Yu Jin,† Venkata S. Kotakadi,† Lei Ying, Anne B. Hofseth, Xiangli Cui, Patricia A. Wood,1 Anthony Windust,2 Lydia E. Matesic,3 Edsel A. Pena,4 Codruta Chiuzan,4 Narendra P. Singh,5 Mitzi Nagarkatti,5 Prakash S. Nagarkatti,5 Michael J. Wargovich,6 and Lorne J. Hofseth*
Ulcerative colitis (UC) is a dynamic, idiopathic, chronic inflammatory condition associated with a high colon cancer risk. American ginseng has antioxidant properties and targets many of the players in inflammation. The aim of this study was to test whether American ginseng extract prevents and treats colitis. Colitis in mice was induced by the presence of 1% dextran sulfate sodium (DSS) in the drinking water or by 1% oxazolone rectally. American ginseng extract was mixed in the chow at levels consistent with that currently consumed by humans as a supplement (75 p.p.m., equivalent to 58 mg daily). To test prevention of colitis, American ginseng extract was given prior to colitis induction. To test treatment of colitis, American ginseng extract was given after the onset of colitis. In vitro studies were performed to examine mechanisms. Results indicate that American ginseng extract not only prevents but it also treats colitis. Inducible nitric oxide synthase and cyclooxygenase-2 (markers of inflammation) and p53 (induced by inflammatory stress) are also downregulated by American ginseng. Mucosal and DNA damage associated with colitis is at least in part a result of an oxidative burst from overactive leukocytes. We therefore tested the hypothesis that American ginseng extract can inhibit leukocyte activation and subsequent epithelial cell DNA damage in vitro and in vivo. Results are consistent with this hypothesis. The use of American ginseng extract represents a novel therapeutic approach for the prevention and treatment of UC.
Ulcerative colitis (UC) is a heterogenous, chronic and relapsing inflammatory condition that has a significant impact on the quality of life. Millions of people have this disease and have an increased colon cancer risk. Despite a wide variety of causes (e.g. environmental factors, genetic susceptibility and imbalanced enteric bacteria), the end result is an abnormal immune response with repeated episodes of colonic inflammation. While not everyone with colitis will develop colon cancer, risk increases when length of disease exceeds 10 years, on the order of 0.5–1.0% per year (1). Conventional treatment of colitis can reduce periods of active disease and help to maintain remission, but these treatments often bring marginal results, patients become refractory and there are side effects. For this reason, many colitis sufferers turn to unconventional treatments in hopes of abating symptoms of active disease and it is estimated that ∼40% of UC patients use some form of megavitamin therapy of herbal/dietary supplement (2,3).
The natural herbal American ginseng (Panax quinquefolius) improves mental performance and detrimental end points associated with diseases such as cardiovascular disease, diabetes and influenza (4,5). These diseases are all associated with inflammation, and American ginseng is a putative antioxidant in that it targets many of the key players involved in inflammation. Since UC is a chronic inflammatory disease, we hypothesized that American ginseng can be used to treat colitis. Here, we describe a role for American ginseng extract in the prevention and treatment of colitis.
In recent years, there have been major advances and promising strategies for the treatment of inflammatory bowel disease (23). Conventional treatment of colitis can reduce periods of active disease and help to maintain remission. However, most treatments often bring side effects with marginal results and have population-specific efficacy. Many patients therefore turn to alternative treatment strategies, including Tai chi, probiotics, flax seed, aloe vera and garlic (2). The frequency of UC patients taking American ginseng is not currently documented in the literature. Given the ability of American ginseng to suppress key inflammatory players such as Cox-2, iNOS and NF-κB (5), we hypothesized that this agent will suppress colitis. Here, we show that American ginseng extract inhibits the onset of colitis (Tables I and andII;II; Figure 1; supplementary Figures 3 and 4 are available at Carcinogenesis Online) and can be used to treat colitis (Table III; supplementary Figure 5 is available at Carcinogenesis Online). The doses we used of the extract were the human equivalent of 58 mg daily. This is significantly under that currently recommended for human consumption and doses used in clinical trials. In such trials, 1–3 g American ginseng has been shown to reduce blood sugar levels in both healthy and diabetic people (8,9,24). Four hundred milligrams American ginseng taken daily for 4 weeks reduced skeletal muscle membrane damage during exhaustive exercise (25). Although studies have shown that American ginseng does not appear to affect blood pressure, it is of note that such studies of long-term treatment also showed little side effects, indicating its safety up to 3 g daily (10,26). It appears that one of the few side effects are that American ginseng may reduce the effectiveness of the anticoagulant, warafin (27), and is therefore contraindicated when taking such drugs.
We also show that American ginseng extract suppresses the activation of key inflammatory markers such as iNOS and Cox-2 (Figure 1). These observations are consistent with other studies (6,28) and give insight into the anti-inflammatory mechanisms in vivo. Because p53 is a marker of inflammatory stress, we also probed for this end point. Our finding that p53 is also reduced indicates less inflammatory stress in animals receiving American ginseng extract. Because p53 appears to play a role in the protection from colitis by American ginseng, future experiments will focus on the role of p53 in inflammatory cell apoptosis. To this end, we have recently shown (13) that p53 overexpression in DSS-treated mice occurs mostly in inflammatory cells, providing a possible target for American ginseng as a mechanism to protection from overactive inflammatory cells.
Another interesting observation, represented in Figure 1, is that Cox-2 expression appeared in areas of high inflammation and ulceration near the luminal surface of the mucosa. Although iNOS and p53 expression also appeared in areas of high inflammation and ulceration, expression was mostly within the mucosa away from the luminal surface. The possibility that Cox-2 expression on the borders is due to drying effects can be ruled out because the slides were submerged in antibody solution and rocked during incubation (see Materials and Methods). The rationale for these expression patterns is currently under investigation. Interestingly, the observation that p53 and iNOS light up in similar areas is consistent with previous findings, showing that nitric oxide drives the overexpression of p53 in colitis (6). Overall, there is high expression of all three end points in the DSS-treated group, and in particular, in areas of mucosal damage.
UC and associated mucosal damage is at least partly caused by an increased number and hyperactive inflammatory cells in the colon (17). In this study, we asked whether American ginseng extract inhibits the activity of inflammatory cells and resultant epithelial cell DNA damage. In vitro experiments show that American ginseng extract can act on at least two levels in the colon. It can act at the level of inflammatory cells by inhibiting an oxidative burst. Alternatively, in the presence of an oxidative burst, it can directly protect epithelial cells from DNA damage. Noteworthy, we were also able to show that American ginseng extract reduces the activity of leukocytes and protects from damage to the colonic epithelial cell DNA in vivo. These results are consistent with in vitro studies from other labs. For example, red ginseng appears to protect cells from Helicobacter pylori-driven DNA damage and cytotoxicity (29). In animals, ginseng can inhibit micronucleus frequencies and chromosomal instability (30–32). It can also stimulate DNA repair (32). There is a strong link between chronic DNA damage and increased cancer risk. Based on results presented here, we are carrying out long-term separate studies to explore the hypothesis that American ginseng extract protects mice from inflammation-driven colon cancer.
In summary, we have shown American ginseng extract as a viable treatment strategy for colitis. Our data reveal that American ginseng directly inhibits leukocyte activation and DNA damage in target epithelial cells of the colon. Further studies will explore whether American ginseng extract can work upstream of the colon, in peripheral blood cells or lymphoid tissues. Indeed, other biological therapies can cause cellular apoptosis in the spleens of treated mice (33). A key mechanism for immune suppression is apoptosis of overly aggressive effector T cells and defects in mucosal T cell apoptosis is likely to play a key role in the pathogenesis of colitis (17,22). To this end, we recognize that there are several components of our American ginseng extract that can potentially influence the immune system. These not only include the ginsenosides (34) but also the polysaccharide/oligosaccharide components of the extract may have immunomodulatory activity (35). We can rule out an effect of maltodextrin since levels were almost identical between chows with and without the addition of American ginseng extract. Although, it may turn out that the most active extract is the combination of the active pharmacologic ingredients found in our American ginseng, ultimately the question of bioactivity of the extract with respect to colitis will be determined by bioassay-guided fractionation. Currently, however, we present evidence that the use of American ginseng extract represents a potential therapeutic approach for the abatement of signs and symptoms of inflammation associated with UC.