Potential anti-inflammatory phenolic glycosides from the medicinal plant Moringa oleifera fruits Sarot Cheenpracha a , Eun-Jung Park a , Wesley Y. Yoshida b , Chaz Barit a , Marisa Wall c , John M. Pezzuto a , Leng Chee Chang a, * a Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI 96720, USA b Department of Chemistry, University of Hawaii at Manoa, 2545 The Mall, Honolulu, HI 96822, USA c USDA Pacific Basin Agricultural Research Center, Hilo, HI 96720, USA article info Article history: Received 20 March 2010 Accepted 23 March 2010 Available online 29 March 2010 Keywords: Isothiocyanate Isothiocarbamate Anti-inflammatory activity Moringa oleifera abstract Bioassay-guided isolation and purification of the ethyl acetate extract of Moringa oleifera fruits yielded three new phenolic glycosides; 4-[(2 0 -O-acetyl-a-L-rhamnosyloxy) benzyl]isothiocyanate (1), 4-[(3 0 -O- acetyl-a-L-rhamnosyloxy)benzyl]isothiocyanate (2), and S-methyl-N-{4-[(a-L-rhamnosyloxy)benzyl]}- thiocarbamate (3), together with five known phenolic glycosides (4–8). The structures of the new metab- olites were determined on the basis of spectroscopic analyses including 1D- and 2D-NMR and mass spectrometry. The anti-inflammatory activity of isolated compounds was investigated with the lipopoly- saccharide (LPS)-induced murine macrophage RAW 264.7 cell line. It was found that 4-[(2 0 -O-acetyl-a-L- rhamnosyloxy)benzyl]isothiocyanate (1) possessed potent NO–inhibitory activity with an IC 50 value of 1.67 lM, followed by 2 (IC 50 = 2.66 lM), 4 (IC 50 = 2.71 lM), and 5 (IC 50 = 14.4 lM), respectively. Western blots demonstrated these compounds reduced LPS-mediated iNOS expression. In the concentration range of the IC 50 values, no significant cytotoxicity was noted. Structure–activity relationships following NO- release indicated: (1) the isothiocyanate group was essential for activity, (2) acetylation of the isothiocy- anate derivatives at C-2 0 or at C-3 0 of rhamnose led to higher activity, (3) un-acetylated isothiocyanate derivatives displayed eight times less activity than the acetylated derivatives, and (4) acetylation of the thiocarbamate derivatives enhanced activity. These data indicate compounds 1, 2, 4 and 5 are respon- sible for the reported NO-inhibitory effect of Moringa oleifera fruits, and further studies are warranted. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Nitric oxide (NO) is one of the inflammatory mediators causing inflammation in many organs. This inorganic free radical has been implicated in physiological and pathological processes, such as vasodilation, non-specific host defense and acute or chronic inflammation. Nitric oxide synthase (NOS) isoforms are homodi- mers that catalyze the oxidation of L-arginine to L-citrulline and ni- tric oxide in an NADPH- and O 2 -dependent process. 1 There are at least three distinct isoforms of NOS. Endothelial isoform (eNOS) and neuronal NOS (nNOS) are constitutively expressed and in- volved in the regulation of smooth muscle relaxation and neuro- transmission, respectively. Unlike eNOS and nNOS, inducible NOS (iNOS) is elevated in macrophages after the exposure of various stimuli, such as the bacterial endotoxin lipopolysaccharide (LPS), IL-1, TNF-a and IFN-c, 2 and produces NO in normal immune re- sponses. Since iNOS is responsible for the overproduction in inflammation, it has become a new target for drug development in the treatment of chronic inflammatory diseases. 1 Additionally, NO synthesized by iNOS has also been considered as an important mediator of carcinogenesis. NO can react with reactive oxygen spe- cies producing reactive nitrogen species that contribute to DNA damage and mutagenesis. 3 In addition, endogenous NO appears to cause a neoplastic transformation of mouse fibroblasts. 4 Overex- pressed iNOS has also been detected in several human tumors. 5–7 Moringa oleifera Lam. (Moringaceae), commonly known as ‘Malúñggay’ in Hawai‘I, is a fast-growing ornamental tree that is widely distributed in tropical areas. 8 The plant is also known to possess high nutritional value, because of the presence of protein and vitamins. 9 The young leaves, flowers, and green pods are used as a vegetable and its roots can be a substitute for horseradish in the Filipino diet. The medicinal value of the seeds and the different parts of the plant has long been recognized in folkloric medicine to treat various ailments related to pain and inflammation. 8,10–14 Sev- eral types of bioactive compounds have been isolated from the fruits of Moringa oleifera. The fruits, in particular, have been re- ported to contain antitumor and anti-inflammatory compounds of the glycoside type (i.e., niazirin, niazimicin, niazicin A). 14,15 0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.03.057 * Corresponding author. Tel.: +1 808 933 2906; fax: +1 808 933 2974. E-mail address: lengchee@hawaii.edu (L.C. Chang). Bioorganic & Medicinal Chemistry 18 (2010) 6598–6602 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc