Submitted to Journal of Natural Products
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Natural stilbenoids have anti-inflammatory properties in vivo and down-regulate the production of inflammatory mediators NO, IL6 and MCP1 possibly in a PI3K/Akt- dependent manner
Journal: Journal of Natural Products
Manuscript ID np-2017-00384z.R3
Manuscript Type: Full Paper
Date Submitted by the Author: n/a
Complete List of Authors: Eräsalo, Heikki; University of Tampere, Faculty of Medicine and Life Sciences, Pharmacology Hämäläinen, Mari; University of Tampere, Faculty of Medicine and Life Sciences Leppänen, Tiina; University of Tampere, Faculty of Medicine and Life Sciences, Pharmacology Mäki-Opas, Ilari; University of Tampere, Faculty of Medicine and Life Sciences, Pharmacology Laavola, Mirka; University of Tampere, Faculty of Medicine and Life Sciences, Pharmacology Haavikko, Raisa; University of Helsinki, Department of Chemistry Yli-Kauhaluoma, Jari; University of Helsinki, Faculty of Pharmacy Moilanen, Eeva; University of Tampere, Faculty of Medicine and Life Sciences, Pharmacology
ACS Paragon Plus Environment Page 1 of 32 Submitted to Journal of Natural Products
1 2 3 Natural stilbenoids have anti-inflammatory properties in vivo and down-regulate the 4 5 production of inflammatory mediators NO, IL6 and MCP1 possibly in a PI3K/Akt- 6 7 dependent manner 8 9 10 Heikki Eräsalo†, Mari Hämäläinen†, Tiina Leppänen†, Ilari Mäki Opas†, Mirka Laavola†, 11 12 Raisa Haavikko‡, Jari Yli Kauhaluoma‡, Eeva Moilanen†* 13 14 15 † 16 The Immunopharmacology Research Group, Faculty of Medicine and Life Sciences, 17 18 University of Tampere and Tampere University Hospital, FI 33014 Tampere, Finland 19 20 ‡ 21 Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of 22 23 Helsinki, FI 00014 Helsinki, Finland 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 2 of 32
1 2 3 ABSTRACT 4 5 6 Stilbenoids are a group of polyphenolic compounds found in plants, trees, berries and nuts. 7 8 Stilbenoids have been shown to serve an anti microbial and anti fungal function in plants. 9 10 There is also evidence that as a part of human diet, stilbenoids play an important role as 11 12 antioxidants and may have anti inflammatory effects. The PI3K/Akt pathway is a well 13 14 characterized signaling pathway controlling cellular functions involved in growth and cell 15 16 17 cycle, and in metabolism. There is also increasing evidence to show the involvement of this 18 19 pathway in the regulation of inflammatory responses. In the present study, an attempt was 20 21 made to investigate the anti inflammatory properties of naturally occurring stilbenoids 22 23 pinosylvin (1), monomethyl pinosylvin (2), resveratrol (3), pterostilbene (4), piceatannol (5) 24 25 and rhapontigenin (6). Glycosylated derivatives of piceatannol and rhapontigenin, namely 26 27 astringin (7) and rhaponticin (8), respectively, were also investigated. In addition to the 28 29 30 natural stilbenoids, pinosylvin derivatives (9-13) were synthesized and subjected to the 31 32 testing of their effects on PI3K/Akt pathway in inflammatory conditions. 33 34 35 The investigated natural stilbenoids (except the glycosylated derivatives) were found to 36 37 down regulate Akt phosphorylation, which is a well acknowledged marker for PI3K activity. 38 39 It was also found that all of the studied natural stilbenoids had anti inflammatory effects in 40 41 vitro. The three most potent stilbenoids, piceatannol, pinosylvin and pterostilbene were 42 43 selected for in vivo testing and were found to suppress the inflammatory edema and to down 44 45 regulate the production of inflammatory mediators IL6 and MCP1 in carrageenan induced 46 47 48 paw inflammation in the mouse. When compared to the commercial PI3K inhibitor 49 50 LY294002, the anti inflammatory effects appeared to be quite similar. The results reveal 51 52 hitherto unknown anti inflammatory effects of natural stilbenoids and suggest that those 53 54 effects may be mediated via inhibition of the PI3K/Akt pathway. 55 56 57 58 2 59 60 ACS Paragon Plus Environment Page 3 of 32 Submitted to Journal of Natural Products
1 2 3 Introduction 4 5 6 Stilbenoids are a group of polyphenolic compounds found in nature and particularly in plants. 7 8 Stilbenoids have been shown to have anti microbial and anti fungal properties and it is likely 9 10 that their role in plants is to serve as defensive compounds against microbial pathogens.1 11 12 They are synthesized in trees as secondary metabolites during heartwood biogenesis and they 13 14 are also actively produced as a response to infection or injury. Stilbenoids can therefore be 15 16 2,3 17 also classified as phytoalexins due to their antimicrobial properties. 18 19 20 Due to being abundant in plants, stilbenoids are also present in human nutrition. Their health 21 22 effects and nutritional significance has also been studied. Resveratrol, probably the most 23 24 well known and investigated stilbenoid, is present in grapes, different berries, nuts and also in 25 26 red wine.4 Resveratrol has been reported to have anti inflammatory, anti carcinogenic, anti 27 28 oxidative and cardioprotective properties.5 However, in addition to resveratrol, several other 29 30 stilbenoid compounds are present in human nutrition. For instance, pterostilbenoid, 31 32 rhapontigenin and piceatannol can all be found in different berries, fruits, nuts and other 33 34 6 10 35 edible plants. Structurally very closely related stilbenoids, pinosylvin and monomethyl 36 11 12 37 pinosylvin, are also readily found in pine knots and also in pine nuts. There are 38 39 fragmentary data on the health promoting effects of those stilbenoids, sometimes resembling 40 41 the effects of resveratrol13 including some anti inflammatory effects.14 16 42 43 44 The PI3K/Akt pathway is a cellular signaling pathway involved in various cellular processes, 45 46 such as cell proliferation, migration and survival as well as glucose metabolism. It is also 47 48 considered to have a remarkable role in inflammatory responses17 and its role in cancer is 49 50 18 51 also vastly studied. The PI3K/Akt pathway consists of phosphatidylinositol 3 kinases that 52 53 catalyze the phosphorylation of phosphatidylinositols, a class of structurally important lipid 54 55 molecules in cell membranes. The phosphatidylinositolphosphates (PIPs) generated in the 56 57 58 3 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 4 of 32
1 2 3 process then serve as a docking platform for Akt kinase (also known as PKB). Akt kinase is a 4 5 serine/threonine kinase that has a wide range of downstream targets regulating different 6 7 processes, such as cell proliferation, glucose metabolism, neuronal development as well as 8 9 inflammation.19 21 It has been shown that when the PI3K/Akt pathway is inhibited with 10 11 pharmacological compounds, the cellular inflammatory responses are attenuated due to 12 13 22 24 14 down regulation of inflammatory genes. 15 16 17 The aim of the present study was to investigate natural stilbenoids (Figure 1), including: 18 19 pinosylvin (1), monomethyl pinosylvin (2), resveratrol (3), pterostilbene (4), piceatannol (5) 20 21 and rhapontigenin (6) and test their effects on the PI3K/Akt pathway in activated 22 23 macrophages. Additionally, the glycoside derivatives of piceatannol and rhapontigenin, 24 25 namely, astringin (7) and rhaponticin (8), respectively, were investigated. In addition to the 26 27 natural stilbenoids, pinosylvin derivatives (9 13) were synthesized and subjected to the anti 28 29 30 inflammatory testing. Another aim was to investigate the effects of the aforementioned 31 32 stilbenoids on the production of the inflammatory mediators interleukin 6 (IL6), monocyte 33 34 chemotactic protein 1 (MCP1) and nitric oxide (NO). Further, the anti inflammatory effects 35 36 of three potent stilbenoids (piceatannol, pinosylvin and pterostibene) were also investigated 37 38 in vivo in the carrageenan induced paw inflammation model in the mouse. Additionally the in 39 40 vivo studies were backed up with ex vivo studies by measuring inflammatory cytokines in the 41 42 43 inflamed paw tissue. The results reveal that all of the investigated natural stilbenoids 44 45 (excluding the glycoside derivatives) have an inhibitory effect on the activation of the 46 47 PI3K/Akt pathway. It was also found that these compounds down regulate the production of 48 49 inflammatory mediators, possibly via a PI3K/Akt dependent mechanism. An anti 50 51 inflammatory effect was also observed in the in vivo model and was further elucidated with 52 53 ex vivo studies. 54 55 56 57 58 4 59 60 ACS Paragon Plus Environment Page 5 of 32 Submitted to Journal of Natural Products
1 2 3 RESULTS 4 5 6 Synthesis of pinosylvin derivatives 7 8 9 A panel of pinosylvin derivatives bearing different alkyloxy and allyloxy moieties 10 11 (compounds 9 13) was designed (Figure 1). The compounds were synthesized by a 12 13 nucleophilic substitution reaction between the commercially available pinosylvin and 14 15 different alkyl (Et, n Pr, Bn, and i Bu) and allyl bromide in the presence of potassium 16 17 carbonate as a base in acetone. The novel pinosylvin ethers were produced in low to moderate 18 19 25 1 13 20 yields. Their chemical structures were confirmed by H and C NMR spectroscopy. 21 22 Natural stilbenoids inhibited PI3K/Akt activation in a concentration-dependent manner 23 24 25 in J774 macrophages 26 27 28 Stimulation of macrophages through a TLR4 mediated mechanism by exposing the cells to 29 30 bacterial endotoxin LPS (10 ng/mL) induced PI3K mediated phosphorylation of Akt protein 31 32 on amino acid residue S473 and treating the cells with the commercial PI3K inhibitor 33 34 LY294002 inhibited Akt phosphorylation (Figure 2). Next, the effects of natural stilbenoids 35 36 pinosylvin (1), monomethyl pinosylvin (2), resveratrol (3), pterostilbene (4), piceatannol (5) 37 38 and rhapontigenin (6) on the phosphorylation of Akt in activated macrophages were 39 40 41 investigated. The results showed that all of the investigated stilbenoids inhibited Akt 42 43 phosphorylation in a concentration dependent manner (Fig 3 A F). Piceatannol (5) was the 44 45 most potent of the tested stilbenoids followed by monomethyl pinosylvin (2) and pinosylvin 46 47 (1) which all inhibited Akt phosphorylation in a statistically significant manner already at 3 48 49 µM concentrations while rhapontigenin (6) appeared to be the least potent in the series of the 50 51 natural stilbenoids. The glycoside derivatives of piceatannol and rhapontigenin, i.e., astringin 52 53 54 (7) and rhaponticin (8), respectively, were also investigated. The glycosides had no effect on 55 56 Akt phosphorylation (Fig 3). 57 58 5 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 6 of 32
1 2 3 Semi-synthetic pinosylvin derivatives exhibited a structure-related loss of effect on Akt 4 5 phosphorylation when compared to natural stilbenoids 6 7 8 In order to compare the effects of natural stilbenoids versus synthetic pinosylvin derivatives, 9 10 Akt phosphorylation was stimulated with LPS in J774 macrophages treated with the 11 12 pinosylvin derivatives 9 13. Pinosylvin (1) was used as a reference compound in the 13 14 experiments. It was found that although some of the synthetic stilbenoid derivatives inhibited 15 16 17 Akt phosphorylation the effect was not comparable in magnitude to the effect of the natural 18 19 stilbenoid pinosylvin (Fig 4): Monoethyl pinosylvin (9) and monoallyl pinosylvin (11) 20 21 reduced Akt phosphorylation in a statistically significant manner when used at 30 µM 22 23 concentrations. Monoisobutyl pinosylvin (12) and monobenzyl pinosylvin (13) had no effect 24 25 at 30 µM concentrations while monopropyl pinosylvin (10) appeared to have a small 26 27 inhibitory effect but it did not reach statistical significance. The loss of effect appeared to 28 29 30 correlate with the length of the carbon side chain on the hydroxy group connected to the R1 31 32 position in the phenyl ring. 33 34 35 36 37 38 Natural stilbenoids down-regulated the production of inflammatory mediators NO, IL6 39 40 and MCP1 in J774 macrophages 41 42 43 In order to investigate the effects of the natural stilbenoids 1 8 on the production of 44 45 inflammatory mediators NO, IL6 and MCP1 in activated macrophages, the J774 macrophage 46 47 cells were stimulated with LPS in the presence of increasing concentrations of the stilbenoids 48 49 for 24 hours prior to cell culture media collection. It was found that the investigated 50 51 stilbenoids had a concentration dependent inhibitory effect on the production of the 52 53 54 aforementioned inflammatory mediators, and that was comparable to the effects obtained 55 56 with the known PI3K inhibitor LY294002. (Figures 5 7) 57 58 6 59 60 ACS Paragon Plus Environment Page 7 of 32 Submitted to Journal of Natural Products
1 2 3 The control compound LY294002 inhibited NO production by 50% at 8.9 µM concentration. 4 5 Closely comparable values were obtained for resveratrol (3, 12.15 µM), pterostilbene (4, 16.7 6 7 µM) and pinosylvin (1, 16.6 µM). Clearly higher values were obtained for monomethyl 8 9 pinosylvin (2, 37.0 µM) and rhapontigenin (6, 102.8 µM). For piceatannol (5) the value was 10 11 the lowest (2.6 µM). (Figure 5) 12 13 14 LY294002 inhibited IL6 expression half maximally at 5.1 µM concentration. Comparable to 15 16 17 this were resveratrol (3, 6.8 µM), pterostilbene (4, 8.7 µM) and piceatannol (5, 13.0 µM). 18 19 Higher values were detected with pinosylvin (1, 32.1 µM), monomethyl pinosylvin (2, 30.2 20 21 µM) and rhapontigenin (6, 99.1 µM). (Figure 6) 22 23 24 For MCP1 production, LY294002 had a 50% inhibitory effect at 3.7 µM. The only stilbenoid 25 26 comparable to that was piceatannol (5, 4.0 µM) and perhaps pterostilbene (4, 10.5 µM). 27 28 Clearly higher values were found for pinosylvin (1, 38.7 µM), monomethyl pinosylvin (2, 29 30 23.7 µM), resveratrol (3, 13.6 µM) and rhapontigenin (6, 19.4 µM). (Figure 7) 31 32 33 In contrast, the glycoside derivatives of piceatannol and rhapontigenin, i.e., astringin (7) and 34 35 rhaponticin (8), respectively, appeared to have lost their activity when compared to the 36 37 38 corresponding non glycosylated stilbenoid (Figures 5 7). 39 40 in vivo ex vivo 41 Natural stilbenoids showed anti-inflammatory effects both and 42 43 Carrageenan induced paw edema mouse model was used as an in vivo model of inflammation 44 45 46 to investigate the anti inflammatory effects of natural stilbenoids. Pinosylvin (1), 47 48 pterostilbene (4) and piceatannol (5) were selected for the in vivo studies due to them having 49 50 similar effects and relatively similar inhibition percentages in the in vitro studies than the 51 52 PI3K inhibitor LY294002. Also, based on the literature search pterostilbene (4) and 53 54 piceatannol (5) appeared also to be the least investigated stilbenoids with very little published 55 56 57 58 7 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 8 of 32
1 2 3 data from in vivo experiments. A widely used anti inflammatory drug dexamethasone was 4 5 used as the control compound. It was found that all of the investigated natural stilbenoids, 6 7 i.e., 1, 4 and 5 (when given at the dose of 30 mg/kg), had an anti inflammatory effect in vivo 8 9 comparable to that of LY294002 (15 mg/kg) and dexamethasone (2 mg/kg), (Figure 8). 10 11 12 In addition to the in vivo studies, ex vivo studies were conducted by measuring the levels of 13 14 inflammatory cytokines IL6 and MCP1 in the mouse paw tissue after carrageenan injection. It 15 16 17 was found that the inflamed paw tissue from mice treated with either stilbenoids or the 18 19 control compounds LY294002 or dexamethasone showed a diminished inflammatory 20 21 response, which was seen as lower expression of the inflammatory cytokines IL6 and MCP1 22 23 (Figure 9). 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 8 59 60 ACS Paragon Plus Environment Page 9 of 32 Submitted to Journal of Natural Products
1 2 3 DISCUSSION 4 5 6 In the present study, our aim was to investigate anti inflammatory properties of natural 7 8 stilbenoids pinosylvin (1), monomethyl pinosylvin (2), resveratrol (3), pterostilbene (4), 9 10 piceatannol (5), rhapontigenin (6), astringin (7) and rhaponticin (8).The hypothesis was that 11 12 stilbenoids would down regulate inflammatory gene expression and the activity of the 13 14 PI3K/Akt pathway in relation to inflammation. The results obtained in this study support the 15 16 17 idea that natural stilbenoids have anti inflammatory properties and the effect is possibly 18 19 mediated via inhibition of the PI3K/Akt pathway. Further, it was found that the investigated 20 21 semi synthetic pinosylvin derivatives showed weaker effects on PI3K/Akt pathway in 22 23 inflammatory conditions when compared to their naturally occurring counterparts. 24 25 26 The health beneficial properties of the stilbenoid resveratrol and red wine have been a 27 28 popular research topic ever since the concept of the French paradox was formulated in the 29 30 1980s.26 Reports have shown that resveratrol has anti inflammatory, anti carcinogenic, anti 31 32 oxidative and cardioprotective properties5 as well as beneficial effects on aging, diabetes and 33 34 27 35 neurological dysfunction. The in vitro anti inflammatory effects of resveratrol were also 36 37 reproduced in the present study. However, there exists a plethora of foodstuffs containing 38 39 stilbenoids other than resveratrol, such as different berries, nuts and other edible plants which 40 41 are a natural part of human nutrition worldwide. Therefore, in this study we focused mainly 42 43 on other stilbenoids besides resveratrol, namely pinosylvin (1), monomethyl pinosylvin (2), 44 45 pterostilbene (4) piceatannol (5), and rhapontigenin (6) as well as five synthetic derivatives of 46 47 48 pinosylvin. 49 50 51 The PI3K/Akt signaling pathway controls an array of cellular functions, some of which are 52 53 also involved in inflammation. Regarding inflammation, the PI3K/Akt pathway is 54 55 documented to regulate survival, motility, differentiation and proliferation of leukocytes via 56 57 58 9 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 10 of 32
1 2 21 3 cytokine and chemokine signaling as well as NF κB mediated transcription. Among three 4 5 different classes of PI3Ks there also exists several different PI3K isoforms. The class I PI3Ks 6 7 are considered to be involved in inflammation and particularly class I PI3K subunits PI3Kδ 8 9 and PI3Kγ which are considered to regulate cytokine signaling and chemotaxis of immune 10 11 cells.22,28 12 13 14 Low grade chronic inflammation has been documented to be increasingly prevalent 15 16 17 especially in western countries. Western lifestyle and diet has been suggested as one of the 18 19 main causes of low grade inflammation and the prevalence of metabolic syndrome and type 2 20 21 diabetes is considered to be a direct effect caused by low grade inflammation.29 Obesity is 22 23 also known to up regulate inflammation and it has been reported that macrophages located in 24 25 the adipose tissue produce significant amounts of IL6 and MCP1 as well as other 26 27 inflammatory mediators.30 It has also been documented that a healthy diet containing 28 29 30 phytonutrients from fruits and vegetables modulates low grade inflammation towards a more
31 31 32 healthy level. A recent study also documented that the PI3K/Akt pathway is activated in 33 32 34 low grade inflammation and that the inflammation can be attenuated with PI3K inhibition. 35 36 Based on our present findings we hypothesize that a diet containing natural stilbenoids could 37 38 possibly also attenuate low grade inflammation via a PI3K dependent mechanism. 39 40 41 In this study we used a commercially available PI3K inhibitor LY294002 as a reference 42 43 compound for PI3K inhibition. LY294002 is a well characterized class I PI3K inhibitor.33 44 45 The phosphorylation of Akt is often considered as a marker for PI3K activity. Upon binding 46 47 48 the phosphatidylinositols produced by PI3Ks, Akt is docked to the cell membrane, which 49 20 50 allows PDK1 and MTOR to phosphorylate it. Our results show that the control compound 51 52 PI3K inhibitor LY294002 used in this study, inhibits Akt phosphorylation upon LPS 53 54 stimulation also in activated macrophages and inhibits the production of inflammatory 55 56 cytokines IL6 and MCP1 in vitro and in vivo as expected. 57 58 10 59 60 ACS Paragon Plus Environment Page 11 of 32 Submitted to Journal of Natural Products
1 2 3 Based on our findings, it appears that the investigated stilbenoids also exhibit similar 4 5 inhibitory effects as the PI3K inhibitor LY294002 on the phosphorylation of Akt indicative 6 7 of the inhibition of the activity of the PI3K/Akt pathway. These data are supported by the few 8 9 studies that have focused on the effects of stilbenoids on the PI3K/Akt pathway in other cell 10 11 types and experimental settings.14,34,35 However, the findings reported in this study suggest 12 13 14 that among the investigated stilbenoids there are differences regarding the PI3K/Akt 15 16 inhibition potency. Piceatannol (compound 5) appeared to be the most potent of the tested 17 18 natural stilbenoids, followed by pinosylvin (compound 1) and monomethyl pinosylvin 19 20 (compound 2) while rhapontigenin (compound 6) was the least potent. Very similar pattern of 21 22 activity was also seen in their inhibitory effects on the production of the inflammatory factors 23 24 nitric oxide, IL6 and MCP1, see below. 25 26 27 The semi synthetic pinosylvin derivatives were less potent than the natural stilbenoid 28 29 30 pinosylvin, and the loss of activity seemed to associate with the length of the side chain on 31 32 the R1 position in the left hand side phenyl ring. When the pinosylvin derivatives were 33 34 compared to the natural pinosylvin, it appeared that a small substituent such as methyl on the 35 36 R1 carbon did not significantly effect the potency of PI3K/Akt inhibition as was documented 37 38 with monomethyl pinosylvin (compound 2). However, when the substituent on R1 became 39 40 longer, such as the ethyl, propyl or isobutyl ether group on compounds 9, 10 and 12, 41 42 43 respectively, or bulkier such as the allyl or benzyl ether groups on compounds 11 and 13, 44 45 respectively, the effect observed on PI3K/Akt inhibtion was clearly weakened. In fact, 46 47 compounds 12 and 13 had no effect at 30 µM concentrations, and compound 10 had a minor 48 49 effect which did not reach statistical significance. This is possibly due to the fact that longer 50 51 or bulkier substituents are more likely to cause steric hindrance in the interaction mechanisms 52 53 between these molecules. 54 55 56 57 58 11 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 12 of 32
1 2 3 In addition, the glycosidic derivatives of piceatannol and rhapontigenin, namely astringin and 4 5 rhaponticin, respectively, did not appear to have an effect on the PI3K/Akt pathway. This is 6 7 possibly due to the same mechanism described above and / or related to the fact that 8 9 glycoside derivatives are not readily transported into the cells but rather stay in the cell 10 11 culture medium where they remain inactive. To further elucidate this, it would be meaningful 12 13 14 to investigate other different glycosidic derivatives (eg. glycosides with galactose or fructose 15 16 conjugates) because both of the tested compounds were O beta D glycoside derivatives of 17 18 the stilbenoids in question. 19 20 21 Nitric oxide produced by inducible nitric oxide synthase (iNOS) as well as the cytokines IL6 22 23 and MCP1 are well known inflammatory mediators. Piceatannol (compound 5) was the most 24 25 potent of the tested stilbenoids in decreasing nitric oxide production while rhapontigenin 26 27 (compound 6) was the least potent of the non glycosylated stilbenoids; and the potency of the 28 29 30 other non glycosylated stilbenoids was between those two. The glycosylated stilbenoids 31 32 astringin (compound 7) and rhaponticin (compound 8) did not have any effect. Very similar 33 34 pattern of activity was also seen in their inhibitory effects on Akt phosphorylation, and IL6 35 36 and MCP1 production. Rhapontigenin (6) is the only aglycone which has a methyl group at 37 38 position C4 while piceatannol (5) has hydroxy groups in positions R1, R3, R4 and R5 which 39 40 may contribute to their anti inflammatory activity. The glycosylated stilbenoids remained 41 42 43 ineffective possibly because they are not readily transported into the cells which is necessary 44 45 for their effects on the production of inflammatory mediators. 46 47 48 The production of IL6, MCP 1 and nitric oxide is activated by various inflammatory stimuli 49 50 and also different transcription factors are documented to be involved. The cellular effects of 51 52 PI3K are occasionally reported to be mediated via the NF κB transcription factor and the 53 54 three measured inflammatory mediators are also reported to be NF κB responsive.36 39 Our 55 56 findings document that inhibition of PI3K/Akt pathway with LY294002 down regulates the 57 58 12 59 60 ACS Paragon Plus Environment Page 13 of 32 Submitted to Journal of Natural Products
1 2 3 expression of these inflammatory mediators, which in turn suggests that these inflammatory 4 5 mediators are PI3K/Akt responsive. It was also found that the natural stilbenoid compounds 6 7 investigated in the present study down regulated the expression of these inflammatory 8 9 mediators. When these data are considered alongside with our findings that the stilbenoids 10 11 also inhibited PI3K/Akt activation, it is plausible that the down regulated inflammatory 12 13 14 response is mediated via PI3K inhibition. 15 16 17 Carrageenan induced paw edema is a well characterized in vivo model of acute inflammation. 18 19 It has been widely reported that the inflammatory factors mediating carrageenan induced 20 21 edema include IL6, MCP1 and NO.40 42 The PI3K/Akt pathway is also considered to be one 22 23 of the signaling pathways involved in acute inflammation such as the inflammatory edema 24 25 caused by exposure to carrageenan. We and others have earlier documented that inhibition of 26 27 the PI3K/Akt pathway leads to reduced edema and decreased inflammatory cell migration 28 29 21,24 30 upon carrageenan irritation. These data support the idea that PI3K/Akt signaling is 31 32 involved in mediating the formation of the inflammatory response induced by exposure to 33 34 carrageenan. In the present study, we found that the investigated natural stilbenoids also 35 36 reduced the carrageenan induced edema and suppressed the expression of the inflammatory 37 38 cytokines IL6 and MCP1 in the inflamed paw tissue. According to our knowledge, this is a 39 40 novel in vivo effect for pterostilbene (4) and piceatannol (5) that has not been previously 41 42 43 reported. The effect appears to be comparable to the effect obtained with the commercial 44 45 PI3K inhibitor LY294002. This in turn suggests that the investigated stilbenoids would have 46 47 anti inflammatory properties in vivo and that the mechanism of the effect possibly includes 48 49 an inhibitory interaction with the PI3K/Akt pathway. 50 51 52 In this study we document our findings regarding several naturally occurring stilbenoid 53 54 compounds, i.e. pinosylvin, monomethyl pinosylvin, resveratrol, pterostilbene, piceatannol 55 56 57 58 13 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 14 of 32
1 2 3 and rhapontigenin, which are all known to be included in a normal healthy human diet. It was 4 5 found that these stilbenoids have anti inflammatory properties when investigated in vitro and 6 7 in vivo along with their inhibitory action on the PI3K/Akt signaling pathway. The effects 8 9 were comparable to the effects obtained with the known PI3K/Akt inhibitor LY294002. 10 11 Additionally, we found that the tested glycoside derivatives of these stilbenoids did not have 12 13 14 a significant effect in the inflammatory models. Based on these findings it appears that the 15 16 naturally occurring stilbenoids included in human nutrition have anti inflammatory properties 17 18 and it also appears that these effects could possibly be mediated via the PI3K/Akt pathway. 19 20 This may be a significant finding that could be exploited to alleviate inflammatory conditions 21 22 by a stilbenoids containing dietary intervention but further studies are needed to understand 23 24 the mechanisms of action and to prove bioavailability and actual clinical efficacy in man. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 14 59 60 ACS Paragon Plus Environment Page 15 of 32 Submitted to Journal of Natural Products
1 2 3 EXPERIMENTAL SECTION 4 5 6 Materials 7 8 9 The reagents used in the study were obtained as following: pAkt S473 and Akt antibodies 10 11 from Cell Signaling Technology, Danvers, MA, USA; β actin antibodies and HRP 12 13 conjugated goat polyclonal anti rabbit IgG antibodies from Santa Cruz Biotechnology, Santa 14 15 Cruz, CA, USA; PI3K inhibitor LY294002 from Merck Millipore, Billerica, MA, USA; 16 17 pinosylvin, monomethyl pinosylvin and resveratrol from Sequoia Research Products, 18 19 20 Pangbourne, UK; pterostilbene, piceatannol and dexamethasone from Orion Pharma, Finland; 21 22 rhapontigenin from Cayman Chemical, MI, USA; astringin from Polyphenols Laboratories, 23 24 Norway, and rhaponticin from Sigma Aldrich, MO, USA. 25 26 27 Chemistry 28 29 30 General experimental details 31 32 33 Commercially available reagents were used without further purification: all solvents were 34 35 anhydrous and HPLC grade, purchased from Sigma Aldrich (St. Gallen, Switzerland). All 36 37 reactions were performed in oven dried glassware under an inert atmosphere of dry argon. 38 39 Thin layer chromatography (TLC) was performed on E. Merck Silica Gel 60 aluminium 40 41 packed plates, visualization accomplished by UV illumination and staining with H2SO4 (5% 42 43 v/v) in MeOH. 1H NMR spectra were recorded on a Varian Mercury VX 300 MHz with 44 45 46 chemical shifts reported as parts per million (in acetone d6 at 23 °C, solvent peak at 2.05 ppm 47 13 48 as an internal standard). C NMR spectra were obtained on a Varian Mercury VX 75 MHz 49 50 spectrometer with chemical shifts reported as parts per million (acetone d6 at 23 °C, solvent 51 52 peaks at 29.84, and 206.26 ppm as an internal standard). 53 54 55 56 57 58 15 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 16 of 32
1 2 3 4 5 6 Synthesis of pinosylvin monoethers 7 8 9 General procedure: A mixture of pinosylvin (0.050 g, 0.24 mmol), potassium carbonate 10 11 (0.050 g, 0.36 mmol), and alkyl or allyl bromide (0.36 mmol) in acetone (3 5 mL) was 12 13 refluxed under argon for 24 hours. The reaction mixture was poured into water and extracted 14 15 with diethyl ether. The organic layer was washed with brine, dried over anhydrous Na2SO4, 16 17 and concentrated in vacuo. The crude products were purified with silica column eluting with 18 19 20 10 30 % hexanes/EtOAc to give monoethers in 8 28 % yields. Pinosylvin monoethyl ether
21 1 22 (9): Off white crystals (10.7 mg, 29%). Rf = 0.76 (n hexane/EtOAc = 1:1). H NMR (300 23 24 MHz, acetone d6) δ 8.32 (s, 1H), 7.58 (m, 2H), 7.27 (m, 1H), 7.36 (m, 2H), 7.15 (d, J = 4.5 25 26 Hz, 2H), 6.68 (m, 2H), 6.35 (t, J = 2.2 Hz, 1H), 4.04 (q, J = 7 Hz, 2H), 1.35 (t, J = 7 Hz, 3H). 27 28 Pinosylvin monopropyl ether (10): Starting from 69 mg (0.33 mmol) of pinosylvin as well 29 30 as using 44 L (0.49 mmol, 1.5 equiv) of 1 bromopropane, and 0.22 g of K CO (1.6 mmol): 31 2 3 32 1 33 White crystals (9.8 mg, 12%). Rf = 0.70 (n hexane/EtOAc = 1:1). H NMR (300 MHz, 34 35 acetone d6) δ 8.31 (s, 1H), 7.59 (m, 2H), 7.36 (m, 2H), 7.25 (m, 1H), 7.15 (d, J = 4.2 Hz, 2H), 36 37 6.68 (m, 2H), 6.36 (t, J = 2.2 Hz, 1H), 3.95 (t, J = 6.5 Hz 2H), 1.78 (m, 2H), 1.03 (t, J = 7.4 38 13 39 Hz, 3H); C NMR (75 MHz, acetone d6) δ 161.0, 158.9, 139.7, 137.7, 129.0, 128.8, 128.8, 40 41 127.8, 127.7, 126.7, 106.3, 104.3, 101.7, 69.3, 22.6, 10.1. Pinosylvin monoallyl ether (11): 42 43 White crystals (17 mg, 28%). R = 0.82 (n hexane/EtOAc = 1:1). 1H NMR (300 MHz, 44 f 45 46 acetone d6) δ 8.34 (s, 1H), 7.58 (m, 2H), 7.37 (m, 2H), 7.25 (m, 1H), 7.15 (d, J = 4.7 Hz, 2H), 47 48 6.70 (m, 2H), 6.39 (t, J = 2.2 Hz, 1H), 6.06 (m, 1H), 5.43 (dq, J = 17.3, 1.5 Hz, 1H), 5.25 (dq, 49 13 50 J = 10.6, 1.5 Hz, 1H), 4.57 (dt, J = 5.2, 1.6 Hz, 3H); C NMR (75 MHz, acetone d6) δ 160.5, 51 52 158.9, 139.7, 137.7, 134.2, 129.0, 128.9, 128.8, 127.8, 126.7, 116.6, 106.6, 104.5, 102.0, 53 54 68.6. Pinosylvin monoisobutyl ether (12): White crystals (4.8 mg, 7.5%). Rf = 0.77 (n 55 56 57 58 16 59 60 ACS Paragon Plus Environment Page 17 of 32 Submitted to Journal of Natural Products
1 2 1 3 hexane/EtOAc = 1:1). H NMR (300 MHz, acetone d6) δ 8.29 (s, 1H), 7.58 (m, 2H), 7.37 (m, 4 5 2H), 7.25 (m, 1H), 7.15 (d, J = 4.5 Hz, 2H), 6.69 (m, 2H), 6.37 (t, J = 2.2 Hz, 1H), 3.77 (d, J 6 13 7 = 6.5 Hz, 2H), 2.06 (m, 1H), 1.03 (d, J = 6.5 Hz, 6H); C NMR (75 MHz, acetone d6) δ 8 9 161.1, 158.9, 139.7, 137.7, 129.0, 128.8, 128.8, 127.7, 126.7, 106.3, 104.4, 101.8, 74.3, 18.8. 10 11 Pinosylvin monobenzyl ether (13): White crystals (13 mg, 18%). R = 0.75 (n 12 f 13 1 14 hexane/EtOAc = 1:1). H NMR (300 MHz, acetone d6) δ 8.37 (s, 1H), 7.62 – 7.54 (m, 2H), 15 16 7.53 – 7.47 (m, 2H), 7.45 – 7.31 (m, 5H), 7.30 – 7.19 (m, 1H), 7.16 (d, J = 4.7 Hz, 2H), 6.82 17 18 (dd, J = 7.2, 5.5 Hz, 1H), 6.76 – 6.65 (m, 1H), 6.45 (t, J = 2.2 Hz, 1H), 5.22 – 5.03 (m, 2H); 19 13 20 C NMR (75 MHz, acetone d6) δ 161.3, 159.6, 144.6, 140.5, 138.5, 138.3, 132.1, 129.6, 21 22 129.5, 129.3, 128.6, 128.4, 127.4, 124.8, 107.4, 105.3, 102.8, 70.5. 23 24 25 Anti-inflammatory testing 26 27 28 Cell culture 29 30 31 Murine J774 macrophages (American Type Culture Collection, Manassas, VA, USA) were 32 33 cultured at 37 °C in 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium with 34 35 Ultraglutamine 1 (Sigma Aldrich, MO, USA) supplemented with 10% heat inactivated foetal 36 37 38 bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 250 ng/mL amphotericin B 39 40 (all from Invitrogen, Paisley, UK). Cells were cultured on 24 well plates for western blot, 41 42 ELISA, and nitrite measurements and on 96 well plates for cytotoxicity testing. Cells were 43 44 grown for 72 h to confluence prior to the experiments. 45 46 47 Cytotoxicity of tested compounds was ruled out by measuring cell viability using Cell 48 49 Proliferation Kit II (Roche Diagnostics, Mannheim, Germany). 50 51 52 53 54 55 56 57 58 17 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 18 of 32
1 2 3 Nitrite assays 4 5 6 The effects of the tested compounds on cellular NO production was investigated by 7 − 8 measuring the accumulation of nitrite (NO2 , a stable metabolite of NO), in the cell culture 9 10 medium with the Griess test43. 11 12 13 IL6 and MCP1 ELISA 14 15 16 IL6 and MCP1 concentrations in the cell culture medium and paw tissue extracts were 17 18 measured by enzyme linked immunosorbent assay (ELISA) using reagents from R&D 19 20 Systems Europe Ltd (Abingdon, UK). 21 22 23 Western blotting 24 25 26 At indicated time points, cells were rapidly washed with ice cold phosphate buffered saline 27 28 (PBS, pH 7.4) and lysed with a buffer containing 10 mM Tris base (pH 7.4), 5 mM EDTA, 29 30 31 50 mM NaCl, 1% Triton X 100, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium 32 33 orthovanadate, 20 µg/mL leupeptin, 50 µg/mL aprotinin, 5 mM NaF, 2 mM sodium 34 35 pyrophosphate and 10 µM n octyl β D glucopyranoside. After incubation for 15 min on ice, 36 37 cell lysates were centrifuged (13 000g, 4 °C, 10 min), supernatants were collected and stored 38 39 in 20 °C in SDS sample buffer containing 0.5 M Tris (pH 6.8), glycerol (10% v/v), SDS (2% 40 41 v/v), bromophenol blue (0.25‰ v/v) and 2 mercaptoethanol (5% v/v). An aliquot of the 42 43 44 44 supernatant was used to determine protein concentration with the Bradford protein assay . 45 46 47 Protein samples (20 µg of total protein from lysates) were analysed according to standard 48 45 49 western blotting protocol as described previously. The membrane was incubated with the 50 51 primary antibody in the blocking solution at 4 °C overnight, and with the secondary antibody 52 53 in the blocking solution for 1 h at room temperature. Bound antibody was detected using 54 55 Super Signal® West Pico or Dura chemiluminescent substrate (Pierce, Rockford, USA) and 56 57 58 18 59 60 ACS Paragon Plus Environment Page 19 of 32 Submitted to Journal of Natural Products
1 2 3 Image Quant LAS 4000 imaging system (GE Healthcare Bio Sciences, Buckinghamshire 4 5 UK). The quantitation of the chemiluminescent signal was carried out with the use of Image 6 7 Quant TL software (GE Healthcare). 8 9 10 Carrageenan induced paw edema in mice 11 12 13 The in vivo anti inflammatory effects of PI3K inhibitor LY294002 and different stilbenoids 14 15 were studied by measuring carrageenan induced paw edema in male C57BL/6 mice. The 16 17 study was carried out in accordance with the legislation for the protection of animals used for 18 19 20 scientific purposes (directive 2010/63/EU) and approved by the National Animal Experiment 21 22 Board (approval number ESAVI/5019/04.10.03/2012, granted September 3, 2012). Paw 23 24 edema was induced under anaesthesia and all efforts were made to minimize suffering. 25 26 27 Animals were housed under conditions of optimum light, temperature and humidity (12:12 h 28 29 light:dark cycle, 22±1 °C, 50–60% humidity) with food and water provided ad libitum. One 30 31 hour prior to carrageenan injection the investigated compounds were delivered i.p. dissolved 32 33 in a vehicle containing 10% DMSO in PBS (pH 7.4). The mice were anesthetised by 34 35 intraperitoneal injection of 0.5 mg/kg of medetomide (Domitor® 1 mg/mL, Orion Oyj, 36 37 38 Espoo, Finland) and 75 mg/kg of ketamine (Ketalar® 10 mg/mL, Pfizer Oy Animal Health, 39 40 Helsinki, Finland), and thereafter the mice received a 50 µL intradermal injection in one 41 42 hindpaw of normal saline containing λ carrageenan (1.5% w/v). The contralateral paw 43 44 received 50 µL of saline only and it was used as a control. Edema was measured before and 45 46 three and six hours after carrageenan injection with a plethysmometer (Ugo Basile, Comerio, 47 48 Italy). 49 50 51 At six hours the mice were sacrificed and the paw tissue injected with carrageenan and the 52 53 54 contralateral tissue injected with the vehicle was collected for analysis into a buffer 55 56 containing Tris (50 mM, pH 7.4), NaCl (150 mM), and protease and phosphatase inhibitors 57 58 19 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 20 of 32
1 2 3 phenylmethylsulfonyl fluoride (0.5 mM), sodium orthovanadate (2 mM), leupeptin (0.10 4 5 µg/mL), aprotinin (0.25 µg/mL) and NaF (1.25 mM). The tissue was minced and centrifuged 6 7 (10 min, 10 000g) and the supernatant was collected for ELISA measurements. 8 9 10 Statistics 11 12 13 Results are expressed as mean+standard error of mean (S.E.M.). Statistical significance of the 14 15 results was calculated by one way ANOVA with Dunnett’s or Bonferroni’s post test. 16 17 Differences were considered significant at *p<0.05, **p<0.01, ***p<0.001. 18 19 20
21 22 23 24 AUTHOR INFORMATION 25 26 27 Corresponding Author 28 29 30 *E mail: [email protected] 31 32 33 ACKNOWLEDGEMENTS 34 35 36 This study was funded by the Finnish Bioeconomy Cluster (FIBIC Ltd), the Finnish Funding 37 38 Agency for Technology and Innovation (TEKES) and the Medical Research Fund of 39 40 Tampere University Hospital. We wish to thank Meiju Kukkonen, Salla Hietakangas and 41 42 Terhi Salonen for excellent technical assistance and Heli Määttä for skillful secretarial help. 43 44 45
46 47 48 49 50 51 52 53 54 55 56 57 58 20 59 60 ACS Paragon Plus Environment Page 21 of 32 Submitted to Journal of Natural Products
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 1: The molecular structures of the studied natural and semi-synthetic stilbenoids. 46 47 170x236mm (300 x 300 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 25 of 32 Submitted to Journal of Natural Products
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 2: LPS stimulated phosphorylation of Akt in activated macrophages. J774 macrophages were 46 stimulated with bacterial lipopolysaccharide (LPS, 10 ng/mL) in the absence or in the presence of 10 µM 47 LY294002 for 4 hours. Thereafter cellular proteins were extracted and the level of Akt phosphorylation was 48 measured by Western blotting with an antibody specific towards phosphorylated S473 residue. Values are 49 mean + S.E.M., n = 4, **p < 0.01. 50 89x129mm (300 x 300 DPI) 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 26 of 32
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 3: The concentration-dependent effect of natural stilbenoids and stilbenoid glycosides on Akt 46 phosphorylation in activated macrophages. J774 macrophages were stimulated with bacterial 47 lipopolysaccharide (LPS, 10 ng/mL) for 4 hours in the presence of increasing concentrations of (A) pinosylvin 48 (1), (B) monomethyl pinosylvin (2), (C) resveratrol (3), (D) pterostilbene (4), (E) piceatannol (5) and 49 astringin (7), and (F) rhapontigenin (6) and rhaponticin (8). Thereafter cellular proteins were extracted and 50 the level of Akt phosphorylation was investigated by western blotting with an antibody specific towards phosphorylated S473 residue. Values are mean + S.E.M., n = 4-6, *p < 0.05 and **p < 0.01. 51 52 207x322mm (300 x 300 DPI) 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 27 of 32 Submitted to Journal of Natural Products
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Figure 4. The effect of semi synthetic pinosylvin derivatives on Akt phosphorylation in activated 40 macrophages. J774 macrophages were stimulated with bacterial lipopolysaccharide (LPS, 10 ng/mL) for 4 41 hours in the presence of the semi synthetic stilbenoids 9 13 (30 µM). Pinosylvin and LY294002 were used as 42 reference compounds. After incubation cellular proteins were extracted and Akt phosphorylation was 43 investigated by Western blotting with an antibody specific towards phosphorylated S473 residue. Values are mean + S.E.M., n = 4, **p < 0.01. 44 45 126x126mm (300 x 300 DPI) 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 28 of 32
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 5: The effects of natural stilbenoids on NO production in activated macrophages. J774 macrophages 46 were stimulated with bacterial lipopolysaccharide (LPS, 10 ng/mL) in the presence of increasing 47 concentrations of (A) pinosylvin (1), (B) monomethyl pinosylvin (2), (C) resveratrol (3), (D) pterostilbene 48 (4), (E) piceatannol (5) and (F) rhapontigenin (6) for 24 hours. After incubation the cell culture media were 49 collected. NO production was investigated by measuring the accumulation of its stabile metabolite nitrite in 50 the cell culture media with the Griess method. In addition, the effects of stilbenoid glycosides astringin (7) and rhaponticin (8) on NO production were investigated and compared to their aglycones (G). LY29004 was 51 used as a reference compound (H). Values are mean + S.E.M., n = 4, *p < 0.05 and **p < 0.01. 52 53 186x249mm (300 x 300 DPI) 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 29 of 32 Submitted to Journal of Natural Products
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 6: The effects of natural stilbenoids on IL6 expression in activated macrophages. J774 macrophages 46 were stimulated with bacterial lipopolysaccharide (LPS, 10 ng/mL) in the presence of increasing 47 concentrations of (A) pinosylvin (1), (B) monomethyl pinosylvin (2), (C) resveratrol (3), (D) pterostilbene 48 (4), (E) piceatannol (5) and (F) rhapontigenin (6) for 24 hours. After incubation the cell culture media were 49 collected and IL6 concentrations were measured with ELISA. In addition, the effects of stilbenoid glycosides 50 astringin (7) and rhaponticin (8) on IL6 production were measured investigated and compared to their aglycones (G). LY29004 was used as a reference compound (H). Values are mean + S.E.M., n = 4, *p < 51 0.05 and **p < 0.01. 52 53 198x258mm (300 x 300 DPI) 54 55 56 57 58 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 30 of 32
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 7: The effects of natural stilbenoids on MCP1 expression in activated macrophages J774 macrophages 46 were stimulated with bacterial lipopolysaccharide (LPS, 10 ng/mL) in the presence of increasing 47 concentrations of (A) pinosylvin (1), (B) monomethyl pinosylvin (2), (C) resveratrol (3), (D) pterostilbene 48 (4), (E) piceatannol (5) and (F) rhapontigenin (6) for 24 hours. After incubation the cell culture media were 49 collected and the concentrations of MCP1 were measured with ELISA. In addition, the effects of stilbenoid 50 glycosides astringin (7) and rhaponticin (8) on MCP1 production were measured and compared to their aglycones (G). LY29004 was used as a reference compound (H). Values are mean + S.E.M., n = 4, **p < 51 0.01. 52 53 201x258mm (300 x 300 DPI) 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 31 of 32 Submitted to Journal of Natural Products
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Figure 8: The effects of natural stilbenoids in the carrageenan-induced paw inflammation model in the 24 mouse. The investigated stilbenoids pterostilbene (4), pinosylvin (1) and piceatannol (5) (all 30 mg/kg) or 25 the PI3K inhibitor LY294002 (15 mg/kg) or dexamethasone (2 mg/kg) were administered to the animals 26 intraperitoneally 2 hours prior to carrageenan (1.5% in saline) was injected into the paw, and saline was injected into the contralateral paw to serve as a control. Paw edema was measured before, and 3 and 6 27 hours after carrageenan injection. Edema is expressed as the difference between the volume changes of the 28 carrageenan-treated paw and the control paw (injected with saline). Values are mean + S.E.M., n = 6 29 (stilbenoids) or 7 (dexamethasone and LY294002), *p < 0.05 and **p < 0.01. 30 31 90x44mm (300 x 300 DPI) 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Submitted to Journal of Natural Products Page 32 of 32
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Figure 9: The effects of natural stilbenoids on the expression of the inflammatory cytokines IL6 and MCP1 in 21 the carrageenan-induced paw inflammation model in the mouse. The investigated stilbenoids pterostilbene (4), pinosylvin (1) and piceatannol (5) (all 30 mg/kg) or the PI3K inhibitor LY294002 (15 mg/kg) or 22 dexamethasone (2 mg/kg) were administered to the animals intraperitoneally 2 hours prior to carrageenan 23 (1.5% in saline) was injected into the paw, and saline was injected into the contralateral paw to serve as a 24 control. Six hours after carrageenan exposure the animals were sacrificed and the paw tissue was extracted. 25 The expression of IL6 and MCP1 in the tissue was measured with ELISA. Values are mean + S.E.M., n = 6 26 (stilbenoids) or 7 (dexamethasone and LY294002), *p < 0.05 and **p < 0.01. 27 87x33mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment