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Skin Lightening and Sebum Control Efficacy of a Cosmetic Emulsion Containing Extract of Tamarind Seeds on Asian Skin Type

Article in LATIN AMERICAN JOURNAL OF PHARMACY ? January 2015

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Latin American Journal of Pharmacy (formerly Acta Farmac?utica Bonaerense) Lat. Am. J. Pharm. 34 (3): 570-5 (2015)

Regular article Received: September 5, 2014 Revised version: January 28, 2015

Accepted: January 29, 2015

Skin Lightening and Sebum Control Efficacy of a Cosmetic Emulsion Containing Extract of Tamarind Seeds on Asian Skin Type

Muhammad K. WAQAS 1, Naveed AKHTAR 1, Sattar BAKHSH 2, Eduardo J. CALDEIRA 3 & Barkat A. KHAN 2,4 *

1 Department of Pharmacy, Faculty of Pharmacy & Alternative Medicine, The Islamia University of Bahawalpur, Punjab, Pakistan

2 Department of Pharmaceutics, Faculty of Pharmacy, Gomal University D.I Khan, KPK, Pakistan 3 Tissue Morphology Laboratory, Department of Morphology and Basic Pathology, Faculty of Medicine of Jundia?, FMJ, Jundia?, S?o Paulo, Brazil 4 School of Pharmacy, Kampala International University-WC, Uganda

SUMMARY. Cosmetic W/O emulsion containing 4% w/w tamarind (Tamarindus indica) seeds extract was formulated and investigated its dermatological effects on functional skin parameters like skin melanin and skin sebum contents. The study was performed using 11 male volunteers and consisted of a home used, single-blinded placebo controlled trial. Tamarind seeds extract was entrapped in the internal aqueous phase of the cosmetic emulsion. The test product (emulsion with tamarind seeds extract) and the placebo product (emulsion without tamarind seeds extract) was applied and gently massaged on each side of the cheeks for one minute twice daily (at night and morning) for a duration of 12 weeks and difference in skin color and skin sebum contents between test and placebo side of cheeks was measured fortnightly by using a Mexameter MPA5 and a Sebumeter MPA5 to evaluate the effects produced. We observed that significant (p 0.05) decrease in skin melanin and sebum contents when the test product was applied while the placebo showed insignificant (p > 0.05) increase in skin melanin and skin sebum contents. The newly formulated cosmetic emulsion containing extract of tamarind seeds can therefore be used safely as skin sebum control and skin lightening agent on Asian skin type. RESUMEN. Una emulsi?n cosm?tica W/O conteniendo 4% w/w de extracto de semillas de tamarindo (Tamarindus indica) se formul? e investigaron sus efectos dermatol?gicos en la piel con par?metros funcionales tales como contenido de melanina y de sebo de la piel. El estudio se realiz? con 11 voluntarios masculinos y consist?a en un ensayo casero de simple ciego controlado con placebo. El extracto de semillas de tamarindo fue atrapado en la fase acuosa interna de la emulsi?n cosm?tica. El producto de prueba (emulsi?n con semillas de de tamarindo) y el producto placebo (emulsi?n sin extracto de semillas de tamarindo) se aplic? con suave masaje en cada lado de las mejillas durante un minuto dos veces al d?a (por la noche y por la ma?ana) con una duraci?n de 12 semanas y la diferencia en el contenido de color de la piel y sebo de la piel entre la prueba y placebo a cada lado de las mejillas se midi? cada dos semanas usando un MPA5 Mexameter y un Sebumeter MPA5 para evaluar los efectos producidos. Hemos observado una disminuci?n significativa (p 0,05) en la melanina de la piel y el contenido de sebo cuando se aplic? el producto de prueba, mientras que el placebo mostr? aumento insignificante (p > 0,05) del contenido de melanina y sebo de la piel. Por consiguiente, la emulsi?n cosm?tica de nueva formulaci?n que contiene extracto de semillas de tamarindo se puede utilizar de forma segura como control de sebo de la piel y agente de aclaramiento de piel del tipo asi?tica.

INTRODUCTION Emulsions are thermodynamically unstable

systems which split into two distinguishable phases. The instability is manifested by a number of processes such as flocculation, sedimentation or creaming, phase inversion or coalescence that would destabilize them. Oil-in-water or water-in-oil emulsions are the examples of colloidal systems that are frequently used now a

day in various fields as pharmaceuticals, cosmetics, paints, food and petrochemicals etc. All these emulsions evolve gradually with time 1. A wide variety of cosmetic emulsions are used as bases for skincare products for healthy and diseased skin. These products can range in consistency from a cream to a lotion or body milk and even a fluid for normal, oily or dry skin 2. Water-in-oil emulsions consists of the water phase,

KEY WORDS: cosmetic emulsion, mexameter, sebumeter, skin lightening, Tamarindus indica. * Author to whom correspondence should be addressed. E-mail: barki.gold@

ISSN 0326 2383 (printed ed.)

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ISSN 2362-3853 (on line ed.)

Latin American Journal of Pharmacy - 34 (3) - 2015

which is internal /dispersed phase, mixed with oil, which is continuous phase .This emulsion type is often more difficult to prepare and stabilize since it is most often based on totally nonemulsifiers. However recent advances in silicon chemistry and polymer chemistry have allowed preparation of excellent water-in-oil (W/O) emulsions. A real benefit of these vehicle emulsions is that they are readily spread on to the lipophilic skin and provide a film which is resistant to water wash off 3.

Plants represent a class of frequently used active agents in cosmeceuticals, containig terpenoids, alkaloids, and phenolics, which have been chemically characterized for their biologic effects. Herb based products usually also provide multiple functionalities and stable formulations of highly reactive ingredients such as antioxidants. Plant extracts have been used for centuries and are present in today's products either for their own properties or as substitutes of animal materials that may have to be removed from products because of pressure from animal rights associations or diseases like bovine spongiform encephalopathy 4. Plant secrets have been passed down through generations as herbal folklore, and now-a-days botanical extracts are playing an increasingly important role in cosmetics. For the cosmetics industry, isolation and purification of the active ingredient within the crude extract are sometimes not needed because such isolation and purification may lead to a loss in the biological activity 5.

Tamarindus indica L. seeds are important sources of antioxidant activity as 2-hydroxy3',4'-dihydroxyacetophenone, metdihydroxybenzoate,3,4dihydroxyphenylacetate and (-)-epicatechin, in addition to oligomeric proanthocyanidins (OPC). OPCs are potent antioxidant, antiinflammatory, antihistaminic agent and ultraviolet protection. OPCs also stabilize elastin, collagen and ground substances 6. The detailed study of the composition of Tamarindus indica L. seeds will bring contribution to health professionals not only regarding its composition and to evaluate its antioxidant potential, fatty acid profile and content of tocopherols. Biological activity assessment of tamarind seed was reported on the radical scavenging, lipid peroxidation reducing and anti-microbial activities including anti-inflammatory potential. These findings led to the interest in using the extract derived from the seeds of tamarind for cosmetics 7.

In this study dermatological evaluation of test product (emulsion with tamarind seeds ex-

tract) and placebo product (emulsion without tamarind seeds extract) was done for its effects on various skin functional parameters.

MATERIALS AND METHODS Materials

Tamarindus indica seeds were obtained from a local market of Bahawalpur, Pakistan and authenticated by the CIDS (Cholistan Institute of Desert studies), The Islamia University of Bahawalpur, Pakistan. For future reference, a voucher specimen (Voucher no. TI- SD-6-15-87) has been kept in the herbarium at CIDS, The Islamia University of Bahawalpur, Pakistan. All materials were used as received and were cosmetics grade. ABIL EM? 90 was purchased from the Franken Chemicals Germany, n. Hexane & paraffin oil was purchased from Merk KGaA Darmstadt (Germany). Ethanol & acetone were taken from BDH England. Distilled water was prepared in the Cosmetics Laboratory, Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan.

Plant material and preparation of the extract

The crushed seeds were extracted with hexane-ethanol-acetone (50:25:25) for 30 min, in a 1:3 seed: solvent ratio, under continuous agitation at room temperature. The mixture was 1st filtered through 8 layers of muslin cloth and then filtered through Whatman No.1 filter paper. It was then subjected to rotary evaporation under pressure reduced to 40 ?C for solvent removal. Concentrated extract was stored in refrigerator for further studies.

Preparation of cosmetic water-in-oil emulsions

Oily phase composed of paraffin oil (14%) and emulsifier Abil-EM?90 (2.5%) were heated together up to 75 ? 1 ?C. At the same time distilled water (quantity sufficient to make 100%) was heated at the same temperature and then 4% tamarind seeds extract was added in it. After that, aqueous phase was added to the oil phase drop by drop. Stirring was continued at 2000 rpm by the mechanical mixer for about 15 min until complete aqueous phase was added, 2 to 3 drops of rose oil were added during this stirring time to give good fragrance to the formulation. After the complete addition of the aqueous phase, the speed of the mixer was reduced to 1000 rpm for homogenization, for a period of 5 min and then the speed of the mixer was fur-

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ther reduced to 500 rpm for 5 min for complete homogenization until the emulsion cooled to room temperature. For the placebo product, the preparation process was the same as the above except the addition of plant extract. Both test and placebo products were found stable after evaluating pH, centrifugation, electrical conductivity, phase separation, organoleptic and physical characteristics (color, creaming and liquefaction) and the temperature stability tests by keeping the emulsions to storage at 8 ? 0.5, 25? 0.5, 40 ? 0.5 ?C, and at 40 ? 0.5 ?C with 70% RH (relative humidity) for the period of three months.

Skin irritation assessment To determine the skin irritation assessment,

Patch tests were performed on forearms of each volunteer. An area of 5 ? 4 cm was marked on both forearms of all the volunteers. Basic values for erythema and melanin were measured with the help of Mexameter. Approximately 1.0 g of test and placebo was applied to the 5 ? 4 cm marked regions separately on each forearm. Surgical dressing was used to cover the marked area of right and left forearm. After 48 h, it was removed and the forearms were washed with physiological saline and were observed for any skin redness/irritation by a dermatologist. The measurements of erythema and melanin were repeated on both forearms.

Non-invasive techniques The evaluation of skin sebum and skin

melanin contents was done by using Mexameter and Sebumeter MPA 5 (Courage + Khazaka Electronic GmbH, Germany.)

Study design The study was designed as a home-use, sin-

gle-blind, randomized and placebo controlled trial. The study was conducted in accordance of Helsinki Declaration and was consistent with Good Clinical Practice guidelines. The protocol was approved by the Board of Advanced Studies and Research (BASR) and institutional ethical committee for in vivo studies (Reference No. 4710/Acad.). Written informed consent was obtained from all participating volunteers.

Subjects Eleven volunteers were selected whose ages

were 25-35 years. Exclusion criteria included exposure to topical steroid, volunteers who had skin disease or wounds on the face, a history of

atopic dermatitis, skin hypersensitivity reaction or a history of allergic reactions to cosmetic ingredients.

Application of the products Each volunteer received two products (test

and placebo). Prior to the tests, a dermatologist for any serious skin disease or damage especially on cheeks and forearms examined the volunteers. They were asked to apply one type of product on one side of their cheeks and the other on another side of cheek twice daily for 12 weeks. Volunteers were asked to use approximately 1 g (i.e., 0.5-1.5 g) of each product, gently massaging it in for one minute. During the study period they were not allowed to continue using their personal cosmetic products. Values for different parameters were taken in controlled room temperature 25 ? 1 ?C and 45 ? 2 % relative humidity. The site of measurement was the whole cheek. Every volunteer was instructed to come for measurement on 2nd, 4th, 6th, 8th, 10th, and 12th week. Values for each parameter was measured three times (n = 3) and average of the values has been noted.

Mathematical analysis The percentage changes for the individual

values of different parameters, taken every week of volunteers were calculated by the following formula: Percentage Change = [(A ? B) / B] ? 100, where A = individual value of any parameter of 2nd, 4th, 6th, 8th, 10th and 12th week and B = zero hour value of that parameter

Statistical analysis The evaluation of data was done by using

SPSS version 19.0 according to two-way ANOVA for variation between different time intervals defining a 5% level of significance and Paired samples t-test for variation between the two preparations. Standard error of means (SEM) was calculated for every mean value.

RESULTS AND DISCUSSION Skin melanin contents

Melanin is responsible for the diversity in human skin tones. Darker skin does not contain more melanocytes, the cells are simply more active. Variation in human skin colour is mainly due to the presence of four pigments, namely, Melanin, Haemoglobin, Carotene and Melanoid. Pigmentation of the skin is controlled by hormones which are synthesized and distributed by the pituitary gland 8.

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The metabolic pathway involved in melanin synthesis is extremely complicated involving several intermediate steps. It starts with the amino acid tyrosine oxidized by the copper containing enzyme tyrosinase to dihydroxyphenylalanine (DOPA) and then to dopaquinone. Dopaquinone undergoes a series of non-enzymatic reactions and rearrangements forming the different molecules that are copolymerized to make one of the two types of melanin, Eumelanin, which is the dark brown/ purple/ black compound found in skin/hair and phaeomelanin, which is yellow to reddish brown pigment present in red hair and rarely in human skin. Both forms of melanin combine with other proteins to form the melanosome that is distributed from the melanocytes to the surrounding cells.

Maturation is categorized in four stages: namely stages I and II include un-melanized immature pre-melanosomes, while melanized melanosomes are classified as stages III and IV. Activation of the melanocortin I receptor promotes the synthesis of eumelanin at the expense of phaeomelanin, although oxidation of tyrosine by tyrosinase is needed for synthesis of both the types of pigments. Both the light and the dark skinned individuals have similar number of melanocytes for the same body region, but melanosomes that contain the pigment are more numerous and more pigmented in darker people than in light skinned people 9. Melanin biosynthesis can be inhibited by avoiding ultraviolet (UV) exposure by the inhibition of melanocyte metabolism and proliferation. Application of tyrosinase inhibitors may be the least invasive procedure for maintaining skin whiteness; such agents are increasingly used in cosmetic products. Thus, the inhibition of melanogenesis has been the focus on medicinal and cosmetic treatments for skin depigmenting and lightening.

Ultraviolet (UV) radiation can increase the melanization and the proliferation of melanocytes by acting on melanocytes directly or indirectly through the release of keratinocyte derived factors. UV radiation also induces the formation of reactive oxygen species (ROS) in the skin; these ROS assist melanin biosynthesis and damage DNA and then may induce the proliferation and/or apoptosis of melanocytes. H2O2, which is one of the ROS generated, causes an increase in the level of tyrosinase mRNA. The dose of UV irradiation seems to regulate and control the UV-induced proliferation of

melanocytes 10. It is reported that UV radiation induced proliferation and melanogenesis of melanocytes was reduced by the topical application of antioxidants such as vitamins C and E to the skin of hairless mice. UV irradiation induced 8-OHdG (a representative DNA basemodified product generated by ROS) within DNA of cultured mouse keratinocytes. It also induced the proliferation of keratinocytes in human skin 11. Therefore, ROS are considered to play important role in regulating the proliferation of melanocytes as well as keratinocytes and melanogenesis of melanocytes, while ROS scavengers and inhibitors of ROS production, such as antioxidants, may reduce hyperpigmentation or prevent new UV-induced melanogenesis 12.

In this study, the placebo improves the skin melanin contents but in case of test product there is regular decline in skin melanin values throughout the study duration (Fig. 1). With the help of two-way ANOVA test defining a 5% level of significance, it was observed that the placebo insignificantly (0.05 < p) increased the skin melanin values while in case of test product there was significant (p 0.05) decrease in skin melanin contents with respect to time. By applying paired sample t-test, it was observed that test product presented significant (p 0.05) effects with respect to placebo. The increase in melanin content by the placebo may be credited to presence of paraffin oil. The preparations containing paraffin oil found to increase the melanin level in the skin when applied topically.

The reduction in the skin melanin content by the formulation may be attributed to the presence of phenolic antioxidants, such as 2-hydroxy-30, 40-dihydroxyacetophenone, methyl 3,4-dihydroxybenzoate, 3,4-dihydroxyphenyl acetate and epicatechin. Extracts exhibit antioxi-

Figure 1. Percentage changes in skin melanin contents after application of placebo and test product.

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