Fatty acid analysis and regulatory effects of citron (Citrus junos Sieb. ex TANAKA) seed oil on nitric oxide production, lipid accumulation, and leptin secretion

Tae Woo Kim; Kyoung Kon Kim; Yun Hwan Kang; Dae Jung Kim; Myeon Choe

doi:10.4163/jnh.2014.47.4.221

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Kim, Kim, Kang, Kim, and Choe: Fatty acid analysis and regulatory effects of citron (Citrus junos Sieb. ex TANAKA) seed oil on nitric oxide production, lipid accumulation, and leptin secretion

Research Article

Journal of Nutrition and Health 2014; 47(4): 221-228.

Published online: 31 August 2014

DOI: https://doi.org/10.4163/jnh.2014.47.4.221

Fatty acid analysis and regulatory effects of citron (Citrus junos Sieb. ex TANAKA) seed oil on nitric oxide production, lipid accumulation, and leptin secretion

Tae Woo Kim^1,^**, Kyoung Kon Kim^2,^**, Yun Hwan Kang^1,^**, Dae Jung Kim¹, Myeon Choe^1,²

¹Well-being Bioproducts RIC, Kangwon National University, Gangwon 200-701, Korea.

²Department of Bio-Health Technology, Kangwon National University, Gangwon 200-701, Korea.

To whom correspondence should be addressed. tel: +82-33-250-8645, mchoe@kangwon.ac.kr

Equal contributor:

**These authors contributed equally to this work.

Received 24 June 2014 Revised 11 July 2014 Accepted 31 July 2014

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

Citron seed oil (CSO) has been reported to have high antioxidant activity. However, the composition and other biologically activities of CSO have not been reported. In this study, we confirmed the fatty acid composition of CSO, which may be beneficial to vascular disease and obesity.

Methods

We investigated the oil composition of CSO using gas chromatography coupled with mass spectrometry (GC-MS) analysis, and cytotoxicity was confirmed by Cell Counting Kit-8 (CCK-8) assay. Nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) was measured using Griess reagent, and lipid accumulation and leptin secretion in 3T3-L1 cells were measured by Oil-Red O staining and commercial ELISA kit, respectively.

Results

GC-MS analysis indicated that CSO contains several components, including linoleic acid, oleic acid, palmitic acid, stearic acid, linolenic acid, palmitoleic acid, and arachidic acid. In physiological activity analysis, CSO did not induce cytotoxic effects in HUVECs and 3T3-L1 cells. Further, CSO significantly induced nitric oxide and leptin secretion as well as inhibited lipid accumulation.

Conclusion

CSO increased NO release, inhibited lipid accumulation, and induced leptin secretion, suggesting it may be useful for the management of vessels and weight gain. Although further studies are required to investigate the safety and mechanism of action of CSO, our results show that the composition and physiological activity of CSO are sufficient for its use as functional edible oil.

Keywords: citron seed oil, fatty acid composition, nitric oxide, leptin, lipid accumulation

Figures and Tables

Fig. 1

Concentration-dependent effects of citron seed oil (CSO) on human umbilical vein endothelial cells (HUVECs) and 3T3-L1 cell growth. A: Cytotoxicity of CSO on HUVECs. B: Cytotoxicity of CSO on 3T3-L1. Cell viability was analyzed using the CCK-8 assay kit. Each bar is the Mean ± S.D. from three independent experiment.

Fig. 2

GC chromatograms and MS fragments of individual fatty acids extracted from citron seed oil (CSO). 1: undecanoic acid, 2: palmitic acid, 3: palmitoleic acid, 4: stearic acid, 5: oleic acid, 6: Unknown, 7: linoleic acid, 8: arachidic acid, 9: linolenic acid.

Fig. 3

Effect of citron seed oil (CSO) on nitric oxide (NO) secretion in HUVACs. Reductions of NO by TNF-α were recovered by CSO (0.1 mg/ml) treatment. Results are presented as Mean ± S.D. of three independent experiments. #: p < 0.05, ^*: p < 0.01 as compared to the TNF-α (100 ng/ml), different letters on the bars (a and b) indicate significant differences (p < 0.05) by Duncan's multiple range test.

Fig. 4

Citron seed oil (CSO) inhibits lipid accumulation in 3T3-L1 cells. Adipocytes were incubated with two concentrations (0.05 and 0.1 mg/ml) of CSO and lipid accumulation was determined by Oil Red O staining. Results are presented as Mean ± S.D. of three independent experiments. ^**: p < 0.01 as compared to the control by unpaired two-tailed t-tests.

Fig. 5

Citron seed oil (CSO) induces leptin secretion in 3T3-L1 cells. Adipocytes were incubated with two concentrations (0.05 and 0.1 mg/ml) of CSO. Leptin secretion was determined by enzyme-linked immunosorbent assay (ELISA). Results are presented as Mean ± S.D. of three independent experiments. ^***: p < 0.001 as compared to the control by unpaired two-tailed t-tests.

Table 1

Analysis condition of GC and GC/MSD

Table 2

Fatty acid profiles of citron seed oil (CSO)

1) Undecanoic acid (C 11 : 0, 29.065 min) was used as a internal standard.

Notes

This work was supported by grants of High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (312001-03-01-HD040), Well-being Bioproducts Regional Innovation Center project (B0009702) and Kangwon National University Institute of Biosciences and Biotechnology (320130015).

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