Nutr Res Pract.  2017 Aug;11(4):265-274. 10.4162/nrp.2017.11.4.265.

Anti-inflammatory effects of Nelumbo leaf extracts and identification of their metabolites

• Park E
• Kim GD
• Go MS
• Kwon D
• Jung IK
• Auh JH
• Kim JH

• KMID: 2385464
• DOI: http://doi.org/10.4162/nrp.2017.11.4.265

Abstract

BACKGROUND/OBJECTIVES
Nelumbo leaves have been used in traditional medicine to treat bleeding, gastritis, hemorrhoids, and halitosis. However, their mechanisms have not been elucidated.
MATERIALS/METHODS
The present study prepared two Nelumbo leaf extracts (NLEs) using water or 50% ethanol. Inflammatory response was induced with LPS treatment, and expression of pro-inflammatory mediators (inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 and nitric oxide (NO) and prostaglandin E₂ (PGE₂) productions were assessed. To determine the anti-inflammatory mechanism of NLEs, we measured nuclear factor-κB (NF-κB) activity. Major metabolites of NLEs were also analyzed and quantified.
RESULTS
NLEs effectively reduced the expression and productions of pro-inflammatory mediators such as IL-1β, IL-6, TNF-α, PGE₂, and NO. NLEs also reduced NF-κB activity by inhibiting inhibitor of NF-κB phosphorylation. Both extracts contained catechin and quercetin, bioactive compounds of NLEs.
CONCLUSIONS
In this study, we showed that NLEs could be used to inhibit NF-κB-mediated inflammatory responses. In addition, our data support the idea that NLEs can ameliorate disease conditions involving chronic inflammation.

Keyword

Nelumbo leaf; inflammation; metabolomics; macrophage

MeSH Terms

Catechin
Cyclooxygenase 2
Dinoprostone
Ethanol
Gastritis
Halitosis
Hemorrhage
Hemorrhoids
Inflammation
Interleukin-6
Interleukins
Macrophages
Medicine, Traditional
Metabolomics
Necrosis
Nelumbo*
Nitric Oxide
Nitric Oxide Synthase
Phosphorylation
Quercetin
Water
Catechin
Cyclooxygenase 2
Dinoprostone
Ethanol
Interleukin-6
Interleukins
Nitric Oxide
Nitric Oxide Synthase
Quercetin
Water

Figure

• 

  Fig. 1 Effects of NLEs on the viability of RAW 264.7 cells, determined by MTT assay.The cells were treated with NLEs for 24 h and then stimulated with LPS for 24 h. The values are expressed as the means ± SE of three individual experiments. LPS, lipopolysaccharide; NLEE, Nelumbo leaf ethanol extract; NLWE, Nelumbo leaf water extract.

• 

  Fig. 2 Effects of NLEs on NO levels, and iNOS expression in LPS-induced RAW 264.7 cells.The cells were pre-treated with various concentrations of NLE for 24 h, and then stimulated with LPS (1 µg/ml). (A) NO levels in cell culture media after 16-h incubation with LPS. (B) iNOS mRNA expression, determined by semi-quantitative RT-PCR after 4 h incubation with LPS. (C) iNOS protein expression in cell lysates, determined by western blot analysis. The values are expressed as the mean ± SD of three individual experiments. Values not sharing the same superscript were significantly (P < 0.05) different. LPS, lipopolysaccharide; NLEE, Nelumbo leaf ethanol extract; NLWE, Nelumbo leaf water extract; NO, nitric oxide; iNOS, inducible nitric oxide synthase.

• 

  Fig. 3 Effects of NLEs on PGE2 levels and COX-2 expression in LPS-induced RAW 264.7 cells.Cells were pre-treated with NLE for 24 h and then stimulated with LPS (1 µg/ml). (A) PGE2 levels were measured in the culture medium after 16 h incubation with LPS. (B) COX-2 mRNA expression was measured by semi-quantitative RT-PCR after 4 h incubation with LPS. (C) COX-2 protein levels in cell lysates were measured by western blot analysis after 16-h LPS stimulation. The values are expressed as the mean ± SE of three individual experiments. Values not sharing the same superscript were significantly (P < 0.05) different. LPS, lipopolysaccharide; NLEE, Nelumbo leaf ethanol extract; NLWE, Nelumbo leaf water extract; PGE2, prostaglandin E2; COX-2, cyclooxygenase 2.

• 

  Fig. 4 Effects of NLEs on cytokine expression.RAW 264.7 cells were pre-treated with NLEs for 24 h and then stimulated with LPS (1 µg/ml) for 4 or 16 h. Total RNA and protein lysates were subjected to semi-quantitative RT-PCR and western blot analysis to analyze IL-1β (A), IL-6 (B), and TNF-α (C). The values are expressed as the mean ± SD of three individual experiments. Values not sharing the same superscript were significantly (P < 0.05) different. LPS, lipopolysaccharide; NLEE, Nelumbo leaf ethanol extract; NLWE, Nelumbo leaf water extract; IL, interleukin; TNF-α, tumor necrosis factor alpha; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 5 Effects of NLE on NF-µB signaling.(A) NF-µB transcriptional activity was determined in nuclear extracts of RAW 264.7 cells that were pre-treated with NLEs for 24 h prior to stimulation with LPS (1 µg/ml) for 20 min. (B) IµB phosphorylation was determined in cell lysate proteins from RAW 264.7 cells that were pre-treated with NLEs for 24 h and then induced with LPS (1 µg/ml) for 15 min. The values are expressed as the mean ± SD of three individual experiments. Values not sharing the same superscript were significantly (P < 0.05) different. LPS, lipopolysaccharide; NLEE, Nelumbo leaf ethanol extract; NLWE, Nelumbo leaf water extract; NF-κB, nuclear factor kappa-light-chainenhancer of activated B cells; I-κB, inhibitor of NF-κB.

• 

  Fig. 6 PCA of the metabolites in NLWE (black circle) and NLEE (Red pentagon), analyzed by LC-MS/MS in (A) negative ion mode and (B) positive ion mode.NLEE, Nelumbo leaf ethanol extract; NLWE, Nelumbo leaf water extract.

• 

  Fig. 7 S-plot generated by OPLS-DA for the metabolites in NLWE and NLEE, analyzed by LC-ESI-ion trap-MS/MS.(A) negative ion mode; (B) positive ion mode NLEE, Nelumbo leaf ethanol extract; NLWE, Nelumbo leaf water extract.

• 

  Fig. 8 Quantitative analysis of quercetin (A and B) and catechin (C and D) in NLWE and NLEE using HPLC.Quercetin hydrate standard (A) and quercetin peak from NLEE and NLWE (B). Catechin hydrate standard (C) and catechin peak from NLEE and NLWE (D). NLEE, Nelumbo leaf ethanol extract; NLWE, Nelumbo leaf water extract.

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