Ann Dermatol.  2015 Apr;27(2):142-151. 10.5021/ad.2015.27.2.142.

Ethanol Extract of Peanut Sprout Exhibits a Potent Anti-Inflammatory Activity in Both an Oxazolone-Induced Contact Dermatitis Mouse Model and Compound 48/80-Treated HaCaT Cells

• Choi DI
• Choi JY
• Kim YJ
• Lee JB
• Kim SO
• Shin HT
• Lee SC

• KMID: 2264802
• DOI: http://doi.org/10.5021/ad.2015.27.2.142

Abstract

BACKGROUND
We developed an ethanol extract of peanut sprouts (EPS), a peanut sprout-derived natural product, which contains a high level of trans-resveratrol (176.75 microg/ml) and was shown to have potent antioxidant activity.
OBJECTIVE
We evaluated the potential anti-inflammatory activity of EPS by measuring its antioxidant potential in skin.
METHODS
The anti-inflammatory activity of EPS was tested using two models of skin inflammation: oxazolone (OX)-induced contact dermatitis in mice and compound 48/80-treated HaCaT cells. As biomarkers of skin inflammation, cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) levels were measured.
RESULTS
OX-induced contact dermatitis was suppressed markedly in mice that were treated with an ointment containing 5% EPS as evidenced by a decrease in the extent of scaling and thickening (p<0.05) and supported by a histological study. COX-2 (messenger RNA [mRNA] and protein) and NGF (mRNA) levels, which were upregulated in the skin of OX-treated mice, were suppressed markedly in the skin of OX+EPS-treated mice. Consistent with this, compound 48/80-induced expression of COX-2 (mRNA and protein) and NGF (mRNA) in HaCaT cells were suppressed by EPS treatment in a dose-dependent manner. As an inhibitor of NF-kappaB, IkappaB protein levels were dose-dependently upregulated by EPS. Fluorescence-activated cell sorting (FACS) analysis revealed that EPS scavenged compound 48/80-induced reactive oxygen species (ROS) in HaCaT cells.
CONCLUSION
EPS exerts a potent anti-inflammatory activity via its anti-oxidant activity in both mouse skin and compound 48/80-treated HaCaT cells in vitro. Compound 48/80-treated HaCaT cells are a useful new in vitro model of skin inflammation.

Keyword

Anti-inflammatory activity; Anti-oxidant activity; p-Methoxy-N-methylphenethylamine; Ethanol extract of peanut sprouts; Oxazolone-induced contact dermatitis

MeSH Terms

Animals
Biomarkers
Cyclooxygenase 2
Dermatitis, Contact*
Ethanol*
Flow Cytometry
Inflammation
Mice*
Nerve Growth Factor
NF-kappa B
Oxazolone
p-Methoxy-N-methylphenethylamine
Reactive Oxygen Species
RNA
Skin
Cyclooxygenase 2
Ethanol
NF-kappa B
Nerve Growth Factor
Oxazolone
RNA
Reactive Oxygen Species
p-Methoxy-N-methylphenethylamine

Figure

• 

  Fig. 1 Oxazolone (OX)-induced inflammatory changes in mouse skin are suppressed by application of 5% ethanol extract of peanut sprouts (EPS). (A) Representative pictures of untreated control mouse, OX-treated mouse, and OX+EPS-treated mouse. (B) Histological results of OX-treated and OX+EPS-treated mice are shown (H&E, ×100).

• 

  Fig. 2 Anti-inflammatory activity of ethanol extract of peanut sprouts (EPS) reduces the oxazolone (OX)-induced increase of skin thickness in hairless mice. Skin thickness was measured with a dial thickness gauge to semi-quantify the degree of skin inflammation in vivo. Skin flaps were obtained from the dorsal skin of untreated control, OX-treated, and OX+EPS-treated mice. Statistical significance was assessed between the OX-treated and OX+EPS-treated mice groups.

• 

  Fig. 3 Anti-inflammatory activity of ethanol extract of peanut sprouts (EPS) on oxazolone (OX)-induced skin inflammation in hairless mice. (A) In reverse transcriptase-polymerase chain reaction (RT-PCR) and (B) western blot analyses for COX-2, as well as (A) RT-PCR for nerve growth factor (NGF) mRNA expression, OX-treated mice (n=4) showed increased expression levels of the markers tested, but the increase was suppressed markedly in OX+EPS-treated mice (n=4). (C) Densitometric analysis of mRNA and protein bands of A, B was performed, and data represent mean±standard deviation of relative density of each group versus control mice without OX and EPS treatment (n=4 for each group). #p<0.01 between OX-treated and OX+EPS-treated mice groups. Control refers to the OX-untreated mice group.

• 

  Fig. 4 Anti-inflammatory activity of ethanol extract of peanut sprouts (EPS) via reactive oxygen species (ROS)-scavenging effect in compound 48/80-treated HaCaT cells. MTT assay was used to detect non-cytotoxic concentrations of (A) compound 48/80 and (B) EPS in HaCaT cells. (C) Fluorescence-activated cell sorting (FACS) analysis demonstrated that compound 48/80 induced ROS production in HaCaT cells, which was suppressed markedly by EPS treatment.

• 

  Fig. 5 Inhibitory effect of ethanol extract of peanut sprouts (EPS) on compound 48/80-induced expression of cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) in HaCaT cells. In (A) reverse transcriptase-polymerase chain reaction (RT-PCR) and (B) western blot analyses for COX-2, and (C) RT-PCR for NGF mRNA expression, compound 48/80 (10 µg/ml) increased expression levels of COX-2 and NGF, but this induction was suppressed markedly in compound 48/80+EPS-treated HaCaT cells in a dose-dependent manner between 0.01 and 1.0 mg/ml at the mRNA level and 0.1 and 1.0 mg/ml at the protein level. (D) Densitometric analysis for mRNA and protein bands of (A~C) was performed, and data represent means±standard deviations of three independent experiments. #p<0.01 between compound 48/80-treated and compound 48/80+EPS-treated sample with different EPS concentration. N.S. means no significance between samples. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 6 Ethanol extract of peanut sprouts (EPS) exerts an anti-inflammatory activity by upregulating IκBα expression in compound 48/80-treated HaCaT cells. (A) In western blot analysis, compound 48/80 down-regulated IκBα expression, which was reversed by EPS treatment at a dose of 10 µg/ml in HaCaT cells. The EPS-induced upregulation of IκBα occurred in a time-dependent manner between 15 and 60 min. (B) Densitometric analysis of western blot protein bands was performed, and data represent means±standard deviations of three independent experiments. #p<0.01 between compound 48/80-treated and compound 48/80+EPS-treated samples at each time point (15, 30, and 60 min).

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