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, 5 (4), 685-96

A Hexane Fraction of American Ginseng Suppresses Mouse Colitis and Associated Colon Cancer: Anti-Inflammatory and Proapoptotic Mechanisms

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A Hexane Fraction of American Ginseng Suppresses Mouse Colitis and Associated Colon Cancer: Anti-Inflammatory and Proapoptotic Mechanisms

Deepak Poudyal et al. Cancer Prev Res (Phila).

Abstract

Ulcerative colitis is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American ginseng extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of American ginseng that suppress colitis and prevent colon cancer. Among five different fractions of American ginseng (butanol, hexane, ethylacetate, dichloromethane, and water), a hexane fraction has particularly potent antioxidant and proapoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex vivo model (CD4(+)/CD25(-) primary effector T cells). A key in vivo finding was that compared with the whole American ginseng extract, the hexane fraction of American ginseng was more potent in treating colitis in a dextran sodium sulfate (DSS) mouse model, as well as suppressing azoxymethane/DSS-induced colon cancer. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling of inflammatory cells within the colonic mesenteric lymph nodes was elevated in mice consuming DSS + the hexane fraction of American ginseng. Results are consistent with our in vitro data and with the hypothesis that the hexane fraction of American ginseng has anti-inflammatory properties and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of American ginseng that suppress colitis and prevent colon cancer associated with colitis.

Figures

Figure 1
Figure 1. The Hexane Fraction of AG suppresses the induced expression of iNOS and Cox-2 at the protein and mRNA level in ANA-1 mouse macrophages
(A) Effect of whole AG extract and the Hexane Fraction of AG on IFN-γ-induced iNOS protein expression. The murine macrophage cell line (ANA-1 cells) was incubated for 12 hr with No AG (media only), the whole AG extract (260 μg/ml), or the indicated AG Fraction (260 μg/ml), washed, then exposed to IFN-γ (100 U/ml) for 0, 2, 4 and 8 hrs. Cell lysates were analyzed by western blot analysis. C+, indicates the positive control, which was an archived ANA-1 cell lysate previously induced by IFN-γ, and known to have iNOS induction. (B) Effect of whole AG extract and the Hexane Fraction of AG on LPS-induced Cox-2 protein expression. Cells were treated as was described in (A). Numbers under the bands indicate densitometry values as a ratio relative to control (time 0 hr) for each treatment. (C) Densitometric quantification of iNOS and Cox-2 bands shown in (A) and (B), respectively, and adjusted for Actin levels. (D) Effect of the Hexane Fraction of AG on IFN-γ-induced iNOS mRNA expression. Cells were treated as was described in (A). (E) Effect of whole AG extract and the Hexane Fraction of AG on LPS-induced Cox-2 mRNA expression. All treatments were repeated 3 times to ensure consistency. *, indicates significant (p < 0.05) reduction in mRNA expression, relative to the untreated sample (no AG).
Figure 2
Figure 2. Effects of whole AG extract (AG) and the Hexane Fraction of AG on the colon histology score in the DSS mouse model of colitis
Results suggest the Hexane Fraction of AG is more potent in treating colitis than the whole AG extract. Values represent the mean ± SE. Representative H&E-stained colons are shown for each group. Arrows point to areas of inflammation and ulceration. Significant differences are indicated.
Figure 3
Figure 3. iNOS, Cox-2 and p53, markers of inflammation and inflammatory stress, are reduced in DSS + Hexane Fraction of AG-treated mice
Tissues from experiments performed for Figure 3 (3.5 Cycles) were examined for iNOS, COX-2, and p53 by immunohistochemistry, using the Antibody Amplifier™ (ProHisto, LLC) rocked on a laboratory rocker to ensure even staining and reproducible results. (A) Representative staining of indicated end points in serial sections from water (n = 11), DSS (n = 15) and DSS + Hexane Fraction of AG (n = 11) groups. Positive staining is brown colored. 100X magnification. (B) Quantification of indicated end points. All three markers were elevated in the DSS-treated group and suppressed when the DSS-treated group was fed the Hexane Fraction of AG. p-values are indicated.
Figure 4
Figure 4. Effects of the Hexane Fraction of AG on apoptosis in cells of the epithelium (A, C) and the MLNs (B, D)
(A) IRS (TUNEL staining) in epithelial cells of indicated groups. (B) IRS (TUNEL staining) in MLNs cells of indicated groups. (C) Quantification of staining in the epithelium. (D) Quantification of staining in the MLNs.

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