Physalactone and 4β-Hydroxywithanolide E Isolated from Physalis peruviana Inhibit LPS-Induced Expression of COX‑2 and iNOS Accompanied by Abatement of Akt and STAT1 Eun-Jung Park, †,‡ Mayuramas Sang-Ngern, ‡,§ Leng Chee Chang, ‡ and John M. Pezzuto* ,†,‡ † Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, New York 11201, United States ‡ The Daniel K. Inouye College of Pharmacy, University of Hawaìi at Hilo, Hilo, Hawaii 96720, United States § School of Cosmetic Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai, Thailand ABSTRACT: In previous studies, withanolides isolated from Physalis peruviana were found to exhibit anti-inflammatory potential by suppressing nitrite production induced by lipopolysaccharide (LPS) treatment. Currently, we selected two of the most potent compounds, 4β- hydroxywithanolide E (1) and physalactone (2), to examine the underlying mechanism of action. With LPS-stimulated RAW 264.7 cells in culture, the compounds inhibited the mRNA and protein expression of iNOS and COX-2. To determine which upstream signaling proteins were involved in these effects, phosphorylation levels of three mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK1/2, and p38, were examined, but found unaffected. Similarly, the degradation of IκBα was not attenuated by the compounds. However, phosphorylation of Akt at the Ser-473 residue was inhibited, as was the phosphorylation of STAT1. Interestingly, the compounds also reduced the protein level of total STAT1, possibly by ubiquitin-dependent protein degradation. In sum, these results indicate the potential of 1 and 2 to mediate anti-inflammatory effects through the unexpected mechanism of inhibiting the transcription of iNOS and COX-2 via Akt- and STAT1-related signaling pathways. I nflammation is an essential defense mechanism to protect the host from various exogenous and endogenous stimuli. However, if the inflammatory response is not curtailed in a timely manner, sustained inflammatory signals can damage adjacent tissues (e.g., causing tissue fibrosis) or serve as an etiological factor in the genesis of conditions such as asthma, atherosclerosis, cancer, cardiovascular diseases, inflammatory bowel diseases, mood disorders, neurological disorders, and periodontal disease. 1,2 Accordingly, it is meaningful to explore novel anti-inflammatory compounds, including those associ- ated with edible sources, which may be of relevance for preventing or reducing the risk of the aforementioned diseases. One method for establishing the potential of anti- inflammatory activity involves a nitrite colorimetric assay. This assay allows for the indirect estimation of nitrite concentration utilizing the Griess reaction, first described in 1879, 3 in which nitrite forms a purple azo chromophore with an aryl amine. In 1982, employing the Griess reaction, Green et al. established an automated system to measure nitrate and nitrite in biological fluids, including gastric juice, milk, plasma, and saliva. 4 Meanwhile, it was reported that the RAW 264.7 cell line (Abelson murine leukemia virus-transformed macro- phage cells derived from male BALB/c mice) showed sensitivity to an endotoxin from Gram-negative bacteria termed lipopolysaccharide (LPS), along with cell growth inhibition. 5 Subsequently, treatment of RAW 264.7 with LPS was shown to induce the production of nitrite/nitrate, thereby suggesting the use of an in vitro cell line based model to measure the endogenous nitrite/nitrate synthesis in nitros- amine-induced carcinogenesis. 6 Of further importance, nitric oxide (NO) was established as an unstable intermediate in the conversion of L-arginine to nitrite and nitrate, 7 and therefore this procedure is known interchangeably as a NO or nitrite assay, even though a nitrite/nitrate assay involves one additional step in which nitrate is converted to nitrite catalyzed by nitrate reductase. Over the years, the assay has been frequently used to evaluate the anti-inflammatory activity of samples since macrophages (RAW 264.7) represent the first line of defense against bacterial infection (LPS from Gram-negative bacteria) by inducing an acute “inflammatory” immune response. In fact, based on a search of PubMed (“RAW 264.7, LPS, and nitric oxide”), 2352 articles have been published using this methodology (Figure 1). Since 2008, more than 20 articles have appeared each year in two natural-product-related Special Issue: Special Issue in Honor of Drs. Rachel Mata and Barbara Timmermann Received: October 15, 2018 Article pubs.acs.org/jnp Cite This: J. Nat. Prod. XXXX, XXX, XXX-XXX © XXXX American Chemical Society and American Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.8b00861 J. Nat. Prod. XXXX, XXX, XXX-XXX J. Nat. Prod. Downloaded from pubs.acs.org by TULANE UNIV on 01/16/19. For personal use only.