Inhibitory effects of Angelica sinensis ethyl acetate extract and major compounds on NF-κB trans-activation activity and LPS-induced inflammation

https://doi.org/10.1016/j.jep.2010.03.022Get rights and content

Abstract

Aim of the study

Previous study showed that the ethyl acetate (EtOAc) fraction from Angelica sinensis (Oliv.) Diels (Apiaceae) (AS) inhibited nitric oxide (NO) and prostaglandin E2 secretions in vitro. This study was to evaluate anti-inflammatory activity of AS EtOAc extract and its major compounds in vivo and in vitro.

Materials and methods

NF-κB luciferase activity and pro-inflammatory cytokine secretions from lipopolysaccharide (LPS) plus interferon (IFN)-γ-stimulated RAW 264.7 cells pre-treated with EtOAc extract or compounds were analyzed. For further in vivo study, BALB/c mice were tube-fed with 1.56 (AS1 group), 6.25 (AS2 group) mg/kg body weight/day in 100 μl soybean oil, while the control and PDTC (pyrrolidine dithiocarbamate, an anti-inflammatory agent) groups were tube-fed with 100 μl soybean oil/day only. After 1 week of tube-feeding, the PDTC group was injected with 50 mg/kg BW PDTC and 1 h later, all of the mice were injected with 15 mg/kg BW LPS. The pro-inflammatory cytokine levels and lifespan of LPS-challenged mice were determined.

Results

The results showed that AS EtOAc extract significantly inhibited NF-κB luciferase activity and TNF-α, IL-6, macrophage inflammatory protein-2 (MIP-2) and NO secretions from LPS/IFN-γ-stimulated RAW 264.7 cells. The AS1 and PDTC groups, but not AS2, had significantly higher survival rate than the control group. This was characterized by the inhibition of the serum TNF-α and IL-12p40 levels after LPS injection (p < 0.05). The major compounds of AS, ferulic acid and Z-ligustilide, also significantly decreased NF-κB luciferase activity, which may contribute to the anti-inflammatory activity of AS.

Conclusions

Low dose of AS EtOAc extract that inhibits the production of inflammatory mediators alleviates acute inflammatory hazards and protect mice from endotoxic shock.

Graphical abstract

The survival rate in the AS1 group (44%) was also significantly higher than the LPS group according to the COX's proportion hazards regression test. Overall, the AS2 group had low survival rate (18%). Our data show that the EtOAc fraction of the AS low dose group was found to significantly protect mice against lethal endotoxemia.

  1. Download : Download full-size image

Introduction

Inflammation, an important component of innate immunity, is dependent on the activation of nuclear factor kappa B (NF-κB). Expression of various pro-inflammatory cytokines and chemokines, such as TNF-α, IL-6, IL-12, macrophage inflammatory protein-2 (MIP-2) and nitric oxide (NO) are induced by NF-κB activation. The proper production of these mediators helps the innate immune response, but excessive inflammation may cause conditions such as chronic inflammation, sepsis and even death (Das, 2000). Sepsis is generally considered a systemic inflammatory disorder and a serious clinical problem with high mortality (Bone et al., 1997).

Traditional Chinese medicine herbs are used in many medicines as well as in dietary supplements in Asia. Our previous study on pre-screening of 22 commonly used Chinese herbs by NF-κB-dependent activity implies that the EtOAc fraction of Angelica sinensis (Oliv.) Diels (Apiaceae) (AS), or Dang gui in Chinese, exerts an anti-inflammatory effect (Chao et al., 2007). Further in vivo study indicates that NF-κB-dependent luciferase reporter assay may serve as pre-screen tool to identify anti-inflammatory Chinese medicine herbs (Chao et al., 2009a). AS, one of the herbs screened out by the suppression of NF-κB luciferase activity, decreased NO and PGE2 production in LPS/IFN-γ-stimulated peritoneal macrophages (Chao et al., 2009b), suggesting that AS is worthy of further in vivo evidence-based research on anti-inflammation.

The main chemical constituents of Angelica roots are ferulic acid, Z-ligustilide (the main lipophilic component of the essential oil of AS), angelicide, butylidenephthalide and butyphthalide. Ferulic acid has been suggested as one of the major bioactive components in AS (Zhao et al., 2003, Dong et al., 2005). The components of AS EtOAC extract, which showed higher inhibitory activity of NF-κB transactivation than hexane or water fractions in our previous study (Chao et al., 2007), were also identified as ferulic acid and Z-ligustilide. The amounts of ferulic acid and Z-ligustilide in AS EtOAc fraction were calculated as 3.75 and 15.95 mg/g dry weight of whole plant (Chao et al., 2009b).

Therefore, we further evaluated the pharmacology effects of the EtOAc extract from AS, ferulic acid and Z-ligustilide for their ability to modulate NF-κB trans-activation activity in this study. In addition, a murine model of LPS-induced endotoxic shock was used to evaluate in vivo anti-inflammatory effect of AS EtOAc extract.

Section snippets

Extraction and semi-purification of Angelica sinensis

Angelica sinensis (Oliv.) Diels (Apiaceae) (AS) was purchased from a licensed Chinese herbal drug store in Taipei City (voucher no. 05-03-10, Yong-Sheng Pharmacy) and was authenticated (Sheng Chang Pharmaceutical, Co., Ltd., Taiwan) (Chao et al., 2009b). In total, 10 g of AS was extracted with 300 ml of 95% ethanol at 50 °C for 3 h twice. The total crude extract was evaporated under vacuum to yield a residue, and then the residue was suspended in 90% ethanol and successively partitioned with hexane

AS EtOAc extract suppresses NF-κB transcriptional activity and pro-inflammatory mediators production in LPS/IFN-γ-activated macrophages

Activation of RAW 264.7 macrophages with LPS/IFN-γ resulted in a significant increase in NF-κB trans-activation activity (Fig. 1). Addition of helenalin, a naturally occurring cell permeable pseudoguainolide sesquiterpenoid lactone that inhibits NF-κB-DNA binding activity (Siedle et al., 2004), thoroughly suppressed reporter activity. AS EtOAc extract pre-treatment significantly and dose-dependently reduced LPS/IFN-γ-activated NF-κB luciferase activity. No cytotoxicity of AS EtOAc extract up to

Acknowledgements

The authors had no conflict of interest to this report. This work was kindly supported by a grant from the Committee on Chinese Medicine and Pharmacy of Department of Health, Republic of China (CCMP95-RD-105).

References (35)

Cited by (82)

  • Traditional Chinese medicine for food allergy and eczema

    2021, Annals of Allergy, Asthma and Immunology
View all citing articles on Scopus
View full text