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

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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
  See discussions, stats, and author profiles for this publication at:   Skin Lightening and Sebum Control Efficacy of a Cosmetic Emulsion Containing Extract of Tamarind Seeds on Asian...  Article   in  LATIN AMERICAN JOURNAL OF PHARMACY · January 2015 CITATIONS 0 READS 342 5 authors , including:Eduardo José CaldeiraFaculdade de Medicina de Jundiaí 39   PUBLICATIONS   90   CITATIONS   SEE PROFILE Barkat KhanGomal University 8   PUBLICATIONS   12   CITATIONS   SEE PROFILE All content following this page was uploaded by Eduardo José Caldeira on 03 April 2015. The user has requested enhancement of the downloaded file.  570 KEY WORDS: cosmetic emulsion, mexameter, sebumeter, skin lightening, Tamarindus indica. *Author to whom correspondence should be addressed.  E-mail:  Latin American Journal of Pharmacy (formerly  Acta Farmacéutica Bonaerense) Lat. Am. J. Pharm. 34 (3): 570-5 (2015) Regular articleReceived: September 5, 2014 Revised version: January 28, 2015Accepted: January 29, 2015 Skin Lightening and Sebum Control Efficacy of a Cosmetic EmulsionContaining 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 Jundiaí, FMJ, Jundiaí, São 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 wasformulated and investigated its dermatological effects on functional skin parameters like skin melanin andskin 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 aqueousphase of the cosmetic emulsion. The test product (emulsion with tamarind seeds extract) and the placeboproduct (emulsion without tamarind seeds extract) was applied and gently massaged on each side of thecheeks for one minute twice daily (at night and morning) for a duration of 12 weeks and difference in skincolor and skin sebum contents between test and placebo side of cheeks was measured fortnightly by usinga Mexameter MPA5 and a Sebumeter MPA5 to evaluate the effects produced. We observed that signifi-cant (  p  ≤  0.05) decrease in skin melanin and sebum contents when the test product was applied while theplacebo showed insignificant (  p > 0.05) increase in skin melanin and skin sebum contents. The newly for-mulated cosmetic emulsion containing extract of tamarind seeds can therefore be used safely as skin se-bum control and skin lightening agent on Asian skin type.  RESUMEN  . Una emulsión cosmética W/O conteniendo 4% w/w de extracto de semillas de tamarindo (Tamarin-dus indica) se formuló e investigaron sus efectos dermatológicos en la piel con parámetros funcionales tales co-mo contenido de melanina y de sebo de la piel. El estudio se realizó con 11 voluntarios masculinos y consistía enun ensayo casero de simple ciego controlado con placebo. El extracto de semillas de tamarindo fue atrapado enla fase acuosa interna de la emulsión cosmética. El producto de prueba (emulsión con semillas de de tamarindo)y el producto placebo (emulsión sin extracto de semillas de tamarindo) se aplicó con suave masaje en cada ladode las mejillas durante un minuto dos veces al día (por la noche y por la mañana) con una duración de 12 sema-nas 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 lasmejillas se midió cada dos semanas usando un MPA5 Mexameter y un Sebumeter MPA5 para evaluar los efectosproducidos. Hemos observado una disminución significativa (  p  ≤  0,05) en la melanina de la piel y el contenidode sebo cuando se aplicó el producto de prueba, mientras que el placebo mostró aumento insignificante (p >0,05) del contenido de melanina y sebo de la piel. Por consiguiente, la emulsión cosmética de nueva formulaciónque contiene extracto de semillas de tamarindo se puede utilizar de forma segura como control de sebo de la piely agente de aclaramiento de piel del tipo asiática. INTRODUCTION Emulsions are thermodynamically unstablesystems which split into two distinguishablephases. The instability is manifested by a num-ber of processes such as flocculation, sedimen-tation or creaming, phase inversion or coales-cence that would destabilize them. Oil-in-wateror water-in-oil emulsions are the examples of colloidal systems that are frequently used now aday in various fields as pharmaceuticals, cosmet-ics, paints, food and petrochemicals etc. Allthese emulsions evolve gradually with time 1 . A wide variety of cosmetic emulsions are used asbases for skincare products for healthy and dis-eased skin. These products can range in consis-tency from a cream to a lotion or body milk andeven a fluid for normal, oily or dry skin 2 . Wa-ter-in-oil emulsions consists of the water phase, ISSN 0326 2383 (printed ed.)ISSN 2362-3853 (on line ed.)  571  Latin American Journal of Pharmacy - 34 (3) - 2015  which is internal /dispersed phase, mixed withoil, which is continuous phase .This emulsiontype is often more difficult to prepare and stabi-lize since it is most often based on totally non-emulsifiers. However recent advances in siliconchemistry and polymer chemistry have allowedpreparation of excellent water-in-oil (W/O)emulsions. A real benefit of these vehicle emul-sions is that they are readily spread on to thelipophilic skin and provide a film which is resis-tant to water wash off 3 .Plants represent a class of frequently usedactive agents in cosmeceuticals, containig ter-penoids, alkaloids, and phenolics, which havebeen chemically characterized for their biologiceffects. Herb based products usually also pro- vide multiple functionalities and stable formula-tions of highly reactive ingredients such as an-tioxidants. Plant extracts have been used forcenturies and are present in today’s products ei-ther for their own properties or as substitutes of animal materials that may have to be removedfrom products because of pressure from animalrights associations or diseases like bovinespongiform encephalopathy 4 . Plant secrets havebeen passed down through generations asherbal folklore, and now-a-days botanical ex-tracts are playing an increasingly important rolein cosmetics. For the cosmetics industry, isola-tion and purification of the active ingredient within the crude extract are sometimes notneeded because such isolation and purificationmay lead to a loss in the biological activity 5 . Tamarindus indica  L. seeds are importantsources of antioxidant activity as 2-hydroxy-3’,4’-dihydroxyacetophenone, metdihydroxyben-zoate,3,4dihydroxyphenylacetate and (-)-epicat-echin, in addition to oligomeric proanthocyani-dins (OPC). OPCs are potent antioxidant, anti-inflammatory, antihistaminic agent and ultravio-let protection. OPCs also stabilize elastin, colla-gen and ground substances 6 . The detailed study of the composition of Tamarindus indica  L.seeds will bring contribution to health profes-sionals not only regarding its composition andto evaluate its antioxidant potential, fatty acidprofile and content of tocopherols. Biologicalactivity assessment of tamarind seed was report-ed on the radical scavenging, lipid peroxidationreducing and anti-microbial activities includinganti-inflammatory potential. These findings ledto the interest in using the extract derived fromthe seeds of tamarind for cosmetics 7 . In this study dermatological evaluation of test product (emulsion with tamarind seeds ex-tract) and placebo product (emulsion withouttamarind seeds extract) was done for its effectson various skin functional parameters. MATERIALS AND METHODSMaterials Tamarindus indica  seeds were obtainedfrom a local market of Bahawalpur, Pakistanand authenticated by the CIDS (Cholistan Insti-tute 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 Is-lamia University of Bahawalpur, Pakistan. Allmaterials were used as received and were cos-metics grade. ABIL EM®90 was purchased fromthe Franken Chemicals Germany, n. Hexane &paraffin oil was purchased from Merk KGaADarmstadt (Germany). Ethanol & acetone weretaken from BDH England. Distilled water wasprepared in the Cosmetics Laboratory, Depart-ment of Pharmacy, The Islamia University of Ba-hawalpur, Pakistan. Plant material and preparation of the extract  The crushed seeds were extracted with hex-ane-ethanol-acetone (50:25:25) for 30 min, in a1:3 seed: solvent ratio, under continuous agita-tion at room temperature. The mixture was 1stfiltered through 8 layers of muslin cloth andthen filtered through Whatman No.1 filter paper.It was then subjected to rotary evaporation un-der pressure reduced to 40 °C for solvent re-moval. Concentrated extract was stored in refrig-erator 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 heatedtogether up to 75 ± 1 °C. At the same time dis-tilled water (quantity sufficient to make 100%) was heated at the same temperature and then4% tamarind seeds extract was added in it. Afterthat, aqueous phase was added to the oil phasedrop by drop. Stirring was continued at 2000rpm by the mechanical mixer for about 15 minuntil complete aqueous phase was added, 2 to 3drops of rose oil were added during this stirringtime to give good fragrance to the formulation. After the complete addition of the aqueousphase, the speed of the mixer was reduced to1000 rpm for homogenization, for a period of 5min and then the speed of the mixer was fur-  572 WAQAS M.K., AKHTAR N., BAKHSH S., CALDEIRA E.J. & KHAN B.A. ther reduced to 500 rpm for 5 min for completehomogenization until the emulsion cooled toroom temperature. For the placebo product, thepreparation process was the same as the aboveexcept the addition of plant extract. Both testand placebo products were found stable afterevaluating pH, centrifugation, electrical conduc-tivity, phase separation, organoleptic and physi-cal characteristics (color, creaming and liquefac-tion) and the temperature stability tests by keep-ing 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 threemonths. 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 onboth forearms of all the volunteers. Basic valuesfor erythema and melanin were measured withthe help of Mexameter. Approximately 1.0 g of test and placebo was applied to the 5 × 4 cmmarked regions separately on each forearm. Sur-gical dressing was used to cover the markedarea of right and left forearm. After 48 h, it wasremoved and the forearms were washed withphysiological saline and were observed for any skin redness/irritation by a dermatologist. Themeasurements of erythema and melanin wererepeated on both forearms. Non-invasive techniques The evaluation of skin sebum and skinmelanin contents was done by using Mexameterand Sebumeter MPA 5 (Courage + KhazakaElectronic GmbH, Germany.) Study design  The study was designed as a home-use, sin-gle-blind, randomized and placebo controlledtrial. The study was conducted in accordance of Helsinki Declaration and was consistent withGood Clinical Practice guidelines. The protocol was approved by the Board of Advanced Stud-ies and Research (BASR) and institutional ethicalcommittee for in vivo studies (Reference No.4710/Acad.). Written informed consent was ob-tained from all participating volunteers. Subjects Eleven volunteers were selected whose ages were 25-35 years. Exclusion criteria included ex-posure to topical steroid, volunteers who hadskin disease or wounds on the face, a history of atopic dermatitis, skin hypersensitivity reactionor a history of allergic reactions to cosmetic in-gredients.  Application of the products Each volunteer received two products (testand placebo). Prior to the tests, a dermatologistfor any serious skin disease or damage especial-ly on cheeks and forearms examined the volun-teers. They were asked to apply one type of product on one side of their cheeks and theother on another side of cheek twice daily for12 weeks. Volunteers were asked to use ap-proximately 1 g ( i.e., 0.5-1.5 g) of each product,gently massaging it in for one minute. Duringthe study period they were not allowed to con-tinue using their personal cosmetic products. Values for different parameters were taken incontrolled room temperature 25 ± 1 °C and 45 ±2 % relative humidity. The site of measurement was the whole cheek. Every volunteer was in-structed to come for measurement on 2 nd , 4 th ,6 th , 8 th , 10 th , and 12 th  week. Values for each pa-rameter was measured three times (n = 3) andaverage 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 fol-lowing formula: Percentage Change = [(A – B) / B] × 100,  where A = individual value of any pa-rameter of 2 nd , 4 th , 6 th , 8 th , 10 th and 12 th  weekand B  = zero hour value of that parameter Statistical analysis The evaluation of data was done by usingSPSS version 19.0 according to two-way ANOVAfor variation between different time intervalsdefining a 5% level of significance and Pairedsamples t-  test for variation between the twopreparations. Standard error of means (SEM) was calculated for every mean value. RESULTS AND DISCUSSIONSkin melanin contents Melanin is responsible for the diversity in hu-man skin tones. Darker skin does not containmore melanocytes, the cells are simply more ac-tive. 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 hor-mones which are synthesized and distributed by the pituitary gland 8 .  573  Latin American Journal of Pharmacy - 34 (3) - 2015 The metabolic pathway involved in melaninsynthesis is extremely complicated involvingseveral intermediate steps. It starts with theamino acid tyrosine oxidized by the coppercontaining enzyme tyrosinase to dihydrox- yphenylalanine (DOPA) and then todopaquinone. Dopaquinone undergoes a seriesof non-enzymatic reactions and rearrangementsforming the different molecules that are copoly-merized to make one of the two types of melanin, Eumelanin, which is the dark brown/purple/ black compound found in skin/hair andphaeomelanin, which is yellow to reddishbrown pigment present in red hair and rarely inhuman skin. Both forms of melanin combine with other proteins to form the melanosomethat is distributed from the melanocytes to thesurrounding cells.Maturation is categorized in four stages:namely stages I and II include un-melanized im-mature pre-melanosomes, while melanizedmelanosomes are classified as stages III and IV. Activation of the melanocortin I receptor pro-motes the synthesis of eumelanin at the expenseof phaeomelanin, although oxidation of tyrosineby tyrosinase is needed for synthesis of both thetypes of pigments. Both the light and the darkskinned individuals have similar number of melanocytes for the same body region, butmelanosomes that contain the pigment are morenumerous and more pigmented in darker peo-ple than in light skinned people 9 . Melaninbiosynthesis can be inhibited by avoiding ultra- violet (UV) exposure by the inhibition of melanocyte metabolism and proliferation. Appli-cation of tyrosinase inhibitors may be the leastinvasive procedure for maintaining skin white-ness; such agents are increasingly used in cos-metic products. Thus, the inhibition of melano-genesis has been the focus on medicinal andcosmetic treatments for skin depigmenting andlightening.Ultraviolet (UV) radiation can increase themelanization and the proliferation of melanocytes by acting on melanocytes directly or indirectly through the release of keratinocytederived factors. UV radiation also induces theformation of reactive oxygen species (ROS) inthe skin; these ROS assist melanin biosynthesisand damage DNA and then may induce the pro-liferation and/or apoptosis of melanocytes.H 2 O 2 , which is one of the ROS generated, caus-es an increase in the level of tyrosinase mRNA.The dose of UV irradiation seems to regulateand control the UV-induced proliferation of  Figure 1 . Percentage changes in skin melanin con-tents after application of placebo and test product. melanocytes 10 . It is reported that UV radiationinduced proliferation and melanogenesis of melanocytes was reduced by the topical appli-cation of antioxidants such as vitamins C and Eto the skin of hairless mice. UV irradiation in-duced 8-OHdG (a representative DNA base-modified product generated by ROS) withinDNA of cultured mouse keratinocytes. It also in-duced the proliferation of keratinocytes in hu-man skin 11 . Therefore, ROS are considered toplay important role in regulating the prolifera-tion of melanocytes as well as keratinocytes andmelanogenesis of melanocytes, while ROS scav-engers and inhibitors of ROS production, suchas antioxidants, may reduce hyperpigmentationor prevent new UV-induced melanogenesis 12 . In this study, the placebo improves the skinmelanin contents but in case of test productthere is regular decline in skin melanin valuesthroughout the study duration (Fig. 1). With thehelp of two-way ANOVA test defining a 5% lev-el of significance, it was observed that theplacebo insignificantly (0.05 < p) increased theskin melanin values while in case of test prod-uct there was significant (  p ≤  0.05) decrease inskin melanin contents with respect to time. By applying paired sample t-  test, it was observedthat test product presented significant (  p   ≤  0.05)effects with respect to placebo. The increase inmelanin content by the placebo may be creditedto presence of paraffin oil. The preparationscontaining paraffin oil found to increase themelanin level in the skin when applied topical-ly. The reduction in the skin melanin contentby the formulation may be attributed to thepresence of phenolic antioxidants, such as 2-hy-droxy-30, 40-dihydroxyacetophenone, methyl3,4-dihydroxybenzoate, 3,4-dihydroxyphenyl ac-etate and epicatechin. Extracts exhibit antioxi-
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