Isolation and synergism of in vitro anti-inflammatory and quinone reductase (QR) inducing agents from the fruits of Morinda citrifolia (noni)
Introduction
Morinda citrifolia, commonly called noni, is a plant typically found in the Pacific Islands, Southeast Asia, and other tropical areas. Noni fruits have been used as folk medicine for thousands of years for the alleviation of many diseases including cancer, colds, diabetes, flu, hypertension, and pain (Wang et al., 2002). Furthermore, noni juice, which is commonly prepared as a drip/exudate from senescing fruits held in fermentation vessels, is widely consumed today as a dietary supplement or food for the purported prevention of several diseases such as diabetes, high blood pressure and arteriosclerosis. So far, over 100 compounds have been identified in noni fruits. The structures of many of these are classified as anthraquinones, coumarins, glycosyl-fatty acid esters, flavonoids, polysaccharides, sterols and sulfur-containing compounds. Coumarins and flavonoids are specific chemotypes of the broader chemical group of polyphenols, which are abundant antioxidant components of fruits and vegetables (Faller & Fialho, 2009). Biological testing of both crude extracts and several pure constituents of noni fruit has been performed, revealing anti-inflammatory (Akihisa et al., 2007, Deng et al., 2007), antioxidant (Su et al., 2005), antibacterial, and Phase II enzyme inducing (Pawlus, Su, Keller, & Kinghorn, 2005) activities. However, many of the isolated compounds remain unexamined in this regard. Published reports suggest that noni fruits have chemopreventive activity against some types of cancers (Taskin et al., 2009, Wang et al., 2009) but which compound(s) from noni are responsible has not been determined.
Two biological effects that influence the disease status of animals includes the up-regulation of “vitagenes”, predominantly coded for by the antioxidant response element (ARE) (Calabrese et al., 2008, Song et al., 1999, Talalay et al., 1995), and down-regulation of signaling pathways related to chronic inflammation, as inflammation is considered a pathological factor for many diseases (Surh & Na, 2008). Phase II enzyme induction provides for protection against oxidative stress in general and carcinogenesis in particular. Phase II enzymes are involved in metabolism of potentially harmful xenobiotics by reduction of electrophilic quinones through quinone reductase (QR; NAD(P)H oxidoreductase), or conjugation with polar groups through glutathione-, glucuronidyl- and sulfo-transferase enzymes (Song et al., 1999, Talalay et al., 1995). Other ARE-encoded enzymes and proteins that defend cells against oxidative stress include hemeoxygenase-1 (HO-1), thioredoxin reductase, sirtuins, glutamate cysteine ligase, and several heat shock proteins (Calabrese et al., 2008, Russo, 2007). The regulatory regions of inducible ARE genes are activated upon binding of the nuclear factor E2-related protein 2 (Nrf2) transcription factor. Nuclear translocation of Nrf2 has been shown to be essential in the up-regulation of these protective genes in response to oxidative stress, electrophiles, and some phytochemicals (ARE inducers) (Eggler, Gay, & Mesecar, 2008).
Up-regulation of the ARE pathway is also believed to attenuate activation of the inflammatory pathway (Chen and Kong, 2005, Li et al., 2008). This “cross-talk” relationship was further revealed in a study that correlated potency among members of a series of established Phase II enzyme inducers with the relative ability to suppress inflammatory signaling (Liu, Dinkova-Kostova, & Talalay, 2008). Nitric oxide (NO) and prostaglandin E2 (PGE2) are two mediators of the inflammatory process, and are respectively synthesized by the inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Induction of these enzymes occurs largely through the nuclear factor kappaB (NF-κB) pathway, mediated by various pro-inflammatory cytokines, including interferon-γ, tumor necrosis factor-α, and interleukins-1 and -6 (Clancy & Abramson, 1995). The expression of the COX-2 and iNOS is up-regulated in acute/chronic inflammatory diseases, and this link is particularly strong for COX-2 induction and colorectal cancer (Crew, Elder, & Paraskeva, 2000). Moreover, NO has been found to activate COX-2, further increasing prostaglandin production that may enhance inflammation or promote tumorigenesis (Cianchi & Masini, 2005). Therefore, suppression of iNOS/COX-2 expression and their activities is a widely recognized target for preventing inflammatory diseases and cancer (Surh & Na, 2008). Many natural constituents from fruits and vegetables have been identified as in vitro inhibitors of COX-2 and iNOS activities, and this provides a basis for continuing to assess the potential for evaluating their anti-inflammatory effects (Lantz et al., 2007).
In the present study, bioassays for potential anti-inflammatory and Phase II inducing activities were used to direct the isolation of bioactive constituents from noni fruits. These isolated noni compounds were then tested alone and in combination in cultured cells to determine mechanistic features of their action related to potential anti-inflammatory and cancer chemopreventive effects.
Section snippets
Chemicals
Scopoletin, quercetin, rutin, alizarin, lipopolysaccharide (LPS) and anti-β-actin antibody were obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI). HPLC-grade ethyl acetate (EtOAc), ethanol (EtOH), methanol (MeOH) and acetonitrile (ACN) were purchased from Fisher Scientific (Chicago, IL). Anti-iNOS, anti-COX-2, anti-NQO1 antibodies and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture
Mouse macrophage cells (RAW 264.7), a commonly used model for
Noni extraction and bioassay-guided fractionation
The yield of the EtOAc crude extract was ~ 2% from noni (puree) powder on a dry matter basis. Initial results using Hepa and macrophage bioassays indicated the EtOAc-extracted material was over an order of magnitude more potent than crude EtOH or water extracts. Thus, anti-inflammatory and QR inducing components obtained from dried noni puree became enriched in EtOAc extractable solids. The EtOAc-extracted material inhibited the production of NO in LPS-activated RAW 264.7 macrophage cells with
Conclusion
In this study, scopoletin and quercetin were isolated from noni fruit puree as potentially beneficial components related to anti-inflammatory and anti-cancer activities. In the anti-inflammatory bioassay, a synergistic relationship between these two components (at ~ 5 μM each) at the same ratio they are present in the active extract of noni puree. The combined actions of these compounds likely involve multiple mechanisms of biological effect. Given the presence of many other bioactive components
Acknowledgements
This work was supported by the Thai Government Science and Technology Scholarship (to VN), the College of Agricultural and Life Sciences in part through a Hatch grant (WIS01184), and the School of Veterinary Medicine, of the University of Wisconsin-Madison.
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Present address: Department of Entomology, University of California, Davis, CA 95616, United States.