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Edible Bird’s Nest attenuates high fat diet-induced oxidative stress and inflammation via regulation of hepatic antioxidant and inflammatory genes
© Yida et al. 2015
Received: 1 April 2015
Accepted: 2 September 2015
Published: 4 September 2015
Edible Bird’s nest (EBN) is an antioxidant-rich supplement that is popular in many parts of Asia. Its antioxidant and anti-inflammatory properties have been reported using in vitro system. This paper aimed to determine the antioxidant and anti-inflammatory effects of EBN in in high fat diet induced rats model.
We evaluate if those properties can be translated in rats. High fat diet (HFD) was fed to rats for 12 weeks to determine its effects on oxidative stress and inflammation, and compared with HFD + Simvastatin and HFD + EBN (2.5 or 20 %). Weights were measured weekly, while serum and hepatic markers of oxidative stress (total antioxidant status and TBARS) and inflammation (interleukin 6 [IL-6], C-reactive protein [CRP] and tumor necrosis factor alpha [TNF-α]) were determined at the end of the intervention. In addition, transcriptional changes in hepatic antioxidant (superoxide dismutase, glutathione reductase, glutathione peroxidase) and inflammation (C-reactive protein, chemokine [C-C] motif 2, nuclear factor kappa beta 1 and tumor necrosis factor alpha) genes were evaluated.
The results showed increases in oxidative stress (raised TBARS and lowered total antioxidant status) and inflammatory markers (raised CRP, IL-6 and TNF-α) in HFD induced rats with corresponding attenuation of antioxidant gene expression and potentiation of inflammatory gene expression. EBN on the other hand attenuated the HFD-induced inflammation and oxidative stress and produced overall better outcomes in comparison with simvastatin.
In aggregate, the results support the evidence-based utilization of EBN as a supplement for preventing obesity-related inflammation and oxidative stress in rats. These promising results can open up opportunities for translating the benefits of EBN to humans.
The contribution of inflammation to many disease processes is well established. In fact, many chronic diseases have been associated with low grade inflammation . Cardiometabolic diseases have been growing steadily over the years and their burden is expected to rise significantly in the near future if no urgent action is taken. Despite advances in medical healthcare, the currently available strategies for the management of cardiometabolic diseases have not been completely effective in controlling the burden of the diseases. Moreover, side effects are limiting the use of some of these therapies, and hence, the search for newer alternatives. Additionally, links between diet and chronic disease development are now strongly acknowledged, and this new understanding has widened the search for alternative therapies to closely consider dietary methods of managing cardiometabolic diseases .
Edible bird’s nest (EBN) is a traditional supplement consumed for its perceived health-promoting properties, especially among Asians. However, there is lack of scientific research to support the evidence-based use of EBN as a supplement. Recent reports indicate that it possesses antioxidative and anti-inflammatory effects [3–5] but the precise mechanisms underlying such effects are not completely understood. Moreover, these reports are from in vitro systems, which sometimes do not reflect what happens in in vivo systems. Furthermore, the bioactivity of foods with multiple constituents is often attributed to their synergistic effects . This is true of EBN, which has many different constituents including proteins, carbohydrates, fatty acids and other minerals . Additionally, we have recently reported that sialic acid is a major protein constituent of EBN (11 % of EBN), and may in fact be contributing significantly to its bioactivity . However, the contribution of its multiple bioactives towards any of its effects cannot be ruled out in view of the importance of food synergy in foods with multiple constituents like EBN . Thus, in view of the lack of information on the effects of EBN on in vivo oxidative stress and inflammation and the underlying mechanisms involved, we tested the hypothesis that EBN regulated hepatic antioxidant and anti-inflammatory genes in rats as the basis for its antioxidative and anti-inflammatory properties.
C-reactive protein (CRP), interleukin 6 (IL6) and tumor necrosis factor alpha (Tnf-α) ELISA kits were purchased from Elabscience Biotechnology Co., Ltd (Wuhan, China), while 1,1,3,3-tetramethoxypropane (TMP), thiobarbituric acid, potassium persulphate (K2S2O8), 2,2’-azino-bis [3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) reagent, trolox standard and trichloroacetic acid were purchased from Sigma Aldrich (St. Loius, MO, USA). RNA extraction kit was from RBC Bioscience Corp. (Taipei, Taiwan) and GenomeLab™ GeXP Start Kit was from Beckman Coulter Inc (Miami, FL, USA). Simvastatin, RCL2 solution and lipid profile kits were from Pfizer (New York, NY, USA), Alphelys (Toulouse, France) and Randox Laboratories Ltd (Crumlin, County Antrim, UK), respectively. Cholesterol and Cholic acid were purchased from Amresco (Solon, OH, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Standard rat pellet was from Specialty feeds (Glen Forrest, WA, USA), while palm oil was supplied by Yee Lee Edible oils Sdn. Bhd. (Perak, Malaysia). All other solvents were of analytical grade and purchased from Merck (Darmstadt, Germany). EBN supplied by Blossom View Sdn. Bhd (Terrengganu, Malaysia) was cleaned under tap water for 5 mins, dried at room temperature and ground into powder manually using mortar and pestle before incorporating it into rat pellet.
Food composition of rat groups and intake
Normal pellet (%)
Cholesterol/cholic acid (%)
Palm oil (%)
Food intake (Kcal/kg/day)
Initial weight (g)
Final weight (g)
215.5 ± 33.5a
260.4 ± 10.7a
384 ± 22.9a
High fat diet
215.0 ± 37.5a
262.6 ± 17.7a
395.2 ± 16.8a
High fat diet + Simvastatin
Simvastatin (10 mg/kg)
215.7 ± 36.6a
267.7 ± 21a
375.7 ± 53.4a
High fat diet + 20 % EBN
20 % EBN
216.1 ± 36.8a
261.7 ± 15.4a
380.7 ± 25.6a
High fat diet + 2.5 % EBN
2.5 % EBN
216.5 ± 35.8a
257 ± 20.1a
368 ± 29.3a
Liver Thiobarbituric acid reactive species (TBARS)
Liver tissue TBARS was measured as reported previously with some modifications . Briefly, liver tissue (80 mg) was homogenized in 250 uL of 0.25Hcl and mixed with 250 uL of 0.375 % thiobarbituric acid and 250 uL of 15 % tricholoroacetic acid. Then, the mixture was incubated at 100 °C for 10 min and cooled before centrifuging at 3000 rpm for 15 min. Finally, absorbance of 100 uL of each sample and standard (TMP) were measured on BioTeK Synergy H1 Hybrid Reader (BioTek Instruments Inc., Winooski, VT, USA) at 532 nm, and the results expressed as uM MDA/mg tissue.
Serum total Antioxidant status
ABTS radical scavenging activity was used as a marker of total antioxidant status . Briefly, 6.62 mg of potassium persulphate, K2S2O8 was dissolved in 10 mL of distilled water to prepare a solution of 2.45 mM. Then, 7 mM ABTS was prepared by dissolving 38.4 mg in 10 mL distilled water. The two solutions were mixed and incubated in the dark for 16 h prior to use, and diluted with distilled water until a spectrophotometric absorbance of 0.700 ± 0.005 at 735 nm was obtained. Serum samples (100 μL) were reacted with 900 μL of the diluted ABTS solution and vortexed. The absorbance was read at 734 nm. ABTS radical cation scavenging activity was calculated as the percentage reduction in absorbance.
Serum C-reactive protein, Interleukin-6 and Tumor necrosis factor-alpha
Serum from blood collected in plain tubes was used for measurements of CRP, IL6 and Tnf-α using the respective ELISA kits according to the manufacturers’ instructions. Absorbances were read on BioTeK Synergy H1 Hybrid Reader (BioTek Instruments Inc., Winooski, VT, USA) at the 450 nm. The results were finally expressed as fold change in comparison to normal group:
Fold change = absorbance for the intervention group/absorbance for the normal group
Gene expression study
Names, accession number and primer sequences used in the study
The means ± standard deviation (n = 6) of the groups were used for the analyses. One-way analysis of variance (ANOVA) was performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) to assess the level of significance of differences between means with a cutoff of p < 0.05.
Results and discussions
EBN attenuated high fat diet-induced oxidative stress
EBN attenuated high fat diet induced inflammation
EBN regulated hepatic antioxidant and inflammatory genes
In aggregate, the ability of EBN to prevent high fat diet-induced weight gain, oxidative stress and inflammation suggests that it may be used as supplement to prevent obesity-associated inflammation and consequent cardiometabolic problems. Already, there are indications that inflammation due to obesity may underlie the cardiovascular and other metabolic diseases that are sequelae of excessive weight gain , and hence EBN may be a good supplement for these problems since it can prevent inflammation, oxidative stress and weight gain. Recently, we demonstrated that the EBN used in this study is predominantly made up of proteins and its effects were likely mediated through synergistic effects of its protein bioactives including sialic acid, lactoferrin and ovotransferrin . Other bioactives may be contributing to this synergistic effect, and these findings are worth evaluating further. The presence of contaminants has also been reported in EBN, which may counter any beneficial effects of EBN. Therefore, future endeavours at studying the health effects of EBN and especially its commercialization must employ stringent screening methods to ensure no toxicity from consumption of EBN.
The data demonstrated that EBN attenuated high fat diet-induced oxidative stress and inflammation partly through transcriptional regulation of hepatic antioxidant and inflammation-related genes, better than Simvastatin. The results also indicated that other post-transcriptional mechanisms may be involved in antioxidant and anti-inflammatory effects of EBN in rats. It is hoped that these findings will open up opportunities for translating the benefits of EBN to humans, and are worth evaluating further.
The authors thank Ministry of Science, Technology and Innovation (MOSTI), Malaysia for sponsoring this research (e-Sciencefund 02-01-04-SF1453) and the staff of the Laboratory of Molecular Biomedicine for their assistance during the study.
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