From The August 2000 Issue of Nutrition Science News
by Anthony Almada
Considering that most health conditions from a skinned knee to cancer involve some level of pain, it only makes sense to decipher the mechanism behind this feeling and find a means to alleviate it. For centuries, natural remedies such as white willow bark (Salix alba) and myrtle (Myrtus communis) provided some pain relief. Then a breakthrough occurred when it was discovered that the hormone like compounds called prostaglandins, which are the cause of inflammation and thus the producers of pain, could be blocked. Out of this new knowledge came the manufacture and use of pain-relieving pharmaceuticals known as nonsteroidal anti-inflammatory drugs (NSAIDs), which are effective but cause unwanted side effects. Over the last 20 months, the FDA has approved two members of a second, more advanced class of prescription drugs that works by inhibiting an enzyme known as cyclooxygenase-2 (COX-2), which triggers the release of prostaglandins. These new productscalled COX-2 inhibitorsare indeed superior to NSAIDs.
The discovery of COX-2 occurred in 1991, when W. L. Xie and colleagues from Brigham Young University in Provo, Utah, found the existence of two different isoforms (chemical derivatives) of the enzyme cyclooxygenase.  The first of these, COX-1, is a constant, physiological "housekeeper" isoform. It is produced widely throughout the body and is involved in the regulation of day-to-day cellular and metabolic activities such as maintaining stomach lining integrity, regulating blood flow within the kidneys and balancing platelet function. In contrast, COX-2 was determined to be an "inducible" isoform, expressed in response to a variety of proinflammatory stimuli and found in the brain, male and female reproductive organs, the kidneys and, in bone-forming cells called osteoblasts. [2 ] Unlike COX-1, COX-2 expression is usually minimal, but when activated COX-2 regulates prostaglandin production primarily within inflammatory cells. This inflammatory response is a vital part of healing and repairing. Aspirin, by virtue of its ability to inhibit COXand thus inhibit the release of prostaglandinscan suppress this biochemical inflammatory response. NSAIDs also exert their pain-relieving effects through COX inhibition. 
Although NSAIDs are effective, their anti-inflammatory, analgesic, anti-fever and anti-thrombotic results can come at a high price. They can inhibit COX-2, but they inhibit COX-1 as well. This is problematic because COX-1 inhibition "turns off" some important functions such as the repair and maintenance of stomach lining, which results in varying degrees of gastric ulcerations, perforations or obstructions in one-third to almost one-half of patients taking them.  Moreover, on the extreme end, more than 16,500 people die in the United States each year from NSAID-related gastrointestinal bleeding.  Out of these statistics emerged a quest for an analgesic/anti-inflammatory that could provide therapeutic efficacy equivalent to that of traditional NSAIDs but without the gastrotoxicity. Such a drug would inhibit COX-2 without affecting COX-1. Thus came the development of COX-2 inhibitors.
From this point, it did not take long before the approval of synthetic COX-2 inhibitors such as celecoxib (Celebrex) and rofecoxib (Vioxx) heralded a new era in NSAIDs in 19981999. Manufacturers promised their products would relieve the signs and symptoms of osteo- and rheumatoid arthritis, acute pain and primary dysmenorrheaand they had the science to back such claims.
Confirmatory testing of such COX-2 inhibitors began with in vivo animal studies. In one such trial, a model of pain was established by injecting the polysaccharide carrageenan into the footpad of rats to create inflammation. Then an oral COX-2 inhibitor was administered, which produced both anti-inflammatory and analgesic effects without gastrotoxicity. Such results confirmed the benefits of COX-2 inhibitors for pain and inflammation. 
More recently, human clinical trials with COX-2 inhibitor drugs have shown similar anti-inflammatory and analgesic efficacy to traditional NSAIDs, yet with significantly less gastrotoxicity.  However, though these products offer some advantage in terms of side effects, they are nine times more expensive on a daily dose comparison.  Fortunately, there is now some evidence that natural, less-pricey COX-2 inhibitors may obstruct the production of pain and inflammation, and do so in a more gentle manner, and for far less money.
Natural COX-2 Inhibitors' Promise
While COX-2 inhibition may seem to clearly describe one pharmacological effect of several common and widely recognized natural products, such a benefit cannot be confirmed until each is systematically tested for such activity. However, the odds seem in favor of the following compounds serving as natural modulators of pain.
Curcumin is one of the pungent active ingredients of turmeric (Curcuma longa), the deep-yellow powder found in virtually every curry dish made in the world. Besides being a culinary delight, several clinical trials have found curcumin to be a notable anti-inflammatory and analgesic compound.  Moreover, recent in vitro studies have explored whether curcumin, a chemopreventive agent, inhibits the expression and activity of COX-2 in several different gastrointestinal cell lines: colon, esophagus and small intestine. 
In one study, Fan Zhang, Ph.D., and colleagues from the Cornell University campus in New York City, exposed gastrointestinal (GI) cells to two known tumor promoters, either bile acids (BA) or phorbol esters (PMA). The team found COX-2 to be induced in several of the cell lines, accompanied by a 10-fold increase in the synthesis of inflammatory-causing prostaglandin E2.  However, dose-dependent treatment of the cells with curcumin suppressed both BA- and PMA-mediated induction of COX-2 protein, genetic COX-2 expression (as measured by mRNA), and the synthesis of prostaglandin E2. Most impressive, however, was the discovery that curcumin directly inhibited the enzymatic activity of COX-2.
Although the Zhang study did not examine the action of curcumin on cells mediating chronic joint inflammation, it does offer a provocative suggestion that curcumin may modulate chronic inflammatory GI events such as Crohn's disease and ulcerative colitis.
An additional study presented at the 1999 American Association for Cancer Research (AACR) conference also examined the pain-relieving properties of curcumin. Researchers at the University of California, San Diego, and the Veterans Administration Medical Center, San Diego, investigated whether curcumin could suppress COX-2 expression in human colon cancer cells.  After exposing such cells to curcumin, the researchers found the compound not only inhibited cell growth but also reduced the expression of COX-2 mRNA in a time- and dose-dependent manner.  Therefore, curcumin would appear to be a safe, natural COX-2 inhibitor in humans, given its safety profiles and demonstrated anti-inflammatory activity.
Thunder God Vine, (Tripterygium wilfordii) is an unfamiliar plant to many. However, extracts of this traditional Chinese herbal medicine have shown preliminary promise in treating rheumatoid arthritis patients. [12, 13] Recent lab studies using a glycoside-rich extract of thunder god vine (plant parts used in the study were not revealed, nor was the method of extraction) found it blunts prostaglandin production in rheumatoid arthritis knee tissue cells.  Additionally, it was found that COX-2 protein expression was reduced significantly without any appreciable effect on COX-2 mRNA expression or COX-2 activity. Moreover, COX-1 protein expression was unaltered by the plant extract. Together, these findings suggest the T. wilfordii extract used in this study reduced pros-taglandin production by selectively reducing the synthesis of the COX-2 enzyme itself without inhibiting the activity of COX-2. Less COX-2 enzyme means less prostaglandins and, thus, less pain.
Omega-3 Fatty Acids, such as those found in fish oils, have been recommended for managing chronic inflammatory conditions given their ability to alter prostaglandin production and to yield measurable changes in certain disease parameters in rheumatoid arthritis patients.  Mechanistically, fish oils modulate the production of proinflammatory mediators such as prostaglandins, thereby altering their function. New research from Clare Curtis, Ph.D., and colleagues in the Connective Tissue Biology Laboratories at Cardiff University in Wales has directed light upon a novel-operating pathway of omega-3 fatty acids. 
In their study, bovine articular cartilage cell (chondrocyte) cultures were grown in the presence of a variety of pure fatty acids of various series and saturations. The concentrations used reflected those found circulating in human plasma. Incubation of chondrocyte cultures with interleukin-1-alpha (IL-1a)a proinflammatory cytokineinduced expression of both isoforms of the proteolytic protein-digesting enzyme called aggrecanase. This enzyme degrades the cartilage protein aggrecan, which imparts both compressibility and elasticity features to cartilage. Its degradation is a hallmark process of arthritic conditions.
Interestingly, by coincubating the IL-1a-treated cartilage cell cultures with the omega-3 fatty acids alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), the team found that they individually and dose-dependently reduced aggrecanase activity and mRNA. In contrast, the saturated fatty acid palmitic acid, the omega-6 polyunsaturate linoleic acid and the omega-9 monounsaturate oleic acid all failed to suppress aggrecanase activity or mRNA.
Finally, the study showed that each of the three omega-3 fatty acids (and none of the other fatty acids) dose-dependently inhibited the production of COX-2 mRNA without affecting COX-1 expression. This last finding suggests omega-3 fatty acids can selectively modulate the expression of COX-2 in inflamed joints while also down-regulating joint degradation processes at the level of the involved genes.
Resveratrol, a phytochemical derived from grape skin, is also abundant in members of the Polygonum genus, widely used in traditional Chinese herbal medicine. The anti-inflammatory effects of resveratrol were first described in 1997 after an animal model determined its primary activity to be the inhibition of COX-1.  Then a study led by some of the same researchers from Cornell Medical College in New York City revealed resveratrol's COX-2 inhibitory effects. 
In this study, K. Subbaramaiah, Ph.D., and colleagues exposed human mammary and oral epithelial cells to phorbol esters, which induce COX-2 expression and the production of prostaglandin E2. The addition of pure resveratrol inhibited both these effects, reversing the increases in COX-2 mRNA and protein. In addition to modifying gene expression, they also found resveratrol to directly inhibit COX-2 activity.
A second resveratrol study, performed by Javier Martinez and Juan Moreno, both Ph.D.s at Barcelona University in Spain, found resveratrol treatment of mouse macrophages inhibited COX-2 protein expression and dose-dependently suppressed prostaglandin E2 production without affecting COX-1 protein expression.  Exposing these cells to superoxide anion radicals or hydrogen peroxide also induced COX-2 expression, which was similarly blocked by resveratrol. However, a 1998 study by Meishiang Jang and John Pezzutto, Ph.D.s, from the University of Illinois at Chicago, found resveratrol to have no effect on phorbol ester-mediated induction of COX-2 in mouse skin.  This suggests that resveratrol may operate in a tissue- and/or species-specific manner. That is, resveratrol could display COX-2 inhibition in one organ (breast) or a specific animal model (rat) but not in another tissue (skin) or species (mouse). Whether this operates in humans remains to be seen.
Flavonoids/Phenolics are plant-based chemicals that also hold great promise as COX-2 inhibitors. In 1998, Michihiro Mutoh, Ph.D., and colleagues of Tsukuba University in Japan attempted to identify the structural requirements of COX-2 inhibitory phytochemicals.  In the study, the team used transforming growth factor-alpha (TGF-a) an inflammatory-mediating cytokine as the COX-2 induction stimulus, against which 14 natural-product compounds were tested (all were characterized as antioxidants and chemopreventive agents). Of these, only five produced significant, dose-dependent decreases in TGF-a-induced COX-2 reporter activity: genistein, kaempferol, quercetin, resorcinol and resveratrol, with quercetin being the most potent. Curcumin, DHA, daidzein, epigallocatechin gallate (EGCG), alpha-tocopherol, ascorbic acid, beta-carotene, tannic acid and reduced glutathione had no effect. These data on curcumin run contrary to the Zhang study described above. This may be due to the different experimental systems used in this study (reporter gene in human colon cancer cells) contrasted to the system used by Zhang, et al. (direct measurement of COX-2 and its activity in various GI tract cells).
The unifying chemical theme shared by the five inhibitory phenolics was the presence of a resorcin component within the chemical structure of each compound (including of course resorcinol itself). All five of these phenolics are present in a variety of plant-derived foods.
Interestingly, EGCG, the principal catechin in green tea that also contains a resorcin component in its structure, failed to exhibit inhibitory effects in this study. Previous work from researchers at Case Western Reserve University in Cleveland, Ohio, has shown that oral consumption of an EGCG-rich green tea polyphenol (GTP) extract by mice with collagen-induced arthritis produces significant reductions in the incidence of arthritis and expression of COX-2 compared to mice not given GTP.  It remains to be explored whether this apparent disparity is due to the activity of other green tea polyphenols, synergistic interactions, or a downstream metabolic product of one or more green tea polyphenols after oral ingestion.
Future Needs and Directions
For many therapeutic targets, the majority of natural products offer only the hope of efficacy and safety. This is due almost exclusively to the absence of well-designed clinical trials, which can elucidate the necessary data to support the use of such products, especially if they are being compared to a standard drug. Although in vitro and animal studies lend substantial credibility to natural COX-2 inhibitors, the influence of digestion, absorption, metabolism distribution and excretion, in addition to species specificity of the effects, cannot be overlooked. That is why well-designed human trials are so necessary.
A second factor holding back the progress of natural COX-2 inhibitors is their slow onset of relief. Unfortunately, there do not appear to be any natural anti-inflammatory/analgesic products that offer almost instantaneous relief of a magnitude close to aspirin or other NSAIDs. The consumer expectation of "relief in minutes" is destined to produce disappointment and derision if not adequately offset by education about these natural products. But if explained to them, the public will surely understand the virtue of natural products that provide gentle, effective, long-term relief.
Anthony Almada is a Aptos, Calif.-based nutritional and exercise biochemist who has collaborated on more than 45 university clinical trials. He is the co-founder of Experimental and Applied Science Inc. (EAS) and founder and chief scientific officer of IMAGINutrition (www.imaginutrition.com).
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