Nutr Res Pract.  2014 Jun;8(3):267-271.

The anti-inflammatory effect of Indonesian Areca catechu leaf extract in vitro and in vivo

• Lee KP
• Sudjarwo G
• Kim JS
• Dirgantara S
• Maeng WJ
• Hong H

Abstract

BACKGROUND/OBJECTIVES
Overproduction of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) enzyme can cause inflammation. Cyclooxygenase-2 (COX-2) is also involved in the inflammatory response through regulation of nuclear factor-kappa B (NF-kappaB). Areca catechu is one of the known fruit plants of the Palmaceae family. It has been used for a long time as a source of herbal medicine in Indonesia. In this study, we explored the effect of Indonesian Areca catechu leaf ethanol extract (ACE) in lipopolysaccharide (LPS)-induced inflammation and carrageenan-induced paw edema models. Recently, this natural extract has been in the spotlight because of its efficacy and limited or no toxic side effects. However, the mechanism underlying its anti-inflammatory effect remains to be elucidated.
MATERIALS/METHODS
We measured NO production by using the Griess reagent, and determined the expression levels of inflammation-related proteins, such as iNOS, COX2, and NF-kappaB, by western blot. To confirm the effect of ACE in vivo, we used the carrageenan-induced paw edema model.
RESULTS
Compared to untreated cells, LPS-stimulated RAW 264.7 cells treated with ACE showed reduced NO generation and reduced iNOS and COX-2 expression. We found that the acute inflammatory response was significantly reduced by ACE in the carrageenan-induced paw edema model.
CONCLUSION
Taken together, these results suggest that ACE can inhibit inflammation and modulate NO generation via downregulation of iNOS levels and NF-kappaB signaling in vitro and in vivo. ACE may have a potential medical benefit as an anti-inflammation agent.

Keyword

Areca catechu; anti-inflammation; lipopolysaccharide (LPS); carrageenan; RAW 264.7 cell; paw edema

MeSH Terms

Areca*
Blotting, Western
Carrageenan
Cyclooxygenase 2
Down-Regulation
Edema
Ethanol
Fruit
Herbal Medicine
Humans
Indonesia
Inflammation
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Carrageenan
Cyclooxygenase 2
Ethanol
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II

Figure

• 

  Fig. 1 Effect of Areca catechu leaf ethanol extract (ACE) on cell viability and production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. (A) RAW 264.7 cells were treated with 0.3-10 µg/ml ACE for 24 h and cell viability was determined with the XTT assay. Values are expressed as a percentage of control. Each value represents the mean from three independent experiments ± SD. *indicates a significant difference compared with untreated controls (P < 0.05). (B) RAW 264.7 cells were treated with 0.3-3 µg/ml ACE and 1 µg/ml LPS for 24 h. NO production was determined with the Griess reagent assay. The values were determined and quantified from the sodium nitrate standard curve. The nitrate concentrations (µM) were determined in three independent experiments. Data were expressed as mean ± SD. *indicates a significant difference compared with LPS treatment only (P < 0.05).

• 

  Fig. 2 Effect of Areca catechu leaf ethanol extract (ACE) on inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, inhibitor of kappa B (IκB), and nuclear factor-kappa B (NF-κB) expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. (A) RAW 264.7 cells were treated with 0.3-3 µg/ml ACE and 1 µg/ml LPS for 24 h. The levels of iNOS, COX-2, IκB, and NF-κB proteins were examined by immunoblot analysis. (B-E) The graphs show the band intensity from each immunoblot with specific antibodies. Value changes from three independent experiments are expressed relative to those in LPS-treated controls. *indicates a significant difference compared with LPS only treatment (P < 0.05).

• 

  Fig. 3 Effect of Areca catechu leaf ethanol extract (ACE) on carrageenan (CR)-induced paw edema in rats. To test the effect of ACE (2.5, 5, or 10 mg/kg) on CR-induced paw edema, rats were divided into 4 groups. Water (control) and ACE (dissolved in 500 µl of normal saline) were administered orally during a 7-day regular schedule. To explore the anti-inflammatory effect of ACE in each group, 50 µl of 1% carrageenan was injected into the sub-plantar side of the hind paw of all animals. The paw edema volumes were recorded at 0, 1, 2, 3, 4, and 5 h post-injection. Values are expressed as the increase in paw edema volume (%).

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