Nutr Res Pract.  2016 Apr;10(2):131-138. 10.4162/nrp.2016.10.2.131.

Bioconverted Jeju Hallabong tangor (Citrus kiyomi × ponkan) peel extracts by cytolase enhance antioxidant and anti-inflammatory capacity in RAW 264.7 cells

• Chang YH
• Seo J
• Song E
• Choi HJ
• Shim E
• Lee O
• Hwang J

• KMID: 2313909
• DOI: http://doi.org/10.4162/nrp.2016.10.2.131

Abstract

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 × 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 µg/ml of citrus peel extracts for 4 h, followed by stimulation with 1 µg/ml lipopolysaccharide (LPS) for 8 h. Cell viability, DPPH radical scavenging activity, nitric oxide (NO), and prostagladin E2 (PGE2) 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 PGE2 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 µg/ml of CKP-C markedly decreased mRNA and protein expression of cyclooxygenase-2 in LPS-stimulated RAW 264.7 cells. Both 100 and 200 µg/ml of CKP-C notably inhibited mRNA levels of interleukin-1β (IL-1β) and IL-6, whereas 200 µg/ml CKP-C significantly inhibited mRNA levels of TNF-α.
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.

Keyword

Hallabong tangor; bioconversion; antioxidant; anti-inflammation; RAW 264.7 cells

MeSH Terms

Asian Continental Ancestry Group
Blotting, Western
Cell Survival
Chromatography, High Pressure Liquid
Chronic Disease
Citrus
Cyclooxygenase 2
Cytokines
Dinoprostone
Flavanones
Flavonoids
Functional Food
Glycosides
Humans
Inflammation
Interleukin-6
Medicine, Traditional
Nitric Oxide
Nitric Oxide Synthase Type II
RNA, Messenger
Cyclooxygenase 2
Cytokines
Dinoprostone
Flavanones
Flavonoids
Glycosides
Interleukin-6
Nitric Oxide
Nitric Oxide Synthase Type II
RNA, Messenger

Figure

• 

  Fig. 1 Effects of CKP and CKP-C on cell viability. RAW 264.7 cells were treated with different concentrations (0-500 µg/ml) of CKP or CKP-C for 24 h. Cell viability was measured by MTT assay. Viability of untreated control cells was defined as 100%. CKP: Citrus kiyomi × ponkan peel extract; CKP-C: Citrus kiyomi × ponkan peel extract after bioconversion with cytolase. Each bar represents the mean ± SD (n = 3). Different letters mean significant difference according to ANOVA, Tukey test (P < 0.05).

• 

  Fig. 2 Effects of CKP and CKP-C on DPPH free radical scavenging activity. Quercetin (50 µg/ml) was used as a positive control. Confluent cells were treated with 0-200 µg/ml of CKP and CKP-C and 0-50 µg/ml of quercetin for 4 h. CKP: Citrus kiyomi×ponkan peel extract; CKP-C: Citrus kiyomi × ponkan peel extract after bioconversion with cytolase. Each bar represents the mean ± SD (n = 3). Different letters mean significant difference according to ANOVA, Tukey test (P < 0.05).

• 

  Fig. 3 Effects of CKP and CKP-C on nitric oxide (NO) and PGE2 production in LPS-stimulated RAW 264.7 cells. Cells were pre-treated with 0, 100, or 200 µg/ml of CKP or CKP-C for 4 h, followed by stimulation with 1 µg/ml of LPS for 8 h. The treated culture media were used to measure the production of NO and PGE2. CKP: Citrus kiyomi × ponkan peel extract; CKP-C: Citrus kiyomi × ponkan peel extract after bioconversion with cytolase. Each bar represents the mean ± SD (n = 3). Different letters mean significant difference according to ANOVA, Tukey test (P < 0.05).

• 

  Fig. 4 Effects of CKP and CKP-C on mRNA levels of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. Cells were pre-treated with 0, 100, or 200 µg/ml of CKP or CKP-C for 4 h, followed by stimulation with 1 µg/ml of LPS for 8 h. Real time-PCR was performed using each cDNA and gene-specific primers. CKP: Citrus kiyomi × ponkan peel extract; CKP-C: Citrus kiyomi × ponkan peel extract after bioconversion with cytolase. Each bar represents the mean ± SD (n = 3). Different letters mean significant difference according to ANOVA, Tukey test (P < 0.05).

• 

  Fig. 5 Effects of CKP and CKP-C on protein expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. Cells were pre-treated with 0, 100, or 200 µg/ml of CKP or CKP-C for 4 h, followed by stimulation with 1 µg/ml of LPS for 8 h. Immunoblotting was performed with whole cell lysates and specific antibodies. CKP: Citrus kiyomi × ponkan peel extract; CKP-C: Citrus kiyomi × ponkan peel extract after bioconversion with cytolase; iNOS: inducible nitric oxide synthase; COX-2: cyclooxygenase-2.

• 

  Fig. 6 Effects of CKP and CKP-C on mRNA levels of IL-1β, IL-6, and TNF-α in LPS-stimulated RAW 264.7 cells. Cells were pre-treated with 0, 100, or 200 µg/ml of CKP or CKP-C for 4 h, followed by stimulation with 1 µg/ml of LPS for 8 h. Real time-PCR was performed using each cDNA and gene-specific primers. CKP: Citrus kiyomi × ponkan peel extract; CKP-C: Citrus kiyomi × ponkan peel extract after bioconversion with cytolase. Each bar represents the mean ± SD (n = 3). Different letters mean significant difference according to ANOVA, Tukey test (P < 0.05).

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