Bioactive Food as Dietary Interventions for Arthritis and Related Inflammatory Diseases: Bioactive Food in Chronic Disease States

USA

Chapter 1

Antioxidant Flavonoids for Arthritis Treatment

Human and Animal Models

S.G. Somasundaram*,† and B. Oommen*

*University of Houston – Victoria, Sugar Land, TX, USA

†Texas A&M University, College Station, TX, USA

Abbreviations

ASU    Avocado-soybean unsaponifiable

COX    Cyclooxygenase

GTP    Green tea polyphenols

H and E    Hematoxylin and eosin

HES    Hesperidin

IL    Interleukin

LD    Lethal dose

MC    Malpighian corpuscles

Mg    Magnesium

NSAID    Nonsteroidal anti-inflammatory drug

RA    Rheumatoid arthritis

THM    Traditional herbal medicine

TNF    Tumor necrosis factor

1 Introduction to Phytoflavonoids

Plants have been used in many cultures and regions as forms of medicine for many centuries. Before pharmaceutical companies came into existence, holistic medicine was practiced. For years, plants and their extracts have been shown to be effective in treating a variety of ailments. However, people still choose traditional medications in spite of numerous adverse effects. With advanced technology, many drugs are being made synthetically after elucidating a few mechanisms of action. In the past, accurate information and studies of herbal medicine and its effect on different diseases were not easily accessible. Today, we know that there are hundreds of phytochemicals, including flavonoids, derived from indigenous plants that we consider important drugs. These medicinal plants are used in the forms of powders, crude powders, compressed tablets, tinctures, decoctions, pastes, etc. These preparations are rarely based on a single herb but are mostly prepared from several plants.

From the report of Latiff et al., there are 1676 plant species that have been reported to cure various diseases. Among them, about 60 plant species are reported to provide a cure for rheumatism or joint pains (Latiff, 1980). There are over 41 plants that were reported to possess anti-inflammatory properties. An antioxidant-rich polyphenolic flavonoid fraction isolated from green tea has been shown to possess anti-inflammatory and anticarcinogenic properties in experimental animals. In three independent studies, experimental mice which were given green tea polyphenols (GTPs) in water exhibited significantly reduced incidences of arthritis, as compared to mice not given GTP in water (Ahmed, 2010). Also, the juice and leaves of Clerodendron inerme were used as an alternative febrifuge and to resolve buboes. When boiled in oil, the roots of this plant yield a liniment that is useful in rheumatism (Chopra et al., 1956). The aqueous extract was found effective in reducing edema of formalin-induced arthritis in rats. External application of the alcoholic extract of the leaf also reduced the edema. Among the 12 Clerodendron species, C. phlomidis, C. indicium, and C. inerme are reported to possess antiarthritic activity.

2 What Is Arthritis?

Can you imagine waking up one morning and not being able to move any limbs, being awakened by excruciating pain, or being restricted from doing daily routines such as brushing your teeth, writing, or even opening a bottle cap? Arthritis is the leading cause of disability in the United States, affecting 43 million US adults (Bolen et al., 2005). This burden will continue to increase, with the prevalence of arthritis in the United States projected to reach 67 million (25% of the adult population) by 2030 (Hootman and Helmick, 2006). Arthritis is most often progressive, and the demineralization of joints may have occurred years before the symptoms even appear. Arthritis’s main targets are the synovial joints. These joints are encased in a tough fibrous capsule lined with a membrane that secretes a clear synovial fluid that helps absorb shock and reduce friction caused by the two articulating bones when the bones rub against each other. Arthritis can involve either inadequate amounts of synovial fluid resulting in stiffness or excessive amounts of fluid causing swelling and edema. In both cases, the result is excruciating pain that patients primarily complain about. In this chapter, we focus on how flavonoids affect arthritis by using data from animal models and clinical trials.

The purpose of this chapter is to put together an organized collection of plant-derived flavonoids to display their use for treating arthritis. Arthritis is an inflammation involving the joints which is categorized into different types, the most common being osteoarthritis, which is a degenerative arthritis, and rheumatoid arthritis, which is an immunological reactive arthritis. Up until now, nonsteroidal anti-inflammatory drugs (NSAIDs) and other anti-inflammatory medications were the only escape that patients could find to relieve themselves of pain. But these medications all have adverse side effects. Among NSAIDs, cyclooxygenase (COX)-1 inhibitors induce lethal side effects. COX-2 inhibitors were promising at first but have led to cardiovascular side effects such as vasoconstriction. Recent work done by our group hypothesized that COX-1 inhibitor-induced side effects are reduced by beta-calcitonin gene-related peptide (CGRP; Somasundaram et al., 2009). In addition, our group has done extensive work in phytoflavonoids for arthritis without causing unfavorable side effects (Somasundaram and Edwards, 2009).

3 Osteoarthritis

The most common arthritis is osteoarthritis, also known as degenerative arthritis. As obesity increases in our country, weight causes more pressure to be placed on the joints, and erosion of cartilage and bone occurs. Almost 60% of Americans who are over the age of 65 have this type of arthritis. Osteoarthritis is not only very uncomfortable to live with, but it is also one of the leading causes of disabilities in America now. It limits the daily activities of one’s life. There is no known cure for arthritis. Medications are only prescribed to alleviate the pain and inflammation. Some drug treatments in current use are analgesics, NSAIDs, COX-2 inhibitors, nonacetylated salicylates, corticosteroid, and other drugs such as methotrexate, cyclosporine, cyclophosphamide, and penicillamine. Because osteoarthritis is incurable, doctors try to maintain a comfortable lifestyle for their patients. Medications are given not to cure the individual from pain but to alleviate the pain temporarily. Osteoarthritis is termed as a degenerative disease or the ‘wear and tear’ arthritis. This arthritis is the most common chronic disease affecting older people, especially those 65 and older. It can involve one or more joints. In osteoarthritis, the normal cartilage is there to absorb the shock and allow less friction between the bones erodes, and it is replaced by thick bone tissue that is rough and gritty. Pain is the primary complaint of many patients, and it can involve almost any joint in the body, through it most often affects the knees and back. Stiffness, limited range of motion, swelling, and an increased incidence of fractures occur. Sometimes in extreme cases, steroidal cortisone injections or even narcotics are administered. This therapy does not fix the underlying problem. Instead, it covers it up and the disease just gets worse and worse. Contrary to the belief that only the elderly suffer from osteoarthritis as an aging process, many people under the age of 65 also can suffer from osteoarthritis. The cartilage that protects the joint begins to wear down over time, finally exposing the bones and causing friction between them. That ‘cushion’ is no longer there. Over time, this can cause inflammation, redness, and swelling and can be very painful. Many elderly will complain that the affected joints are stiff and less flexible.

4 Rheumatoid Arthritis

Unlike osteoarthritis, rheumatoid arthritis (RA) is an autoimmune disease. Autoantibodies attack the body’s very own tissues. About two million Americans suffer from this condition. It is most commonly caused by inflammation, leading to severe joint deformity, and can also affect tissue of the body other than the joints. In RA, T cells and macrophages in the synovial joints become overactivated causing inflammation. This inflammation may cause deformities of the joints. Spurs, which are new bone growth, may grow making it even harder to mobilize the joints. RA only involves limited joints, most often the wrists.

5 Flavonoids

Flavonoids are polyhydroxy chemical compounds synthesized in various plants. Some of them possess medicinal properties. Depending on their mechanism of action on biological tissues, they may be classified as potent therapeutic agents. They also possess free radical scavenging properties. Some of the plants rich in phytoflavonoids provide not only nutrition but also a highly therapeutic value for human beings who suffer from various illnesses. Figures 1.1–1.5 describe the different chemical structures of flavonoids as per the USDA Database for the Flavonoid Content of Selected Foods(2003) from selected foods. All of them contain polyhydroxy phenolic groups. The position of the hydroxy groups plays a major role in expressing their biochemical mechanisms as per Table 1.1.

Figure 1.1 Chemical structure of flavonoids (quercetin, kaempferol, myricetin, and isorhamnetin).

Figure 1.2 Chemical structure of flavones (luteolin, and apigenin).

Figure 1.3 Chemical structure of flavonones (eriodictyol, hesperetin, and naringenin).

Figure 1.4 Structure of flavan-3-ols (catechins, epicatechins, theaflavins, and thearubigins).

Figure 1.5 Chemical structure of anthocyanidins (cyanidins, delphinidin, malvidin, pelargonidin, peonidin, and petunidin).

Table 1.1 The Effects of Compounds Tested on Archidonate Metabolism

Source: Bauman, J., Bruchhausen, F.V., Wurm, G., 1980. Flavonoids and related compounds as inhibitors of arachidonic acid peroxidation. Prostaglandins 30, 627–639.

6 Animal Models of Acute and Chronic Inflammation

Table 1.2 describes various animal models of arthritis for both acute and chronic arthritis. In this chapter, we focus mainly on an adjuvant-induced arthritis model.

Table 1.2 Animal Models for Screening New Anti-Inflammatory Drugs

aNorthover, B.J., Subramanian, G., 1961. Analgesic and anti-pyretic drugs as inhibitors of kallikrein. British Journal of Pharmacology 17, 107–115.

bPearson, C.M., 1956. Development of arthritis, periarthritis, and periostitis in rats given adjuvant. Proceedings of the Society for Experimental Biology and Medicine 91, 95.

cWinter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan-induced edema in the hind paw of the rat as an assay for anti inflammatory drugs. Proceedings of the Society for Experimental Biology and Medicine 111, 544.

dFukuhara, M., Tsurufuji Susumu, 1969. The effect of locally injected anti-inflammatory drugs on the carrageenin granuloma in rats. Biochemical Pharmacology 18, 475–484.

eArrigoni-Martelli, E., Schiatti, P., Selva, D., 1971. The influence of anti-inflammatory and immunosuppressant drugs on nystatin-induced oedema. Pharmacology 5, 215.

7 Flavonoids and Its Effect on Animal Model Arthritis

Among several phytoflavonoids, we intend to use flavonoids from C. inerme for our discussion of the animal model. Ethylcholesta-5, 22,25-trien-3B-ol, and 7-O-glucuronides of apigenin, scutellarein, and pectinolinergenin (flavonoidal compound) were found in C. inerme plants (Subramanian, 1973).

The pharmacological activity of this plant was analyzed, and it was reported to possess anti-inflammatory properties through the studies of the aqueous extract of the plant leaves against formalin-induced edema in rats. The albumin/protein, albumin/globulin ratios, the serum cationic levels (Na, K, Ca), and mucoprotein contents in normal, inflamed, and drug-treated groups have also been investigated. The serum protein profile was also studied using polyacrylamide gel electrophoresis.

A systematic biochemical approach to this plant is absolutely necessary to understand the mechanism of action of this drug and also to know whether it provides long-term relief. Since recently available anti-inflammatory drugs are ulcerogenic along with other side effects, it is essential to investigate its toxic effects. Such a study throws light on the safety of C. inerme. The biochemical reactions were studied on inflammation-induced animal models (male albino rats) after treatment with/without drugs.

Several experimental models have been employed for this purpose. Among these models, the inhibition of swelling, redness, pain, and the loss of function have been taken as criteria. The most popular methods for new anti-inflammatory substances were based on the inhibition of an induced swelling of the rat’s paw. Edema was induced by injecting small amounts of a suspension of edemogen in the plantar tissues of the rat’s hind paw. The volume of swelling was measured by determining the weight of the paw or by the displacement of mercury (Winder et al., 1962), using an edema meter.

Among the different fractions of C. inerme, the alcoholic fraction was found to contain flavonoidal compounds such as apigenin, scutellarein, and pectinolinergenin. The alcoholic fraction was prepared by grinding 2% gum acacia at different concentrations and was administered to the animals orally at intervals of 1 and 24 h before the induction of inflammation. The animals in the control group received orally the same volume of vehicle, that is, 2% gum acacia alone. For adjuvant-induced arthritic rats, the drugs were given daily for 15 days. Likewise, the standard drugs such as oxyphenbutazone (10 mg/100 g body weight) and hydrocortisone (1.5 mg/100 g body weight orally) were administered at their effective doses for the same period for comparison. In this chapter, we discuss the radiological changes that can occur during the development of chronic arthritis with subsequent flavonoidal glycosides and hydrocortisone treatment.

8 Radiographic Analysis of Adjuvant-Induced Arthritis

The radiological analyses of animals with adjuvant-induced arthritis were studied (Clark et al., 1979). Each radiograph of the lower extremities was evaluated for the presence and severity of each of the following factors:

1. Soft tissue swelling (including joint effusion)

2. Osteoporosis

3. Erosions

4. Subperiosteal new bone

5. Joint space narrowing

6. Degenerative changes

7. Alignment alteration

A grade of 0–4 (0 representing normal and 4 representing severe changes) was assigned for each of the above seven possible findings. A total score of the sum of each individual grade for any given animal was then obtained. Thus, the maximum grade any individual animal could receive would be 28.

The C. inerme effective fraction of flavonoid glycosides may play a significant role in inhibiting acute phase reactants such as mucoprotein, stimulated by various inflammatory mediators that are brought on by adjuvant-induced arthritis. Furthermore, the long-term therapeutic effects were reflected in our studies on the arthritic scores. It has been demonstrated that both C. inerme and hydrocortisone prevented the paw swelling in arthritic rats compared to the untreated controls. However, the C. inerme-treated group exhibited stronger antiarthritic activity on the forty-ninth day than the hydrocortisone-treated group (Figure 1.6). This was due to drug treatments in both injected and noninjected paws of rats at various days. X-Ray analysis of rats exhibited severities of periostitis, osteoporosis, and other bone degenerative changes with the radiological results. When compared to C. inerme, treated groups showed greater improvements in bone erosion on the forty-ninth day compared to the hydrocortisone-treated group. Though the drugs were administered for only 15 days, the long-lasting antiarthritic activity persisted, even up to the forty-ninth day. This suggested that plant-derived flavonoid glycosides act on improving the health status of the individual rather than merely suppressing symptoms of the disease.

Figure 1.6 Evidence of changes in bones, especially the soft tissues that were predominately affected and resulted in swelling in the primary phase of arthritis. The left panel of each drug-treated group indicates the noninjected paw, and the right panel indicates the Freund’s injected paw on different days of arthritic development.

Reproduced from Somasundaram, S., Edwards, C., 2009. Flavonoidal glycosides of the Clerodendron inerme confer long term relief for experimental arthritis in rats. Acta Horticulturae 841, 403–409, Published with permission of ISHS.

In other animal models, additional flavonoids have proven to be effective in suppressing arthritis. Traditional herbal medicines (THMs) such as Kampo found mostly in Japan are often used as an alternative remedy for serious diseases such as RA. Generally, RA treatment with THM has been performed as adjunctive therapy (Kogure et al., 2005). Although the main target molecules remain unclear, it has been demonstrated that the Kampo formula suppresses the production of interleukin (IL)-6 from macrophages and fibroblasts. The serum levels of anti-type II collagen antibody titer are decreased in a collagen-induced mouse model (Hai le et al., 2002). Administration of a citrus flavonoid hesperidin (HES) to mice before lipopolysaccharide significantly reduced tumor necrosis factor (TNF)-α production in a dose-dependent manner. HES markedly suppressed plasma levels of TNF-α and high mobility group box chromosomal protein-1, decreased the number of apoptotic cells in livers, and normalized the activated states of blood coagulation factors such as prothrombin time and platelet numbers caused by infection (Kwaguchi et al., 2004).

9 Flavonoids Devoid of Toxic Effects

Many of the plant flavonoids listed are not without side effects or toxic doses. Because the chemical contents in plants of the same species vary, it is difficult to pinpoint the potency of each plant. At this time, there are very limited data about the suitability or contraindications of flavonoids when they are combined. If you were on a prescribed medication, it would be best to consult your doctor before taking any supplements. It is very common to note that there were various toxic effects owing to the misuse of medicinal plants, sometimes resulting in death.

The reason for the approach of alternative medicine is that disease-causing organisms have developed resistance to chemical drugs. Researchers are looking for various plant extracts that can work as replacement for or concomitantly with the drugs in better use against arthritis. According to our research, it has been observed that after 15 days of treatment to normal healthy rats with standard dose, there was no sign of morphological changes in histology of spleen, stomach, kidney, and liver (Figures 1.7–1.10). This further demonstrates the flavonoids are devoid of any toxic side effects (Somasundaram, 1983).

Figure 1.7 The results of histopathology of rat spleen. (a) Normal rat spleen section (H and E 200 ×). It shows capsule with malpighian corpuscles (MCs). There is congestion in the sinusoids. (b) Alcoholic fraction of C. inerme -treated rat spleen section (hematoxylin and eosin – H and E 200 ×). It shows the intense congestion of the sinusoids. Some MCs show hypertrophy while others are normal.

Figure 1.8 The results of histopathology on ulcerogenic activity of rat stomach. (a) The results of studies of ulcerogenic activity indicate the everted stomachs of normal group of rats showing no abnormalities in both the cardiac (c) and the pyloric (p) portion. (b) Everted stomachs of alcoholic fraction of C. Inerme -treated rats showing less hyperemic condition in the cardiac portion (c). Pyloric (p) region shows no abnormality. (c) Everted stomachs of aspirin-treated rats showing more hyperemic condition in the cardiac region (c). Pyloric (p) region shows less ulceration.

Figure 1.9 The results of histopathology of rat kidney. (a) Normal rat kidney section (H and E 200 ×). It shows normal kidney structure without any abnormality. (b) Alcoholic fraction of C. inerme -treated rat kidney section (H and E 200 ×). It shows glomeruli (G) with intense congestion of the capillaries. The tubules (T) show cloudy swelling. The interstitium shows no abnormalities.

Figure 1.10 The results of histopathology studies of rat liver. (a) Normal rat liver section (H and E 200 ×). It shows normal liver parenchyma cells with congestion of sinusoids. (b) Alcoholic fraction of C. inerme -treated rat liver section (H and E 200 ×). It shows congestion of the sinusoids with mild chronic inflammatory infiltrate around the portal area (P).

However, additional mechanistic studies are warranted. Though there are several promising phytoflavonoids that have been tested in animal models, for the clinical studies, this chapter only focuses on specific flavonoids from Tripterygium wilfordii, Senecio scandens, and Crataegus monogyna.

10 Clinical Trial of Flavonoids on Arthritis

Glucosamine, chondroitin, and avocado-soybean unsaponifiable (ASU) are supplements that have helped with arthritis. They have been used in France for several years and the benefits of these supplements are fewer side effects. Vast amounts of clinical research have proved that glucosamine, chondroitin, and ASU work in both humans and animals. S. scandens is a plant found in Southern China that is used as a folk medicine for the treatment of inflammation, bacterial infection, arthritis, and rheumatic disease. The ethanol extract of S. scandens was found to demonstrate significant cytotoxic effects (Xiao-Y et al., 2009).

T. wilfordii also known as Thunder God Vine is currently in clinical trials as a botanical drug for RA. It contains C-methylated flavonoids (Zeng et al., 2010). T. wilfordii is a perennial vine-like plant and has a history of use in China for inflammatory and autoimmune diseases such as RA, systemic lupus erythematosus, Behcet’s disease, and psoriatic arthritis. A well-known and significant toxicity profile (lethal dose, LD50 in mice as low as 160 mg kg−1) can be addressed by removing the outer stem bark layer of the roots and extracting it with ethanol followed by ethyl acetate partitioning (LD50 in mice 860–1300 mg kg−1; Lipsky et al., 1996).

The main activities of the extract and the bioactive compounds involve transcriptional inhibition of proinflammatory genes, including IL-2, TNF-α, inducible nitric oxide synthase (i-NOS), IL-1α, and COX-2. The extract also has a steroid-sparing effect, reducing the need for the corticosteroid prednisone when coadministered with it in clinical trials. The extract also has shown a better side effect profile than conventional steroids in clinical trials. The long history of human use and preclinical and clinical trials all suggest that T. wilfordii extracts may be used safely. In studies in which various extracts were compared with conventional RA drugs such as methotrexate, the extracts generally had fewer side effects, although in studies with some extracts up to 30% of subjects reported side effects (Yao and Nian, 2004).

According to the study, the extract binds to the glucocorticoid receptor (GR) in T cells preventing the glucocorticoid pathway. This is in contrast to corticosteroid medications such as dexamethasone, which bind to the receptor that works on the pathway but triggers unwanted typical steroid side effects such as hypoglycemia, weight gain, osteoporosis, and endocrine feedback system. This accounts for the reduced risk of side effects of the extract relative to steroid medications. In a reported phase I study of the extract that began in 1993 in 13 patients with established RA, nine patients tolerated the extract in doses of up to 570 mg day−1, although three withdrew early in the trial, receiving only 180 mg day−1 maximum. Only one withdrawal was related to an adverse effect, namely, diastolic hypertension at a dose of 180 mg day−1 extract. Six of ten subjects showed disease improvement at doses of 180 mg day−1, while eight of ten subjects who received over 360 mg day−1 experienced improvement in both clinical and laboratory findings. One patient experienced remission (Tao et al., 2001).

A prospective double-blind placebo-controlled trial was conducted in RA patients in whom conventional therapy had failed. Subjects were randomized to receive a placebo or low-dose (180 mg day−1) or high-dose (360 mg day−1) extract for 20 weeks, after which an open-label observational period was instituted. Of the 35 subjects enrolled in the trial, 21 completed the 20-week study. One patient in each group experienced side effects that precipitated withdrawal. Among the subjects who completed at least 4 weeks of treatment, eight in the high-dose and four in the low-dose group experienced clinical response. Fifteen subjects were enrolled in the open-label intervention, of whom 11 experienced response. Diarrhea was the most common side effect and resulted in one withdrawal in the high-dose group; there were no withdrawals due to adverse effects in the open-label extension (Tao et al., 2002).

C. monogyna is one of the most common species used as in traditional medicine for the treatment of many anti-inflammatory diseases. The entire plants, including the leaves, flowers, and fruits, are all used medicinally. The list of flavonoids in C. monogyna includes hyperoside, quercetin, vitexins, rutin, and more. It also contains other compounds such as proanthocyanidins, various vitamins and minerals, fructose, beta-sitosterol, xanthine derivatives, etc. C. monogyna is known to be beneficial for arthritis because it is known to stabilize collagen, which is destroyed in many inflammatory diseases.

Studies have shown that boswellic acids contain anti-inflammatory effects similar to the NSAIDs. Boswellia inhibits proinflammatory mediators in the body, such as leukotrienes (Singh and Atal, 1986). As opposed to NSAIDs, long-term use of boswellia does not appear to cause irritation or ulceration of the stomach.

Trials in people with RA found curcumin to be somewhat useful in the reduction of inflammation and unwanted symptoms. A separate double-blind trial found that curcumin was superior to placebo or phenylbutazone for alleviating postsurgical inflammation (Satoskar et al., 1986).

Bioavailability of flavonoids is a challenging problem in administering the phytoflavonoids. However, it has been shown that the methoxy derivatives or naturally occurring polymethoxy flavonoids increased the bioavailability and were more potent than the nonmethylated polyhydroxy flavonoids (Walle, 2007).

11 The Mechanism of Actions of Flavonoids

The flavonoids can be classified according to their molecular structure and site of actions. Recently, it has been documented that apigenin reduces the oxidative stress and increases superoxide dismutase and glutathione reductase (Pillai, 2010). It has also been reported that several flavonoids containing O-dihydroxy groups inhibit the COX reaction during prostaglandin formation in the renal medulla of the rat. These flavonoids are strong scavengers for peroxide anion radicals (Baumann et al., 1980). In the present investigation, the alcoholic fraction of C. inerme contains three flavonoidal glycosides like apigenin, scutellarein, and pectinolinergenin. Among them, apigenin and scutellarein have O-dihydroxy functional groups similar to galangin, rutin, and luteolin. From Table 1.1, it can be observed that among galangin, rutin, and luteolin, galangin and luteolin inhibit lipoxygenase and prostaglandin biosynthesis, but rutin predominately inhibits lipoxygenation. So the compounds apigenin and scutellarin may be involved in the inhibition of both lipoxygenase and COX activity and exert a long-term relief in adjuvant-induced arthritis. Moreover, it has also been reported that this dual inhibition of COX and lipoxygenase could form the basis of an improved anti-inflammatory activity (Higgs et al., 1979). So the longtime anti-inflammatory activity of the alcoholic fraction of C. inerme may also be included in the above-mentioned category. Hence the overall mechanisms of flavonoids may be summarized in the Figure 1.11.

Figure 1.11 The chart explains the role of flavonoids in both anti-inflammatory and antioxidative mechanisms by increasing antioxidant enzymes and decreasing prostaglandin pathways.

Acknowledgment

This work is partially supported by CSIR, MK University, India, and UHV Faculty Development grant. This project is partially funded by the USDA-CSREES # 2006-34402-17121 Designing Foods for Health through the Vegetable & Fruit Improvement Center, Texas A&M University, College Station.

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Chapter 2

Inflammation in Arthritis

N.J. Correa-Matos and S.B. Vaghefi

University of North Florida, Jacksonville, FL, USA

Abbreviations

COX    Cyclooxygenase

CRP    C-reactive protein

DHA    Docosahexaenoic acid

EPA    Eicosapentaenoic acid

IL    Interleukin

NFκB    Nuclear factor kappa B

OA    Osteoarthritis

PUFA    Polyunsaturated fatty acid

RA    Rheumatoid arthritis

RANKL    Receptor activator of NFκB ligand

TNF    Tumor necrosis factor

1 Introduction

Arthritis is one of the inflammatory diseases affecting 50 millions of Americans. Between 2005 and 2009, 50% of adults over 65 years of age were diagnosed with any types of arthritis including rheumatoid arthritis (RA), osteoarthritis (OA), gout, and/or fibromyalgia (CDC, 2010), being higher in obese and minorities. It is considered an autoimmune disease and is characterized by an inflammation of the synovial area and bone and joint destruction, accompanied by severe pain and immobility (Viswanathan and Sylvester, 2008). Smoking, alcohol consumption, lack of fruits and vegetables in the diet, sedentary life, and obesity are among the risk factors for arthritis considering that all of them have shown to produce a low-grade inflammation (Lu et al., 2010).

Several pathways within the inflammatory cascade are activated in arthritis. All of them result in the upregulation of proinflammatory cytokines interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF)-alpha, and nuclear factor kappa B (NFκB), among others, macrophages, neutrophils, adhesion molecules, and clotting factors. The local and systemic immune responses lead to the development of other low-grade inflammatory conditions in cardiovascular diseases. In obese patients, arthritis is not only a consequence but also a risk factor for inflammation.

2 Mechanism of Inflammation in Arthritis

Bone homeostasis involves a balance between osteoblasts (bone formation cells) and osteoclasts (bone destruction cells). The release of inflammatory components (mediated by T cells) is the trigger for osteoclasts activation through upregulation of receptor activator of NFκB (RANK) nuclear factor and the consequent binding to the ligand (RANKL). The bone cells will be destroyed at a faster rate compared to the process of bone formation leading to severe bone resorption, bone destruction, and tissue damage, which consequently, contribute to further inflammation and destruction of the bone joints causing severe pain (Lacativa and Farias, 2010).

In vitro studies have shown that macrophages and lymphocytes increase the cytokines activation of osteoclasts: IL-1, IL-6, IL-11, IL-15, and IL-17, TNF-alpha, NFκB (Mundy, 2007). Conversely, the release of cytokines promotes the release of macrophage colony-stimulating factors, which increase macrophages and also induce osteoclasts activation (Gillespie, 2007). They do the same action through different metabolic pathways: (1) TNF-alpha and IL-6 together inhibit apoptosis of the osteoclasts and stimulate the activation of intercellular adhesion molecules, which causes blockage of blood vessels and formation of clots; (2) some of these proinflammatory cytokines respond to low levels of hormones related to bone homeostasis, such as estrogen, parathyroid hormone (PTH), thyroxin, and 1,25 vitamin D (Papanicolaou et al., 1998).

A study with 74 adult patients diagnosed with RA showed elevated levels of a serum biomarker of inflammation C-reactive protein (CRP) when compared to controls. IL-1, IL-6, TNF-alpha, and cell adhesion molecules (ICAM, VAM, and ECAM) were also elevated (Dessein et al., 2005).

3 Dairy Products and Inflammation

Many dietary factors have been shown to be associated with the activation of RANK/RANKL. The lack of calcium in the diet, and eventually in the blood, leads to the release of PTH and osteoclasts activation. Consequently, the lack of calcium stimulates the release of 1,25 vitamin D, which causes the calcium influx from the bones to the cell, reducing available calcium in blood and elevating calcium levels inside the cell to a point that it stimulates the activation of the enzyme fatty acid synthase (FAS) and lipogenesis, obesity, and further inflammation caused by the effect of the enlarged adipocyte over the adjacent tissues (Zemel, 2001). A mice model with low-calcium (0.4%) versus high-calcium diets (~1.2%) found a 40% reduction in intracellular calcium, an inhibition in FAS, and a reduction in weight gain and adiposity in the high-calcium diets compared to low-calcium diets. When calorie restrictive diets were supplemented with calcium-fortified cereals, adiposity and weight loss were observed, and moreover, when milk was added, these benefits were synergistically improved (Zemel, 2002, 2003).

It seems that other components of milk besides calcium have a potential effect in the reduction of adiposity, which eventually reduces inflammation and the symptoms related to RA.

Protein components in milk, whey, and casein, have shown to reduce inflammation. Whey has been linked to its antioxidant function against the cyclooxygenase (COX) pathway (Brown et al., 2004), inhibiting phospholipase A2 and preventing inflammation (Smith, 2006). Other components of whey, alpha-lactalbumin and lactoferrin, provide benefits for patients with arthritis by reducing inflammation via the inhibition of cytokines (Yamaguchi et al., 2001, 2009) and reducing pain (Hayashida et al., 2003), respectively. Most recent laboratory cell culture data found that proteins and fat components in dairy products reduce IL expression (Dalbeth et al., 2010).

Yogurt is a probiotic food containing high amounts of Lactobacillus and has shown to reduce inflammation in animal models (Baharav et al., 2004) via the inhibition of proinflammatory cytokines. Moreover, it has been shown that inflammation is less intense and duration is reduced in animals fed with yogurt when compared to control animals.

4 Effects of Food and Spices in Inflammation

The bioactive components in several spices have shown to reduce inflammation by different mechanisms. When garlic cloves are crushed or chopped, the enzyme alliinase is activated releasing diallyl disulfide. This compound has been shown to reduce inflammation and prevent cartilage destruction by the inhibition of histone deacetylase in the cell. This action reduces the synthesis and activity of the matrix metalloproteinases (MMPs), which are involved in cartilage and bone destruction and inflammation (Williams et al., 2010; Young et al., 2005). In this way, the reduction and further inhibition of MMP eventually inhibit the proinflammatory cytokine IL-1, reducing symptoms associated with inflammation like pain, swelling, redness, and fever.

The inhibition of NFκB, which is involved in the transcription of the genes related to the synthesis of proinflammatory cytokines, contributes to the inhibition of inflammation in patients with arthritis. A review of the data available regarding the effects of spices on inflammation by Aggarwal and Shishodia (2004) confirmed that turmeric, red pepper, cloves, ginger, anise, and garlic inhibited the NFκB pathway and reduced inflammation by inhibiting proinflammatory cytokines TNF-alpha, IL-1, and IL-6 (Funk et al., 2009; Jagetia and Aggarwal, 2007; Ramadan et al., 2011) in different cells and animal models. However, the amounts needed to see the benefits in humans are yet to be determined.

The food additive carrageenan is a potent activator of the NFκB pathway. Cell culture and experiments with rodents found two different mechanisms of activation of inflammation risk factors, one via the activation of NFκB inducing kinase and the other via the phosphorylation of intermediates that eventually activate it (Bhattacharyya et al., 2010). In patients receiving anti-inflammatory therapy, the consumption of products with this food additive can interfere with this treatment and should be avoided.

Research is still elucidating the effects of coffee consumption on the development of arthritis. The increased risk for RA arthritis was observed when consuming more than four cups of coffee a day; however, the mechanism was not elucidated (Heliövaara et al., 2000). It seems that the effect of caffeine in increasing calcium excretion may develop the inflammatory response when calcium levels were low. This was discussed earlier in this chapter. Conversely, a study conducted using data from the Iowa Women’s Health Study (Mikuls et al., 2002) looking at caffeinated and decaffeinated coffee and tea found that decaffeinated coffee (equal to or more than four cups per day) and tea (more than three cups per day) were associated with RA development risk; however, more information is needed to explain its mechanism. Similar findings in reduction of TNF-alpha were observed when adding cocoa polyphenol extracts to the diet of arthritis-induced mouse models as compared to controls (Kim et al., 2010).

Studies of patients with knee OA taking passion fruit extracts (150 mg day−1) for 2 months found a reduction in inflammation and pain; however, it was not clear if the mechanism of this benefit was their anti-inflammatory or antioxidant properties (Farid et al., 2010).

Meat consumption has been associated with risk of development of RA and gout. A multicountry data examined by Grant (2000) in Europe found association between high meat consumption and risk for RA; however, the mechanism responsible for the inflammation needs to be elucidated. One possible hypothesis could be the levels of saturated fat and the presence of nitrites (a potent carcinogen) and the high concentration of omega-6 polyunsaturated fatty acid (PUFA), which act as proinflammatory mediators.

Olive oil and fish oil contain oleic acid (n-9 PUFA), which inhibits the activation of the proinflammatory compound leukotriene (Evans et al., 1985). Olive oil contains vitamin E and can also increase the levels of omega-3 PUFA, which are both anti-inflammatory components (Linos et al., 1999). It is possible that these components in olive oil will synergistically contribute to the anti-inflammatory properties that reduce symptoms in RA (Linos et al., 1999). A meta-analysis of randomized controlled trials confirmed the effects of fish oil inhibiting proinflammatory cytokines in arthritis (James et al., 2010; Proudman et al., 2008). More studies are needed to confirm the adequate dose for such benefits.

5 The Role of PUFA in Arthritis

Omega-3 and omega-6 fatty acids are essential to humans because of lack of the enzyme desaturase that can make unsaturated fatty acids linkages before carbon 9. The relationship between the anti-inflammatory effects of omega-3 PUFAs and the omega-6 fatty acids (proinflammatory) has been important in arthritis. It has been shown that the omega-6 to omega-3 ratio impacts the symptoms and the development of the disease. Although it is known that inflammation is a response needed to start the immune response cascade, a too prolonged or too strong response can be damaging to bone tissues by increasing action of osteoclasts and promoting the augmented expression of proinflammatory cytokines in the synovial cells. This will cause swelling, pain, and bone destruction.

Omega-3 PUFAs are very long-chain fatty acids. The most common ones are eicosapentaenoic acid (EPA) (20 carbons) and docosahexaenoic acid (DHA) (22 carbons). These two have been associated with the reduction in the gene expression of proinflammatory cytokines with an increase in anti-inflammatory cytokines (Simopoulos, 2008), cell membrane fluidity, and the immune response (Calder, 2008). Studies in vitro (Fujikawa et al., 1992) in mice models (Huang et al., 1992) and in humans (Hughes et al., 1996) have confirmed the reduction in the expression of antigen-presenting cells major histocompatibility complex class II molecules (MHC-II), macrophages, and interferon-gamma with fish oil-rich diets, leading to a reduced inflammatory response less damaging to cell membrane. However, the immunosuppression caused by large amounts of fish oil leading to less protection to the host is yet to be determined.

Other studies showed a reduction in T cell helper-1 expression and in the synthesis of the proinflammatory arachidonic acid (AA) (Calder et al., 1992). EPA and DHA have shown to produce a less potent inflammatory response via the activation of the 5-lipoxygenase pathway (Calder, 2008) and resolvins, which are more potent anti-inflammatory mediators (Hong et al., 2003). This reduction in inflammation can either reduce the development of arthritis or reduce the severity of the symptoms and bone damage in the patients.

A meta-analyses conducted by Calder et al. (2007) showed that 12 studies in humans consuming fish oils in the amount between 1.4 and 3.0 g day−1 for 12–15 weeks provided a reduction in joint swelling, duration of stiffness, and pain. These amounts are equivalent to 2–3.5 oz of fatty fish (salmon, mackerel, herring, lake trout, sardines, and albacore tuna) twice a week, as recommended by The American Heart Association. Most recently, the use of flaxseed oil reduced IL-1 and TNF-alpha, reducing inflammation (James and Cleland, 1992).

The Mediterranean diet which is low in meats and high in fruits, fish, olive oil, garlic, and vegetables has been shown to provide a healthier balance between omega-3 and omega-6 PUFA when compared to the Western diets rich in meats and fats and has been shown to prevent chronic disease via a reduction in inflammatory markers.

Most omega-6 fatty acids in the diet come from vegetable oils as linoleic acid (LA). Omega-6 PUFA is also long chains of fatty acids that produce proinflammatory AA, resulting in production of prostaglandins, thromboxanes, leukotrienes, and cyclooxygenases. Several animal and human studies have shown the presence of these compounds in the joint fluids of patients and animals with arthritis (Sano et al., 1992; Sperling, 1995).

Trans fats are fatty acids that have been commercially created by hydrogenation of oils to increase the shelf life of products such as margarine, cookies, and other baked goods; however, humans do not have the ability to use trans linkages to activate desaturase enzymes in the elongation and desaturation process of omega-3 and omega-6 that results in a reduction in the markers of immune response and prevention of inflammation (Simopoulos, 2002). The excess of omega-6 also competes with omega-3 for the enzyme desaturase, reducing the amount of EPA and DHA that can be made. The major sources of omega-6 are vegetable oils such as safflower, mayonnaise, hydrogenated margarine, and meats.

The typical American diet tends to contain 14–25 times more omega-6 fatty acids than omega-3 fatty acids (Raper et al., 1992). It was found that Western diet contains a ratio of omega-6 to omega-3 of 15:1. The high concentration of omega-6 in the diet has been related to low-grade inflammation seen in chronic disease such as cardiovascular, obesity, and arthritis, among others.

The opposing effects of omega-3 (anti-inflammatory) and omega-6 (proinflammatory) lead to the question of what will be the best ratio of omega-6 to omega-3 that can provide enough immune response to promote a mild inflammation preventing an immunosuppressed state in the patient.

Several studies have looked at different ratios in relation with diseases (Simopoulos, 2008). A ratio of 1:1 has shown to decrease markers of inflammation of CRP (Zampelas et al., 2003). However, in a study comparing Swedish diet with Mediterranean diets, where the Mediterranean diet is low in meats and high in fruits and vegetables, a higher omega-6 to omega-3 ratio was found in the Swedish diet (6:1) compared to a lower ratio, 2:1, in the Mediterranean diet (Ambring et al., 2006). A study with humans consuming ratios of omega-6 to omega-3 fatty acids found that a higher ratio of omega-3 to omega-6 was correlated with a reduction in proinflammatory cytokines IL-6, IL-1, TNF-alpha, and CRP (Ferrucci et al., 2006). A study with arthritis patients found that the ratio of omega-6 to omega-3 of 4:1 reduced proinflammatory cytokines, TNF-alpha, and IL-1B (James and Cleland, 1997).

Animal studies showing that a lower omega-6 to omega-3 ratio (more omega-3) is beneficial for bone health because when adding fish oil to mice, they found less bone loss in a menopausal model compared to higher levels of omega-6 (Simopoulos, 2008). In studies with bone cells, an increase in bone formation markers when EPA (omega-3) was added to the media was found (Watkins et al., 2003).

There are some controversies with gamma-LA (GLA), which is an intermediate in the production of AA. GLA is found in evening primrose oil and black currant seed oil. Although it is elongated to AA, studies have shown that it reduced inflammation by activation of other pathways that are less inflammatory than the prostaglandins, leukotrienes, and thromboxanes. Studies show GLA helped to reduce pain and swelling but not to prevent arthritis after 3 months of treatment.

6 Antioxidants and Inflammation in Arthritis

The role of antioxidants in arthritis has been studied by looking at the functions of the vitamins and minerals. Antioxidants can act as free radical scavengers as well as inhibitors of the oxidative pathways, nitric oxide, reactive oxygen species, and COX (Canter et al., 2007). Oxidation can lead to cell damage and eventually to inflammation (Hitchon and El-Gabalawy, 2004).

Most specifically to arthritis, the inflammation results from the damage to cartilage and synovial spaces. Studies with arthritis-induced mice have shown a reduction in inflammation with the addition of selenium by activating superoxidase dismutase and vitamin E (Hagfors et al., 2003; Hitchon and El-Gabalawy, 2004). Epidemiological and clinical data have shown that a low consumption of antioxidants by including fruit and vegetables is associated with arthritis (RA) incidence (Canter et al., 2007).

Twenty clinical trials were reviewed by Canter et al. (2007) to evaluate the significance of the data available in the role of vitamins and minerals in arthritis. Several studies in humans performed by Peretz et al. (1992) failed to show statistically significant data on the effects of selenium in reducing oxidation and inflammation in RA patients. Similar results were observed in the role of vitamin E, vitamin C, and vitamin A (Jäntti et al., 1991).

Epidemiological data and results from The Women’s Health Study (a placebo-controlled trial which included more than 39 000 participants from 1992 to 2004) found that patients with RA had lower levels of vitamin E compared to controls; however, other studies were not able to confirm these results (Karlson et al., 2008).

Similar results were found in a study with mice where the experimental group was vitamin E deficient displaying elevated levels of TNF-alpha, adhesion molecules (ICAM), and IL-1 beta. When vitamin E was supplemented, the gene expression of these proinflammatory cytokines was significantly reduced, demonstrating a protective effect of vitamin E in reducing inflammation (Choi et al.,