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Phytochemicals as Modulators of Signaling in Inflammation |
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e ects in cardiovascular disease.12,13 However, questions have arisen recently,
regarding the safety and e cacy of PPARg agonists in cardiovascular disease.14,15 Although they are e ective, they may also have their own associated
toxicity in susceptible individuals.
Another study implicated LXR in the immune response, showing that the downregulation of LXR led directly to decreased phagocytosis of Ab peptides, and increased inflammation in the brains of mice in Alzheimer disease models.16 The same study showed that LXR agonists can increase Ab phagocytosis, while decreasing inflammatory pathways. In addition, thiazolidinediones are known for improving outcomes in Alzheimer’s disease,17–19 diabetes20 and other chronic inflammatory conditions. Here, phytochemicals and natural product inhibitors of NF-kB will be considered, as well as PPARg agonists and LXR agonists as alternatives to the current synthetic pharmacologic strategies.
12.5Natural Products and Phytochemical Inhibitors of NF-jB
How do phytochemicals interact with NF-kB? Many of the phytochemicals discussed below intervene at the stage of IkB kinase to inhibit the action of this enzyme. Other phytochemicals intervene upstream or downstream of IkB kinase. Still others intervene indirectly, such as the PPAR or LXR agonists, which, when stimulated, have an indirect but dramatic inhibitory e ect on NF-kB pathways. Most of these polyphenol compounds are considered to be poorly absorbed and rapidly eliminated. However, human plasma levels of these agents can reach mM levels in normal dietary regimens, thereby approaching levels that may inhibit IkB kinase. The following descriptions of phytochemicals are a small sampling of common inhibitors of inflammation, their mechanisms of action with NF-kB and data as to their potential bioavailability.
12.5.1Anthocyanins
Some of the more powerful inhibitors of NF-kB-mediated inflammatory pathways are anthocyanins. Anthocyanins are glucosides of anthocyanidins (Figure 12.2). Anthocyanins are often responsible for the blue, red or purple pigment found in fruits and plants and belong to the class of flavonoid molecules. They also protect cells by absorbing blue-green and UV light. Plants that contain high amounts of anthocyanins are blueberry, cranberry, bilberry,
O+
Figure 12.2 Basic structure of an anthocyanin.
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raspberry, blackberry, blackcurrant, cherry, grape, red |
cabbage and |
eggplant peel. Although they are considered powerful antioxidants, their antiinflammatory properties in signal transduction mechanisms are emphasized here.
Pomegranate fruit extracts contain anthocyanins and tannins, which inhibit NF-kB-dependent inflammatory pathways.21 This study exposed mast cells and basophils to the inflammatory stimuli, phorbol-12-myristate 13-acetate and calcium inophore A23187, and found that pomegranate extracts, in concentrations of 20–100 mg/ml, can decrease the levels of inflammatory cytokines IL-6 and IL-8 promoted under these conditions. These pomegranate extracts contained ellagitannins, punicalagins, punicalins, ellagic acid and gallic acid. The anti-inflammatory e ect was found to be c-jun N-terminal kinase (JNK) and extracellular regulated kinase (ERK) dependent. NF-kB activation was also inhibited by pomegranate extracts by inhibiting IkB-degradation in basophils.
Another study looked at pomegranate extracts and found that a prostate cell cancer line, dependent on activation of TNFa and NF-kB for cell growth, was inhibited by these extracts in a dose-dependent fashion.22 Under their conditions, TNFa decreased IkBa protein levels and increased NF-kB activity directly, but pomegranate extracts were able to block the degradation of IkBa, thereby inhibiting NF-kB. In this particular study, the authors then went on to examine the e ects in an in vivo mouse model. Murine xeonografts of LAPC4 tumor cells, with known endogenous NF-kB activity, were placed in immunodeficient mice. Pomegranate extracts were able to reduce tumor size to about one-third compared to controls with no treatment. The amount of ellagitannins and punicalagins in a single serving of pomegranate juice is 80 mg. Ten times this dose was used and adjusted according to mouse body weight to make 0.8 mg of pomegranate extracts.
In addition, anthocyanins appear to be bioavailable. For instance, one report documented that individuals drinking 480 ml of cranberry juice had plasma concentrations of various anthocyanins that ranged from 0.56–4.64 nM after four hours.23
12.5.2Gallates
Gallates (gallic acid esters) are found in wine, red tea and green tea and have been found to inhibit NF-kB pathways in human umbilical vein endothelial cells (HUVEC).24 Treatment of HUVEC cells with cytokines promotes inflammation. However, pre-treatment with ethyl gallate in 10-mM concentrations inhibited NF-kB activation, which resulted in suppression of IL-1a, TNFa, VCAM-1, ICAM-1 and E-selectin. This results in decreased adhesion of leukocytes to HUVEC cells. Suppression of NF-kB occurred through the blocking of NF-kB–p65 translocation into the nucleus and not by binding to the promoter region of NF-kB.24
Galloyl compounds are often found in plants in the form of gallic acid, alkyl esters (methyl gallate or ethyl gallate) and galloyl tannins (galloyl glucose, epicatechin gallate and procyanidin gallate). One study found that the ingestion
Phytochemicals as Modulators of Signaling in Inflammation |
237 |
OH
OH
HO O
OH
O
OH |
OH |
O
OH
OH
Figure 12.3 Epigallocatechin gallate.
by human subjects of 200 mg of epicatechin gallate (Figure 12.3) resulted in a plasma concentration of 0.15 mM epicatechin gallate two hours after ingestion.25
12.5.3Quercetin
Quercetin is a plant-derived flavonoid found in onions, shallots, garlic, leeks, black and green tea, capers, apples and various berries. Quercetin (Figure 12.4) has been found to be a direct inhibitor of IkB Kinase.26 The cell lines used in this study were histiocytic lymphoma (U-937), HeLa or T (Jurkat) cells. These cells were treated with 80 mg/ml of quercetin for one hour followed by TNFa. This concentration of quercetin completely abolished TNFa activation of NF- kB-mediated inflammation. This study also observed by Western blot that quercetin abolished IkBa degradation, thereby inhibiting inflammation.
Another study looked at the direct inhibition by enzyme kinetics studies.27 They found that IkB kinase and activation of NF-kB-mediated inflammation is strongly inhibited by quercetin. This study found that IkB Kinase a and b were inhibited with apparent Ki values of 11 and 4 mM.
Quercetin has reasonable bioavailability in humans. In one study, healthy humans ingested 50, 100 or 150 mg/day of quercetin for two weeks. After
150 mg/day of quercetin, plasma levels of quercetin were observed to be 0.43 mM.28
12.5.4Isoflavones
Genistein is an isoflavone found in soy and fava beans. It has been demonstrated to be an inhibitor of IkB kinase.26 This study (Figure 12.5) compared inhibition of IkB kinase by quercetin or genistein, and found that 40 mg/ml of genistein was enough to abolish TNFa activation of NF-kB. Downstream
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OH
OH
HO |
O |
OH
OH O
Figure 12.4 Quercetin.
HO |
O |
OH O
OH
Figure 12.5 Genistein.
e ects were found such as that genistein inhibits TNFa induced adhesion of neutrophils to endothelial cells and inhibits the expression of cell adhesion molecules ICAM-1, VCAM-1 and E-selectin. Genistein has other useful purposes such as inhibiting parasitic infection.
Bioavailability studies have been performed using genistein contained in soy foods. It was found that 96 mg of isoflavones/day contained in soy products resulted in plasma levels of 4 mM.29
12.5.5Piperine
Piperine, the active ingredient in black pepper (Figure 12.6), has been shown to block the translocation of NF-kB by blocking the degradation of its inhibitory protein, IkBa, and, therefore, transport into the nucleus in endothelial cells.30 In this study, endothelial cells were stimulated by TNFa, which resulted in increased IkB kinase activity. However, pretreatment of cells with piperine before TNFa inhibited IkB kinase activity, with maximal inhibition of activity occurring at 40 mg/ml.30
Piperine is known for increasing bioavailabilty in the gut of many other medicinal compounds. Piperine itself, after being given in one 50 mg dose, after one hour resulted in peak concentrations of 1 mg/ml in healthy human males, and is highly bioavailable.31
Phytochemicals as Modulators of Signaling in Inflammation |
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N
O
O
O
Figure 12.6 Piperine.
O OH
HO
O
Figure 12.7 Gingerol.
12.5.6Gingerol
Gingerol is another phytochemical, known to inhibit NF-kB-mediated inflammation (Figure 12.7). It is the pungent constituent of ginger. Gingerol has also been found to inhibit IkB kinase activity.32 In this study, macrophages (RAW 264 cells) were stimulated by LPS, which activated the cells and initiated an inflammatory cascade with NF-kB stimulation. It was found that an 80-mM concentration of gingerol can inhibit 70% of the NF-kB activation by LPS. The bioavailability of gingerol is not high, with peak concentrations of metabolites being 0.1 mg/ml to 1.7 mg/ml in human plasma after a 1 g dose.33
12.5.7Curcumin
Curcumin is the main ingredient found in the curry spice tumeric (Figure 12.8). Curcumin has been found to inhibit NF-kB. One study found that 10–20 mM amounts of curcumin can inhibit NF-kB in MyD88 and RAW264.7 cells activated by LPS.34 This was shown to occur through blocking the dimerization of TLR-4 and by inhibiting IkB kinase. Curcumin inhibits numerous inflammatory pathways upstream and downstream of NF-kB, such as mitogenic pathways, AP-1
transcription and expression of COX-2, anti-apoptotic proteins and growth factors.35,36 Curcumin was confirmed to inhibit IkB kinase activity.36 The con-
centration needed to inhibit NF-kB from IL-1 activation in U937 cells was 50 mM. However, curcumin has very poor bioavailability. The concentrations found in human plasma were 0.4–3.6 mM after the ingestion of 4–8 g of
240 Chapter 12
curcumin.37 The bioavailability is greatly improved with the addition of piperine.
12.5.8Guggulsterone
Guggulsterone has been used for centuries in Ayurvedic medicine for its anti-inflammatory activity in arthritis, cardiovascular disease, obesity and bone fractures38 (Figure 12.9). It is found in the guggul plant in northern India. Guggulsterone has been found to inhibit NF-kB activation by RANKL (TNFa cytokine family) in RAW-269.7 cells.38 This study demonstrated that guggulsterone was a direct inhibitor of IkB kinase. The doses found to inhibit NF-kB entirely were 10 mM in this cell system. Another study similarly found that NF-kB activation by IL- 1b could also be abolished at 10 mM concentrations of guggulsterone in fibroblastlike synoviocytes, a cell model for arthritis,39 by blocking the degradation of IkBa.
It is known that mM amounts of guggulsterone inhibit inflammation in vivo, and guggulsterone has numerous beneficial e ects in vivo,40 such as in the cardiovascular system in humans. However, there are few data as to the bioavailability of this agent.
O |
OH |
O |
O |
HO |
OH |
Figure 12.8 Curcumin.
H O
H H
O
H O
H H
O
Figure 12.9 E- and Z-Guggulsterone.