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. 2018 May 23;27(6):1801–1809. doi: 10.1007/s10068-018-0396-z

Protective effects of citrus based mixture drinks (CBMDs) on oxidative stress and restraint stress

MyoungLae Cho 1, Dan-Bi Kim 2, Gi-Hae Shin 6, Jae-Min Kim 6, Yoonhee Seo 3, Soo Young Choe 3, Ju Hyun Cho 4, Young-Cheul Kim 5, Jin-Ha Lee 6, Ok-Hwan Lee 6,✉
PMCID: PMC6233418  PMID: 30483445

Abstract

In the current study investigated the protective effects of citrus based mixture drinks (CBMDs) using oxidative stress in human dermal fibroblast (HDF) cells and restraint-stressed rats. The CBMDs contained citrus bioflavonoids including narirutin and hesperidin. The cell viability of HDF cells treated with H2O2 was observed at 53.9% but treated with CBMD-1 and CBMD-2 (500 μg/mL) on H2O2 exposed HDF cells significantly increased the relative cell viability at 65.0 and 72.2%, respectively. In the treadmill test, the time spent on the electrode plate in the restraint-stressed group was analyzed 24.1 s, but restraint-stressed rats with administered CBMDs (300 mg/kg) had significantly decreased the time at 2.4 (CBMD-1) and 4.7 (CBMD-2) s, respectively. In addition, number of touches the electrode plate in restraint-stressed group was observed at 42.4 ea, but, restraint-stressed rats with administered CBMD-1 and CBMD-2 (300 mg/kg) were significantly decreased at 7.0 and 10.2 ea, respectively.

Keywords: Citrus based mixture drinks, Narirutin, Hesperidin, Oxidative stress, Restraint stress

Introduction

Stress is a defensive reaction to harmful stressors such as physical, psychical, chemical, and social factors. The behavioral responses to stress depend on the stress type, duration, and event (Chrousos, 2009; Jaggi et al., 2011). These physiological and biological behavioral alterations induced by stress affect various organs, which may contribute to depression, anxiety and reduced immunity as well as heart disease (Gregus et al., 2005; Novío et al., 2011). The main symptoms of depression reported besides tiredness include nervousness, irritability, sleep disturbance, and decreased weight (Nestler et al., 2002). In addition, several instances of depression were accompanied by anxiety, and depression with co-occurring anxiety may cause various diseases such as arousal, vigilance, and increased blood pressure (Gold and Chrousos, 2002; Nestler et al., 2002). Therefore, many studies on stress-induced depression have reported potentially negative parameters. Psychological stress including immobilization stress and/or restraint stress has induced excessive amount of nitric oxide (NO) and nitric oxide synthase (iNOS), and acute restraint stress has led to hyperalgesia via non-involvement of adrenergic mechanisms in rats (Leza et al., 1998; Olivenza et al., 2000). Several studies have reported the beneficial effects of the use of natural resources such as Withania somnifera, Ginkgo biloba, and Panax ginseng against acute and chronic stresses, including immobilization stress (Bhattacharya et al., 1987; Rai et al., 2003).

Citrus fruits are one of the most popular fruits in the world, and are also commonly used for juices, pies, ice creams, and jams. Among them, the citrus juices have beneficial effects on humans because of various nutrients, polyphenols, flavonoids, and their low-fat content (Bocco et al., 1998). Recent studies have demonstrated that drinking citrus juices reduced oxidative stress via inhibition of superoxide anion production (Ohnishi et al., 2015), and the intake of citrus juice exhibited antioxidant activity by decreasing reactive oxygen species (ROS) levels in mildly hypercholesterolemic men (Constans et al., 2015). Consumption of citrus juice has also shown vascular protective effects in human clinical research (Morand et al., 2011).

Our previous study reported that citrus based mixture drink (CBMD) has strong oxygen radical absorbance capacity (ORAC) and anti-aging activities in human dermal fibroblasts (HDF) cells and hairless mice (Kim et al., 2016). However, the beneficial effects of CBMDs against various stresses and evaluation of their toxic effects have not been studied yet. Therefore, the objective of this study was to determine the citrus flavonoid content of CBMDs using high performance liquid chromatography (HPLC) and to investigate the anti-stress effects of CBMDs on oxidative stress in HDF cells and restraint-stressed rats. In addition, the toxic effects of CBMDs on rats and their organs, such as the liver, spleen and adrenal gland, were investigated by the administration of CBMDs for 4 weeks.

Materials and methods

Sample preparation

The citrus based mixture drink powder (CBMD-1 and CBMD-2) made using Citrus sunki, Citrus sphaerocarpa, and Vitis vinifera were obtained from Hurum Co. (Hurum, Jeju, Korea). These CBMDs were selected as representative of 20 different juice mixtures with high antioxidant activity from a pilot study. The mixtures of CBMDs were freeze-dried and kept at − 20 °C until use.

Determination of citrus flavonoids in the CBMDs

The citrus flavonoids were analyzed using high performance liquid chromatography system (Waters 2695 Separation Module, Waters Co., Milford, MA, USA) with Waters 996 photodiode array detector (Waters Co.) in the 200–400 nm range. The HPLC chromatograms of citrus bioflavonoids were acquired at 280 nm. The sample volume of 10 μL was injected into the Sunfire™ C18 column (250 × 4.6 mm, 5 μm particle size) (Waters Co.) and eluted at a constant flow rate of 1.0 mL/min. The mobile phase was composed of acetonitrile and water (60:40 in 0.1% phosphoric acid, v/v) using isocratic condition. The narirutin and hesperidin contents were calculated using the standard curves.

Cell viability and protective effects on oxidative stress in HDF cells

Cytotoxicity was measured using a modification of the method of Kim et al. (2016), which utilizes the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H tetrazolium-5-carboxanilide inner salt (XTT) assay (WelGene, Seoul, Korea). The HDF cells were provided by Professor E.K. Hong (Kangwon National University, Chuncheon, Republic of Korea). The cells were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified Eagles medium (DMEM, Gibco BRL, Grand Island, NJ, USA) containing 10% (v/v) fetal bovine serum (FBS, Invitrogen Life Technologies, Carlsbad, CA, USA) and 1% antibiotics (penicillin/streptomycin, v/v). To determine the viability of HDF cells, cells were seeded in a 96-well plate (1 × 106 cells/well), then added with an aliquot of each samples (100, 200 and 500 μg/mL of CBMDs), and incubated for 24 h at 37 °C in the presence of 5% CO2. The XTT reagent was mixed with the phenazine methosulfate (PMS) reagent (WEL GENE, Seoul, Korea) and incubated for 4 h at 37 °C in the presence of 5% CO2. The absorbance was then detected with a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm. To determine the protective effect of CBMDs, 1 mM of hydrogen peroxide (H2O2) was added to induce oxidative stress to the cells after treating with the control and samples. The absorbance was measured after 3 h using a microplate reader at 450 nm (test wavelength) and 690 nm (reference wavelength).

Animals

Male Sprague-Dawley (SD)-rats (8 weeks old) were purchased from Daehan Biolink Ltd. (Ochang, Korea). Rats were housed in a climate-controlled room (22 °C at 50% humidity) with 12 h light/dark cycles and with free access to food and water. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Chungbuk Technopark (IACUC number OMCA-2013-011). The rats were allocated to nine groups, which included normal (n = 6), restraint-stressed before administered vehicle (n = 6), and restraint-stressed before administered diazepam and CBMDs groups using the following compounds and concentrations: 0.5 mg/kg of diazepam (n = 6), 33 mg/kg of CBMD-1 (n = 6), 100 mg/kg of CBMD-1 (n = 6), 300 mg/kg of CBMD-1 (n = 6), 33 mg/kg of CBMD-2 (n = 6), 100 mg/kg of CBMD-2 (n = 6) and 300 mg/kg of CBMD-2 (n = 6). The CBMD compounds were dissolved in distilled water and administered once daily by oral gavage for each concentration (33, 100 and 300 mg/kg/10 mL). The commercial anti-anxiety drug, diazepam (Valium, Roche Pharmaceuticals, Nutley, New Jersey) was used as a positive control (0.5 mg/kg/10 mL).

Restraint stress procedure

The restraint stress protocol was developed by Poleszak et al. (2006). The animals were divided into nine groups as mentioned above. Normal and experimental groups were orally administered a vehicle, diazepam or CBMDs for 4 weeks and then they were submitted to restraint stress for a period of 24 h. The restraint stress restricted all physical movement without causing pain. The rats were deprived of food and water during the restraint stress period.

Treadmill test

The treadmill test was performed after 30 min following the restraint-stress. The rats were placed on a motorized treadmill with a mild motivational shock bar (Eck-6 M treadmill, Columbus Instruments, Columbus, OH, USA) at a speed of 25 cm/s for 6 min, and the total distance travelled on the treadmill was measured. A gentle touch was sufficient to keep the rats running, and electro-stimulation was applied by an electrode plate at 0.4 mA during treadmill training.

Tissue preparation and histological assays

One hour after the restraint-stress procedure, the animals were anesthetized by ether inhalation, and then blood samples were rapidly taken from the abdominal aorta. Other tissue specimens such as liver, spleen and adrenal gland were removed and kept in 10% formaldehyde solution.

The liver, spleen and adrenal gland tissues were fixed with 30% formaldehyde (v/w), dehydrated using ethanol and embedded in paraffin. Tissue slices were cut 3.5–4 mm thick using paraffin blocks with a microtome (SM2000R, Leica Instruments, Wetzlar, Germany), stained with hematoxylin–eosin before drying, and observed with light microscopy (Nikon, Labophot-2, Tokyo, Japan).

Statistical analysis

All experiments were repeated three times (n = 3). The data are presented as the mean value ± standard deviation. All statistical analyses were performed using both SAS (SAS Institute; Cary, NC, USA) and SPSS software (Version 7.5, Chicago, IL, USA). Significant differences were tested by ANOVA and Duncan’s multiple range tests. Statistical significant was considered at p < 0.05. In the treadmill test (distance, time spent on electrode plate, and number of touches of electrode plate) was analyzed by Dunnet’s test for the homogeneity of variances; when the variances were considered significantly different by Duncan’s test, the data were analyzed by Dunnett’s T test (two-sided) (Dunnett, 1955). Statistical significant difference from restraint-stress group by Dunnet’s t test was considered at *p < 0.05 and **p < 0.01.

Results and discussion

Citrus flavonoid content in CBMDs

Citrus based mixture drinks included excellent nutritional compounds such as ascorbic acid, polyphenols and citrus flavonoids (Kim et al., 2016). In addition, the citrus flavonoids exhibited various biological activities, including neuro-protective (Hwang et al., 2012), antioxidative, and anti-carcinogenic activities (Nakajima et al., 2013). Moreover, intake of citrus juices observed beneficial effects on humans including reduced oxidative stress, decreasing ROS levels, and vascular protective effects (Constans et al., 2015; Morand et al., 2011; Ohnishi et al., 2015). Therefore, in the current study, the content of citrus flavonoids was determined by HPLC. Among the various citrus flavonoids, narirutin and hesperidin were detected in the CBMDs (Fig. 1). The peaks of narirutin and hesperidin were detected at 9.8 and 12 min, respectively. Comparing the two samples, CBMD-2 showed higher peaks of narirutin and hesperidin than CBMD-1. As shown in Table 1, the content of narirutin and hesperidin in CBMD-1 were 3.16 and 4.67 μg/g sample, respectively. The content of narirutin and hesperidin in CBMD-2 were 5.70 and 8.58 μg/g sample, respectively. These results suggest that the major citrus flavonoids in CBMDs were correctly identified as narirutin and hesperidin.

Fig. 1.

Fig. 1

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HPLC chromatograms of standard compounds and CBMDs. (A) Structure and HPLC chromatograms of narirutin and hesperidin, (B) HPLC chromatogram of CBMD-1, and (C) HPLC chromatogram of CBMD-2

Table 1.

Content of citrus flavonoids hesperidin and narirutin in CBMDs

Sample Narirutin Hesperidin
Mean ± SD (μg/g) RSD (%) Mean ± SD (μg/g) RSD (%)
CBMD-1 3.16 ± 0.02 0.6 4.67 ± 0.01 0.1
CBMD-2 5.70 ± 0.05 0.8 8.58 ± 0.01 0.1
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Cell viability and protective effects on oxidative stress in HDF cells

The cell viability of HDF cells was related to the cell toxicity of CBMDs. Samples treated with CBMD-1 and CBMD-2 had over 100% cell viability until a CBMD concentration of 500 μg/mL was reached [Fig. 2(A)]. Therefore, we proceeded to investigate the protective effects of CBMDs on HDF cells under oxidative stress induced by H2O2. In Fig. 2(B), treatment with H2O2 on HDF cells decreased cell viability (53.9%) compared with control (100%), but, cells treated with CBMD-1 (500 μg/mL) before exposure to H2O2 significantly increased (65.0%) relative cell viability compared to control (p < 0.05). On the other hand, HDF cells treated with low concentration of CBMD-2 (100 μg/mL) also significantly increased (64.8%) relative cell viability. The treated with CBMD-2 (500 μg/mL) on H2O2 exposed cells showed the greatest relative cell viability (72.2%) against oxidative stress. However, the relative cell viabilities of CBMDs were lower than the relative cell viability of our positive control NAC (83.0%).

Fig. 2.

Fig. 2

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Effects of cell viability and protective effects against oxidative stress of CBMDs. (A) Relative cell viability of HDF cells treated with CBMDs, (B) protective effects of CBMDs on oxidative stress-induced damage in HDF cells. Means with different letters differ significantly at p < 0.05

Body and organ weights and histological assays

The body weights of rats were measured every 3 days after administration of CBMDs and exhibited time-dependent change during the course of the experiment. However, no significant changes in body weight were observed among the control and CBMDs treated groups (Fig. 3). Figure 4 shows the representative section and weight of various organs after administration of CBMDs. The representative image of the liver stained with hematoxylin and eosin shows that relative vesicular liver steatosis did not significantly differ among groups, suggesting the CBMDs did not affect hepatic steatosis in restraint-stressed rats [Fig. 4(A)]. In addition, the final liver weight of the restraint-stressed group gained 3.36 g, and the groups treated with 33 and 100 mg/kg of CBMDs exhibited quite similar liver weights compared with the restraint-stressed (3.23–3.29 g) and positive control (3.24 g) groups. However, little low liver weights were observed for groups administered at 300 mg/kg of CBMD-1 (3.03 g) and CBMD-2 (3.11 g). Figure 4(B) shows the stained spleen sections and final spleen weight for each group. The stained spleens of control, restraint-stressed, and treated with CBMDs groups were not in alveolar space; moreover, significant differences in the spleen weight in restraint-stress and treated with CBMDs were negligible. However, final spleen weight of normal group showed little high and treated with positive control, diazepam was little low spleen weight. The effect of CBMDs on adrenal gland is shown in Fig. 4(C). The histology result of the adrenal gland revealed a normal arrangement and no distinct differences between experimental groups. The final adrenal glands weight of all groups did not differ very much among the restraint-stress rats without treated with 100 μg/mL of CBMD-1 group. The such small differences of 100 μg/mL of CBMD-1 compared with other CBMDs groups was considered experimental error. These results indicate that the rat’s body and organs including liver, spleen, and adrenal glands were not affected by administration of CBMDs.

Fig. 3.

Fig. 3

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Changes in body weights of rats during 4 weeks. Normal, non-restraint-stressed group; RS, administered vehicle with restraint-stressed group; RS + Dia., administered diazepam at 0.5 mg/kg with restraint-stressed group; RS + CBMD-1, administered CBMD-1 at 300 mg/kg with restraint-stressed group; RS + CBMD-2, administered CBMD-2 at 300 mg/kg with restraint-stressed group

Fig. 4.

Fig. 4

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Effects of CBMDs on restraint-stressed rat organs. (A) Liver representative sections and final liver weight, (B) spleen representative sections and final spleen weight, and (C) adrenal gland representative sections and final adrenal gland weight in restraint-stressed rats. Means with different letters differ significantly at p < 0.05

Treadmill exercise test

The comparison between the effects of CBMDs and the positive control (diazepam) on treadmill exercise in restraint-stressed rats is shown in Table 2. Each treadmill exercise group, including those administered diazepam or CBMDs, showed slight increases in the average running distance (85.8–88.7 m) compared with the restraint-stressed group (80.6 m). In addition, a significant (p < 0.05) decrease in both total time spent and number of touches of the electrode plate were observed in those treated with CBMDs and diazepam after restraint stress. The time spent on the electrode plate in the restraint-stressed group was at 24.1 s, but, restraint-stressed groups treated with CBMD-1 (8.7–2.4 s) and CBMD-2 (6.7–4.7 s) showed dose-dependent decreases at concentrations ranging from 33 to 300 mg/kg. In addition, the groups treated with 300 mg/kg of CBMD-1 (2.4 s) and CBMD-2 (4.7 s) were much lower than that of the positive control, diazepam (7.3 s) group. On the other hand, a similar trend was observed in the number of touches of the electrode plate. The groups treated with CBMDs (16.8–7.0 ea) were significantly decreased touches number of the electrode plate compared with RS group (42.4 ea). Moreover, administration of the highest concentrations (300 mg/kg) of CBMD-1 (7.0 ea) and CBMD-2 (10.2 ea) were more decreased the number of touches of the electrode plate than diazepam (15.8 ea). However, when tested at low concentrations (33 mg/kg), the number of touches the electrode plate in the CBMD-1 (16.8 ea) and CBMD-2 (14.6 ea) groups were comparable to that of the positive control, diazepam group.

Table 2.

Effects of CBMDs on treadmill exercise in restraint-stressed rats

Group Treadmill test
Distance (m) Time spent on electrode plate (s) Number of touches of electrode plate (ea)
Normal 86.5 ± 2.2** 4.5 ± 2.7** 10.5 ± 5.6**
RS 80.6 ± 7.2 24.1 ± 10.9 42.4 ± 24.3
RS + Dia. 85.8 ± 3.1* 7.3 ± 5.8** 15.8 ± 11.1**
RS + CBMD 1 (mg/kg)
 33 87.3 ± 2.4** 8.7 ± 4.4** 16.8 ± 9.2**
 100 88.2 ± 1.6** 8.6 ± 5.0** 8.3 ± 4.5**
 300 88.3 ± 0.8** 2.4 ± 1.7** 7.0 ± 3.3**
RS + CBMD 2 (mg/kg)
 33 86.5 ± 0.8** 6.7 ± 2.9** 14.6 ± 3.0**
 100 87.5 ± 1.6** 5.1 ± 3.8** 14.0 ± 13.8**
 300 88.7 ± 0.8** 4.7 ± 2.7** 10.2 ± 6.0**
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Values are presented as the mean ± SD (n = 6)

Diazepam was administered at 0.5 mg/kg

Statistical significant difference from RS group by Dunnet’s t test was considered at *p < 0.05 and **p < 0.01

In the current study, we investigated that the CBMDs contained citrus bioflavonoids, including narirutin and hesperidin, and have protective effects on oxidative stress in HDF cells. In addition, the administration of CBMDs to restraint-stressed rats produced non-toxic effects and significantly decreased the amount of time spent and the number of touches of the electrode plate.

CBMDs are complex mixtures made by Citrus sunki, Citrus sphaerocarpa, and Vitis vinifera, and these fruits contain various bioactive flavonoids such as naringin, naringenin, hesperidin, hesperetin, rutin, nobiletin, and tangeretin (Choi et al., 2007; Kim et al., 2016). The citrus flavonoid nobiletin isolated from citrus fruit peels improved cognitive impairment and reduced oxidative stress in mouse model study (Nakajima et al., 2013). In addition, Hwang et al. (2012) reported that dietary citrus flavonoids could mediate neuro-protective effects in mouse cortical neurons. Moreover, our previous study also found that the citrus based mixture juices exhibited antioxidant and anti-aging activities because they contain various citrus flavonoids (Kim et al., 2016). In the current study, the CBMDs had protective effects against oxidative stress induced by hydrogen peroxide oxidation, and it was considered that the major citrus bioflavonoids, hesperidin and narirutin in the CBMDs may have biological activities. Narirutin and hesperidin are abundant citrus flavonoids and are major compounds in sweet oranges, lemons, and grape-fruit (Garg et al., 2001; Park et al., 2013). Furthermore, bioflavonoids including narirutin and hesperidin have been reported to have health benefits such as antioxidant, anti-inflammatory, and vascular protective activities (Manach et al., 2003). It was reported that narirutin showed anti-inflammatory activity on allergic eosinophilic airway inflammation (Funaguchi et al., 2007), and protective effects against ethanol-induced liver damage (Garg et al., 2001). Additionally, hesperidin reduced plasma and hepatic cholesterols in rats (Monforte et al., 1995), and inhibited N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced carcinogenesis and 12-O-tetradecanoyl-13-phorbol acetate (TPA)-induced tumor promotion in mice (Yang et al., 1997).

Chronic exposure to restraint stress may be generate ROS in the mitochondria, chloroplasts, and peroxisomes (Zafir and Banu, 2009), and an excessive amount of ROS may cause cellular oxidative stress, which is related to various diseases such as cancer, stroke, and aging (Cho et al., 2011). In addition, restraint-stressed rats have alterations in antioxidant defense in their plasma (Al-Qirim et al., 2002). Protection against restraint stress also increases the immune system and has neuro-protective effects (Okimura et al., 1986). Therefore, the protective effect of oxidative stress is related to anti-stress activity in restraint stress. In the current study, we determined the beneficial effects of CBMDs against psychologically stressed (restraint-stressed) rats. Administration of CBMDs for 4-weeks in the rats did not much affect the rat body or organs. Park et al. (2013) also reported small changes in body and liver weights after administration of citrus narirutin fraction. Therefore, such a small change in liver, spleen, and adrenal gland weights suggest that feeding rats at high concentrations of CBMDs (300 mg/kg) is not toxic. In addition, there were beneficial effects in restraint-stressed rats post CBMDs treatment, which included decreased levels of both total time spent and total number of touches on the electrode plate. The restraint-stressed rats changed in emotional behavior and increased anxiety behavior in the open-field test (Gregus et al., 2005). However, administration of CBMDs ameliorated restraint stress induced behavior in rats. Sub-chronic administration of the citrus bioflavonoid apigenin also decreased immobility time on the forced swimming test in mice (Yi et al., 2008). Therefore, the above results indicate that the sub-chronic intake of CBMDs may be effective on restraint-stressed rat behavior.

The overall results demonstrated that the CBMDs contained the citrus bioflavonoids, such as narirutin and hesperidin, and had protective effects on oxidative stress in HDF cells. Furthermore, the CBMDs were not toxic to the rat body or organs including the liver, spleen, and adrenal gland. The oral administration of CBMDs for 4 weeks decreased levels of total time spent and total number of touches on the electrode plate. The current study, therefore, suggests that the sub-chronic administration of CBMDs may have protective effects on oxidative stress and restraint stress. The results also imply that the CBMDs may have potential applications in pharmaceutical and functional food industries.

Acknowledgements

This research was financially supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) and the Jeju Institute for Regional Program Evaluation (GWIRPE) through the Leading Industry Development for Economic Region and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A3B06028469).

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interest.

References

  1. Al-Qirim TM, Shahwan M, Zaidi KR, Uddin Q, Banu N. Effect of khat, its constituents and restraint stress on free radical metabolism of rats. J. Ethnopharmacol. 2002;83:245–250. doi: 10.1016/S0378-8741(02)00251-9. [DOI] [PubMed] [Google Scholar]
  2. Bhattacharya SK, Goel RK, Kaur R, Ghosal S. Anti-stress activity of sitoindosides VII and VIII, new acylsterylglucosides from Withania somnifera. Phytother. Res. 1987;1:32–37. doi: 10.1002/ptr.2650010108. [DOI] [Google Scholar]
  3. Bocco A, Cuvelier ME, Richard H, Berset C. Antioxidant activity and phenolic composition of citrus peel and seed extracts. J. Agric. Food Chem. 1998;46:2123–2129. doi: 10.1021/jf9709562. [DOI] [Google Scholar]
  4. Cho ML, Lee HS, Kang IJ, Won MH, You SG. Antioxidant properties of extract and fractions from Enteromorpha prolifera, a type of green seaweed. Food. Chem. 2011;127:999–1006. doi: 10.1016/j.foodchem.2011.01.072. [DOI] [PubMed] [Google Scholar]
  5. Choi SY, Ko HC, Ko SY, Hwang JH, Park JG, Kang SH, Han SH, Yun SH, Kim SJ. Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits. Biol. Pharm. Bull. 2007;30:772–778. doi: 10.1248/bpb.30.772. [DOI] [PubMed] [Google Scholar]
  6. Chrousos GP. Stress and disorders of the stress system. Nat. Rev. Endocrinol. 2009;5:374–381. doi: 10.1038/nrendo.2009.106. [DOI] [PubMed] [Google Scholar]
  7. Constans J, Bennetau-Pelissero C, Martin JF, Rock E, Mazur A, Bedel A, Morand C, Bérard AM. Marked antioxidant effect of orange juice intake and its phytomicronutrients in a preliminary randomized cross-over trial on mild hypercholesterolemic men. Clin. Nutr. 2015;34:1093–1100. doi: 10.1016/j.clnu.2014.12.016. [DOI] [PubMed] [Google Scholar]
  8. Dunnett CW. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 1955;50:1096–1121. doi: 10.1080/01621459.1955.10501294. [DOI] [Google Scholar]
  9. Funaguchi N, Ohno Y, La BLB, Asai T, Yuhgetsu H, Sawada M, Takemura G, Minatoguchi S, Fujiwara T, Fujiwara H. Narirutin inhibits airway inflammation in an allergic mouse model. Clin. Exp. Pharmacol. Physiol. 2007;34:766–770. doi: 10.1111/j.1440-1681.2007.04636.x. [DOI] [PubMed] [Google Scholar]
  10. Garg A, Garg S, Zaneveld LJD, Singla AK. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother. Res. 2001;15:655–669. doi: 10.1002/ptr.1074. [DOI] [PubMed] [Google Scholar]
  11. Gold PW, Chrousos GP. Organization of the stress system and its dysregulation in melancholic and atypical depression: high vs low CRH/NE states. Mol. Psychiatry. 2002;7:254–275. doi: 10.1038/sj.mp.4001032. [DOI] [PubMed] [Google Scholar]
  12. Gregus A, Wintink AJ, Davis AC, Kalynchuk LE. Effect of repeated corticosterone injections and restraint stress on anxiety and depression-like behavior in male rats. Behav. Brain Res. 2005;156:105–114. doi: 10.1016/j.bbr.2004.05.013. [DOI] [PubMed] [Google Scholar]
  13. Hwang SL, Shih PH, Yen GC. Neuroprotective effects of citrus flavonoids. J. Agric. Food Chem. 2012;60:877–885. doi: 10.1021/jf204452y. [DOI] [PubMed] [Google Scholar]
  14. Jaggi AS, Bhatia N, Kumar N, Singh N, Anand P, Dhawan R. A review on animal models for screening potential anti-stress agents. Neurol. Sci. 2011;32:993–1005. doi: 10.1007/s10072-011-0770-6. [DOI] [PubMed] [Google Scholar]
  15. Kim DB, Shin GH, Kim JM, Kim YH, Lee JH, Lee JS, Song HJ, Choe SY, Park IJ, Cho JH, Lee OH. Antioxidant and antiaging activities of citrus-based juice mixture. Food Chem. 2016;194:920–927. doi: 10.1016/j.foodchem.2015.08.094. [DOI] [PubMed] [Google Scholar]
  16. Leza JC, Salas E, Sawicki G, Russell JC, Radomski MW. The effect of stress on homeostasis in JCR: LA-cp rats: the role of nitric oxide. J. Pharmacol. Exp. Ther. 1998;286:1397–1403. [PubMed] [Google Scholar]
  17. Manach C, Morand C, Gil-Izquierdo A, Bouteloup-Demange C, Remesy C. Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. Eur. J. Clin. Nutr. 2003;57:235–242. doi: 10.1038/sj.ejcn.1601547. [DOI] [PubMed] [Google Scholar]
  18. Monforte MT, Trovato A, Kirjavainen S, Forestieri AM, Galati EM, Lo Curto RB. Biological effects of hesperidin, a Citrus flavonoid (note II): hypolipidemic activity on experimental hypercholesterolemia in rat. Farmaco. 1995;50:595–599. [PubMed] [Google Scholar]
  19. Morand C, Dubray C, Milenkovic D, Lioger D, Martin JF, Scalbert A, Mazur A. Hesperidin contributes to the vascular protective effects of orange juice: a randomized crossover study in healthy volunteers. Am. J. Clin. Nutr. 2011;93:73–80. doi: 10.3945/ajcn.110.004945. [DOI] [PubMed] [Google Scholar]
  20. Nakajima A, Aoyama Y, Nguyen TT, Shin EJ, Kim HC, Yamada S, Nakai T, Nagai T, Yokosuka A, Mimaki Y, Ohizumi Y, Yamada K. Nobiletin, a citrus flavonoid, ameliorates cognitive impairment, oxidative burden, and hyperphosphorylation of tau in senescence-accelerated mouse. Behav. Brain Res. 2013;250:351–360. doi: 10.1016/j.bbr.2013.05.025. [DOI] [PubMed] [Google Scholar]
  21. Nestler EJ, Barrot M, DiLeone RJ, Eisch AJ, Gold SJ, Monteggia LM. Neurobiology of depression. Neuron. 2002;34:13–25. doi: 10.1016/S0896-6273(02)00653-0. [DOI] [PubMed] [Google Scholar]
  22. Novío S, Núñez MJ, Amigo G, Freire-Garabal M. Effects of fluoxetine on the oxidative status of peripheral blood leucocytes of restraint-stressed mice. Basic Clin. Pharmacol. Toxicol. 2011;109:365–371. doi: 10.1111/j.1742-7843.2011.00736.x. [DOI] [PubMed] [Google Scholar]
  23. Ohnishi A, Asayama R, Mogi M, Nakaoka H, Kan-no H, Tsukuda K, Chisaka T, Wang XL, Bai HY, Shan BS, Kukida M, Iwanami J, Horiuchi M. Drinking citrus fruit juice inhibits vascular remodeling in cuff-induced vascular injury mouse model. PloS one. 2015;10:e0117616. doi: 10.1371/journal.pone.0117616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okimura T, Ogawa M, Yamauchi T. Stress and immune responses. III. Effect of restraint stress on delayed type hypersensitivity (DTH) response, natural killer (NK) activity and phagocytosis in mice. Jpn. J. Pharmacol. 1986;41:229–235. doi: 10.1254/jjp.41.229. [DOI] [PubMed] [Google Scholar]
  25. Olivenza R, Moro MA, Lizasoain L, Lorenzo P, Fernandez AP, Rodrigo J, Boscá L, Leza JC. Chronic stress induces the expression of inducible nitric oxide synthase in rat brain cortex. J. Neurochem. 2000;74:785–791. doi: 10.1046/j.1471-4159.2000.740785.x. [DOI] [PubMed] [Google Scholar]
  26. Park HY, Ha SK, Eom H, Choi I. Narirutin fraction from citrus peels attenuates alcoholic liver disease in mice. Food Chem. Toxicol. 2013;55:637–644. doi: 10.1016/j.fct.2013.01.060. [DOI] [PubMed] [Google Scholar]
  27. Poleszak E, Wlaz P, Kedzierska E, Nieoczym D, Wyska E, Szymura-Oleksiak J, Fidecka S, Radziwoń-Zaleska M, Nowak G. Immobility stress induces depression-like behavior in the forced swim test in mice: effect of magnesium and imipramine. Pharmacol. Rep. 2006;58:746–752. [PubMed] [Google Scholar]
  28. Rai D, Bhatia G, Sen T, Palit G. Anti-stress effects of Ginkgo biloba and Panax ginseng: a comparative study. J. Pharmacol. Sci. 2003;93:458–464. doi: 10.1254/jphs.93.458. [DOI] [PubMed] [Google Scholar]
  29. Yang M, Tanaka T, Hirose Y, Deguchi T, Mori H, Kawaka Y. Chemopreventive effects of diosmin and hesperidin on N-butyl-N-(4-hydroxybutyl) nitrosamineinduced urinary-bladder carcinogenesis in male ICR mice. Int. J. Cancer. 1997;73:719–724. doi: 10.1002/(SICI)1097-0215(19971127)73:5&#x0003c;719::AID-IJC18&#x0003e;3.0.CO;2-0. [DOI] [PubMed] [Google Scholar]
  30. Yi LT, Li JM, Li YC, Pan Y, Xu Q, Kong LD. Antidepressant-like behavioral and neurochemical effects of the citrus-associated chemical apigenin. Life Sci. 2008;82:741–751. doi: 10.1016/j.lfs.2008.01.007. [DOI] [PubMed] [Google Scholar]
  31. Zafir A, Banu N. Induction of oxidative stress by restraint stress and corticosterone treatments in rats. Indian J. Biochem. Biophys. 2009;46:53–58. [PubMed] [Google Scholar]

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