Evid Based Integrative Med 2005; 2 (3): 147-166 1176-2330/05/0003-0147/$34.95/0

REVIEW ARTICLE

© 2005 Adis Data Information BV. All rights reserved.

Capsaicin A Promising Multifaceted Drug from Capsicum spp. B.C.N. Prasad, Richa Shrivastava and Gokare A. Ravishankar Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore, India

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 1. Biosynthesis of Capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 2. Metabolism of Capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 3. Analogues of Capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 4. Medicinal Properties of Capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 4.1 Antioxidant Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 4.2 Anticancer Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 4.3 Gastric Protection and Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 4.4 Antiarthritic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 4.5 Analgesic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 5. Pain Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 6. Characterisation of the Transient Receptor Potential Vanilloid Type (TRPV1) Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 7. TRPV1 Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Abstract

Capsaicin, the pungency-imparting alkaloid, is found only in the Capsicum genus. Capsaicin biosynthesis involves the phenyl propanoid and valine pathways and a final condensation step to involve vanillylamine and 8-methyl nonenoic acid catalysed by capsaicin synthase. In recent times, capsaicin has been one of the most highly exploited biomolecules because of its versatility in application. So far, researchers have reported capsaicin as an antioxidant, antiarthritic, gastroprotective agent, anticancer (also procancer) agent and an analgesic. This review is a compilation of reports on the biosynthesis of capsaicin in Capsicum spp., the exploitation of capsaicin in pharmacy and the molecular mechanisms involved in capsaicin receptors.

Secondary metabolites in plants help in the fight against animal predators and pests and perhaps even competing plant species. Capsaicin in chillies (Capsicum spp.) is one such secondary metabolite, which prevents animals from eating the chilli fruits so that fruit-eating birds can consume them.[1] Medicinal powers of pepper have made it a favourite plant for researchers from ancient times to the modern day. Capsaicin is the common name for 8methyl N-vanillyl-6-nonamide, a pungent substance from the fruit of Capsicum that has been used as a medicine for centuries. Capsicum is well known as a vegetable and spice, and is commonly called hot pepper, paprika, sweet pepper and bell pepper. It was

considered helpful for various conditions of the gastrointestinal tract, including stomach aches, cramping pains and flatulence. Green pepper is frequently used as a tonic to treat diseases of the circulatory system. Exploiting the counter-irritant effect of pepper, it is used as a remedy for rheumatic pains and arthritis. From the physiological point of view, capsaicin (the active agent found in hot chilli peppers) is perhaps one of the most enigmatic deterrent molecules ever produced by plants.[2] Capsaicin accomplishes its effect by evoking a sharp burning pain sensation when it comes in contact with mucous membranes. It has been speculated that capsaicin production gives a biological ad-

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vantage to hot chilli peppers over non-hot peppers, as mammals, which reduce the germinating capability of seeds, are discouraged from eating the hot fruit, while other animals, such as birds, which pass the seeds without affecting their germinating ability, can eat them freely. Thus, seed disposal is more effective for hot than for non-hot peppers.[3] Despite the burning pain we experience by eating hot dishes, the majority of us still find eating hot chilli pepper-containing meals highly enjoyable. Although it is undoubtedly an intriguing dilemma why most people enjoy the capsaicininduced burning pain sensation, for more than a century scientists have been rather puzzled over other biological effects of capsaicin. Capsaicin relieves pain and itching by acting on sensory nerves by temporarily stimulating the release of various neurotransmitters from these nerves, leading to their depletion. Capsaicin and other constituents in cayenne pepper have been shown to have several actions, including antioxidant, anticancer, antiarthritic, analgesic and counter-irritant properties (figure 1). Medical studies have shown that capsaicin significantly lowers cholesterol and is a factor in warding off strokes and heart attacks. Capsaicin is also reported to increase blood flow in stomach mucous lining, which may help in healing of the stomach tissue.[4] The functionality of capsaicin seems to be significantly dependent on the properties of its receptor (vanilloid receptor, VR1).[5] Researchers have discovered the interesting ability of the receptor to integrate multiple pain stimuli, such as temperature, mechanical disruption and capsaicin activation. This integration is the reason eating foods containing capsaicin is perceived as being ‘hot’. The depleting effect that capsaicin has on cell stores of the painproducing substance P and causing cell death evokes a gradual desensitisation to the stimulation of the nociceptors, thus causing a numbing effect in the area of targeted sensory neurons. This numbing, or analgesia, has spurred much interest in the potential use of capsaicin as a treatment for chronic pain, such as that associated with arthritis, oral mucositis and atypical facial pain. Furthermore, history shows that the qualities of chilli pepper engendered by the physiological effect of capsaicin on the body go beyond its analgesic properties to having an effect on ailments spanning everything from the common cold to diabetes mellitus. Anti-inflammatory

Capsaicin actually works as a noxious chemical signal activating the peripheral terminals of sensory neurons. The sensory neurons are fired as a result of capsaicin increasing membrane permeability to cations such as calcium and sodium. More specifically, capsaicin binds to and activates the thinly myelinated A primary sensory neurons and unmyelinated C fibres.[6] The nociceptor neurons transmit information into the CNS and release neuropeptide, including substance P. The release of substance P is responsible for the sensations of burning and irritating pain. Thus, this chemical pathway is the explanation for the burning pain experienced when biting into a chilli, a defensive mechanism – “chemical warfare armoury… designed to protect plants from being eaten.”[1] The therapeutic potential of spices/spice principles have been known for a long time in Indian medicine. Their antimicrobial and anti-inflammatory properties have been exploited over centuries. Spice principles have also been shown to inhibit the formation of many proinflammatory compounds. Capsaicin is shown to lower the formation of superoxide anions, hydrogen peroxide and nitric oxide (NO) in activated macrophages. These effects of spice principles may enable them to act as good anti-inflammatory compounds. Spices are nontoxic and nonmutagenic, with good antioxidant and anti-inflammatory properties. They are promising viable alternatives for drugs in the treatment of arthritis. Capsaicin is currently experiencing a renaissance in that a number of recent studies have emerged, adding to its already impressive list of actions. Scientists are taking notice and looking at Capsicum with new respect and interest. Perhaps what sets capsaicin apart is that, unlike powerful pharmaceutical stimulants and painkillers, it possesses potency without deleterious adverse effects. This article reviews a compilation of published scientific studies on the role of capsaicin and discusses its potential as a medicinal agent and the various uses of capsaicin. The literature search was conducted via websites including ScienceDirect and PubMed, and gene sequences were searched for in the National Center for Biotechnology Information (NCBI) databases. We in no way claim originality in the work, except where our references are cited. Analgesic

CH3O

Antiarthritic

Antioxidant O C N H

Anticarcinogen

Gastroprotective

Fig. 1. Capsaicin – a multifaceted drug. © 2005 Adis Data Information BV. All rights reserved.

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Phenyl propanoid pathway

Fatty acid pathway O

Phenylalanine

CH3

C

Valine/leucine pathway HO

HC CH

CH3

NH2

CH2CHCOOHNH2

PAL O

α-Keto isovalerate

Cinnamic acid

CH3

C HO

HC C

CH3

O2

CH=CHCOOH

Ca4H HO

CH3

Coumaric acid

Isobutyryl CoA

HC

CoA−S C O

CH3

CH=CHCOOH

Ca3H

OH HO

Caffeic acid

3× Malanyl CoA

KAS

CH=CHCOOH

CoMT OCH3 O

HO

Ferulic acid

8-Methyl-6 nonenoic acid

CH3

CoA−SC(CH2)4CH=CHCH CH3

CH=CHCOOH

?

OH

OCH3

CH3O HO

Vanillin

CS

O

CH=O

N H OCH3

C

PAMT ? Capsaicin

HO

Vanillylamine CH2NH2

Fig. 2. Capsaicin biosynthetic pathway. Ca3H = coumaric acid 3-hydroxylase; Ca4H = cinnamic acid 4-hydroxylase; CoA = coenzyme A; CoMT = caffeic acid O-methyl transferase; CS = capsaicin synthase; KAS = keto-acyl synthase; PAL = phenylalanine ammonia lyase; PAMT = putative amino transferase; ? between ferulic acid and vanillin indicates that the biochemical steps during conversion of ferulic acid to vanillin are not reported in the capsaicin biosynthetic pathway in Capsicum; ? after PAMT indicates that its functionality is not yet confirmed and it is putative.

1. Biosynthesis of Capsaicin Capsaicin is a unique plant alkaloid with a biosynthesis that is restricted to the Capsicum genus. In pepper fruits, capsaicin is biosynthesised by capsaicin synthase through condensation of © 2005 Adis Data Information BV. All rights reserved.

vanillylamine, a phenyl propanoid pathway intermediate, and fatty acid moieties in placental tissues of Capsicum fruits (figure 2).[7,8] There are reports on biotransformation of phenyl propanoid pathway intermediates to capsaicinoids,[9-11] elicitation[9,12] of capsaicin in immobilised cell cultures leading to biochemical regulation, Evid Based Integrative Med 2005; 2 (3)

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and also on involvement of calcium calmodulin in regulation of capsaicin production mediated by capsaicin synthase.[13,14]

4. Medicinal Properties of Capsaicin

The first report on capsaicin biosynthesis mainly emphasised the condensation of vanillylamine and fatty acid moieties by capsaicin synthase[7] (figure 2). To date, many genes (AF081215, AF088847, AF081214, AF085148, AF085149, AAC99311, AY819026, AY819030, AY819027, AY819031, AY819028, AY819032, AY819029) involved in capsaicin biosynthesis have been isolated through suppression subtractive hybridisation.[15-17] The two most fiery capsaicinoid compounds are capsaicin and dihydrocapsaicin, which produce burning everywhere from the mid-tongue and palate down into the throat. Evidently, all of the capsaicinoids work together to produce the pungency of peppers but capsaicin is rated the strongest.[18]

4.1 Antioxidant Properties

2. Metabolism of Capsaicin Three modes of capsaicin metabolism have been investigated: (i) oxidation by hepatic mixed-function oxidase systems; (ii) oxidation through radical formation; and (iii) non-oxidative metabolism. It has been postulated that the liver mixed-function oxidase system converts capsaicin to an electrophile intermediate, a ring epoxide, that is capable of covalently bonding to nucleophilic sites of hepatic protein.[19] This could account for the hepatotoxicity of capsaicin. Recently, it was shown that liver cytochrome P450 (CYP) 2E1 activity was responsible for converting capsaicin into a phenoxy radical that either dimerises or covalently binds to CYP2E1, inactivating the enzyme.[19] Finally, capsaicin-hydrolysing activity was found highest in the liver, followed by the kidney, lung, and small intestine.[19] Hydrolysis of the amide linkage produces vanillylamine, which is later reduced to vanillyl alcohol for excretion. Thus, capsaicin absorbed in the gastrointestinal tract reaches the CNS almost exclusively in the form of degradation products. 3. Analogues of Capsaicinoids Capsaicinoids are divided into two groups, based on the chain lengths of their branched chain fatty acid moieties (figure 3).[20] Capsaicin and dihydrocapsaicin (which contribute nearly 90% of the total pungency of Capsicum) have an even number of branched chain fatty acid moieties, whereas nordihydrocapsaicin, homodihydrocapsaicin and homocapsaicin have an odd number of branched chain fatty acid moieties. © 2005 Adis Data Information BV. All rights reserved.

In recent years, phenolic compounds have attracted the interest of researchers because their antioxidant properties can protect the human body from free radicals, the formation of which is associated with the normal natural metabolism of aerobic cells.[21] Epidemiological data have indicated beneficial effects of antioxidant compounds in the prevention of a multitude of disease states, including cancer, cardiovascular disease and neurodegenerative disorders.[22-24] Phenolic compounds cannot be produced by the human body, and thus must be taken in mainly through the diet. Knowledge about the nutritional and therapeutic role of dietary phenolic antioxidants is essential for the development of functional foods, i.e. the improvement of conventional foods with added health benefits.[22] Capsaicin inhibited lipid peroxidation significantly,[25] being more effective than the well known antioxidant α-tocopherol.[26] Capsaicin was also found to scavenge 1,1V-diphenyl-2-picrylhydrazyl (DPPH) radicals both in solution and in membranes, especially the latter. Capsaicin was found to scavenge radicals both at/ near the membrane surface and in the interior of the membrane. The phenolic OH group of capsaicin remained intact after reaction with DPPH radicals, indicating that the hydroxyl group is not associated with the radical scavenging reaction. From the results of quantum chemical calculations of various radical intermediates derived from the model compound N-vanillylacetamide, and the findings that vanillin and 8-methyl-6-noneamide were major reaction products of capsaicin with DPPH radicals, it was concluded that the radical scavenging site of capsaicin is the C7-benzyl carbon.[26] Lee et al.[27] demonstrated that blood flow measurements in rats were negatively correlated with low-density lipoprotein (LDL) after treatment with capsaicin. Although capsaicin did not show a noticeable effect on the serum total cholesterol level, LDL decreased while high-density lipoprotein (HDL) and triglyceride increased in rats treated with capsaicin 3 mg/kg for 3 days. The antioxidant effect of capsaicin was not evident when the rats were treated with capsaicin 1 mg/kg bodyweight. However, rats treated with capsaicin 3 mg/kg bodyweight for 3 days showed a reduction of oxidative stress measured as malondialdehyde in the liver, lung, kidney and muscle. Liver glycogen was found to decrease after 3 days’ treatment with capsaicin 3 mg/kg bodyweight. From this study, the authors have concluded that capsaicin can be a potent antioxidant and aid in lowering LDL even when consumed for a short period. Small-dose capsaicin treatment also increased antiinflammatory interleukin-10 levels and attenuated the increases in Evid Based Integrative Med 2005; 2 (3)

Capsaicin: A Promising Multifaceted Drug

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Capsaicinoids

Group I Even number branched chain fatty acid moieties

Group II Odd number branched chain fatty acid moieties

OH

OH

CH3O

CH3O

CH3

O

CH3

O

C

C CH3

N H

CH3

N H

Capsaicin

Nordihydrocapsaicin

OH

OH

CH3O

CH3O

CH3

O

O

C

C

CH3

N H

N H

Dihydrocapsaicin

Homodihydrocapsaicin OH CH3O

CH3

O C

CH3

N H

Homocapsaicin

Fig. 3. Analogues of capsaicinoids.

proinflammatory cytokines, NO and tissue malondialdehyde in septic rats.[28] The study by Rosa et al.[29] showed that capsaicin has remarkable antioxidant activity. These analogues of capsaicin could protect linoleic acid against free radical attack in simple in vitro systems, inhibiting both its auto-oxidation and its iron- or ethylene diamine tetraacetic acid (EDTA)-mediated oxidation. These properties were retained in some simple synthetic analogues (vanillyl nonanoate and its dimerisation products). Capsiate, dihydrocapsiate, and their analogues were also devoid of prooxidant activity and showed a highly significant antioxidant activity in all systems investigated. Materska and Perucka[30] studied the antioxidant properties of capsaicin and dihydrocapsaicin in different species of Capsicum with varying in pungency levels. They revealed the differences © 2005 Adis Data Information BV. All rights reserved.

between antioxidant activity of capsaicinoid fractions from green and red fruits. In general, red fruits showed activity (38–80%) higher than green fruits. Comparison of antioxidant activities of fractions of capsaicinoids with flavonoid and phenolic acid derivatives showed that both fractions from red pepper had similar values, and the flavonoid fraction from hot pepper exhibited a higher antioxidant activity than that from semi-hot pepper, especially by the capsaicinoids present in the fruit pericarp. They opined that the process of capsaicin biosynthesis in hot pepper cultivars may occur at the cost of flavonoid. The most acceptable explanation of the antioxidant property of capsaicin is the presence of a methoxy group in the ortho position to OH but not due to the amide group present in capsaicin. The main product of capsaicin oxidation is its dimer dicapsaicin. Compared with dihydrocapEvid Based Integrative Med 2005; 2 (3)

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saicin, capsaicin has more antioxidant activities, which is attributed to its double bond in the lipid chain. Very recently, capsaicin has been reported[30] to inhibit constitutive activation of nuclear factor-κB (NF-κB) mediated by reactive oxygen intermediates endogenously generated via the nicotinamide adenine dinucleotide [NAD](P)H quinone oxidoreductase system in malignant melanoma cells. Topical application of capsaicin to dorsal skin of female ICR mice strongly suppressed 12O-tetradecanoylphorbol-13-acetate (TPA)-stimulated activation of NF-κB by blocking degradation of the inhibitory protein IκB and subsequent nuclear translocation of the free NF-κB dimer. Capsaicin also inhibited epidermal activation of AP-1 in mice.[30] 4.2 Anticancer Properties

Chemoprevention of cancer by ingestion of chemicals is thought to be a major way to reduce the risk of carcinogenesis, and natural dietary products may be of special relevance because of their long record of safe human consumption. Eukaryotic cells continuously produce reactive oxygen species (ROS) as side products of redox reactions. Generation of ROS is mainly under mitochondrial control, and involves the production of hydrogen peroxide, hydroxyl radicals, and superoxide anions. Production of ROS by aerobic organisms is a ‘double-edged sword’, since these species are required for signal transduction in pathways controlling cell growth, but can also damage structures essential for cell integrity and homeostasis.[31] The plasma membrane of certain types of cells contains a further electron transport chain, apparently essential to control cell growth and differentiation. This plasma membrane oxidoreductase (PMOR) system has been found in animal, plant and yeast cells[32] and has been suggested to play a role in the maintenance of the cellular redox state. The constituents of the PMOR system have long remained elusive, but a plasma membrane reduced NAD (NADH)-oxidase (NOX) was eventually characterised as a 32 kDa ectoenzyme located on the outer surface of the plasma membrane.[33,34] This NADH oxidase activity is inhibited by the capsaicin, and may be an important target for anticancer drugs. Capsaicin, and possibly other vanilloids as well, are endowed with a pleiotropic pattern of biological activities, some of which are mediated by the activation of cellular targets different from VR1. Thus, capsaicin can inhibit the plasma membrane NADH oxidase[34,35] and induce apoptosis in tumoural cells and in activated T cells in a VR1-independent way.[36-38] Analogues of capsaicin, devoid of VR1 activity but retaining the ability of the natural product to induce apoptosis in cancer cells, have potentially farreaching therapeutic applications. It is crystal clear that capsiates (capsaicin derivatives without pungency) lack significant vanilloid © 2005 Adis Data Information BV. All rights reserved.

activity, but can nevertheless exert pro-oxidant activity and induce apoptosis in cancer cells. These properties were retained in the simplified analogue nor-dihydrocapsiate (3c), which could strongly prevent tumourogenesis in athymic mice.[39] The antitumoural activity of capsaicin has been documented in vivo not only in tumour prevention but also when injected directly on the tumour.[19] Hail and Lotan[40] have conducted a series of elegant experiments that afford important insights into mechanisms underlying the apoptogenic action of capsaicin at the cellular level. They reported that capsaicin-induced apoptosis in cultured cells derived from human cutaneous squamous cell carcinoma occurs through inhibition of mitochondrial respiration. Studies by several investigators have also demonstrated that capsaicin-induced apoptosis in some transformed cells[34,38] and in activated T cells[41] is associated with the suppression of PMOR, an enzyme that transfers electrons from cytoplasmic NADH via coenzyme Q (ubiquinone) to external electron acceptors such as oxygen. PMOR is thought to be involved in the control of cell growth and proliferation[32] by maintaining the proper NAD+/ NADH ratio required for cell viability. However, PMOR activity in normal tissues and in nontransformed cells is responsive to growth factors and hormones, whereas PMOR activity in tumour tissues and transformed cells is not.[42] Apoptogenic activity of capsaicin does not appear to be mediated by the vanilloid receptor, at least in human blood cell lines, because treatment with capsazepin, a prototype vanilloid receptor antagonist, failed to block the apoptosis induced by those compounds.[37,38] The possible involvement of ROS in vanilloid-induced apoptosis has also been reported by Macho et al.[36,37,41] They showed that capsaicin- or resiniferatoxin-treated transformed human T cells (i.e. Jurkat cells) displayed a loss of nuclear DNA and a concomitant increase in the proportion of subdiploid (apoptotic) cells, and that this vanilloid-induced apoptosis was preceded by an increase in ROS generation and disruption of the mitochondrial transmembrane potential – both invariant features of early programmed cell death. The study by Morr´e et al.[34] also suggested that the NADH oxidase activity of rat liver plasma membrane was largely unaffected by capsaicin, whereas the NADH oxidase activity of HeLa plasma membranes was strongly inhibited. This indicates the possible role of capsaicin as an anticancer agent with the fundamental response to NADH oxidase activity of normal and transformed cells and tissues that was associated with inhibition of growth and induction of apoptosis in the transformed cells. Growth of HL-60 cells was inhibited by capsaicin. A growing number of recent studies have focused on anticarcinogenic or Evid Based Integrative Med 2005; 2 (3)

Capsaicin: A Promising Multifaceted Drug

antimutagenic phytochemicals, particularly those included in human diet.[43] Capsaicin has been suggested to exert such chemoprotective effects through modulation of metabolism of carcinogens/mutagens and their interactions with target DNA.[19] Inhibition by capsaicin of arylhydrocarbon hydroxylase responsible for the metabolism of many carcinogenic polycyclic aromatic hydrocarbons including benzo[a]pyrene. In line with this finding, capsaicin suppressed the metabolism and covalent DNA binding of benzo[a]pyrene in human and mouse keratinocytes,[44,45] showing that dihydrocapsaicin, the saturated analogue of capsaicin, is a suicidal or mechanism-based inhibitor of CYP2E1, an isoform that has an important role in metabolic activation as well as detoxification of a wide array of chemical carcinogens of relatively small molecular size.[46] Capsaicin also inhibits rodent hepatic CYP2E1 activity in vitro.[19] In this context, it is noteworthy that pretreatment of mice with a topical dose of capsaicin attenuates skin carcinogenesis induced by vinyl carbamate,[19] which is preferentially activated by CYP2E1 to an ultimate electrophilic and carcinogenic epoxide metabolite.[46]

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Fig. 4. Representation of signalling cascades mediating oxidative and proinflammatory stimuli such as capsaicin and other phytochemicals. COX = cyclo-oxygenase; IκB = inhibitory κB; MAP = mitogen-activated protein; NF-κB = nuclear factor-κB.

The chemopreventive effects exerted by curcumin, zingarol and capsaicin phytochemicals are often associated with their antioxidative and anti-inflammatory activities. Cyclo-oxygenase (COX)2 has been recognised as a molecular target of many chemopreventive as well as anti-inflammatory agents. Recent studies have shown that COX-2 is regulated by the eukaryotic transcription factor NF-κB (figure 4). The molecular mechanisms underlying the chemopreventive effects of the capsaicin involving intracellular signalling cascades such as NF-κB, mitogen-activated protein kinases[47] and COX-2 indicates that COX-2 is a key enzyme catalysing the production of prostaglandins in response to inflammatory stimuli (figure 4).

activity in rats were ameliorated by intragastric capsaicin treatment, which was associated with suppression of COX-2.[52] Regarding the association of tumour promotion with oxidative and inflammatory tissue damage, it would be worthwhile determining whether capsaicin has antioxidant and anti-inflammatory activities which could inhibit tumour promotion. While lacking an intrinsic tumour-promoting activity, capsaicin inhibited TPA-promoted mouse skin papillomagenesis.[52,53]

Multiple lines of evidence support the notion that COX-2 plays a role in the development of tumours.[48] Capsaicin can act as an antioxidant, as revealed by attenuation of oxidative damage or lipid peroxidation in various organs of experimental animals.[25] Anti-inflammatory properties of capsaicin have also been reported. Capsaicin was shown to antagonise mouse ear oedema induced by croton oil.[49] In another study, capsaicin ameliorated carrageenan-induced inflammation in rats.[50] Moreover, capsaicin has the ability to inhibit platelet aggregation, possibly through blockade of phospholipase A2, a key enzyme responsible for formation of arachidonic acid from the membrane lipid. In addition, capsaicin repressed calcium-ionophore-stimulated pro-inflammatory responses, such as generation of superoxide anion, phospholipase A2 activity, and membrane lipid peroxidation in macrophages.[50] Capsaicin exerted protective effects against ethanol-induced gastric mucosal injury in rats.[51] Ethanol-induced haemorrhagic erosion, lipid peroxidation and myeloperoxidase

The study by Keisuke et al.[54] showed that capsaicin suppressed the growth of leukaemic cells via induction in G0-G1 phase cell cycle arrest and apoptosis. Capsaicin-induced apoptosis was in association with the elevation of intracellular ROS production. Capsaicin-sensitive leukaemic cells were possessed of wildtype p53, resulting in the phosphorylation of p53 at the Ser-15 residue as a result of capsaicin treatment. These authors concluded that capsaicin has potential as a novel therapeutic agent for the treatment of leukaemia. Capsaicin was reported to preferentially repress the growth of various immortalised or malignant cell lines via induction of apoptosis.[45,55,56] However, its biological and biochemical activities have not been elucidated in endothelial cells. Capsaicin inhibited vascular endothelial growth factor (VEGF)-induced proliferation, DNA synthesis, migration and tube formation of endothelial cells in vitro. Moreover, the compound markedly inhibited tumour- or VEGF-induced new blood vessel formation in chick chorioallantoic membrane and in vivo atrigel

© 2005 Adis Data Information BV. All rights reserved.

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plug assays. Capsaicin also caused G1 arrest of endothelial cells through downregulation of cyclin D1, and inhibited VEGF-induced angiogenic signalling pathways. Taken together, these results suggest that capsaicin may inhibit tumour growth via its antiangiogenic activity.[57] A number of independent studies have demonstrated that capsaicin can promote or prevent carcinogenic and mutagenic processes in vitro and in vivo.[19] In epidemiological observations, it is suggested that capsaicin consumption may reduce the risk of colon cancer in humans. On the contrary, high capsaicin pepper use in some ethnic-cultural groups is considered to be a risk factor for developing stomach cancer.[58] A study at Purdue University (West Lafayette, IN, USA)[59] has revealed that a synergistic formulation of Capsibiol-T®,1 – a unique, synergistic formulation of modified capsaicin and decaffeinated green tea – inhibits tNOX (a member of the ECTO-NOX family). When tNOX activity is inhibited, cancer cells fail to grow to the size required for division and undergo apoptosis. Before the proprietary process to modify capsaicin for exclusive use in Capsibiol-T® was developed, the dosage required for capsaicin to be effective (its therapeutic dosage) had exceeded the level at which its pungency and neurological discomfort could be tolerated. The amount of modified capsaicin (12.5mg per capsule) in synergy with decaffeinated green tea (637.5mg per capsule) enables Capsibiol-T® to achieve a 10-fold increase in potency. 4.3 Gastric Protection and Stimulation

All tested plant-originated substances including capsaicin afforded gastroprotection against ethanol-induced damage and this was accompanied by an increase in gastric microcirculation.[60] Plant-originated substances are highly gastroprotective probably due to enhancement of the expression of NO synthase (NOS) NO release and an increase in gastric microcirculation. The discovery of specific actions of capsaicin on a subset of mammalian afferent nerves, called capsaicin-sensitive afferent nerves, raised the possibility of using this drug to approach the possible role of these nerves in the different functions of different organs both physiologically and pathologically. Moderate concentrations of the sensory stimulant drug capsaicin caused relaxation in human and animal intestinal circular muscle preparations (guinea-pig proximal colon, mouse distal colon, human small intestine and appendix) in vitro. With the exception of the guinea-pig colon, the NOS inhibitor N (G)-nitroL-arginine (L-NOARG; 10–4 mol/L) strongly inhibited the relaxant effect of capsaicin.[61] Tetrodotoxin, an inhibitor of voltagesensitive Na+ channels, failed to significantly reduce the inhibitory effect of capsaicin in the guinea-pig colon, human ileum and 1

appendix; it caused an approximately 50% reduction in the mouse colon. The relaxant effect of capsaicin was strongly reduced in colonic preparations from transient receptor potential vanilloid type (TRPV1) receptor knockout mice compared with their wildtype controls. It is concluded that NO, possibly of sensory origin, is involved in the relaxant action of capsaicin in the circular muscle of the mouse and human intestine.[61] The mechanisms underlying the capsaicin-induced relaxation of the acetylcholine contraction as well as potassium chloride (KCl) contraction were studied[62] by measuring isometric force and phosphorylation of a 20 kDa regulatory light chain subunit of myosin [MLC(20)] in ileal longitudinal smooth muscles of rats. Capsaicin relaxed acetylcholine- and KCl-stimulated preparations in a concentration dependent manner; the former was less sensitive to capsaicin than the latter and maximum responses to capsaicin (a percentage of papaverine-induced relaxation) were 70.6 ± 7.5% and 97.1 ± 0.9%, respectively. The response showed no desensitisation. Like nifedipine, capsaicin relaxed the tissue precontracted with an agonist of L-type Ca2+ channels as well. The relaxant effect of capsaicin was not inhibited by capsazepine (a selective antagonist of vanilloid VR1 receptors), nitro-l-arginine, indomethacin and guanethidine, nor by inhibitors of soluble guanylate cyclase. Capsaicin inhibited acetylcholine-induced transient contraction in a Ca2+-free, EGTA (ethylene glycol-O,O′-bis[2-amino-ethyl]-N,N,N′,N′,-tetraacetic acid) solution. Phosphorylation of MLC(20) [a percentage of phosphorylated to total MLC(20)] was increased 1 minute after application of acetylcholine 10 μmol/L (7.8 ± 2.0% [n = 6] vs 22.6 ± 3.2% [n = 6]) and of KCl 65.9 mmol/L (2.2 ± 0.3% [n = 8] vs 10.7 ± 1.7% [n = 12]). Capsaicin reduced the KCl-induced increase more markedly than acetylcholine-induced increase in MLC(20) phosphorylation. When the tissue was contracted for 20 minutes with acetylcholine, MLC(20) phosphorylation was increased, and capsaicin markedly reduced the contraction and abolished MLC(20) phosphorylation both elicited by acetylcholine. It is suggested that capsaicin relaxes the rat ileum via its direct action on smooth muscle, and that capsaicin inhibits contractile mechanisms involving extracellular Ca2+ influx via non-L-type Ca2+ channels, possibly via storeoperated Ca2+ channels and Ca2+ release from intracellular storage sites. The effects of capsaicin on acetylcholine- and KCl-induced contraction could be explained by a decrease in MLC(20) phosphorylation. An alternative explanation for the effect of capsaicin on the human preparations could be that capsaicin-sensitive pharmacological receptors[63] are situated on myenteric (partly ‘nitrergic’) neurons, an arrangement for which there is morphological evidence in the pig and guinea-pig ileum.[64] Capsaicin most

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Evid Based Integrative Med 2005; 2 (3)

Capsaicin: A Promising Multifaceted Drug

probably induces the release of both relaxant and contractile neurotransmitters from sensory nerves; moreover, intrinsic enteric neurons are activated in the course of the action of capsaicin. From the latter, acetylcholine is released; at the same time, tachykinins are probably liberated from both extrinsic sensory and enteric motor neurons.[65] Another study, by Horei et al.,[66] also showed that intragastric administration of capsaicin inhibited the formation of gastric lesions in a dose-dependent manner (0.1–2.5 mg/kg) and VR1 plays a protective role in the gastric defensive mechanism in rats. The immunohistochemical studies revealed that nerve fibres expressing VR1 exist along gastric glands in the mucosa, around blood vessels in the submucosa, in the myenteric plexus, and in the smooth muscle layers, especially the circular muscle layer.[66] Aihara et al.[67] have previously reported that intraluminal application of capsaicin also stimulates the secretion of HCO3– in these tissues through the activation of capsaicin-sensitive afferent neurons. In their study they observed that both acid and capsaicin produced an increase of HCO3– secretion in the stomach mediated by these afferent neurons, and clearly showed the difference in their modes of action in terms of sensitivity to TRPV1 and prostanoid receptors TRPV1 is a nonselective cation channel responsive to protons as well as capsaicin.[5] The binding sites of capsaicin are located at the intracellular site of the receptor protein[68] whereas the target of protons is thought to be located on the extracellular surface of the receptor protein.[69] When the TRPV1 antagonist capsazepine was applied to the mucosa together with capsaicin or acid, it was found that this agent completely blocked the increase in gastric HCO3– secretion induced by capsaicin but not acid, despite both responses being mediated by capsaicin-sensitive afferent neurons. The important role of capsaicin-sensitive afferents in gastroprotection has been summarised recently in various studies.[70,71] Intragastric capsaicin (10–6 to 10–5 g/mL), inhibited ethanol (50%)-induced gastric ulceration in the rat. Furthermore, in pylorus-ligated rats, capsaicin (130 nmol/L to 1.3 mmol/L) inhibited the gastric ulcers provoked by 0.6N HCl, acidified acetylsalicylic acid, ethanol (50%, 96% of capsaicin, respectively) or indomethacin (20 mg/kg subcutaneously). The gastroprotective effect of capsaicin is prevented by immunoneutralisation of calcitonin gene-related peptide (CGRP), by the CGRP1-R antagonist CGRP8-37, or by NOS inhibitors. It is also attenuated by the tachykinins neurokinin 2 receptor (NK2-R) antagonist MEN10627. These data indicate that the release of CGRP acting on CGRP1-R, tachykinins acting on NK2-R, and NO all play mediating roles in capsaicin-induced gastroprotection. Early studies and more recent publications[72,73] on the gastric actions of different chilli extracts resulted in controversial conclusions about the role © 2005 Adis Data Information BV. All rights reserved.

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of capsaicin-sensitive nerves in gastroprotection. Prolongation of gastric emptying was observed in seven healthy volunteers in response to red pepper sauce (tabasco).[74] In the other study, 20g of chilli given orally in 200mL of a saline solution (0.1N NaOH pH 7) inhibited the gastroduodenal mucosal damage evoked by acetylsalicylic acid 600mg.[73] It has been shown that capsaicin given intragastrically in a concentration range of 1–8 mg/mL in 200mL of a saline solution (0.1N NaOH pH 7) inhibited in a dosedependent manner the H+ output and total secreted volume of the gastric juice for about 1 hour.[75] The mechanism by which capsaicin causes relaxation seems to vary slightly among these preparations. Tetrodotoxin, an inhibitor of fast Na+ channels, caused a significant, yet partial, reduction in the effect of capsaicin in the mouse colon but no inhibition in the human preparations. Of what is known about the intestinal effects of capsaicin, it is probable that this drug acts by stimulating endings of extrinsic, sensory nerves.[71,76] There is ample evidence that the release of smooth muscle-active substances from sensory nerve endings by capsaicin is a tetrodotoxin-resistant process,[77] since the opening of the TRPV1 receptors in response to capsaicin does not depend on tetrodotoxin-sensitive Na+ channels. Hence, in the mouse, intrinsic enteric neurons may be activated by capsaicinsensitive nerve endings, and the source of NO may be the capsaicin-sensitive extrinsic neurons, the enteric neurons, or both populations. It is, however, also possible that there is a ‘nitrergic’ link between sensory and enteric neurons and that the latter also utilise other inhibitory neurotransmitter(s). NO is able to activate enteric neurons. A third possibility is that capsaicin activates part of sensory nerve endings directly (via TRPV1 receptors) and these latter activate some further sensory nerve endings indirectly, through an ‘axon reflex’ arrangement in the mouse colon. Finally, the possibility cannot be ruled out that the mucosa is involved in the relaxant response to capsaicin in the mouse colon. Transcripts for TRPV1 have been detected in the mouse small intestinal mucosa by reverse transcription-polymerase chain reaction (RTPCR).[78] As mentioned earlier, various structures seem to express TRPV1 in the gastrointestinal tract: peripheral terminals of primary and vagal sensory neurons, intrinsic enteric neurons in the myenteric plexi, and gastric epithelial cells.[79-81] Regarding the stomach and duodenum, one of the most prominent functions of TRPV1-expressing sensory nerves is the maintenance of the integrity of the tissues against protons and activated enzymes.[82] A major barrier protecting gastric and duodenal tissues is a viscous mucus layer covering the entire luminar surface of the stomach and duodenum. Activation of TRPV1-expressing primary afferents has been shown to contribute to the thickening of this protective layer.[83] While selective elimination of capsaicin-sensitive Evid Based Integrative Med 2005; 2 (3)

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nerve fibres aggravates the chemically induced mucosal damage in the stomach, low concentrations of the TRPV1 ligands, such as capsaicin, acids and alcohol results in increased resistance of the gastric mucosa towards chemical injury.[84] Capsaicin-sensitive primary afferents seem to contribute to the tissue protection through more than one mechanism. On the one hand, capsaicin induces hyperaemia through vasorelaxation produced by CGRP release from capsaicin-sensitive primary sensory fibres, which increases the metabolic activity of the cells. On the other hand, the capsaicin-induced CGRP release has been shown to activate COX1 enzymes, inducing the production of prostaglandin E2.[85] This latter compound activates secretory cells, which produce the protective layer.[86] Recent findings indicate that activation of TRPV1 expressed on gastric epithelial cells may also play some role in the defence mechanism.[80] Kato et al.[80] found that two TRPV1 activators – protons (pH 4.0) and alcohol (10%) – induce cell damage, while activators such as the vanilloids, capsaicin (10–9 to 10–6 mol/L) and resiniferatoxin (10–12 to 10–9 mol/L) concentrationdependently prevent the proton- and alcohol-evoked effects. These authors have also found that TRPV1 expressed by gastric epithelial cells is 99.8% identical to that cloned by Caterina et al.[5] (1997). These findings indicate that the subtle difference between TRPV1 in primary sensory neurons and in gastric epithelial cells could be enough to produce major differences in sensitivity to, and in the well-described potentiation effect of, various activators. Alternatively, TRPV1s in the epithelial cells and in primary sensory neurons are indeed identical, but the subcellular distribution is different, e.g. in epithelial cells TRPV1 is expressed only on the endoplasmic reticulum and only capsaicin could reach TRPV1 without producing membrane damage. It has been speculated that the direct capsaicin-evoked protective effect on epithelial cells involves Ca2+-dependent activation of COX-1. TRPV1 expressed by primary sensory neurons in the gastrointestinal tract has also been implicated in the development of inflammation and hypermotility/hyper-reflexia. It has been shown that substance P is a major player mediating inflammation in the intestines.[87] The finding that the TRPV1 antagonist, capsazepine, prevents the development of toxin A-induced inflammation indicates that the capsaicin receptor is involved in the process, and capsaicin-sensitive primary sensory fibres are the major source of substance P. Recent findings suggest that the endogenous substance activating TRPV1 during ileitis is anandamide.[88] Anandamide concentration in the inflamed tissues is increased and this endogenous TRPV1 ligand exacerbates ileitis. However, data from Mang et al.[89] indicating that anandamide induces acetylcholine release from intrinsic enteric neurons expressing TRPV1 receptors suggests that the capsaicin receptor expressed by neurons in the © 2005 Adis Data Information BV. All rights reserved.

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myenteric plexi might also contribute to the development of enhanced intestinal motility and secretion. It should be noted, however, that other groups using different chemicals to induce experimental colitis or enteritis[90,91] reported a rather accentuated inflammation following ablation of capsaicin-sensitive nerve fibres with systemic capsaicin treatment of adult animals. This effect was explained by the possible protective actions of the sensory neuropeptide, CGRP, on the mucous membrane. Activity of TRPV1 has also been implicated in the development of abdominal pain occurring during irritable bowel syndrome, which is the most common form of the pathological conditions termed functional bowel disorders. Since the pain experienced by the patients is not matched with any detectable structural abnormality by conventional diagnostic methods, the concept of an altered nociceptive function as the main aetiological factor of irritable bowel syndrome-associated pain has been developed.[63,92] According to the proposed mechanism, sensitisation of TRPV1 by a variety of ligands – including the protease activated receptor 2 expressed by primary sensory neurons[93,94] and by paracrine/endocrine substances produced by the enterochromaffin (e.g. serotonin) or enteroendocrine (e.g. cholecystokinin) cells – underly the development of abdominal pain in irritable bowel syndrome.[95] Although the involvement of TRPV1 in the development of visceral hyperalgesia has been demonstrated in humans, the contribution of this mechanism to the pathogenesis of the irritable bowel syndrome is still debated. Histological investigations also revealed that neonatal capsaicin treatment significantly reduces tissue damage occurring in pancreatitis.[96] Since previous observations showed a substantial role for substance P in the pathophysiology of acute pancreatitis, it has been suggested, that substance P release from capsaicinsensitive sensory fibres is responsible for the development of the neurogenic component of pancreatitis.[97,98] The mechanism by which capsaicin-sensitive sensory fibres are stimulated is not known, but the involvement of TRPV1 receptor activation was confirmed by showing that capsazepine administration significantly decreases substance P release and alleviates parenchymal damage and myeloperoxidase production in acute pancreatitis.[97] 4.4 Antiarthritic Properties

Capsaicin extracted from red chilli peppers are being used in antiarthritic ointments. Capsaicin is known to increase and then to deplete a messenger substance that transmits pain signals to the brain. Although it is quite difficult to conduct double-blind trials because of the burning sensation that capsaicin initially causes, applying capsaicin ointments to painful joints appears to ease pain. However, capsaicin is not reported to cause redness like counterEvid Based Integrative Med 2005; 2 (3)

Capsaicin: A Promising Multifaceted Drug

irritant ointments. Pain relief from capsaicin applied topically may occur gradually, as the substance P is decreased in the nerve cells. Rheumatoid arthritis is an autoinflammatory disease of unknown aetiology, which is common in patients experiencing high levels of circulating immune complexes. These antigen-antibody complexes accumulate in the joints, in turn triggering an inflammatory response at the joints, causing erosion and destruction of the synovial membrane and the underlying cartilage. Such uncontrolled inflammatory reactions can lead to the destruction of the joint.[99] Topical application of capsaicin (from red pepper) has been reported to alleviate the painful osteoarthritis of the hands.[100] In addition, dietary curcumin and capsaicin lower the generation of proinflammatory mediators such as ROS and NO released by macrophages. Joe and Lokesh[101] showed the therapeutic effect of capsaicin on rheumatoid arthritis in rats. They found that capsaicin, can decrease the incidence, delay the onset and reduce the extent of inflammation of adjuvant-induced arthritis in rats. Temporomandibular joint (TMJ) arthritis was induced in female Lewis rats by unilateral injection of a suspension of heatkilled Mycobacterium butyricum in paraffin oil into the TMJ. Carleson et al.[102] studied the arthritic and control rats pretreated with either capsaicin or denervation on the mandibular branch of the trigeminal nerve. In all groups, the levels of substance P-like immunoreactivity (SP-LI), CGRP-like immunoreactivity (CGRPLI) and neuropeptide Y-like immunoreactivity (NPY-LI) were higher in the trigeminal ganglia than in the TMJs. In control rats, capsaicin significantly lowered the levels of SP-LI in the trigeminal ganglia and TMJ but not CGRP-LI and NPY-LI. In the arthritic rats, capsaicin pretreatment significantly lowered the SP-LI and CGRP-LI in the trigeminal ganglia and TMJ, but not the NPY-LI. In the trigeminal ganglia the unilateral denervation significantly lowered SP-LI in control rats, and in arthritic rats SP-LI and CGRP-LI. On the denervated side of the arthritic TMJ, NPY-LI, SP-LI and CGRP- LI were significantly lowered compared with the arthritic control rats and compared with the contralateral side. In this rat model, pretreatment with capsaicin and surgical denervation decreased the neuropeptide content in the trigeminal ganglia and the TMJ. The results clearly demonstrate a close interaction between increased neuropeptide release from sensory and sympathetic neurons after induction of arthritis in the rat.[102] Prior treatment with systemic capsaicin attenuates the severity of adjuvant arthritis, an effect also reported by other workers.[103,104] When capsaicin was given to rats after the 21 days induction of arthritis, footpad swelling was reduced by approximately 46% compared with that seen in untreated arthritic rats.[105] Interestingly, in the sciatic nerves of those same rats a reduction of approximately 41% in substance P levels at the same time-point of © 2005 Adis Data Information BV. All rights reserved.

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arthritis has now been shown.[105] This suggests that capsaicin may attenuate inflammation, at least in part, by reducing tissue levels of substance P. Alternatively, any arthritis-induced increase in substance P levels may be inhibited by capsaicin treatment and may represent a degree of functional impairment of sensory neurons. Indeed, mustard oil-induced neurogenic plasma extravasations in the rat hind paw, considered a functional parameter for peptidergic sensory C-fibres, is significantly attenuated after treatment with capsaicin.[106] The 1995 study of Ahmed et al.[103] revealed that capsaicin administration in adjuvant-induced arthritis reduces the otherwise up-regulated levels of sensory neuropeptides in dorsal root ganglia and ankle joints. However, capsaicin can only mitigate, not completely prevent, the development of joint inflammation. Nonetheless, the findings suggest that antineuronal therapy targeted against specific neurotransmitters may prove useful in inflammatory joint disease. In patients with osteoarthrosis of finger joints, topical capsaicin treatment was recently reported to reduce the signs of inflammation.[100,107] Intradermal injections of capsaicin in human skin produce a well defined response comprising pain, axon reflex vasodilatation and surrounding areas of hyperalgesia.[108] Many of these features mirror those observed in musculoskeletal disease, and intradermal capsaicin has been used to investigate peripheral sensory fibre activity in these conditions.[109,110] Capsaicin also exhibited beneficial effects in modulating arthritis.[111] Administration of spice principles delayed the onset and severity of adjuvant-induced arthritis. This effect was observed irrespective of the nature of the diet. Clinical tests have confirmed that topical capsaicin ointments substantially alleviate the pain that characterises osteoarthritis and rheumatoid arthritis. These studies revealed that using capsaicin 0.075% cream reduced tenderness and pain. Seventy patients with osteoarthritis and 31 with rheumatoid arthritis received capsaicin or placebo for 4 weeks. The patients were instructed to apply capsaicin 0.025% cream or placebo to painful knees four times daily. Significantly more relief of pain was reported by the capsaicin-treated patients than by the placebo patients throughout the study.[100,107] 4.5 Analgesic Properties

Blumstein and Gorevic[112] used topicals such as 5% lignocaine (lidocaine) patches or capsaicin, or orally administered analgesics such as acetaminophen, tramadol, NSAIDs and opiates. Although attractive because of the reduced incidence of serious gastrointestinal adverse reactions, selective COX-2 inhibitors may have significant renal and cardiovascular toxicities and, thus, they sugEvid Based Integrative Med 2005; 2 (3)

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gested using with caution in the older patient with co-morbid diseases affecting these organs. Investigations by Oshita et al.[113] suggest that the CB1 cannabinoid receptor could inhibit either the capsaicin-induced Ca2+ influx or the potentiation of capsaicin-induced SP-LI release by long-term treatment with bradykinin through involvement of a cyclic-adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway. In conclusion, CB1-receptor stimulation modulates the activities of transient receptor potential vanilloid receptor 1 in cultured rat DRG cells. Intranasal medications for the treatment of headache have recently received increased attention. Experimental medication using civamide, a cis-isomer of capsaicin, was used for the treatment of migraine and cluster headache. Although the efficacy of intranasal agents varies with the product used, intranasal delivery was found convenient and more effective than other modes of drug delivery for a variety of reasons: (i) intranasal administration bypasses small bowel gastrointestinal tract absorption, which is often significantly delayed during the acute phase of a migraine attack; (ii) nauseated patients may prefer non-oral formulations, as they decrease the chance of vomiting and are more rapidly effective; (iii) intranasal administration causes no pain or injection site reaction and is easier and more convenient to administer than injection or suppository, and so may be used earlier in a migraine attack, resulting in better efficacy; (iv) intranasal medication produces the same number or fewer adverse events than injections; and (v) intranasal formulations offer a more rapid onset of action than oral medications, for some of the above reasons and, as such, may be more useful in patients with cluster headache, although this needs to be verified. However, it is important to emphasise that a preference study showed that most patients prefer oral tablets to an intranasal formulation.[114] Intraplantar injection of small doses of capsaicin has been shown to produce hyperalgesia and upregulation of the levels of proinflammatory cytokines, suggesting the possible mediation of these effects by sensory neuropeptides and mast cells. Various groups of rats received intraplantar injection of capsaicin alone or preceded by the injection of antagonists to substance P, CGRP and histamine (H1, H2) or the mast cell antagonist ketotifen. All pretreatments prevented, in a dose-related manner, the capsaicininduced hyperalgesia. Substance P, H2-antagonists and ketotifen prevented the upregulation of all cytokines and nerve growth factor (NGF) levels, while the CGRP and H1-antagonists showed only attenuation of the NGF level.[115] Of the characteristics studied, variation in the risk of common adverse effects and gastrointestinal ulcer had the greatest impact on patients’ choice. Assuming patients are responsible for the full cost of their medications, over 40% prefer capsaicin. COX-2 © 2005 Adis Data Information BV. All rights reserved.

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inhibitors become patients’ preferred choice only if they are described as being 3-fold more effective than capsaicin and are covered by insurance. Nonselective NSAIDs are among the least preferred options across all simulations.[111] Nelson et al.[116] examined the effects of capsaicin application on pressor responses evoked by muscle contraction (which are mediated by group III and IV muscle afferents) by monitoring heart rate, blood pressure and end-tidal CO2 in cats decelerated under halothane. They found that changes in peak mean arterial pressure induced by static ipsilateral muscle contraction were significantly attenuated at 20 minutes and tended to approach baseline levels at 40 minutes after capsaicin application. However, there were no significant changes in heart rate. Gottrup and co-workers[117] examined the capsaicin-induced flux in the primary and secondary hyperalgesic area after pretreating the capsaicin injection site with local ketamine, lignocaine or saline 10 minutes prior to injection in humans. Ketamine reduced flux significantly both in the primary and secondary hyperalgesia area. Lignocaine reduced flux significantly in the primary hyperalgesic area, but no effect was observed on flux in the secondary hyperalgesic area. Only lignocaine reduced spontaneous pain, evoked pain and areas of hyperalgesia, whereas ketamine had no effect. These results suggest that there is no simple and close relation between vascular and sensory reactions to pharmacological manipulation following intradermal capsaicin injection. Mason et al.[118] concluded that though topically applied capsaicin has moderate to poor efficacy in the treatment of chronic musculoskeletal or neuropathic pain, it may be useful as an adjunct or sole therapy for a small number of patients who are unresponsive to, or intolerant of, other treatments. Burning mouth syndrome is a major diagnostic and therapeutic problem. Systemic and topical treatments (capsaicin, lignocaine, antihistamines, sucralfate and benzydiamine) have been tried, but they appear to be inadequate. Topical capsaicin is bitter, may cause burning and has low therapeutic efficacy. Petruzzi et al.[119] hypothesised in 2004 that systemic administration of capsaicin could reduce the limitations of topical administration and have better therapeutic efficacy; this hypothesis was tested in a controlled trial. A systemic oral capsaicin dose of 0.25% was used for patients with burning mouth syndrome. After diagnosis, patients were dentally and medically examined. They were assigned either to treatment with capsaicin or to a shape/smell/taste/colour-matched placebo. The severity of symptoms was scored at trial entry and 30 days thereafter by investigators who were unaware of the assigned intervention. The use of systemic capsaicin implied significant gastric toxicity (referred gastric pain), with eight cases (32%) documented in the treatment group compared with zero cases (0%) Evid Based Integrative Med 2005; 2 (3)

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Outer plasma membrane

in the placebo control group. Hence, it was concluded that systemic capsaicin is therapeutically effective for the short-term treatment of burning mouth syndrome but major gastrointestinal side effects may threaten its large-scale, long-term use.[119]

Inner plasma membrane

5. Pain Induction

C A

P´orsz´asz and Jancs´o[120] were the first to show that capsaicin selectively and specifically excites a subpopulation of primary sensory fibres, which can also be activated by noxious stimuli, and that capsaicin sensitivity of the fibres is quickly reduced following capsaicin exposure. Jancs´o-G´abor et al.[121] reported that capsaicin also activated and induced damage in a group of hypothalamic neurons that are responsible for thermoregulation. Later, studies on animals injected with capsaicin neonatally indicated that capsaicin evokes its effects through a specific receptor, and that the capsaicin receptor might be involved in the development of various pathological events, such as that of pain, visceral hyperreflexia and neurogenic inflammation.[122-124] Szallasi and Blumberg[125] provided unquestionable evidence for the existence of a capsaicin-responsive receptor. As the capsaicin-sensitive receptor also responds to other related molecules with vanilloid moiety, the putative capsaicin receptor was named the ‘vanilloid receptor’.[125] The work regarding understanding the pathological events of the vanilloid receptor received extra momentum from the recent identification and characterisation of VR1 or TRPV1,[5,126,127] and the development of TRPV1 knockout mice.[128,129] Results of these studies have provided evidence that TRPV1 is indispensable for the development of certain pathological conditions, such as inflammatory heat hyperalgesia or visceral hyper-reflexia.[128-130] However, the list of diseases in which TRPV1 might be involved is increasing. Recent studies[5,126,128] have shed light on the mechanisms through which TRPV1 is activated in pathological conditions. 6. Characterisation of the Transient Receptor Potential Vanilloid Type (TRPV1) Receptor The capsaicin receptor (figure 5) is particularly important in mediating hyperalgesia responses in inflammatory pain states, as demonstrated by research in knockout animals and with smallmolecule antagonists. It is anticipated that TRPV1 antagonists, and perhaps antagonists at other thermosensitive TRP channels, will provide new therapeutic options with which to treat clinical pain.[131] The cloned TRPV1 is a 95 kDa protein[5] with N- and Cterminals that are intracellular. The N-terminus has three ankarin © 2005 Adis Data Information BV. All rights reserved.

A

N

A

Fig. 5. Predicted membrane topology and domain structure of the capsaicin receptor, VR-1. A = amino acid; C = C-terminal; N = N-terminal.

repeat domains, of which only one splice variant of TRPV1, the VR.5′sv, has been found[132] (figure 5). The predicted structure of TRPV1 shows that it has six transmembrane domains, with an additional intramembrane loop connecting the fifth and sixth transmembrane domains.[5] The structure and amino acid sequence of TRPV1 are similar to those of the TRP family of cation channels.[5,133] TRPV1 has consensus phosphorylation sequences with seven for PKA,[134] six for protein kinase C (PKC)[135] and six for Ca2+/ calmodulin-dependent kinase II (CaMkII).[136] TRPV1 also has several glycosylation sites – domains that link it to other proteins.[137] Results from Kedei et al.[138] and Kuzhikandathil et al.[139] reveal that the predominant form of the functioning TRPV1 is probably homotetramer.[5,126] However, it has recently been shown[136] that TRPV1 co-immunoprecipitates with another TRPV molecule, TRPV3, when human embryonic kidney 293 (HEK293) cells are co-transfected with both TRPV1 and TRPV3. The coprecipitation was shown to depend on the presence of TRPV3 in the immune complex, and the result of a specific interaction between TRPV1 and TRPV3. Co-transfection of HEK293 cells with TRPV1 and TRPV3 also showed that TRPV3 increases the response of the heteromer. The heteromeric structure of the capsaicin receptor is particularly interesting, as native TRPV1 might also form heteromers with other TRP channels. These TRPV1-containing heteromers probably have different sensitivity. This assumption is supported by the finding that native capsaicin receptors expressed by cultured primary sensory neurons are heterogeneous in their sensitivity to different stimuli at the single channel level.[126] Recent findings also show that the capsaicin receptor, VR1, is involved in phototransduction in Drosophila similar to the high affinity neurotrophic factor receptor, tyrosine kinase B, enzymes including phospholipase C (PLC) and PKC.[140-142] Data that reduction of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) Evid Based Integrative Med 2005; 2 (3)

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level in the plasma membrane increases the activity of TRPV1[142] suggest that PtdIns(4,5)P2 must also be a member of the capsaicin receptor ‘transducisome’. Prescott and Julius[143] showed that PtdIns(4,5)P2 binds to TRPV1, and this binding regulates the activity of the capsaicin receptor. In addition to tyrosine kinase A (trkA), PLCγ and PtdIns(4,5)P2, PKA, PKC and calmodulin are also (though probably only transiently) members of the capsaicin receptor complex. Recently, Numazaki et al.[144] and Rosenbaum et al.[145] have reported that calmodulin, in a Ca2+-dependent manner, also binds transiently to TRPV1. The transient calmodulin binding, on the other hand, seems to be responsible for the capsaicin receptor activity-induced inhibition, known as desensitisation of the receptor.[144] A recent study revealed that two synaptic vesicle proteins, snapin and synaptotagmin IX also interact with TRPV1[146] temporarily as synaptic vesicle proteins bind to TRPV1 only in the cytoplasm. However, their interactions seem to be pivotal in the trafficking and cytoplasmic membrane expression of TRPV1. Electrophysiological characterisation in expression systems revealed that TRPV1 forms a nonselective cationic channel which – in addition to vanilloids such as capsaicin and the ultrapotent vanilloid, resiniferatoxin – can also be activated by heat above 42°C.[5,147] The characteristics of TRPV1-mediated current recorded in different expression systems closely resemble those mediated by the native capsaicin receptor expressed in primary sensory neurons.[5,126] Depolarisation enhances the TRPV1-mediated current, which indicates that the activity of the channel is voltagedependent, and that depolarisation and agonists act in a synergistic fashion.[148] The Q10 (quotient 10) of the cooling-evoked decrease of the capsaicin-induced current is similar to that of voltage-gated currents.[149] Based on these features, the capsaicin receptor has been called a stimulus integrator.[127] A large body of evidence indicates that post-translational modifications of TRPV1, such as PKA-, PKC- and CaMkII-mediated phosphorylation of, and PtdIns(4,5)P2 hydrolysis from, TRPV1 increase the activity of the capsaicin receptor. The PKA-mediated phosphorylation of the capsaicin receptor was first reported to mediate the sensitising effect of the inflammatory mediator, prostaglandin E2 on capsaicin-induced responses.[150] Later, PKA activation was shown to increase the response of the capsaicin receptor to other exogenous and endogenous activators[151,152] and to be involved in NGF-evoked, and metabotropic glutamate 5 receptor (mglu5)-mediated increase in capsaicin-induced responses. Recent findings suggest, however, that the PKA-mediated phosphorylation of TRPV1 produces the sensitisation effect through the reduction of desensitisation.[134,135] © 2005 Adis Data Information BV. All rights reserved.

Prasad et al.

Although the expression of CaMkII in TRPV1-positive primary sensory neurons has been reported previously,[153] the role of CaMkII-mediated phosphorylation of TRPV1 is just becoming clear. Bonnington and McNaughton[154] found that inhibition of CaMkII reduces the NGF-evoked sensitisation of capsaicinevoked responses in primary sensory neurons. Most recent data show that CaMkII must phosphorylate TRPV1 before the receptor is activated by any activator.[136] Furthermore, these authors found that in contrast to CaMkII-mediated phosphorylation of TRPV1, dephosphorylation of the molecule by calcineurin induces desensitisation of the receptor. Thus, Jung et al.[136] have proposed that CaMkII-mediated phosphorylation and calcineurin-mediated dephosphorylation of TRPV1 can dynamically control the activation/desensitisation states of the capsaicin receptor. Activation of PLC either through trkA, bradykinin B2 or mglu5 receptors enhances the capsaicin receptor-mediated responses.[142,155] Similarly to PKA- and PKC-mediated phosphorylation of TRPV1, PLC activation also significantly reduces the heat threshold of the receptor.[142,143] Thus, bradykinin can decrease the heat threshold of TRPV1 through PKC-mediated phosphorylation and PLC-mediated PtdIns(4,5)P2 hydrolysis.[142,156] Similarly to the inhibition of PKA, PKC and PLC block capsaicin-evoked responses in cultured primary sensory neurons, indicating that constitutional activity of PLC is also necessary to maintain the responding configuration of the capsaicin receptor.[157] The already known putative endogenous TRPV1 activators, which bind to the receptor, include protons, ATP, N-arachidonoylethanolamine (anandamide), N-arachidonoyl-dopamine, Noleoyldopamine, lipoxygenase products – such as 12- and 15(S)hydroperoxyeicosatetraenoic acid (12-(S)-HPETE and 15-(S)HPETE), 5- and 15-(S)-hydroxyeicosatetraenoic acids (5-(S)HETE, 15-(S)-HETE) – and leukotriene B4 receptors.[5,137,158-162] All of these endogenous compounds induce capsaicin receptormediated inward currents both in recombinant TRPV1-expressing cells and in the native capsaicin receptor-expressing primary sensory neurons. Molecular mapping has revealed the function of several TRPV1 residues. Studies on chimeras of TRPV1s cloned from capsaicin-sensitive and insensitive species, such as rat or human, and chicken or rabbit, respectively, and on mutated TRPV1s show that intracellular/intramembranous residues in and adjacent to the third and fourth transmembrane domains (Y511 and T550) are responsible for binding of capsaicin to TRPV1.[163,164] Studies on chimeras of rat TRPV1 and TRPV2, a capsaicin nonsensitive but noxious heat-sensitive TRPV1 homologue[165] and on TRPV1 with various deletions of the C-terminus showed that residues in both the C- and N-termini modify capsaicin-sensitivity and binding.[166,167] Evid Based Integrative Med 2005; 2 (3)

Capsaicin: A Promising Multifaceted Drug

Residues involved in desensitisation are also worth noting. The high Ca2+-permeability of the capsaicin receptor, which is important in the development of desensitisation, is determined by Y671 located on the sixth transmembrane domain. The CaMkIIcalcineurin-mediated regulation of TRPV1 desensitisation involves amino acids S502 and T704.[136] 7. TRPV1 Expression TRPV1 is expressed in at least three cellular compartments, i.e. cytoplasmic membrane, endoplasmic reticulum and cytoplasmic vesicles.[146,168] When TRPV1 is in the cytoplasmic membrane, TRPV1-mediated effects are inward currents or transmitter release, those in the cytoplasmic vesicles seem to serve as a reserve, which can be quickly translocated to the cytoplasmic membrane, for example following PKC activation.[146] However, there are no reports of TRPV1 in endoplasmic reticulum. The finding that activation of these receptors by capsaicin or resiniferatoxin evokes Ca2+ mobilisation from intracellular stores shows that these receptors are also functional and they might be involved in the regulation of Ca2+ homeostasis.[169] Results of radioactive ligand binding, RT-PCR, in situ hybridisation, Western-blotting, immunohistochemical staining and functional studies showed TRPV1 expression by various neurons and non-neuronal cells. However, the functionality of TRPV1 in all cells is dubious. Regarding primary sensory neurons, TRPV1 is expressed by one-third to one-half of dorsal root and trigeminal ganglion neurons.[168,170,171] In agreement with the putative role of TRPV1 in nociception, the great majority of the TRPV1-expressing cells belong to small, nociceptive cells expressing substance P and CGRP, or binding site for the isolectin, IB4. Both somatic and visceral primary afferents express TRPV1, and the molecule is expressed by both the spinal and peripheral terminals.[130] Interestingly, it seems that instead of the TRPV1 protein, the mRNA of the receptor is transported to the terminals and the translation occurs there.[172] Similarly to dorsal root ganglion and trigeminal ganglion neurons, TRPV1 is also expressed by both the peripheral and central terminals.[81] In the skin, TRPV1-expressing fibres can be found in the dermis, along the epidermal/dermal junction and epidermis.[168] Although the capsaicin receptor is associated with nociception, with C-type primary sensory neurons terminating in free nerve endings at the periphery, in the skin, TRPV1-immunopositive fibres have been found to innervate Meissner corpuscles.[173] This indicates that Meissner corpuscles might also be involved in nociception. Recent findings indicate that TRPV1 is expressed in many areas of the CNS. In the spinal cord, TRPV1 has been shown on © 2005 Adis Data Information BV. All rights reserved.

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postsynaptic structures with spinal cord origin. Binding and in situ hybridisation studies on wild-type and TRPV1 knock-out mice confirmed the results of previous findings on the widespread expression of TRPV1 in the brain.[174] TRPV1 expression has also been demonstrated in the inner ear, where TRPV1-expressing cells include inner and outer hair cells, inner and outer pillar cells, Hensen’s cells, spiral ganglion neurons, Scarpa’s ganglionic neurons and satellite cells.[175,176] The findings that both capsaicin and resiniferatoxin increased the threshold for auditory nerve compound action potential generation and reduced the magnitude of cochlear microphonic and electrically evoked otoacoustic emissions suggest that capsaicin receptors are functional in the inner ear.[176] In addition to neurons and cells in the inner ear, other cells of ectodermal origin also seem to express functional capsaicin receptors. Human cultured keratinocytes express TRPV1 and capsaicin induces Ca2+ influx into a subpopulation of these cells.[177,178] Interestingly, other heat-sensitive TRP channels are also expressed by keratinocytes.[179,180] Kato et al.[80] have shown TRPV1 protein and mRNA expression in cultured rat gastric epithelial cells, though it is not clear whether all the cells or just a subpopulation of cells express TRPV1, and they have also shown that capsaicin does not induce desensitisation or degeneration in gastric epithelial cells. Epithelial cells in the urinary bladder have also been shown to express TRPV1 at both the mRNA and the protein level.[181] Immunohistochemical reactions revealed TRPV1 expression both in the basal and superficial layers of the urothelium. Both capsaicin and resiniferatoxin induce a TRPV1-mediated increase in intracellular calcium concentration, indicating that the receptors are functional. Similarly to gastric epithelial cells, however, capsaicin does not induce the characteristic vanilloid-evoked desensitisation in epithelial cells of the urothelium either. This is an important difference between neurons and epithelial cells expressing TRPV1. Cells of mesodermal origin have also been reported to express TRPV1. Birder et al.[181] have reported that bladder smooth muscle cells express TRPV1. However, these findings have not been confirmed either by showing TRPV1 protein expression or capsaicin-induced responses in isolated smooth muscle cells. Cardiomyocytes seem to express TRPV1 transiently during the development in rats between E14 and P30.[182] Whether these receptors are functional also remains to be elucidated. TRPV1 expression was also shown in interstitial cells of the urinary bladder and the prostate recently by using immunohistochemical-staining.[183,184] However, the methods used by these authors suggest that some of the straining they found must be artefactual. Evid Based Integrative Med 2005; 2 (3)

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Table I. List of US patents involving capsaicin as a drug molecule Patent no.

Title

6,867,009

Human capsaicin receptor and uses thereof

6,790,629

Nucleic acid sequences encoding capsaicin receptor and capsaicin receptor-related polypeptides and uses thereof

6,723,730

Capsaicin receptor ligands

6,673,840

Use of lipoxygenase metabolite of arachidonic acid and its derivatives in capsaicin-channel agonist

6,593,370

Topical capsaicin preparation

6,482,611

Human capsaicin receptor and uses thereof

6,348,501

Lotion compositions utilising capsaicin

6,335,180

Nucleic acid sequences encoding capsaicin receptor and uses thereof

6,248,788

Therapeutic method with capsaicin and capsaicin analogues

6,239,180

Transdermal therapeutic device and method with capsaicin and capsaicin analogues

5,955,631

Method for industrial purification of capsaicin

5,910,512

Topical analgesic using water soluble capsaicin

5,869,533

Non-irritating capsaicin formulations and applicators therefor

5,660,830

Solubilising counter-irritants and concurrently extracting capsaicin from the same specific peppers

5,466,459

Wax and capsaicin based pesticide

5,021,450

New class of compounds having a variable spectrum of activities for capsaicin-like responses, compositions and uses thereof

6,440,464

Nutritive composition for cardiovascular health containing fish oil, garlic, rutin, capsaicin, selenium, vitamins and juice concentrates

6,326,031

Method of decreasing cholesterol and triglyceride levels with a composition containing fish oil, garlic, rutin, and capsaicin

6,248,788

Therapeutic method with capsaicin and capsaicin analogues

6,239,180

Transdermal therapeutic device and method with capsaicin and capsaicin analogues

6,022,718

Method of producing capsaicin analogues

5,994,407

Procedure to prepare a solution with capsaicin

5,981,583

Inhibition of nuclear transcription factor NF-.kappa.B by caffeic acid phenethyl ester (CAPE), derivatives of CAPE, capsaicin (8-methyl-N-vanillyl-6-nonenamide) and resiniferatoxin

5,962,532

Therapeutic method with capsaicin and capsaicin analogues

5,910,512

Topical analgesic using water soluble capsaicin

5,869,533

Non-irritating capsaicin formulations and applicators therefor

5,840,720

4-O and 5-aminomethylation of synthetic capsaicin derivatives, a new discovery of capsaicin antagonist

5,821,269

Treated bird seed preferentially palatable to birds but not palatable to animals having capsaicin sensitive receptors

5,788,982

Method and composition for treating oral pain using capsaicin

5,747,052

Aqueous composition to neutralise the irritating effect of capsaicin on eyes, skin and mucous membranes

5,660,830

Solubilising counter-irritants and concurrently extracting capsaicin from the same specific peppers

5,431,914

Method of treating an internal condition by external application of capsaicin without the need for systemic absorption

5,403,868

Capsaicin derivatives

5,397,385

Anti-fouling coating composition containing capsaicin

5,178,879

Capsaicin gel

5,021,450

New class of compounds having a variable spectrum of activities for capsaicin-like responses, compositions and uses thereof

4,997,853

Method and compositions utilising capsaicin as an external analgesic

8. Conclusion The fiery hot red chilli pepper arrived in our hemisphere in the sixteenth century. It is only recently, since science began to validate capsaicin medicinal use, that the bioactive compound has gained the prestige it deserves. Capsaicin is one of the most potent © 2005 Adis Data Information BV. All rights reserved.

whole body stimulants, with a wide array of therapeutic actions. Interestingly, several therapeutic formulations are available as evident from the number of US patents listed in table I. Furthermore, capsaicin may offer us the most curative benefits with the least side effects. We can be certain that when it comes to using Evid Based Integrative Med 2005; 2 (3)

Capsaicin: A Promising Multifaceted Drug

capsaicin for health-related conditions, we have only seen the tip of the iceberg. Clearly, after further study, capsaicin should be utilised more fully as a medicinal staple. In our opinion, chilli pepper should be considered nothing less than a wonder herb that has scientifically proven its worth. The discovery of the remarkable properties of capsaicin exemplifies the rich pharmacological potential of dietary secondary metabolites which go undetected by our senses, and suggests that, while the nutritional aspects of food have been well characterised, their pharmacological properties are still largely unexplored and unexploited. Acknowledgements This work was supported by a competitive grant to GAR during 2002–5 from the Department of Biotechnology, Ministry of Science and Technology, Government of India. BCNP and RS thank the Council of Scientific and Industrial Research and the Indian Council of Medical Research, New Delhi, for research fellowships. The authors have no conflicts of interest that are directly relevant to the content of this review.

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Correspondence and offprints: Dr Gokare A. Ravishankar, Scientist F and Head, Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570020, India. E-mail: [email protected] Evid Based Integrative Med 2005; 2 (3)