterol levels of the O1 and O5 groups were significantly lowered to $64.6{\pm}5.4$ and $59.5{\pm}4.7$ mg/dL, respectively. As for the changes in the LDL-cholesterol level, compared with the control group's $18.4{\pm}3.7$ mg/dL, the LDL-cholesterol levels of the O1 and O5 groups were significantly lowered to $13.0{\pm}3.6$ and $13.0{\pm}1.9$ mg/dL, respectively. For the changes in the serum triglyceride level, compared with the control group's $74.5{\pm}13.2$ mg/dL, the serum triglyceride level of the O5 group was significantly lowered to $48.6{\pm}11.2$ mg/dL. Finally, with regard to the changes in body fat, compared with the control group, the 05 group showed a significant decrease. In conclusion, it was observed that citrus peel ethanol extract was effective in lowering the total-cholesterol, LDL-cholesterol, and triglyceride levels of high-fat-diet-fed rats.