1. CosmeceuticalsSUBMITTED BY: DEEP DAS ROLL NO: 11121EN007 CLASS: B.PHARM, PART II, SEMESTER V DEPARTMENT OF PHARMACEUTICS INDIAN INSTITUTE OF TECHNOLOGY (BHU) COURSE: PHARMACOGNOSY II, PH-3102 COURSE INSTRUCTURE: A. N. SAHU DATE: 29/10/2013

2. CONTENTS A. Introduction ...2 B. Classification of cosmeceutical products......3 1. Moisturizers.5 1.1 Occlusive moisturizers...6 1.2 Humectant moisturizers.7 2. Photoprotection...7 2.1 Luffa cylindrica........................8 2.2 Portulaca oleracea....9 2.3 Terminalia chebula............,..........9 2.4 Piper longum........9 2.5 Aloe Vera...10 2.6 Emblica officinalis.10 2.7 Crocus sativus....10 2.8 Peumus boldus Molina...11 3. Sunscreen Agents...,,,,....11 3.1 Vitamins........12 3.2 Vitamin A,,,...13 3.3 Vitamin E..13 3.4 Vitamin C......14 4. Skin Lightening Agents.........14 4.1Ascorbic acid.15 4.2 Licorice extract.....15 4.3 Alpha-lipoic acid..16 4.4 Kojic acid,.....16 4.5 Aleosin,.....16 4.6 Arbutin..16 4.7 Hydroquinone.......17 5. Antioxidants...17 5.1 Carotenoids,.18 5.2 Astaxanthin.....18 5.3 Lutein..18 5.4 Lycopene...,.....18 5.5 Retinol,....19 5.6 Flavonoids..19 5.7 Soy,.....20 5.8 Curcumin,...20 5.9 Silymarin,...........20 5.10 Pycnogenol,......21 5.11 Ginkgo,.21 5.12 Green tea,,.22 1|Page5.13 Pomegranate......22 6. Other antioxidants used in post resurfacing......23 6.1 Aloevera.23 6.2 Ubiquinone.23 7. Depigmentation agents..........24 7.1 Phenolic Compounds..24 7.2 B Nonphenolic Compounds...25 7.2.1 Azelaic Acid....25 7.2.2 Tretinoin......26 7.2.3 L-ascorbic Acid...26 7.2.4 Kojic Acid...26 7.2.5 Arbutin....27 8. Hydroxy Acids....27 8.1 -Hydroxyacids....27 8.2 -Hydroxyacids....28 8.3 Polyhydroxy acids................28 8.4 Bionic acids..29 9. Retinoids......29 9.1 Tretinoin..30 9.2 Retinol.....31 9.3 Retinyl-palmitate.....31 9.4 Retinaldehyde..31 9.6 Alitretinoin..........31 9.7 Tazarotene...31 9.8 Adapalene...32 10. Peptides..32 10.1 Antimicrobial peptides..32 10.2 Defensins..33 10.3 Psoriasin...33 10.4 RNase 7........34 10.5 Dermicidin 1L..34 11. Peptides involved in wound healing....35 11.1 RGD Peptides..35 11.2 Copper Peptides...35 11.3 Interleukins (IL)...36 12. Growth Factors.....36 13. Conclusion.....37 14. References.....37 3. A. INTRODUCTION Cosmeceuticals are cosmetic products with biologically active ingredients purporting to have medical or drug like benefits. A cosmeceutical is an ingredient with medicinal properties that manifests beneficial topical actions and provides protection against degenerative skin conditions. The word "Cosmeceuticals" was popularized by Albert M. Kligman in the late 1970s. It encompasses cosmetic actives with therapeutic, disease fighting, or healing properties, serving as a bridge between personal care products and pharmaceuticals. Like cosmetics, cosmeceuticals are topically applied, but they contain ingredients that influence the biological function of the skin.1 Cosmeceuticals improve appearance by delivering nutrients necessary for healthy skin. Cosmeceuticals typically claim to improve skin tone, texture, and radiance, while reducing wrinkling. Cosmeceuticals are the fastestgrowing segment of the natural personal care industry. For cosmetic labels, no division between active ingredients and other ingredients is required; they are all listed together. The most important botanicals pertaining to dermatologic uses such as cosmeceuticals include teas, soy, pomegranate, date, grape seed, pycnogenol, horse chestnut, German chamomile, curcumin, comfrey, allantoin, and aloe; only green and black tea, soy, pomegranate, and date have been studied to the extent that clinical trials for the treatment of parameters of extrinsic aging have been published. Few botanicalbased cosmeceuticals have uses that are supported by evidencebased science. Chemoprevention by oral or topical use of dietary or pharmacologic agents to inhibit or reverse the development of cancer is a possibility. Potential cosmeceutical agents in this category include green tea, grape seed extract, vitamin E, and betacarotene. Cosmeceuticals being cosmetic products having medicinal or druglike benefits are able to affect the biological functioning of skin owing to type of functional ingredients they contain. There are skincare products that go beyond coloring and adorning the skin. These products improve the functioning/texture of the skin by encouraging collagen growth by combating harmful effects of free radicals, thus maintaining keratin structure in good condition and making the skin healthier. Most of the eminent multinational companies engaged in cosmetics productions have introduced a wide range of products having therapeutic effects by addition of a single or more active drug like component to enhance the efficacy of products which later on was found to be beneficiary for the required purpose. These products have also mended some flaws associated with existing products leading consumers to discontinue the use of that product due to allergic reactions or excessive skin drying. Many companies add humectants like glycerin as a soothing agent which increases the skin moisture level and keeps it hydrated. Commonly used substances included in cosmeceutical formulations are described as follows. 4. B. CLASSIFICATIONOF COSMECEUTICAL PRODUCTS The cosmeceuticals are mainly divided into seven categories. In choosing an effective Cosmeceuticals regimen it is critical to match patients and their problems with the appropriate products. Most patients have multiple needs, and they should be matched with products that offer ingredients with multifunctional benefits.CLASSIFICATION OF COSMECEUTICALSAlso It can be broadly classified into following categories according to their daily life uses. SL. NOCATEGORIES1Creams, emulsions, lotions, gels and oils for the skin (hands face, feet, etc.).2Face masks (with the exception of chemical peeling products).3Tinted bases (liquids, pastes, powders).4Make-up powders, after-bath powders, hygienic powders, etc.1|Page 5. 5Toilet soaps, deodorant soaps, etc.6Perfumes, toilet waters and eau de Cologne.7Bath and shower preparations (salts, foams, oils, gels, etc.).8Depilatories.9Deodorants and anti-perspirants.10Hair care products: Hair tints and bleaches,Products for waving, straightening and fixing,Setting products,Cleansing products (lotions, powders, shampoos),Conditioning products (lotions, creams, oils),Hair dressing products (lotions, lacquers, brilliantine).11Shaving products (creams, foams, lotions, etc.).12Products for making-up and removing make-up from the face and the eyes.13Products intended for application to the lips.14Products for care of the teeth and the mouth.15Products for nail care and make-up.16Products for external intimate hygiene.17Sunbathing products.18Products for tanning without sun.19Skin-whitening products.20Anti-wrinkle products.2|Page 6. Mechanisms of current cosmeceutical agents. Alpha-hydroxyacid (AHA), post-inflammatory hyperpigmentation (PIH), ultraviolet light (UV).1. Moisturizers The main cutaneous function of cosmeceuticals is to enhance the barrier function of the skin following a resurfacing procedure. Enhancing the barrier decreases stinging and burning from a sensory standpoint and improves the look and feel of the skin. Moisturizers can smooth down desquamating corneocytes and fill in the gaps between the renaming corneocytes to create the impression of tactile smoothness. This effect is temporary, of course, until the moisturizer is removed from the skin surface by wiping or cleansing. From a functional standpoint. Moisturizers can create an optimal environment for healing and rninirnize the appearance of lines of dehydration by decreasing transepidermal water loss. Trans epidermal water loss increases when the brick-and-mortar organization of the protein -rich corneocytes held together by intercellular lipids is damaged. A well-formulated cosmeceutical moisturizer can decrease the water loss until healing occurs following a resurfacing procedure. There are two cosmeceutical ingredient categories that can reduce transepidermal water loss post-resurfacing: occlusive and humectants. The most common method3|Page 7. for reducing transepidermal water loss is the application of occlusive ingredients in combination with humectant ingredients in a thin moisturizer film.1.1 Occlusive moisturizers Occlusive moisturizers contain oily substances that create a barrier to water evaporation. The most popular and effective occlusive ingredient used following resurfacing is petrolatum. Petrolatum is effective because it blocks 99% of water loss from the skin surface. This remaining 1% transepidermal water loss is necessary 10 provide the cellular message for barrier repair initiation following wounding. If the transepidermal water loss is completely halted. The removal of the occlusion results in failure to repair the barrier. And water loss quickly resumes at its pre-application level. Tubs the occlusion does not initiate barrier repair. Petrolatum does not function as an impermeable barrier. Rather it permeates throughout the interstices of the stratum corneum allowing barrier function to be re-established.1.2 Humectant moisturizers Another concept in rehydrating the stratum corneum is the use of humectants. Humectants have been used in cosmetics for many years to increase shelf life by preventing product evaporation and subsequent thickening due to variations in temperature and humidity. Humectants are a necessary in a11oil-in-water creams to maintain the required water content. Substances that function as humectants include glycerin, honey, sodium lactate. urea. propylene glycol, sorbitol, pyrrolidone carboxylic acid, gelatin, hyaluronic acid, vitarnins and some proteins. Following resurfacing, the barrier has been injured, and water must be drawn to the wounded skin to prevent desiccation, which slows healing. Desiccation of the facial skin also leads to an undesirable sensation of pulling and drawing. Humectants attract water from the deeper epidermal and dermal tissues to rehydrate the stratum corneum. This water is trapped by the occlusive moisturizing ingredients in a thin film on top of the stratum comeum . Humectants mayalso allow the skin to feel smoother by filling holes in the stratum eerneum through swelling. Therefore a good moisturizer should combine both occlusive and humectant ingredients. There is no doubt that most anti-aging cosmeceuticals are primarily well constructed moisturizers containing occlusive and humectant ingredients. Furthermore most of the claims associated with cosmeceuticals are moisturizer claims. Keep in mind that the moisturizer is really the vehicle for transporting the special ingredient to the skin surface but the vehicle may actually be the active ingredient in many cosmeceutical formulations. Thus. cosmeceuticals can be used to aid healing and maintain an excellent result following skin resurfacing. 4|Page 8. 2. Photoprotection Many new developments have occurred in the photoprotection cosmeceutical market to increase both efficacy and cosmetic acceptability. Higher sun protection factor (SPF) formulations are more popular as new sunscreen combinations arise that provide better ultraviolet B (UVB) protection. New methods of increasing the longevity of UVA photoprotectants provide better broad-spectrum protection. These advances have improved the ability of sunscreens to prevent post-inflammatory hyperpigmentation following resurfacing. Sunscreen filters can be classified into two major categoties. Chemical and physical. Chemical sunscreens also known as organic filters, undergo a chemical transformation known as resonance delocalization to absorb UV radiation and transform it to heat. This reaction occurs within the phenol ring, which contains an electron-releasing group in the ortho and/or para position, and is irreversible, rendering the sunscreen inactive once it has absorbed the UV radiation. Physical sunscreens, also known as inorganic filters, are usually ground particulates that reflect or scatter UV radiation absorbing relatively little of the energy. For this reason they have longer activity on the skin surface. The most important protection following facial resurfacing is from UVA radiation which is directly responsible for pigment production. Two important UVA filters include oxybenzone and avobenzone. Oxybenzone provides weak UVA photoprotection below 320 nm. It is commonly combined with avobenzone. Avobenzone is also chemically incompatible with other commonly used inorganic filters, such as zinc oxide and titanium dioxide. Another important UVA photoprotection in facial resurfacing is ecarnsule, better known as Mexoryl". Mexoryl (L'Oreal) was originally developed to stabilize avobenzone. It is used in combination with oxybenzone and octocrylene to provide excellent proteetion from post-inflammatory hyperpigmentation following resurfacing. It is available in two forms: Mexoryl SX and Mexoryl XL. Mexoryl SX is a water-soluble form that is suitable for daywear sunscreen formulations. The inorganic UVA/UVB filters titanium dioxide and zinc oxide are also important in preventing postinflammatory hyperpigmentation following resurfacing. Titanium dioxide is usually micronized to contain particles of many sizes to provide optimal UV scattering abilities. Unfortunately, it leaves a white film on the skin and is used mainly for beachwear sunscreens and not cosmeceuticals. Zinc oxide is usually available in a microfine forrn, meaning it contains small particles of one size making it appropriate for day wear. A newly introduced colorless zinc oxide with extrernely small particles is finding its way into many post-resurfacing cosmeceuticals, but there is concern that the nanoparticles may enter the skin through appendageal structures creating a permanent reservoir. The cosmeceutical industry has placed a voluntary hold on nanoparticle inorganic filters and pigments until the penetration issues are better understood.5|Page 9. Effect of ROS on skin2.1 Luffa cylindrica L. cylindrica (Linn) M. Roem. is a climber with a slender, slightly hairy stem with little furrowing. The seeds of L. cylindrica contain oil in which the fatty acids are stearic and linoleic acids - are unsaturated fatty acids. It has been reported that naturally occurring unsaturated fatty acids and phenolic compounds have free radical scavenging properties In a study by Yoganandam et al , it was concluded that the fixed oil isolated from the seed kernels of the plant not only scavenges off the free radicals but also inhibits generation of free radicals. 6|Page 10. 2.2 Portulaca oleracea Portulaca oleracea (Common Purslane; also known as Verdolaga, Pigweed, Little Hogweed or Pusley) is an annual succulent in the family Portulacaceae, which can reach 40 cm in height. It is found throughout India and the Middle East, but is naturalized elsewhere, and in some regions, is considered an invasive weed. The whole plant is considered antiphlogistic (takes the heat out), a bactericide, antidiabetic, anaphrodisiac (opposite to aphrodisiac), emollient, calmative, diuretic, and refreshing agent. Sanja et al has proved the antioxidant activity of the methanol extract using methods such as DPPH free radical scavenging, reducing power estimation by FeCl3, nitric oxide free radical scavenging superoxide scavenging activity. The extract has a tendency to scavenge the free radicals involved in the ageing process and skin wrinkling and thus may provide some photoprotective action rejuvenate the skin. The fresh gel, juice or formulated products have been used for medical and cosmetic purposes and to enhance general health.2.3 Terminalia chebula Terminalia chebula, also called Harde, belongs to the family Combretaceae. It is used commonly in many Ayurvedic preparations as laxative, diuretic and cardiotonic, as well as in some health supplements. Its chemical constituents include ascorbic acid, gallic acid and ellagic acid, which are well known to exert free radical scavenging properties.2.4 Piper longum Piper longum L. belonging to the family Piperaceae, is commonly found in Indonesia, India and the Phillipines. It consists of a spike of fruits forming a structure about 4 cm long and 6 mm in diameter. The fruit (pepper) contains 1 2.5 % volatile oil, 5 95 % of crystalline alkaloid piperine and piperettine, and a resin. Piperine extracted from this plant has been used as an ingredient in Ayurvedic formulations because of its antioxidant potency both in vitro and in vivo in mice. Piperine, due to this antioxidant property, is used topically in a cream base to treat sunburn diseases.7|Page 11. 2.5 Aloe Vera The leaves of Aloe vera (A. barbadensis) (Fam. Liliaceace) are the source of aloe vera gel. The gel does not include the sap of Aloe vera, which contains anthraquinones. Aloe vera gel is widely used in cosmetics and toiletries for its moisturizing and revitalizing action. The whole leaf of Aloe vera is known to aid cellular repair as well as digestion, assimilation of foods, vitamins, minerals and other vital nutrients to rejuvenate the skin. The fresh gel, juice or formulated products have been used for medical and cosmetic purposes and to enhance general health.2.6 Emblica officinalis Emblica officinalis Gaertn., commonly known as amla, is a rich dietary source of vitamin C, minerals and amino acids. It also contains various phenolic compounds. Amla extract is known to exhibit potent antioxidant properties and to provide protection for human dermal fibroblasts against oxidative stress and therefore, it is assumed to be useful for natural dermal care. Recently, it was reported that amla extract has effect on human skin fibroblasts, especially production of procollagen and matrix metalloproteinases (MMPs). The water extract from dried amla powder contains 2 % ascorbic acid and 29.4 % polyphenols including gallic acid and elaeocarpusin. Amla extract elevates the mitochondrial activity of human skin fibroblasts and promotes production of procollagen. Therefore, due to its potential mitigative, therapeutic and cosmetic applications, amla has been used for skin treatment since ancient times.2.7 Crocus sativus Saffron, the dried stigma of the plant Crocus sativus L, popularly used as a spice and food colorant, has been used in traditional medicine for the treatment of many diseases including tumors. The chemical constituents of saffron include the colored carotenoids - crocin and crocetin - and the monoterpene aldehydes picrocrocin and saffranol. Salomi et al reported the anti-promoting and nonmutagenic activity of saffron extract. Lin et al showed that crocins can protect from the adverse effects of hepatocarcinogenic compounds and that crocetin, the deglycosylated crocin derivative, can inhibit intracellular nucleic acid synthesis. The anticarcinogenic effect of the aqueous infusion of saffron, administered orally, using a two-stage skin carcinogenesis model in mice, has 8|Page 12. been evaluated; the protective role of saffron against carcinogenic exposure was attributed to its action on the physiological detoxification processes and this proved that saffron can prevent chemically-induced skin carcinogenesis in Swiss albino mice.2.8 Peumus boldus Molina Peumus boldus belonging to the family Monimiaceae (a tree whose leaves have been traditionally employed in folk medicine) is now widely recognized as an herbal remedy by a number of Pharmacopoeias. Its leaves are rich in several aporphine-like alkaloids, among them, boldine, which is the most abundant one. Research conducted during the early 1990s claimed that boldine is one of the most potent natural antioxidants. Its pharmacological actions, which arise from its antioxidant properties, includes cytoprotective, anti-tumor promoting, antiinflammatory, anti-diabetic and antiatherogenic actions; it has also shown some actions which do not seem to be associated with these activities, e.g., vasorelaxing, anti-trypanocidal, immuno- and neuromodulator, cholagogic and/or choleretic actions. Free radicals are known to participate in either the aetiology or the development of most UV-induced skin lesions. Through the experiments conducted on boldine, it has been proved that boldine has a UV light-filtering property relevant to a photoprotective action. In fact, Hidalgo et al showed boldine to be photo-unstable when irradiated at wavelengths up to 300 nm and to display a photo-protector effect against UV-B, both in vitro and in vivo in mice. Photo-protection was evidenced by the prevention of UVinduced increase in the skin temperature of rodents.3. Sunscreen Agents Use of sunscreen agents and limiting the exposure to sun prevents early wrinkling and skin cancer. Sunscreen agents are used to prevent sunburns. There are two kinds of sunscreen agents: chemical and physical. Chemical sunscreen agents protect the skin from the sun by absorbing the ultraviolet (UV) and visible sun rays, while 9|Page 13. physical sunscreen agents reflect, scatter, absorb, or block the rays. Sunscreen agents often may comprise more than one ingredient. For example, products may contain an ingredient that provides protection against the ultraviolet A (UVA) sun rays and another ingredient that protects from the ultraviolet B (UVB) sun rays, which are more likely to cause sunburns than the UVA sun rays. Ideally, coverage should include protection against both UVA and UVB sun rays. The sun protection factor (SPF) that is present on the label of these products reflects the minimum amount of UVB sunlight that is needed with that product to produce redness on sunscreen protected skin as compared with unprotected skin. Sunscreen products with high SPFs provide more protection against the sun. The following sunscreen agents have been recommended by the U.S. Department of Health: Cycloform (isobutyl pamino benzoate) Propylene glycol pamino benzoate Monoglyceryl pamino benzoate Digalloyl trioleate Benzyl salicylate and benzyl cinnamate (2% each) Besides these, chemical sunscreens mainly based on paraamino benzoic acid, its deivatives, cinnamates, various salicylates and benzophenones, dibenzoylmethanes, anthraline derivatives, octocrylene and homosalate are frequently employed as sun blocking agents. Direct physical blockers include metal containing compounds such as iron, zinc, titanium, and bismuth. Zinc oxide and titanium dioxide are highly reflective white powders, but submicron zinc oxide or titaniumdioxide powder particles transmit visible light while retaining their UV blocking properties, thus rendering the sun block invisible on the skin.3.1 Vitamins Exposure to the UV radiations accelerates the aging effect of the skin. The progressive telomere shortening and finally its disruption by lowgrade oxidative damage are related to the aging. Damage is initiated by the generation of reactive oxygen species (free radicals). It is a progressive process whose consequences are damage to DNA. The topical treatment of acne vulgaris with vitamin A, is very well supported by evidence. Vitamin B3, commonly known as nicotinamide or niacinamide, is available in cosmetic and cosmeceutical products and can be used as a complementary agent for some types of acne, as well as aging skin. Activation of tolllike receptors may also be involved in the scarring process by activating the metalloproteinases the retinoids arem vitamin A derivatives constituting the most effective comedolytic agents. They function by normalizing desquamation of the follicular epithelium, preventing the formation of new microcomedo, and minimizing the formation of comedones and10 | P a g e 14. inflammatory lesions. Nicotinamide is useful as a complementary drug because of its mild antiinflammatory activity and its possible action in the reduction of sebum production and improvement of the skin barrier.3.2 Vitamin A The human epidermis contains significant amounts of vitamin A (alltrans retinol). It has been observed that both UVB and UVA can damage the metabolism and transport of vitamin A. This may lead to vitamin A deficiency in the skin.13 Small amounts of retinol in the body gets converted to alltrans retinoic acid also called tretinoin (active form) & rest of the retinol is converted into retinyl ester (storage form). Topical retinoids have successfully been used to treat acne. The efficacy of topical tretinoin in the treatment of photoaged and intrinsically aged skin is sufficiently established. The effects are believed to be mediated through its binding to the nuclear retinoid acid receptors. It induces type I and type III procollagen gene expression in human skin, resulting in increased deposition of collagen fibrils in the dermis. The effects result in an improvement in the clinical and histologic skin appearance. Tretinoin cream in the appropriated concentrations of 0.025%, 0.05%, and 0.1%, as well as 0.1% isotretinoin and 0.1% tazarotene, frequently produce moderate to severe skin irritation. Retinaldehyde (0.05%) is another useful topical agent for the treatment of photoaged skin. It has a lower frequency of irritation but less efficacy than tretinoin.16 Photosenstivity is another problem to be dealt in case of tretinoin. The useful concentration of topical retinol ranges from 0.3% to 1%. Most of the overthecounter products available usually contain lower levels of retinol (about 0.08% or less), compared with the concentration used in the few clinical studies available. Vitamin A (retinol) is the prototype of all other retinoids and is necessary for proper growth, bone development, and integrity of mucosal and epithelial surfaces. In vitamin A deficiency, the eyes and the skin are severely affected. The conjunctiva and the cornea develop metaplasia and keratinization, leading to night blindness. Vitamin A exists in three isomeric forms among which beta form found to be more active than alpha & gamma isomer. Its deficiency may lead to dry rough skin. The advent of synthetic analogs of vitamin A in the 1970s brought new interest into their biological activity, especially on the skin. Since then, vitamin A and its derivatives have been useful in the treatment of many skin disorders, including ichthyosis, acne, and psoriasis.3.3 Vitamin E The physiological function of vitamin E, if applied dermally is to contribute to the antioxidant defense of the skin, because of its tendency to absorb UV light in the solar spectrum region that is responsible for most of the harmful biologic effects of 11 | P a g e 15. the sun18. Vitamin E blocks lipid peroxidation in cells & tissues & it is a good antioxidant. It helps to enhance the performance of UV filters, softens skin & moisturizes within. Vitamin E is the body's major lipidsoluble antioxidant, if oxidized, vitamin E can be regenerated back to its reduced form by Lascorbic acid. Vitamin E as alphatocopherol or tocopherol acetate is used in topical OTC (overthe counter) products in concentration (1% 5%). Alphatocopherol has been found to be beneficial in reducing minimal erythema and the number of epidermal sunburn cells, which marks skin damage related to oxidative stress caused by UVB19. The effect of vitamin E after sun exposure seems to have no benefit. Vitamin E can reduce UVinduced erythema and edema when it is applied before UV exposure. Topical application of vitamin E may increase stratum corneum hydration and enhance water binding capacity. Alphatocopherol also shows synergistic effect with vitamins A (retinol) and C (ascorbic acid) in combined products, providing an appreciable photo protection and antioxidant action that suggests a potential effect in the protection against photo aging and skin cancer. Nanoemulsions have been formulated containing palm oil esters in water with vitamin E and are promising potential nanocosmeceuticals.3.4 Vitamin C The increase of vitamin C in skin concentration is limited even with huge oral supplementation. Vitamin C has become a popular topically applied cosmeceutical because topical application of Lascorbic acid is the only way to further increase skin concentration. Free radical scavengers have grabbed the attention of researchers on vitamin C. Lascorbic acid is the active form of vitaminC, which was first used as cosmeceutical creams. Previous formulations (Lascorbic acid) were very unstable due to the oxidation of the vitamin exposed to air. So to overcome this problem, esterified derivatives of Lascorbic acid in topical formulations have been used to improve stability. The most commonly used derivatives are ascorbyl 6palmitate and magnesium ascorbyl phosphate. Skinlightening effect has been observed by the demonstration of Magnesium ascorbyl phosphate.4. Skin Lightening Agents Hyperpigmentation is the changing of colour intensity of the skin to darker hue, which is due to an increased amount of melanin in the epidermis, the dermis, or both. This change can be due to 2 pathophysiologic processes: melanocytosis (increased number of melanocytes) and melanosis (increased amount of melanin). Skin lightening agents work best when melanosis or melanocytosis is confined to the epidermis. Patients with Fitzpatrick skin types III have advantage over type IV such as type IIII benefit from local pigment lightening for the treatment of 12 | P a g e 16. hormonally induced melasma and postinflammatory hyperpigmentation caused by acne and trauma, whereas those with Fitzpatrick skin types IV and darker may also seek therapy for pigmentary changes that occur around the eyes, in the intertriginous areas, following dermatitis, or with acne and trauma. Standard dermatologic agent for skin lightening is hydroquinone but its safety is questionable, leading to the use of alternative agents such as retinoids, mequinol, azelaic acid, arbutin, kojic acid, aleosin, licorice extract, ascorbic acid, soy proteins, and N acetyl glucosamine.4.1Ascorbic acid Ascorbic acid, also known as vita min C, is used in cosmeceuticals for the treatment and prevention of hyperpigmentation because it interrupts melanogenesis by interacting with copper ions to reduce dopaquinone and block dihydrochinindol-2-carboxyl acid oxidation. Ascorbic acid, an antioxidant, is rapidly oxidized when exposed to air, with limited stability. For this reason. Many cosmeceuticals are using the more stable magnesium ascorbyl phosphate. Which is metabolized to ascorbic acid in the skin. High concentrations of ascorbic acid must be used with caution, however, as the low pH can be irritating to the skin. Pigment-lightening cosmeceuticals may contain ascorbic acid as a pH adjustor or to function as an antioxidant preservative. It is important to recognize that ascorbic acid is a multifunctional ingredient with very minimal pigmentlightening capabilities.4.2 Licorice extract Licorice extracts are found in cosmeceuticals to decrease facial redness and reduce pigmentation. The extract contains liquiritin and isoliquertin. Which are glycosides containing flavonoids, which induce skin lightening by dispersing melanin. To see clinical results. The liquiritin must be applied in the dose of 1 g per day for 4 weeks. Irritation is not a side effect, as is so frequently observed with hydroquinone and ascorbic acid, but efficacy is Minimal.13 | P a g e 17. 4.3 Alpha-lipoic acid Alpha-lipoic acid is found in a variety of anti-aging cosmeceuticals ramfunction as an antioxidant, but it mayalso have very lirnited pigmentlightening properties. It is a disulfide derivative of octanoic acid that is able to inhibit tyrosinase. However, it is a large molecule and cutaneous penetration to the level of the melanocyte is challenging, significantly reducing its efficacy.4.4 Kojic acid Kojic acid, chemically known as 5-hydroxymethyl-4H-pyrane-4-one, is one of the most popular cosmeceutical skin-lightening agents found in cosmetic-counter skinlightening creams distributed worldwide. It is a hydroph.ilic fungal derivative obtained from Aspergillus and Penicillium species. It is the agent most commonly employed in vast Asia for the treatment of melasma, but it is highly unstable. Newer formulations have incorporated kojic dipalmitate but the efficacy of this derivative has not been well studied. Some research indicates that kojic acid is equivalent to hydroquinone in pigmentlightening ability. The activity of kojic acid is attributed to its ability to prevent tyrosinase activity by binding to copper.4.5 Aleosin Aleosin is a low-molecular-weight glycoprotein obtained from the aloe vera plant. It is a natural hydroxymethylchromone functioning to inhibit tyrosinase by competitive inhibition at the DOPA oxidation site. In contrast to hydroquinone, it shows no cell cytotoxicity, but it has limited ability to penetrate the skin due to its hydrophilic nature. The effects of aleosin have been largely demonstrated in pigmented skin equivalents. Not human-use studies. It is sometimes mixed with arbutin to enhance its Skin-lightening abilities.4.6 Arbutin Arbutin. Chemically known as 4-hydroxyphenyl-beta-glucopyranoside, is obtained from the leaves of the Vaccinium vitis-idaea and other related plants. It is a naturally occurring gluconopyranoside that causes decreased tyrosinase activity without affecting messenger RNA expression. It also inhibits melanosome maturation. Arbutin is not toxic to melanocytes and is used in a variety of pigmentlightening preparations in Japan at concentrations of 3%. Higher concentrations 14 | P a g e 18. are more efficacious than Jower concenrrauons, but a paradoxical pigment darkening may occur. Arbutin-betaglycosides have been produced that are less cytotoxic than arbutin.4.7 Hydroquinone The gold standard for the treatment of hyperpigmentation in the USA remains hydroquinone. This substance is actually quite centroversial having been removed from the aTC markets in Europe and Asia. Concern arose because oral hydroquinone has been reported to cause cancer in mice fed large amounts of the substance. While oral consumption probably is not related to topical application. Hydroquinone remains controversial because, it is toxic to melanocytes. Hydroquinone, a phenolic compound chemically known as lA-dihydroxybenzene, functions by inhibiting the enzymatic oxidation of tyrosine and phenol oxidases. It covalently binds to histidine or interacts with copper at the active site of tyrosinase. It also inhibits RNA and DNA synthesis and may alter melanosome formation. Thus selectively damaging melanocytes. These activities suppress the melanocyte metabolic processes. Inducing gradual decrease of melanin pigment production. Hydroquinone is still the best option for lightening post-inflammatory hyperpigmentation following resurfacing.5. Antioxidants Antioxidants form one of the most popular categories of cosmeceutical ingredients. This is due tGthe fact that the major cause of cutaneous aging is oxidation of skin structures from highly reactive oxygen molecules present in our oxygen-rich environment. It is amazing tG think that the life-giving oxygen required to survive is also the same oxygen responsible for aging the human body. The primary souree of cosmeceutical antioxidant ingredients is botanical extracts, since all plants must protect themselves from oxidation following UV exposure. Antioxidant botanieals function by quenching singlet oxygen and reactive oxygen species, such as superoxide anions. hydroxyl radicals fatty peroxy radicals and hydroperoxides. There are many botanical antioxidants available. Both from raw-material suppliers and from the cosmeceutical industry and they can be classified in three categones, Carotenoids, flavonoids and polyphenols. Carotenoids are chemically related to retinoids, while flavonoids possess a polyphenolic structure that accounts for their antioxidant, UVprotectant, and metal chelation abilities. Lastly, polyphenols represent a chemical subset of flavonoids. Antioxidants are found in many skin lines for use following resurfacing procedures. They are typically placed in moisturizing vehicles that may aid in healing through the prevention of transepidermal water loss. Whether Cosmeceuticals the antioxidant formulation extends the effect of a resurfacing procedure has never been documented, yet their frequent use demands a thorough understanding of their function. 15 | P a g e 19. 5.1 Carotenoids Carotenoids are derivatives of vitamin A and have found widespread use in cosmeceuticals due to the established topical anti-aging benefits associated with the prescription retinoid tretinoin. The carotenoids are a large family of orange, red, and yellow substances that perform vital antioxidant roles when ingested and are less well established as topical antioxidants. The carotenoids are discussed in detail here.5.2 Astaxanthin Astaxanthin is a pink carotenoid found in high concentration in salmon accounting for the characteristic pink color of the fish. This is the rationale for anti-aging diets recommending the ingestion of a serving of salmon fivemtimes weekly. For topical application purposes. Astaxanthin is obtained from the marine rnicroalgae Haematococcus pluvialis. The efficacy of astaxanth in is attributed to its cell mernbrane composed of two external lipid layers, which has been touted to possess stronger antioxidant abilities than vitamin E. Few studies exist to confirm the topical effect of astaxanthin, but it has been studied extensively as an oral supplement. Astaxanthin in concentrations of 0.03-0.07% produces a pink-colored cream. This limits the concentration that can be used, but no topical adverse reactions have been associated with this carotenoid. The topical antioxidant benefits of astaxanthin have not been established.5.3 Lutein Another carotenoid found in topical cosmeceuticals is lutein. It is naturally found in green leafy vegetables. Such as spinach and kale. Lutein is an antioxidant in the plant kingdom, also being used for blue light absorption, in the animal kingdom, lutein is found in egg yolks, animal fats and the corpus luteum. It is a lipophilic molecule, not soluble in water, characterized bya long polyene side chain composed of conjugated double bonds. These double bonds are degraded by light and heat, a universal characteristic of carotenoids to a greater or lesser degree. The topical value of lutein in wound healing has never been evaluated.5.4 Lycopene Lycopene is a potent carotenoid found in most fruits and vegetables with .a red color including tornatces, waterrnelon, pink grapefruit, papaya, gac, red beli pepper and pink guava. Lycopene is a highly unsaturated hydrocarbon containing 11 conjugated 16 | P a g e 20. and two unconjugated double bonds which makes it a longer molecule than any other carotenoid. This makes its absorption into the skin doubtful. It undergoes cis-isomerization when exposed to sunlight. Even though lycopene was the new oral supplement added to many commercial multivitarnins this year its topical value has never been documented. It is safe for skin application, but may stain the skin in high concentration.5.5 Retinol Of all the topical carotenoids retinol is the best understood. Since it is necessary for vision and possesses a weli-characterized skin receptor. It is theoretically possible to interconvert the retinoids from one form to another. For exarnple. retinyl palmitate and retinyl propionate. Chemically known as retinyI esters, can become biologically active following cutaneous enzymatic cleavage of the ester bond and subsequent conversion to retinol. mRetinol is the naturally occurring vitamin A form found in red, yellow, and orange fruits and vegetables. It is the pigment responsible for vision but is highly unstable. Retinol can be oxidized to retinaldehyde and then oxidized to retinoic acid, also known as prescription tretinoin. It is this cutaneous conversion of retinol to retinoic acid that is responsible for the biologie activity of some of the new stabilized OTC vitamin A preparations designed to irnprove the appearance of benign photodamaged skin. Unfortunately, only small amounts of retinyl palrnitate and retinol can be converted by the skin, accounting for the increased efficacy seen with prescription preparations containing retinoic acid. The main problem with prescription retinoids is their irritancy. Unfortunately, as the biological efficacy of the retinoid increases, so does the irritancy. This is also the case with the OTC retinoids. Retinol is more irritating than the retinyl esters and also more unstable. It is for this reason that cosmeceutical formulations not manufactured under strict oxygenfree conditions prefer to add retinyl palrnitate to moisturizing creams. However, the retinyl palrnitate may act as an antioxidant for the lipids present in the moisturizer. The topical benefit of retinol has been documented by weU-controlled studies. It is commonly felt among dermatologists that retinol is of benefit, but it is difficult in moisturizer studies that do not include vehicle control to separate the retinol benefit from the moisturizer benefit. Nevertheless of all the carotenoids available for Iermulation. Retinol has the most evidence to support topical application efficacy following resurfacing.5.6 Flavonoids Flavonoids are aromatic compounds, frequently with a yellow color that occur in higher plants. Five thousand flavonoids have been identified with a sirnilar chemical structure, possessing 15 carbon atoms and a variety of biologie activities. Flavonoids can be divided into flavones. Flavonols, isoflavone and flavones. Each with a slightly different chemical structure. Currently, the most 17 | P a g e 21. common isoflavone incorporated into cosmeceuticals are daidzein and genistein, derived from soybeans. Other sourees of flavonoids include curcumin, silyrnarin. pycnogenol, and gingko.5.7 Soy The soybean-derived isoflavones genistein and daidzein function as phytoestrogens when orally consumed, and have been credited with the decrease in cardiovascular disease and breast cancer seen in Asian women. These isoflavones are present when the soy is fermented. Some of the cutaneous effects of soy have been linked to its estrogenic effect in postmenopausal wornen. Topical estrogens have been shown to increase skin thickness and promote collagen synthesis [35]. It is interesting to note that gemsrein increases collagen gene excressicn in cell culture, out there are no published reports of this collagen-stimulating effect in topical human trials. Genistein has also been reported to function as a potent antioxidant, scavenging peroxyl radicals and protecting against lipid peroxidation in vivo.5.8 Curcumin Curcumin is a popular natural yellow food coloring used in everything from prepackaged snack foods to rneats. It is sometimes used in skin-care products as a natural yellow coloring in products that claim to be tree of artificial ingredients. Curcumin comes from the rhizome of the turmeric plant and is consumed orally as an Asian spice, frequently found in rice dishes to color the otherwise white rice yellow. However, this yellow color is undesirable in cosmetic preparations. Since yellowing of products is typically associated with oxidative spoilage. Tetrahydrocurcumin, a hydrogenated form of curcumin, is off-white in color and can be added to skincare products not only to function as a skin antioxidant but also to prevent the lipids in the moisturizer from becoming rancid, the antioxidant effect of tetrahydrocurcumin is said to be greater than vita min E by cosmetic chemists. It is said to provide antioxidant skin benefits by quenching oxye-gen radicals and inhibiting nuclear factor-KB.5.9 Silymarin Silymarin is an extract of the milk thistle (Silybum marianums, which belongs to the aster family of plants including diasies, thistles. and artichokes. The plant is named milk thistle because the oldest recorded use of the extract was to enhance human lactation, and because the plant produces a white milky sap. The extract consists of three flavonoids derived from the fruit, seeds, and leaves of the plant. These 18 | P a g e 22. flavonoids are silybin, silydianin, and silychristin. Homeopathically, silymarin is used to treat liver discase. But it is a strong antioxidant, preventing lipid peroxidation by scavenging free radical species. Its antioxidant effect have been Cosmeceuticals demonstrated topically in hairless mice by a 92% reduction in skin tumors Icllowing UVB exposure. The mechanism for this decrease in tumor production is unknown but topical silymarin has been shown to decrease the formation of pyrimicline dimers in a mouse model. It has also been found to improve the healing ofburns in albino rats, which is the rationale for its incorporation into some wound-healing and postresurfacing preparations. Silymarin is found in a number of high-end moisturizers for benign photoaging to prevent cutaneous oxidative damage and to re duce facial redness. A double-blind placebo-controlled study in 46 subjects with stage I-UI rosacea found improvement in skin redness, papules, itching, hydration and skin color. This was felt to be due to its direct activity on modulating cytokines and angiokines.5.10 Pycnogenol Pycnogenol is an extract of French marine pine bark iPinus pinasters. The extract is a water-soluble liquid containing several phenolic constituents, inelucling taxifolin, catechin, procyanidins. It also contains several phenolic acids inelucling phydroxybenzoic. Protocatechuic, gallic, vanillic, p-couric, caffeic, and ferulic. It is a trademarked ingredient, a potent tree radical scavenger that can reduce the vita min C radical returning the vitamin C to its active form (VI, C). The active vita min C in turn regenerates vitamin E to its active Iorrn, maintainlng the natural oxygen-scavenging mechanisms of the skin intact. Pycnogenol is the ideal anti-aging additive since it demonstrates no chronic toxicity no mutagenicity, no teratogenicity, and no allergenicity. In B16 melanoma cells. It was shown to inhibit tyrosinase activity and melanin biosynthesis. Many discussions of antioxidant flavonoids include a mention of pycnogenol, but few high-quality data are presented.5.11 Ginkgo Ginkgo biloba, also named the maidenhair tree, is the last member of Ginkgoaceae family which grew on earth some 200-250 million years ago. For this reason, ginkgo contains flavonoids not found in other botanieals. In possesses bilobalide (a sequiterpene), ginkgolides (diterpenes with 20 carbon atoms), and other aromatic substances such as ginkgol, bilobdol, and ginkgolic acid. It is a plant with numerous purported benefits and has been a common part of homeopathic medicine in East Asia for 4000 years. The plant leaves are said to contain unique polyphenols such as terpenoids 19 | P a g e 23. (ginkgolides. bilobalides) flavinoids and flavonol glycosides that have antlinflammatory effects. These antiinflammatory effects have been linked to anti-radical and anti-lipoperoxidant effects in experimental fibroblast models. Ginkgo flavonoid fractions containing quercetin, kaempferol, sciadopitysin, ginlegetin, and isoginkgetin have been demonstrated to induce human skin fibroblast proliferation in vitro. Increased collagen and extracellular fibronectin were also demonstrated by radioisotope assay. Ginkgo extracts are therefore added to many cosmeceuticals to function as antioxidants and promoters of collagen synthesis following resurfacing, based on non-human mode Is of oxidative damage. Polyphenols Polyphenols are a subset of flavonoids used in many cosmeceuticals. Two main sourees of polyphenols are teas and fruits. This section presents green tea and pomegranate as examples of the evidence available to support polyphenol biologie activity.5.12 Green tea Tea (Camellia sinensis) is botanical popular in East Asia for 5000 years. Used both topically and orally. There are several different types of teas: green, black, oolong and white. The different teas come from the same plant, but different processing imparts different properties. Green tea is made from unfermented tea leaves and contains the highest concentration of polyphenol antioxidants. Green tea can be easily added to topical creams and lotions designed to combat the signs of photoaging but it must be stabilized itself with an antioxidant, such as butylated hydroxytoluene. A topically applied green tea Cosmeceuticals extract containing GTP ((-)-epigallocatechin- 3-gallate) was found to reduce UVB-induced inflammation as measured by double skin-Iold swelling. They also found proteetion against UVinduced edema, erythema, and antioxidant depletion in the epidermis. This was further investigated by applying GTP 10 the back of humans 30 minutes prior 10 UV irradiation, which resulted in decreased myeloperoxidase activity and decreased infiltration of leukocytes as compared to untreated skin. The application of topical green tea polyphenols prior to UV exposure has also been shown to decrease the formation of cyclobutane pyrimidine dimers. These dimers are critical in initiating UV-induced mutagenesis and carcinogenesis which represent the end stage of the aging process. Thus green tea polyphenols can function topically as antioxidants, antiinflammatories, and anti-carcinogens, making them a popular cosmeceutical additive.5.13 Pomegranate Pomegranate iPunica granatum) is a deciduous tree bearing a red fruit native to Afghanistan, Pakistan, Iran, and northern India. It was brought to California by the Spanish settlers in 1769 and is commercially cultivated for its juice. Pomegranate juice commonly consurned in the Middle East, provides about 16% of the adult requirement of vitamin C per 100 mg serving. It also contains pantothenic acid, 20 | P a g e 24. also known as vita min B5, potassium. And antioxidant polyphenols. These substances have been demonstrated to proteet against UVA- and UVB-induced cell damage in SKU-1064 human skin fibroblasts. Pomegranate juice has also been purposed to reduce oxidative stress and to affect low-density lipoprotein (LDL) and platelet aggregation in humans and apolipoprotein E-deficient mice. It has also been studied for improving hyperlipidemia in diabetic patients. It is found in some wound-healing preparations to promote healing, but its value has never been demonstrated.6. Other antioxidants used in post-resurfacing Other antioxidants are commonly found in commercial preparations for use after resurfacing procedures. These ingredients are used to promo te healing, and they include aloe vera and ubiquinone.6.1 Aloevera Probably the most widely used cutaneous botanical anti-inflarnmatory is aloe vera. The mucilage is released from the plant leaves as a colorless gel containing 99.5% water and a complex mixture of mucopolysaccharides, amino acids hydroxy quinone glycosides and minerals. Compounds isolated from aloe vera juice inelude aloin, aloe emodin, aletinic acid, choline, and choline salicylate. Reponed cutaneous effects of aloe vera inelude increased blood flow, reduced inflammation, decreased skin bacterial colonization, and enhanced wound healing. The anti-inflarnrnatory effects of aloe vera may result from its ability to inhibit cyclooxygenase as part of the arachidonic acid pathway. Studies have evaluated the effect of aloe vera on burn wounds and acne. Aloe vera cream was found to show no tanning or sunburn proteetion and no efficacy in sunburn treatment as compared to placebo. Rueter and colleagues studied a 97.5% concentration of aloe vera for its antiinflammatory effect and demonstrated positive results in a sunburn cell assay as compared to 1% hydrocortisone. These data provide evidence for the anti-inflammatory effect of pure aloe vera gel. However, most products sold over the counter for under $10 do not contain a high enough percentage of aloe vera to induce clinically relevant inflammation reduction.6.2 Ubiquinone An endogenous antioxidant that has been incorporated into antiaging moisturizers is ubiquinone, also known as Coenzyme Q10 or CoQ10. For a topical I antioxidant to be clinically effective, it must penetrate into the skin. Hoppe and colleagues from Beiersdorf demonstrated the topical penetration of ubiquinone into the viable epidermis and a reduction in oxidation as measured by weak photon emission. They were also able to show a 21 | P a g e 25. significant decrease in the expression of collagenase in human dermal fibroblasts following UVA radiation and improvement in orbital wrinkling. However, oral supplementation had no effect on the main antioxidant defenses or pro-oxidant generation in tissues in mice. It also did not affect the life span in mice, according to Shoal and colleagues. In a .human study, Passi et al. administered 50 mg vitamin E, 50 mg Coenzyme Q10, and 50 mg selenium. An increase in stratum corneum Coenzyme QI0 was noted after 15 and 30 days of ingestion, but the significance of this finding was not evaluated. Other evidence suggests that topical Coenzyme QI0 may provide additive antioxidant benefits when combined with the colorless carotenoids phytoene and phytofluene. This effect was demonstrated in fibroblast cultures.7. DEPIGMENTATION AGENTS Since the introduction of hydroquinone as a skin lightening agent in 1961, several products with bleaching properties have been used for the treatment of pigmentary disorders of the skin. The most important medical indications for the use of these agents are melasma and postinflammatory hyperpigmentation, although they have also been used as alternative options for the treatment of ephelides, solar lentigenes, nevi, and lentigo maligna. This article reviews the most commonly used bleaching agents, discusses their mechanism of action, and focuses on their efficacy and safety in treating unwanted skin pigmentation. Based on our current knowledge, the ideal bleaching agent has to fulfill certain pharmacologic criteria. It should have a potent bleaching effect with a rapid time of onset (less than 2 to 3 months), carry no short- or long-term side effects, and lead to a permanent removal of undesired pigment. Most of the currently available bleaching or depigmenting agents cause a temporary removal of hyperpigmentation, which usually recurs after discontinuation of therapy. Presently, there are three categories of bleaching agents, phenolic compounds, nonphenolic compounds, and combination formulas.7.1 Phenolic Compounds These chemical compounds contain a phenol group. The most important agent of this group is hydroquinone (HQ), which is considered the most commonly prescribed bleaching agent today. HQ derivatives are the monobenzyl ether of hydroquinone, the 4-methoxyphenol, the 4-isopropylcatechol, the 4-hydroxyanisol, and the N-acetyl-4-S-cystaminylphenol. Hydroquinone HQ, a hydroxyphenolic chemical compound, inhibits the conversion of dopa to melanin by inhibiting the tyrosinase enzyme. These cytotoxic compounds are responsible for the destruction of pigment cells, which results in skin depigmentation. However, cells are capable of protecting themselves against cytotoxic agents by intracellular glutathione (GSH). This protection takes place under the enzymatic action of the detoxification enzyme glutathione S-transferase (GST), which is responsible for the 22 | P a g e 26. conjugation of toxic species to GSH. The depigmenting effect of hydroquinone is shown to be potentiated by buthionine sulfoximine (BSO) and cystamine as the result of the reduction of intracellular levels of GSH by these two agents. Additionally, BSO and cystamine are shown to inhibit the activity of GST. The combination of alltrans-retinoic acid (tretinoin, TRA) with hydroquinone or 4-hydroxyanisole is also known to produce synergetic skin depigmentation. TRA serves as a potent inhibitor of mammalian GSTs and is known to make cells more susceptible to the cytotoxic effect of chemicals by inhibiting the activity of this enzyme. This agent is also shown to reduce the level of intracellular GSH in certain cells. We have proposed that the mechanism of action of TRA to synergistically enhance the melanocytotoxic effect of chemicals involves the inhibition of GST and the impairment of glutathione-dependent cytoprotection against melanocytotoxic agents.7.2 B Nonphenolic Compounds 7.2.1 Azelaic Acid Azelaic acid (AZA) is a naturally occurring 9-carbon dicarboxylic acid that was isolated recently from cultures of Pityrosporum ovale and was associated with the hypomelanosis seen in tinea versicolor. It has been shown to have beneficial therapeutic effects in acne vulgaris and certain pigmentary disorders, such as melasma and lentigo maligna. AZA interferes with the function of tyrosinase in vitro and may also inhibit DNA synthesis and mitochondrial oxidoreductase. It does not appear to affect normal melanocytes, and treatment of constitutively pigmented normal skin, freckles, lentigenes, and nevi with AZA did not produce a significant therapeutic result. The drug, however, appears to exert an antiproliferative and cytoxic effect on hyperactive and abnormal melanocytes and may halt the progression of lentigo maligna to lentigo maligna melanoma. AZA has been used at concentrations of 1520% for the treatment of melasma and postinflammatory hyperpigmentation. In double-blind comparative studies, topical AZA achieved a good-toexcellent response in 6070% of melasma patients, and was found to be more effective than 2% HQ and of equivalent efficacy to 4% HQ. A combination regimen of AZA with topical tretinoin 0.05% for the treatment of melasma produced similar results after 6 months compared with AZA monotherapy (approximately 73% of goodto-excellent improvement), although the addition of tretinoin was associated with an earlier and more pronounced lightening of melasma pigmentation and a greater percentage reduction of lesion size during the early phase of treatment. These results suggest that tretinoin augments the efficacy of AZA. In another clinical study, the addition of 1520% glycolic acid lotion to AZA cream 20% was as effective as 4% HQ cream for the treatment of facial hyperpigmentation in darker-skinned individuals. At the usual concentrations, AZA is well tolerated in humans. Adverse reactions such as pruritus, transient erythema, scaling, and irritation are usually mild and subside 23 | P a g e 27. within 2 to 4 weeks. Allergic sensitization and phototoxic reactions can occur, but are rare. No systemic toxicity has been reported.7.2.2 Tretinoin The use of tretinoin for the treatment of cutaneous hyperpigmentation was introduced by Kligman in 1975, stemming from his observation that acne patients receiving tretinoin developed lighter skin after months of treatment. Tretinoin has been shown in vitro to inhibit both constitutive and induced melanin formation in melanoma cell lines. In vivo, tretinoin enhances the epidermal cell turnover, decreasing the contact time between keratinocytes and melanocytes and promoting the rapid loss of pigment through epidermopoesis. Tretinoin has been used in concentrations from 0.0250.1% to treat a variety of pigmentary disorders. The bleaching action is usually very protracted and occurs anywhere within 12 to 44 weeks of continuous daily application. Comparative studies in melasma patients have shown a 6873% degree of improvement by tretinoin at 40 weeks of treatment compared with a control group. Moderate cutaneous side effects, such as erythema and desquamation, are observed in the majority of patients, and in some cases a more severe dermatitis as well as distressing hyperpigmentation have been reported. When prescribing tretinoin, the use of sunscreen is rather important to counteract the possibility of sunburn and photo damage.7.2.3 L-ascorbic Acid Vitamin C, or L-ascorbic acid (AsA), interferes with pigment production at various oxidative steps of melanin synthesis, for example 5, 6 dihydroxyindole oxidation. AsA has a reducing effect on o-quinones and oxidized melanin and it can alter melanin from jet black to light tan. A disadvantage of AsA is its chemical instability in aqueous solution where it becomes quickly oxidized and denatures. AsA esters have been tested in an effort to overcome this problem. A stable derivative of AsA, magnesium L-ascorbyl-2-phosphate (VC-PMG), was used in a 10% cream base and produced a significant lightening effect in 19 of 34 patients with melasma after 3 months of twice daily application.7.2.4 Kojic Acid Kojic acid (5-hydroxy-2-hydroxymethyl-4-H-pyran-4- one) is a fungal metabolic product that is structurally related to maltol. It is a potent tyrosinase inhibitor and functions by chelating copper at the active site of the enzyme. It also acts as an antioxidant and prevents the conversion of the o-quinones of DL-DOPA, norepinephrine, and dopamine to their corresponding melanin. Kojic acid is used in a 14% cream base, alone or 24 | P a g e 28. in combination with tretinoin, hydroquinone, and/or a corticosteroid. It appears to act synergistically with glycolic acid. The addition of 2% kojic acid in a gel containing 10% glycolic acid and 2% HQ was superior to the same gel without kojic acid in improving the epidermal melasma of 40 women after 12 weeks of treatment. Compared with 2% HQ, kojic acid alone appears to be less effective. There are scant data from the literature with regard to its long-term side effects, al-though some investigators have reported a high frequency of contact sensitivity to this product.7.2.5 Arbutin Tyrosinase is the enzyme for conversion of the substrate tyrosine to melanin in melanocytes, providing pigmentation to skin. Various compounds that bind to the active site of tyrosinase to inhibit its activity have been developed as agents to lighten skin and ameliorate unwanted pigmentation. These agents include hydroquinone, kojicacid,andarbutin,amongstothers.Arbutinisderivedfromtheleavesof bearberry, cranberry, mulberry or blueberry shrubs, and also is present in most types of pears. It can have melanin-inhibiting properties. Arbutin and other plant extracts are considered safe alternatives to commonly used depigmenting agents to make the skin fairer. Medical studies have shown the efficiency of Arbutin for skin lightening. There are patents controlling its use for skin lightening. Arbutin actually exists in two conformations, alpha and beta. The alpha conformation offers higher stability over the beta conformation and is the preferred form for skin lightening indications.8. Hydroxy Acids Hydroxy acids are organic carboxylic acids classified into alphahydroxy acids (AHA), betahydroxy acids (BHA), polyhydroxy acids, and bionic acids on the basis of their molecular structure. Hydroxy acids are found in most of the marketed cosmetic preparation but are used in very low concentration.8.1 -Hydroxyacids The AHAs are organic carboxylic acids with one hydroxyl group attached to the position of the carboxyl group. The hydroxyl and carboxyl groups are both directly attached to an aliphatic or alicyclic carbon atom. The hydroxyl group in the AHA is neutral, and only the carboxyl group provides an acidic property. Many AHAs are present in foods and fruits and, therefore, are called fruit acids. Glycolic acid, the smallest AHA, occurs in sugar cane and is the most widely used HA in skin care. Lactic acid, the next smallest AHA, is also widely used in topical formulations to exfoliate and provide antiaging effects. Some AHAs contain a phenyl group as a side-chain substituent. This changes the solubility profile of the 25 | P a g e 29. AHA, providing increased lipophilicity over conventional water-soluble AHAs and can be used to target oily and acne-prone skin. Examples include mandelic acid (phenyl glycolic acid) and benzilic acid (diphenyl glycolic acid).8.2 -Hydroxyacids The BHAs are organic carboxylic acids having one hydroxyl group attached to the -position of the carboxyl group. The hydroxyl group in the BHA is neutral in nature and the carboxyl group provides the acidic property. Some BHAs, such as -hydroxybutanoic acid, are present in body tissues as metabolic intermediates and energy sources; however, they have not yet been commercialized in dermatologic formulations. Some molecules are both an AHA and BHA because they contain a hydroxyl group in the -position to one carboxyl group and in the -position to another carboxyl group. Malic acid (apple acid), for example, contains one hydroxyl and two carboxyl groups, and citric acid contains one hydroxyl and three carboxyl groups, making both molecules an AHA and a BHA. Citric acid is widely used in topical formulations as an antioxidant and pH adjustor, and its antiaging benefits are well established.5 Although some have termed salicylic acid a BHA, we do not consider it to be a BHA; for that reason, it is not included in this discussion. Salicylic acid behaves differently on skin than other HAs, presumably due to its phenolic hydroxyl attachment that renders the hydroxyl acidic rather than neutral.8.3 Polyhydroxy acids The PHAs are organic carboxylic acids with two or more hydroxyl groups in the molecule attached to carbon atoms of an aliphatic or alicyclic chain. All the hydroxyl groups in the PHA are neutral, and only the carboxyl group provides its acidity. To be both an AHA and PHA, also known as a polyhydroxy AHA, it is essential that at least one hydroxyl group be attached to the -position. Many PHAs are naturally occurring, endogenous metabolites, or intermediate products from carbohydrate metabolism in body tissues. For example, gluconic acid and gluconolactone are important metabolites formed in the pentose phosphate pathway from glucose during the biosynthesis of ribose for ribonucleic acid. Gluconolactone is the most commercialized PHA in skin care products, because it is readily available and delivers the antiaging benefits of HAs, in addition to strengthening skin barrier function and being a gentle, moisturizing, antioxidant/ chelating substance. For example, an in vitro cutaneous model of photoaging demonstrated that gluconolactone protects against ultraviolet (UV) radiation. These findings were attributed to the ability of gluconolactone to chelate oxidation-promoting metals and trap free radicals. In addition, pretreatment of skin with gluconolactone does not lead to an increase in sunburn cells after UVB irradiation, as 26 | P a g e 30. has been shown to occur with glycolic acid; this is thought to be due to its antioxidant effects. Gluconolactone can also be formulated with oxidative drugs, such as benzoyl peroxide, to help reduce irritation potential and erythema caused by the oxidative drug.8.4 Bionic acids The BAs are chemically classified as aldobionic acids. They consist of one carbohydrate monomer chemically linked to an aldonic acid PHA; examples are lactobionic acid, maltobionic acid, and cellobionic acid. BAs are commonly obtained from their disaccharide through chemical or enzymatic oxidation; for example, lactobionic acid is obtained from lactose, maltobionic acid from maltose, and cellobionic acid from cellobiose. Although the BAs are larger molecules than traditional AHAs, they are small enough to penetrate skin at approximately 358 daltons, and their pKa is roughly equivalent to smaller AHA molecules; for example, the pKa of lactobionic acid is 3.8, which matches that of glycolic acid. BAs are hygroscopic materials that readily attract and retain water, forming a gel matrix when their aqueous solution is evaporated at room temperature. The transparent gel contains certain amounts of water, forming a clear gel matrix. Formation of a gel matrix may add protective and soothing effects for inflamed skin. Indeed, formulations containing BA are well tolerated and help calm skin when applied after cosmetic procedures that weaken the skins barrier, including superficial HA peels and microdermabrasion. One notable protective use of lactobionic acid, a BA used in some commercial skin care formulations, is as an antioxidant chelator in organ transplantation preservation solutions. Lactobionic acid reportedly inhibits hydroxyl radical production by forming a complex with the oxidationpromoting metal Fe (II). Furthermore, gluconolactone (a PHA) and lactobionic acid and maltobionic acid (BAs) inhibit oxidative degradation of hydroquinone and banana peel. Lactobionic acid also functions as an inhibitor of the matrix metalloproteinase (MMP) enzymes. Excessive activity of MMPs occurs with age and sun exposure, contributing to wrinkle formation, skin laxity, and visible telangiectasia. The use of BAs to inhibit MMPs may provide a significant benefit in the prevention of photodamage.9. RETINOIDS Vitamin A and its derivatives, both natural and synthetic, have been popular additives in topical for years and are recognized as the gold standard for the prevention and treatment of photoaging. The following topical retinoids are recognized as being useful: Natural retinoids Retinol (vitamin A alcohol) 27 | P a g e 31. Retinyl-palmitate (vitamin A ester) Retinyl-acetate (vitamin A ester) Retinaldehyde (vitamin A aldehyde) Tretinoin (all-trans-retinoic acid) Isotretinoin (13-cis-retinoic acid) Alitretinoin (9-cis-retinoic acid) Synthetic retinoids Tazarotene Adapalene Tretinoin, isotretinoin, alitretinoin, tazarotene, and adapalene are registered as drugs; the others are cosmeceuticals (medicinally active cosmetics). Vitamin A and its derivatives exert their action by binding to specific nuclear receptors. The ligand-receptor complex modulates the expression of the genes involved in cellular differentiation and proliferation, normalizing cell keratinization. Retinoids might also act independently from the binding to nuclear receptors. Each of them exerts its own activity, offering a further choice to the dermatologists who deal with topical retinoids. Although there are several studies proving the efficacy of tretinoin as topical treatment of photoaging, few studies are available for the other retinoids.9.1 Tretinoin Kligman and Willis1 first introduced retinoids for use as photoaging agents. After its application, the author noticed improvement of skin depigmentation and rejuvenation. When used on photodamaged skin, tretinoin's clinical effects include improvement of wrinkles, roughness, mottled pigmentation, and skin appearance as a whole. The histologic changes observed are decreased corneocyte adhesion (loss of desmosomes, decreased tonofilaments, increased autolysis of keratinocytes, intracellular glycogen deposition), epidermal hyperplasia, increased number of Langerhans cells, increased synthesis of collagen and elastin, and angiogenesis. Tretinoin enhances epidermal cell turnover, decreasing contact time between keratinocytes and melanocytes and promoting a rapid loss of pigment through epidermopoesis. Tretinoin is available in different concentrations (0.01%, 0.25%, 0.5%, and 0.1%) and as different formulations (cream, gel, solution). Creams are generally prescribed for sensitive skins, whereas gels are prescribed for oily skins. Continuous once-daily application is mandatory to achieve maximum results, in any case not occurring before a 3-month period. The only clinical improvement that appears after only 1 month is skin smoothness. To maintain the results, long-term treatment is necessary. There are no limits to the duration of tretinoin topical use. Moderate cutaneous side effects, especially erythema and 28 | P a g e 32. desquamation, are observed in most patients even if after 2 to 4 weeks these side effects decrease without discontinuing the treatment. It may be necessary to interrupt the treatment for 2 to 3 days, to apply a calming and moisturizing cream, and then to restart treatment once every 2 days. Whenever tretinoin is prescribed, the use of sunscreen is very important to avoid sunburns (the treated skin is thinner) and worsening of photodamage (UV radiations decrease the expression of retinoid receptors in skin cells, thus limiting the effects of retinoids).9.2 Retinol There are no studies comparing its effectiveness with that of tretinoin, although retinol does not appear to be as effective. In photoaging, it is used as a cream in different concentrations, ranging from 0.075% to 1%. It can be considered a light alternative to tretinoin in case of sensitive skins.9.3 Retinyl-palmitate and retinyl-acetate Esters are not considered effective against photoaging if used alone. Most of the products available are, in fact, a combination of esters and hydroxy acids.9.4 Retinaldehyde Retinaldehyde is formulated as cream or gel and in concentrations varying from 0.015% to 0.1%. Its efficacy is similar to that of tretinoin, but it is much less irritating.9.5 Isotretinoin Topical isotretinoin is available as a 0.05% cream or gel. It appears to be less irritating, yet less effective, than tretinoin.9.6 Alitretinoin The theoretic benefit of alitretinoin 0.1% gel in the treatment of photoaging originates from the binding and activation of all nuclear retinoid receptors, but larger, blind, and controlled trials are necessary to better investigate its role.9.7 Tazarotene Tazarotene is an analogue of tretinoin that belongs to the family of acetylenic retinoids. It has a specific binding profile for beta and gamma retinoid receptors.29 | P a g e 33. Tazarotene improves skin roughness, fine wrinkling, and epidermal atrophy. It is available at 0.05% and 0.1% gel and cream.9.8 Adapalene Even if usually indicated for acne, adapalene has also been tested for the treatment of photoaging. Once-daily application of adapalene 0.1% gel for 4 weeks, followed by a twice-daily application for up to 9 months, significantly reduced actinic keratoses and lentigines.10. PEPTIDES 10.1 ANTIMICROBIAL PEPTIDES (AMPS) Cathelicidins and defensins are the two major groups of epidermal AMPs that possess inherent antimicrobial activity. Like most AMPs they are positively charged, with the positively charged amino acids localised to one side of the molecule and opposite to the most hydrophobic groups. Granulysin is also an AMP but whilst it has action in the skin it is not synthesised in the skin. Other peptides and proteins that exert antimicrobial activity within the skin are psoriasin, RNase 7, adrenomedullin (AM), antileukoprotease and DCD. Cathelicidins The expression of cathelicidins and mouse cathelicidin- related antimicrobial peptide (CRAMP) in skin keratinocytes varies with infection and/or injury. Only one cathelicidin, human cationic antibacterial protein has been identified in humans, although about 30 cathelicidin members are present in mammalian species. hCAP expression has been detected in human skin keratinocytes at sites of inflammation in the skin and in specialised keratinocytes of the nail, neonatal skin and in eccrine glands Cathelicidin peptide (human cathelicidin; LL-37) is produced by the neutrophils, mast cells and keratinocytes in response to inflammatory processes. LL-37 also acts as a chemoattractant for neutrophils, monocytes, T cells andmast cells. Mast cells can produce LL-37 thereby leading to a positive feedback cycle. Elevated levels of LL-37 have been reported in psoriatic skin (_304 mM) and wounded skin. LL-37 plays an important role in the repair of damaged tissue and wound closure by promoting wound vascularisation and reepithelisation. It also induces IL-8, IL-18 and IL-20 production by human keratinocytes through MAP kinase pathway. IL-18, an interferon (IFN)-g inducer, is a proinflammatory cytokine intracellularly produced from a biologically inactive precursor, pro-IL-18. It is produced by keratinocytes and its expression is highly enhanced in skin diseases, like psoriasis in which human b-defensins (hbD) and LL-37 are highly expressed.30 | P a g e 34. 10.2 Defensins Defensins are cationic peptides (MW 35 kDa), characterised by six cysteine residues that form characteristic disulphide bridges. They are divided into alpha, beta and theta subfamilies based on the alignment of the disulphide bonds. AMPs of the defensin family exhibit broad activity against gram-negative bacteria, fungi, mycobacteria and enveloped viruses and have been isolated from neutrophil granules, macrophages and some specialised epithelial cells of the small intestine. a-Defensins Six alpha defensins have been identified, of which four are known as human neutrophil peptides (HNP)-1, 2, 3 and 4, as they are associated with human neutrophils. The other two are called human defensins (HD)-5 and 6. They are expressed in the paneth cells of the intestine and in the epithelial cells of the female genitourinary tract.21 Alpha defensins exert their action on both microbes and the host. They have potent antiviral activity and HNPs 13 have been shown to increase the expression of tumour necrosis factor (TNF) a and interleukin-1 in human monocytes activated by Staphylococcus aureus. The expression of alpha defensins has been studied in squamous cell carcinomas of the human tongue and compared with autogenous nontumour tissue. HPLC-MS and amino acid sequencing was utilized for separation, structural identification and quantitation of the HNPs. MALDI-MS analysis of HPLC fractions from both tumour and nontumour tissue detected peptide masses for HNP-1, HNP-2 and HNP-3 which were confirmed by amino acid sequencing. When analysis of paired tumour and nontumour tissue samples was performed, the concentration of defensins in the tumour tissue was about 2 12 times higher than in the nontumour tissue. b-Defensins Beta defensins have been identified in many cell types including epithelial cells and neutrophils. HbD 14 have been identified in humans. HbD-1 (MW 3.9 kDa) is constitutively produced by various epithelial tissues including the urogenital and respiratory tracts. u-Defensins This is a novel class of defensins isolated from rhesus monkey neutrophils and named u-defensins for their circular molecular structure.10.3 Psoriasin Another peptide discovered in psoriatic skin lesions is psoriasin. Psoriasin is an AMP (MW 11 kDa) which is constitutively expressed in healthy skin keratinocytes and is a member of the S100 gene family. Although the physiological function of S100 proteins is not fully understood, a few studies have indicated involvement of S100 proteins in innate host defence. Over-expression of psoriasin may be linked to inflammation which is common in psoriasis. Glaser et al. also identified psoriasin as the principal Escherichia coli-killing AMP in healthy human skin, which is present on the skin surface and focally expressed in healthy skin keratinocytes. Psoriasin was induced in vitro and in vivo in keratinocytes by E. coli, indicating that its focal expression in skin 31 | P a g e 35. may be derived from local microbial induction. Zn2-saturated psoriasin showed diminished antimicrobial activity, suggesting that Zn2 sequestration could be a possible antimicrobial mechanism.10.4 RNase 7 RNase 7 (MW 14.5 kDa) is a basic protein that is abundant in healthy skin and is believed to be one of the principal cationic proteins of healthy human skin.40 The RNase A superfamily has been extensively researched over the last few decades and among the better characterised RNases are RNase 1 and RNase 5. Others include eosinophil cationic protein (ECP) or RNase 3, and RNases 4, 6, 7 and 8. These were identified by Harder and Schroder40 who showed that crude extracts from stratum corneumcontained antimicrobial activity against E. coli and S. aureus. RNase 7 may be an inducible peptide since the levels are high in psoriatic skin and gene expression is increased on contact of keratinocytes with bacteria. Adrenomedullin AM (MW 6 kDa) is a 52 amino acid peptide which is expressed in keratinocytes of the epidermis and hair follicles, cells of the eccrine, apocrine sweat glands and secretory ducts of normal skin, and in skin tumours of different histologies. AM is reported to have numerous physiological roles including vasodilation, renal homeostasis, hormone regulation, neurotransmission and growth modulation. It is also thought to be involved in the wound repair process. Martinez et al. characterised adrenomedullin receptor (AM and AM-R) expression in human skin in order to understand its potential functions in the skin. The presence of AM and its receptor in normal and neoplastic skin were confirmed by RT-PCR and Western blot analysis performed on cell extracts from human skin cell lines. Immunoreactivity for AM and in situ hybridization signal for AM-R was found in all epithelial components of the skin. Many specimens corresponding to the major histological types of skin cancer showed a positive staining for AM and AM-R. Antileukoprotease Antileukoprotease (ALP), also called mucous protease inhibitor or secretory leukoprotease inhibitor, is present in human callus and detected in supernatants of cultured human primary keratinocytes and in various human body fluids. It is a very potent human serine protease able to inhibit the neutrophil derived serine proteases polymorph nuclear leukocyte elastase (HLE) and Cathepsin G (CG). Wiedow et al. described the constitutive production and release of ALP by human keratinocytes and epithelial carcinoma cell lines (KB grown in Dulbeccos Modified Eagle and A431 grown in minimum essential medium). When the antimicrobial activity of ALP was compared with HbD-2, ALP was seen to be constitutively produced and expressed by the keratinocytes whereas HbD-2 is only expressed after stimulation of keratinocytes with bacteria. Recombinant ALP exhibited microbiocidal activity in a dose-dependent manner.10.5 Dermicidin 1L DCD-1L is a novel AMP family with a broad spectrum of activity and no similarities to other known peptides. It is constitutively expressed in eccrine sweat glands and transported to the epidermal surface. DCD expression was not observed in epidermal keratinocytes of healthy human skin. Immunohistochemistry and RTPCR studies 32 | P a g e 36. showed that DCD-1L expression was not induced in keratinocytes in inflammatory conditions such as psoriasis, AD and lichen planus. DCD-1L thus functions by modulating the colonisation of the skin rather than responding to inflammation. Lai et al. further expressed DCD-1L in E. coli as a fusion protein to understand its mechanism and investigate its antimicrobial spectrum. DCD-1L displayed antimicrobicidal activity against nosocomial pathogens, but no haemolytic activity against human erythrocytes. The antimicrobial activity of DCD was not affected by the low pH and high salt concentrations of human sweat. This finding suggests that sweat glands may have a function in the innate immune responses of the skin by secreting these antimicrobial agents.11. PEPTIDES INVOLVED IN WOUND HEALING Wound healing is a localised process which involves a series of specific and coordinated events such as inflammation, wound cell migration and mitosis, neovascularisation, and regeneration of the extracellular matrix (ECM).11.1 RGD Peptides Most cells are attached to the ECM through integrins that link the intracellular cytoskeleton with the ECM. Many of the integrins recognise a tripeptide (arginine-glycine-aspartate) or RGD in target proteins of the ECM. Vigor et al hypothesised that during the proteolytic remodelling of the ECM, small soluble RGD-containing peptides were released into the matrix. Soluble RGD-peptides have previously been found to be internalized into a cell in an integrin-independent manner and are able to directly bind to and activate caspase-3, thus inducing apoptosis. To investigate this hypothesis dermal fibroblasts were embedded into collagen type I or fibrin matrices and viability was assessed by in situ haematoxylin staining. Results indicate that apoptosis was induced specifically in response to collagen matrix remodelling. Small soluble RGD containing peptides were produced by enzymatic cleavage of collagen, which induces apoptosis of dermal fibroblasts through specific caspase-3 cleavage.11.2 Copper Peptides Copper peptides have been used in skin care in a similar way to vitamin C, alpha lipoic acid and green tree extracts. Copper functions as a part of cytochrome c oxidase and superoxide dismutase which are used in energy production and as antioxidants. It is also essential to the normal growth, development and function of the human body. Copper is bound to glycine, histadine and lysine which are used to synthesize a copper based peptide. Copper peptide has a positive influence on the growth and regulation of hair follicles and when used on wounds increases collagen disposition, tensile strength and angiogenesis in healing tissues. A method for stimulating hairgrowth by topically administering or injecting an effective amount of a peptide copper complex has been described and patented. Buffoni et al. examined the effects of Gly-His-Lys-Cu and of three synthetic analogues (I, II and III) on wound healing of guinea-pig dorsal skin and on cultured fibroblasts. Hydroxyproline, proteins, 33 | P a g e 37. DNA and semicarbazide-sensitive amine oxidase were measured and it was found that both the peptides caused a decrease in the activity of semicarbazidesensitive amine oxidase but there was no significant difference between the two peptides .The main effects of these peptide-copper complexes were slower reorganization of the skin and a delayed activation of fibroblasts.11.3 Interleukins (IL) Interleukins are a class of cytokines identified for their role in mediating immunological functions. IL-1 is produced by keratinocytes in two forms: IL-la and IL-lb. IL-6 (MW 26 kDa) is secreted from the cells in multiple glycosylated forms. Like TGF-a, the levels of IL-6 are elevated in psoriasis, cultured keratinocytes and skin tumour cell lines. IL-8, also known as neutrophil activating protein (NAP), mediates both growth stimulatory and inflammatory processes. It is produced by cultured human dermal fibroblasts and keratinocytes in response to IL1 or TNF-a. Rennekampff et al. hypothesised that IL-8 was released from an intracellular preformed pool in keratinocytes in the presence of psoriasis. Sticherling et al. showed that IL-8 is produced de novo by wound cells leading to increased reepithelialisation in vitro and in vivo by stimulating keratinocyte proliferation and migration. They investigated in vitro whether IL-8 upregulates the underlying phenotype of keratinocytes with respect to the expression of integrin subunits a2, a3, a5 and a6. An inverse relationship between IL-8 immunoreactivity and expression of the a6 integrin was found and loss of intracellular IL-8 immunoreactivity was accompanied by an increase in a6 expression. Flow cytometry analysis revealed strong expression of integrin subunits a2 and a3 and weaker expression of a5 and a6 on cultured, unstimulated keratinocytes. IL-8 was shown to be the major bioactive chemoattractant for PMNs in human blister and skin graft donor site wound fluids. In vitro experiments on the effect of recombinant human (rh) IL-8 on keratinocytes proliferation revealed a rise in cell number accompanied by an increase in cells in S phase and over-expression of the integrin. Topical application of IL-8 on human skin grafts in a chimeric mouse model showed enhanced reepithelialisation in IL-8 treated animals over controls.12. Growth Factors Growth factors act as regulators in wound healing and on exogenous application can modify the process. Two peptide growth factors which play a pivotal role in normal wound healing in tissues such as skin, cornea and the gastrointestinal (GI) tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-a). Other peptides such as basic and acidic fibroblast growth factors (bFGF and aFGF), platelet derived growth factors (PDGF-AA, -AB and -BB) and insulin-like growth factor (IGF-I) have been identified as potential wound-healing agents. EGF/TGF-a receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells and epithelial cells of the GI tract. Healing of a variety of wounds in animals and patients has been enhanced by treatment with EGF or TGF-a. EGF also increased the tensile strength of skin incisions in rats and corneal incisions in rabbits, cats and primates. Sorensen 34 | P a g e 38. et al. demonstrated that two of the important growth factors in wound healing, IGF-I and TGF-a, induce the expression of the AMPs/polypeptides hCAP-18, hbD-3, NGAL and SLPI in human keratinocytes.Growth hormone (GH) and prolactin (PRL) are produced in the anterior pituitary gland and skin is one of the target organs for GH and prolactin bioregulation. Dermal fibroblasts in cell culture have been shown to produce PRL and GH mRNA. Slominski et al.64 investigated whether the epidermis expresses the genes for GHand PRL. Detectable levels of GH and PRL were not found in human immortalized keratinocytes or in malignant melanocytes (basal cell carcinoma) but IGF-1 was expressed in malignant specimens. GHmRNA was detected in normal human skin but not in cultured human epidermal keratinocytes. Transforming Growth Factor-b Transforming growth factor-b (TGF-b) can act as a multi-functional regulator of both cell growth and differentiation. Three isotypes of TGF-bs namely TGF-b1, TGF-b2 and TGF-b3, have been found in human tissues. TGF-b2 is usually expressed in the intercellular space of all the layers of the epidermis and TGF-b3 is present in the subepidermal area of the dermis. Falanga et al. could not detect TGF-b1 or TGF-b2 in the epidermis or epithelial structures of forearm skin from healthy human volunteers. The dermal matrix contained minimally detectable amounts of the two isof