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Anti-inflammatory Effects of Extract from Eucommia ulmoides Oliv. Leaves on Macrophage Cells

Hyun Woo Kang
Journal of Food and Nutrition Research. 2017, 5(5), 309-312. DOI: 10.12691/jfnr-5-5-4
Published online: April 21, 2017

Abstract

This study investigated the anti-inflammatory effects of Eucommia ulmoides Oliv. (EUO) leaf extracts on RAW264.7 macrophages. Cell toxicity was determined by MTT assay. We evaluated the anti-inflammatory effects of EUO extracts by measuring nitric oxide (NO), inducible nitric oxide synthase (iNOS) production, and cyclooxygenase-2 (COX-2) expression by western blotting. EUO ethanolic extracts (0.1, 1, 5, and 10 μg/mL) significantly suppressed LPS-stimulated production of NO. EUO ethanolic extracts reduced the expression of iNOS and COX-2 proteins. The present results show that EUO ethanol extract has potent anti-inflammatory effects on RAW264.7 macrophages. These results also suggest that the anti-inflammatory effects of EUO extracts may be related to the inhibition of LPS-stimulated NO production. Therefore, ethanolic extracts of EUO leaves may be utilized as a good source of functional food and pharmaceutical applications for protection against inflammatory diseases.

1. Introduction

Proinflammatory cytokines work in a synergistic manner through a cytokine transcription factor regulatory loop, thereby augmenting the inflammatory response and tissue damage 1. Nitric oxide (NO) is an important inflammatory mediator produced by NOS under physiological and pathophysiological conditions and downregulation of NO is necessary for the treatment of the latter 2. The production of NO and prostaglandins by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, is considered to be the most prominent molecular mechanism in inflammatory processes 3, 4, and is also involved in multistage carcinogenesis, especially at the promotion stage 5. Excessive and prolonged NO generation caused by overexpression of iNOS has been implicated in inflammational tumorigenesis, while COX-2-mediated prostaglandin production has been shown to stimulate cell proliferation, invasion, and angiogenesis in cancer development 6.

Eucommia ulmoides Oliver (EUO) is one of the oldest known and used tonic herbs in Asia. It is an elm-like deciduous and dioecious tree belonging to only one species, one genus (Eucommia) and one family (Eucommiaceae) 7. The tree EUO belongs to the family Eucommiaceae, its cortex and leaves are traditional oriental medicines 8. In traditional Chinese medicine, roasted Eucommiae cortex is utilized to reinforce the muscles and lungs, lower blood pressure, prevent miscarriages, improve the tone of the liver and kidneys and increase longevity 9, 10. The EUO contain various bioactive substances, including lignans, iridoids, phenols, steroids, triterpenes, organicacids, polysaccharides, flavonoids, aminoacids, and microelements 11 as well as nutrients, i.e., amino acids, vitamins and minerals 12.

Our results suggest that EUO extract could have potential applications as a natural anti-inflammation supplement in the food industry.

2. Materials and Methods

2.1. Materials

3-[4,5-dimethy-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay kit, and LPS from Escherichia coli (serotype 0127:B8) were procured from Sigma Aldrich (St. Louis, MO, USA). Tissue culture plates and culture dishes were purchased from Nunc, Inc. (North Aurora Road, IL, USA). The antibodies against COX-2, iNOS, and β-actin were supplied by Cell Signaling (Danvers, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Hyclone (Logan, UT, USA). Reagents for ECL western blot detection were purchased from GE Healthcare Biosciences (Piscataway, NJ). All other reagents were of the highest grade available commercially.

2.2. Cell Culture

Mouse macrophage cell line, RAW264.7, was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM supplemented with penicillin, streptomycin, and 10% heat-inactivated FBS in 5% CO2 and 95% humidified air at 37°C.

2.3. MTT Assay

For evaluating the cytotoxicity of extracts from EUO (0.1, 1, 5, and 10 μg/mL) with LPS (100 ng/mL), the cells were harvested using phosphate buffered saline (PBS) containing 0.15% trypsin and 0.08% EDTA, Cells were incubated in well plates at a density of 1 x 105 cells/well. MTT solution was added to each well. Following incubation for 4 h at 37°C in 5% CO2, the supernatant was removed. The medium was removed and the cells were washed by PBS, and the formazan crystals produced in viable cells were solubilized in 200 μL DMSO. The absorbance was measured using a microplate reader (Tecan Trading AG, Männedorf, Switzerland) at 550 nm. All experiments were performed with three replicates 13.

2.4. NO Assay

The RAW264.7 cells were plated in a 96-well plate at a density of 5 × 104 cells/well in 100 μL of culture medium. Following attachment, cells were pretreated for 30 min with predetermined concentration of extract from EUO (0.1, 1, 5, and 10 μg/mL) and then stimulated with LPS, 100 ng/mL, for 24 h. Following LPS treatment, 50 μL of supernatant from each well was transferred to a corresponding well in another plate containing 50 μL of Griess reagent and incubated for 10 min at room temperature and absorbance measured at 540 nm using a microplate reader (Tecan Trading AG). Experiments were performed in triplicate 14.

2.5. Western Blot Analysis

To obtain the total cell lysate, 50 μL of RIPA buffer was added to the changed cells (3 x 105 cells/mL) cultured in 6-well plates. The cells were harvested, incubated for 10 min on ice, and centrifuged at 14,000 g for 10 min at 4°C. The protein concentration was determined using a DC protein assay from Bio-Rad, and 20 μg of whole cell lysate was separated on 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Electrophoresis was performed and the proteins were transferred to Polyvinylidene difluoride (PVDF) membranes using an electro blotting apparatus. The membranes were blocked for 1 h in a mixture of Tris-Buffered Saline and Tween 20 (TBS-T), containing 0.1% Tween-20 and 5% dry skim milk; the membranes were incubated overnight with primary antibodies (1:1000) followed by incubation for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:1000). The optical densities of the antibody-specific bands were analyzed by a Luminescent Image Analyzer, LAS-3000 (Fuji, Tokyo, Japan) 15.

2.6. Statistical Analysis

Statistical analyses were performed 3 times for all the experiments. The data are expressed as the mean ± one standard error of mean (SEM). Statistical analyses were assessed by Student’s t-test for paired data. Graph Pad Prism software version 4.00 (Graph Pad Software Inc., San Diego, CA) was used. Significant differences (p < 0.05) between the mean values of the triplicate samples were determined for various assays.

3. Results and Discussion

The effect of EUO extract at concentrations of 0.1, 1, 5 and 10 µg/mL on the viability of RAW264.7 cells following exposure for 24 h was examined by performing the MTT assay. As shown in Figure 1A, cell viability was not reduced by various concentration of EUO extract. To investigate whether EUO extract could inhibit LPS-induced NO production, RAW264.7 cells were pretreated for 4 h with various concentration (0.1, 1, 5, and 10 μg/mL) EUO extract followed by stimulation with LPS at 100 ng/mL for 18 h. The level of NO in the culture media was determined using the Griess reagent. Cells treated with LPS alone showed a notable increase in NO production compared to negative control and this upsurge was substantially attenuated in cells that were pretreated with EUO extract (Figure 1B). NO plays a central role in the physiology and pathology of diverse organs including the immune system. Further investigations were carried out to ascertain whether decreased expression of iNOS and COX-2 at the protein level correlated with the EUO extract-mediated suppression of NO production. Western blot analysis of RAW264.7 cells treated as in the NO assay revealed that iNOS (Figure 2A) and COX-2 (Figure 2B) protein levels increased following LPS-stimulation. Moreover, this increase in protein expression was considerably mitigated in the cells pretreated with EUO extract. These results correlate well with the EUO extract-mediated inhibition of NO production in activated macrophages, suggesting that suppression of iNOS and COX-2 at the protein level plays a major role in the anti-inflammatory mechanism of EUO extract.

Excessive and prolonged NO generation caused by overexpression of iNOS has been implicated in inflammational tumorigenesis, while COX-2-mediated prostaglandin production has been shown to stimulate cell proliferation, invasion, and angiogenesis in cancer development 16. Early reported that the hypoglycemic activity of EUO extract 17. Its indicated that EUO could lower hyperglycemia by inhibiting carbohydrate- degrading enzymes and glucose transport during digestion. In addition, another reported that the inhibitory effects on angiotensin converting enzyme (ACE) in vitro compared with captopril from EUO extract. Particularly, among active compounds of EUO, (6R, 7E, 9R)-9-hydroxy-4, 7-megastigmadien-3-one-9-O-[α-L-arabinopyranosyl-(l → 6)-β-D-glucopyranoside, foliasalacioside B1 and eleganoside A showed the highest anti-hypertensive effects 18. However, utilization of EUO activities has a little information until now. In the present study, we showed that EUO extract inhibited the production of NO, iNOS, and COX-2 in LPS-stimulated RAW264.7 cells. However, iNOS, COX-2, and pro-inflammatory cytokine expression may be regulated through different pathways in immunoregulatory signaling. Thus, further studies are warranted to elucidate the molecular mechanisms underlying the anti-inflammatory properties of EUO.

  • Figure 1. Effect of extract of EUO and/or LPS on the cell viability of RAW264.7 cells using the MTT assay. RAW264.7 cells were incubated with 0.1, 1, 5, and 10 μg/mL of extract and LPS (100 ng/mL) for 24 h. The results are shown as percentages of control samples (A). Effects of extract of EUO on NO production. RAW264.7 cells were pre-treated with the indicated concentrations of extract (0.1, 1, 5, and 10 μg/mL) for 30 min before incubating with LPS (100 ng/mL) for 24 h (B). Data are presented as the mean ± S.E.M. (n = 3) for three independent experiments

4. Conclusions

The current study demonstrated that EUO extract has an ameliorative effect on the LPS-induced iNOS expression and NO production in activated macrophages. Natural products have been historically used in traditional medicine and are now proving to be a potential source of new drugs and nutraceuticals. In light of this, our study verifying the immunomodulatory activity of EUO extract and the possible underlying mechanism would contribute to the understanding of the biological properties of EUO and its further consideration for therapeutic applications.

Acknowledgments

This work was supported by a 2016 research grant from Youngsan University, Republic of Korea.

References

[1]  Fiocchi, C., “Inflammatory bowel disease: Etiology and pathogenesis” Gastroenterology 115(1), 182-205. 1998.
In article      View Article
 
[2]  Ma, J.S., Kim, W.J., Kim, J.J., Kim, T.J., Ye, S.K., Song, M.D., Kang, H., Kim, D.W., Moon, W.K., and Lee, K.H., “Gold nanoparticles attenuate LPS-induced NO production through the inhibition of NF-jB and IFN-b/STAT1 pathways in RAW264.7 cells” Nitric Oxide 23(3), 214-219. 2010.
In article      View Article  PubMed
 
[3]  Moncada, S., “Nitric oxide: Discovery and impact on clinical medicine” J Roy Soc Med 92(4), 164-169. 1999.
In article      PubMed
 
[4]  Turini, M.E., and DuBois, R.N., “Cyclooxygenase-2: A therapeutic target” Annu Rev Med 53, 35-57. 2002.
In article      View Article  PubMed
 
[5]  Pan, M.H., and Ho, C.T., “Chemopreventive effects of natural dietary compounds on cancer development” Chem Soc Rev 37(11), 2558-2574. 2008.
In article      View Article  PubMed
 
[6]  Mann, J.R., Backlund, M.G., and DuBois, R.N., “Mechanisms of disease: Inflammatory mediators and cancer prevention” Nat Clin Pract Oncol 2(4), 202-210. 2005.
In article      View Article  PubMed
 
[7]  Chinese Pharmacopoeia Commision “Pharmacopoeia of the People's Republic of China” China Medical Science Press, Beijing, 165-166. 2015.
In article      
 
[8]  Nakazawa, Y., Toda, Y., “Eucommia ulmoides Oliv. (Eucommiaceae): in vitro culture and the production of iridoids, lignans, and other secondary metabolites” In: Bajaj, Y.P.S. (Ed.), Medicinal and Aromatic Plants VIII: Biotechnology in Agriculture and Forestry. Springer, Berlin, Heidelberg, pp. 215-231. 1995.
In article      View Article
 
[9]  Nakasa, T., Yamagchi, M., Okinaka, O., Metori, K., Takashi, S., “Effects of Duzhong leaf extract on plasma and hepatic lipids in rats fed on a high fat plus high cholesterol diet” Nippon Nogeikagaku Kaishi 69, 1491-1498. 1995.
In article      View Article
 
[10]  Watanabe, J., Kawabata, J., Kurihara, H., Niki, R., “Isolation and identification of α-glucosidase inhibitors from tochu-cha (Eucommia ulmoides)” Biosci Biotechnol Biochem 61, 177-178. 1997.
In article      View Article  PubMed
 
[11]  Peng, H., Li, X., “Pharmacological research and application of Duzhong” China J Chin Med 28,72-73. 2013.
In article      
 
[12]  He, X.R., Wang, J.H., Li, M.X., Hao, D.J., Yang, Y., Zhang, C.L., He, R., Tao, R., “Eucommia ulmoides Oliv.: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine” J Ethnopharmacol 151, 78-92. 2014.
In article      View Article  PubMed
 
[13]  Lee, S.J., Lee, S.Y., Ha, H.J., Cha, S.H., Lee, S.K., and Hur, S.J., “Rutin Attenuates lipopolysaccharide-induced nitric oxide production in macrophage cells” J Food Nutr Res 3(3), 202-205. 2015.
In article      View Article
 
[14]  Lee, S.J., Kim, E.K., Oh, H.J., Kwon, H.J., Hwang, J.W., Moon, S.H., Jeon, B.T., Park, P.J., Lim, B.O., “Free radical scavenging activity and protective effect against H2O2-induced stress in neuronal cells of enzymatic extracts from Sarcodon aspratus” Korean J Med Crop Sci 19(2), 77-82. 2011.
In article      View Article
 
[15]  Lee, S.J., Kang, H.W., Lee, S.Y., and Hur, S.J., “Green tea polyphenol epigallocatechin-3-O-gallate attenuates lipopolysaccharide-induced nitric oxide production in RAW264.7 cells” J Food Nutr Res 2(7), 425-428. 2014.
In article      View Article
 
[16]  Mann, J.R., Backlund, M.G., and DuBois, R.N., “Mechanisms of disease: Inflammatory mediators and cancer prevention” Nat Clin Pract Oncol 2(4), 202-210. 2005.
In article      View Article  PubMed
 
[17]  Zhang, Y., Zhang, H., Wang, F., Yang, D., Ding, K., Fan, J., “The ethanol extract of Eucommia ulmoides Oliv. leaves inhibits disaccharidase and glucose transport in Caco-2 cells” J Ethnopharmacol 163, 99-105. 2015.
In article      View Article  PubMed
 
[18]  Yan, J.K., Ding, L.Q., Shi, X.L., Donkor, P.O., Chen, L.X., Qiu, F., “Megastigmane glycosides from leaves of Eucommia ulmoides Oliver with ACE inhibitory activity” Fitoterapia 116, 121-125. 2017.
In article      View Article  PubMed
 

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Normal Style
Hyun Woo Kang. Anti-inflammatory Effects of Extract from Eucommia ulmoides Oliv. Leaves on Macrophage Cells. Journal of Food and Nutrition Research. Vol. 5, No. 5, 2017, pp 309-312. http://pubs.sciepub.com/jfnr/5/5/4
MLA Style
Kang, Hyun Woo. "Anti-inflammatory Effects of Extract from Eucommia ulmoides Oliv. Leaves on Macrophage Cells." Journal of Food and Nutrition Research 5.5 (2017): 309-312.
APA Style
Kang, H. W. (2017). Anti-inflammatory Effects of Extract from Eucommia ulmoides Oliv. Leaves on Macrophage Cells. Journal of Food and Nutrition Research, 5(5), 309-312.
Chicago Style
Kang, Hyun Woo. "Anti-inflammatory Effects of Extract from Eucommia ulmoides Oliv. Leaves on Macrophage Cells." Journal of Food and Nutrition Research 5, no. 5 (2017): 309-312.
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  • Figure 1. Effect of extract of EUO and/or LPS on the cell viability of RAW264.7 cells using the MTT assay. RAW264.7 cells were incubated with 0.1, 1, 5, and 10 μg/mL of extract and LPS (100 ng/mL) for 24 h. The results are shown as percentages of control samples (A). Effects of extract of EUO on NO production. RAW264.7 cells were pre-treated with the indicated concentrations of extract (0.1, 1, 5, and 10 μg/mL) for 30 min before incubating with LPS (100 ng/mL) for 24 h (B). Data are presented as the mean ± S.E.M. (n = 3) for three independent experiments
  • Figure 2. Effect of extract of EUO on LPS-induced iNOS (A) and COX-2 (B) protein expression in RAW264.7 cells. Cell lysates were electrophoresed and the protein expression of iNOS and COX-2 was detected by the specific antibody. β-actin was used as an internal control for the western blot analysis. Quantification data are shown in the lower panel. Data are presented as the mean ± S.E.M. (n = 3) for three independent experiments
[1]  Fiocchi, C., “Inflammatory bowel disease: Etiology and pathogenesis” Gastroenterology 115(1), 182-205. 1998.
In article      View Article
 
[2]  Ma, J.S., Kim, W.J., Kim, J.J., Kim, T.J., Ye, S.K., Song, M.D., Kang, H., Kim, D.W., Moon, W.K., and Lee, K.H., “Gold nanoparticles attenuate LPS-induced NO production through the inhibition of NF-jB and IFN-b/STAT1 pathways in RAW264.7 cells” Nitric Oxide 23(3), 214-219. 2010.
In article      View Article  PubMed
 
[3]  Moncada, S., “Nitric oxide: Discovery and impact on clinical medicine” J Roy Soc Med 92(4), 164-169. 1999.
In article      PubMed
 
[4]  Turini, M.E., and DuBois, R.N., “Cyclooxygenase-2: A therapeutic target” Annu Rev Med 53, 35-57. 2002.
In article      View Article  PubMed
 
[5]  Pan, M.H., and Ho, C.T., “Chemopreventive effects of natural dietary compounds on cancer development” Chem Soc Rev 37(11), 2558-2574. 2008.
In article      View Article  PubMed
 
[6]  Mann, J.R., Backlund, M.G., and DuBois, R.N., “Mechanisms of disease: Inflammatory mediators and cancer prevention” Nat Clin Pract Oncol 2(4), 202-210. 2005.
In article      View Article  PubMed
 
[7]  Chinese Pharmacopoeia Commision “Pharmacopoeia of the People's Republic of China” China Medical Science Press, Beijing, 165-166. 2015.
In article      
 
[8]  Nakazawa, Y., Toda, Y., “Eucommia ulmoides Oliv. (Eucommiaceae): in vitro culture and the production of iridoids, lignans, and other secondary metabolites” In: Bajaj, Y.P.S. (Ed.), Medicinal and Aromatic Plants VIII: Biotechnology in Agriculture and Forestry. Springer, Berlin, Heidelberg, pp. 215-231. 1995.
In article      View Article
 
[9]  Nakasa, T., Yamagchi, M., Okinaka, O., Metori, K., Takashi, S., “Effects of Duzhong leaf extract on plasma and hepatic lipids in rats fed on a high fat plus high cholesterol diet” Nippon Nogeikagaku Kaishi 69, 1491-1498. 1995.
In article      View Article
 
[10]  Watanabe, J., Kawabata, J., Kurihara, H., Niki, R., “Isolation and identification of α-glucosidase inhibitors from tochu-cha (Eucommia ulmoides)” Biosci Biotechnol Biochem 61, 177-178. 1997.
In article      View Article  PubMed
 
[11]  Peng, H., Li, X., “Pharmacological research and application of Duzhong” China J Chin Med 28,72-73. 2013.
In article      
 
[12]  He, X.R., Wang, J.H., Li, M.X., Hao, D.J., Yang, Y., Zhang, C.L., He, R., Tao, R., “Eucommia ulmoides Oliv.: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine” J Ethnopharmacol 151, 78-92. 2014.
In article      View Article  PubMed
 
[13]  Lee, S.J., Lee, S.Y., Ha, H.J., Cha, S.H., Lee, S.K., and Hur, S.J., “Rutin Attenuates lipopolysaccharide-induced nitric oxide production in macrophage cells” J Food Nutr Res 3(3), 202-205. 2015.
In article      View Article
 
[14]  Lee, S.J., Kim, E.K., Oh, H.J., Kwon, H.J., Hwang, J.W., Moon, S.H., Jeon, B.T., Park, P.J., Lim, B.O., “Free radical scavenging activity and protective effect against H2O2-induced stress in neuronal cells of enzymatic extracts from Sarcodon aspratus” Korean J Med Crop Sci 19(2), 77-82. 2011.
In article      View Article
 
[15]  Lee, S.J., Kang, H.W., Lee, S.Y., and Hur, S.J., “Green tea polyphenol epigallocatechin-3-O-gallate attenuates lipopolysaccharide-induced nitric oxide production in RAW264.7 cells” J Food Nutr Res 2(7), 425-428. 2014.
In article      View Article
 
[16]  Mann, J.R., Backlund, M.G., and DuBois, R.N., “Mechanisms of disease: Inflammatory mediators and cancer prevention” Nat Clin Pract Oncol 2(4), 202-210. 2005.
In article      View Article  PubMed
 
[17]  Zhang, Y., Zhang, H., Wang, F., Yang, D., Ding, K., Fan, J., “The ethanol extract of Eucommia ulmoides Oliv. leaves inhibits disaccharidase and glucose transport in Caco-2 cells” J Ethnopharmacol 163, 99-105. 2015.
In article      View Article  PubMed
 
[18]  Yan, J.K., Ding, L.Q., Shi, X.L., Donkor, P.O., Chen, L.X., Qiu, F., “Megastigmane glycosides from leaves of Eucommia ulmoides Oliver with ACE inhibitory activity” Fitoterapia 116, 121-125. 2017.
In article      View Article  PubMed