International Journal of Research in Cosmetic Science

Available online at http://www.urpjournals.com International Journal of Research in Cosmetic Science Universal Research Publications. All rights reserved ISSN 2277 7172 Original Article Evaluation of Sunscreen activity of Cream containing Leaves Extract of Butea monosperma for Topical application B.H. More 1*, S.N. Sakharwade 2, S.V. Tembhurne 1, D.M. Sakarkar 1 1 Sudhakarrao Naik Institute of Pharmacy, Nagpur road, Pusad Dist: Yavatmal 445104 (M.S) India. 2 L.A.D. and SMT R.P. College for Women, Dept. of Cosmetic Technology, Seminary Hills, Nagpur. Received 02 January 2013; accepted 24 January 2013 Abstract Evaluation of sunscreen activity is an important aspect in the cosmetic industry, as exposure to sunlight is a recognized as a major factor in the etiology of the progressive unwanted changes in the skin appearance and physiology due to UV rays present in the sunlight. In the present study, sunscreen activity of formulated cream containing the leaves extract (0.5, 1 and 1.5%) of Butea monosperma respectively was determined by absorption spectroscopy and transmission spectroscopy methods. In absorption spectroscopy, the absorption spectrum of dilute test solution of formulation from 290-320nm was studied to determine SPF against UV-B, most risky UV rays while in the transmittance spectroscopy, UVA and UVB protection and average UVA protection factor were calculated by taking transmission of formulations from 290nm-400nm using PVC as substrate. The results of the present study shows that the formulated creams have potency to protect against UV-rays with good SPF against UVB (Absorption spectroscopy), as well the formulations produced by incorporating different concentration of extracts can be applicable for different type of Skin respectively as per SPF value. Result of transmission spectroscopy also revealed that formulations has satisfied protection against UV-A and B rays with good average UVA protection factor (Transmission spectroscopy) indicating sunscreen activity. From the result of the present study, it concludes that cream of Butea monosperma can be used as sunscreen for different types of skin as per SPF obtained for formulations containing different concentration of extract. 2013 Universal Research Publications. All rights reserved Keywords: Sunscreen activity, Absorption and Transmittance spectroscopy, Butea monosperma. Introduction The skin is the body s first line of defense for external exposure. The signs of ageing are most visible in the skin. Although, ageing skin is not a threat to a person, it can have a detrimental effect on the psychology of a person [1]. Much of the premature ageing occurs as a direct or indirect result of skin s interaction with environment. Exposure to sunlight is a recognized as a major factor in the etiology of the progressive unwanted changes in the skin appearance. The harmful effects of solar radiation are caused predominantly by the ultraviolet (UV) region of the electromagnetic spectrum, which can be divided into three regions: UVA, from 320 to 400 nm; UVB, from 290 to 320 nm and UVC, from 200 to 290 nm Exposure to UV-A radiation results in damage to the elastic and collagen fibers of connective tissue of skin, which causes premature ageing (photo-ageing), while UV-B radiation bring about acute inflammation (sun burn) and intensification of photoageing(unitedstate-environmentalprotectionagency).[2,3]. UVC radiation is filtered by the atmosphere before reaching earth. UVB radiation is not completely filtered out by the ozone layer and is responsible for the skin damage due to sunburn. UVA radiation reaches the deeper layers of the epidermis and dermis and provokes the premature aging of the skin. Ultraviolet radiations have been implicated as a causative factor of skin cancer. Due to these facts, sunscreens substances are now incorporated into everyday products such as moisturizers, creams, lotions, shampoos, mousses, and other hair and skin preparations [2]. Sunscreen use began in the early 20 th century, salicylates were the first agents used in sunscreen preparations. These salicylates have been increasingly reported for allergic and contact dermatitis, phototoxic and photo-allergic reactions, contact urticaria and even solitary cases of severe anaphylactic reactions. Therefore, the researchers have 1

turned their attention towards developing herbal sunscreen agents which are effective with less or no side effects [4]. In Ayurvedic system of medicine, the Butea monosperma commonly known as Flame of Forest or Palas tree and has been used for relieving burning sensation, in treatment of gout, leprosy and other skin diseases. Chemical investigation showed that the plants are rich source of glycosides, linoleic acid, flavanoids etc. It is evident that the plant has great potentials in treating a number of ailments where the free radicals have been reported to major factors contributing to the disorders. Earlier studies have also shown that the plant possesses good potential related to various skin diseases such as anti-inflammatory, antibacterial, antifungal, wound healing [5-9]. In the present study our intention was to formulate and evaluate the sunscreen activity of cream containing the leaves extract of Butea monosperma. Materials and methods Collection of plant material and preparation of extract: The leaves of Butea monosperma were collected from the Sakoli village, Nagpur region in the summer season. The plant was authenticated by Dr. N. M. Dongarwar of Botany Department; RTM Nagpur University, Nagpur India. A voucher specimen no. 9282 was deposited at Herbarium, Department of Botany, RTM Nagpur University Nagpur. The leaves were dried in shade and then ground to produce coarse powder. Previously defatted powder was extracted with ethanol for 72 hrs by soxhlet apparatus. The extract was filtered using Whattman filter paper and then concentrated at 45 0 C. The percent yield was found to be 18 % w/w. The extract were kept in sterile bottle and stored under refrigerated condition for further analysis. Chemicals and Materials: All materials used in formulation and evaluation of formulation were analytical grade Preparation of cream: The different concentrations (0.5, 1 and 1.5%) of leaves extract of Butea monosperma were incorporated in the vanishing cream base and were evaluated for various physicochemical parameters [10]. Spectrophotometric SPF Determination using Mansur equation The in-vitro SPF was determined according to the methods described by Mansur [11-13]. Procedure 1gm of samples was weighed, transferred to a 100 ml volumetric flask, diluted to volume with ethanol, and then filtered through cotton, to give 10000 ppm solution. Rejecting the first 10 ml, a 5.0 ml aliquot was transferred to 50 ml volumetric flask and diluted to volume with ethanol to produce 1000ppm solution. Then a 5.0 ml aliquot was transferred to a 25 ml volumetric flask and the volume completed with ethanol (200 ppm solution). The absorption spectra of each aliquot prepared were determined from 290-400 nm, taking ethanol as a blank. The absorption data were obtained in the range of 290nm to 320nm, every 5 nm, and 2 determinations were made at each point, followed by the application of Mansur equation [11]. The in-vitro SPF of cream equivalent to 200ppm of extract was also determined by same method. Where: CF is correction factor (= 10); EE (λ) erythemal effect of radiation with wavelength λ; I (λ) solar intensity spectrum; Abs (λ) - absorbance of sunscreen product. The values of EE (λ) x I (λ) are constants as given determined by Sayre et al. 1979 [14]. The obtained absorbance values Abs (λ) were multiplied with the respective EE (λ) x I (λ) values and then summation was taken and multiplied with the correction factor 10. SPF Determination by tramission spectrum Sunscreen activity was evaluated by in-vitro method through recording transmission spectrum of formulations in the range 290-400nm.The analysis was carried out in duplicate and the values are recorded as mean vlues of two readings [15-17]. Procedure Sample was applied on polyvinyl chloride (PVC) sheet strip, spread uniformely with help of capillary to form a thin film. The strip was then placed inside UV-Vis cuvette in such that the formulation touches transparent side of cuvette. It was allowed to equilibrate for 15 mins to ensure levelling of the formulation between PVC and wall of the cuvette. The cuvette was placed inside UV spectrophotometer (Shimadzu) and a transmission spectrum was recorded from 290-400 nm, using air as reference. The data was appropriately processed to calculate UVA and UVB protection factors using the following formulas [16]. 1) Average transmittance spectrum of sunscreen in either region is averaged in order to produce one value, which describes UV A, or UVB blocking. 100-T(UVA) or T(UVB) gives % blocking or % protection against UVA or UVB. 2) Average UVA protection factor (PF) Arithmetic mean of monochromatic protection factor (MPF) calculated between 320-400 nm. A(λ) is absorbance at λ T(λ) is transmitanceat λ MPF is monochrmatic protectio factor i.e.1/t Λis measured wavelength interval (5nm) Results Determination of SPF for cream containing leaf extract SPF of developed cream containining leaf extract was checked by absorbtion spectroscopy by Mansur equation method. Spectrum of all formulations were taken from 290nm-400nm and depicted in Fig 1. Absorbances obtained in spectrum were considered for SPF calculations and 2

Table 1: Normalized product function used in the calculation of SPF Wavelength ((λ nm) EE(λ) x I(λ) (normalized) 290 0.0150 295 0.0817 300 0.2874 305 0.3278 310 0.1864 315 0.0839 320 0.0180 Total 1 depicted in Table 2. SPF of cream containing 0.5% leaf extract was found to be 0.187±0.024, 2.59±0.014 and 8.3±0.019 for 1000ppm, 10,000ppm and concentration of cream equivalent to 200ppm extract respectively. SPF of 1% cream at 200ppm, 1000ppm, 10,000ppm and concentration of cream equivalent to 200ppm extract was found to be 0.234±0.011, 0.61±0.014, 4.96±0.0045 and 8.94±0.017 respectively. SPF Table 2: Determination of Sun protection Factor (SPF) Values for Cream of LEBM Sample 0.5% cream 1% cream 1.5% cream Conc. (ppm) 200 1000 10000 Eq. extract 200 1000 10000 Eq. extract 200 1000 10000 Eq. extract of cream containining 1.5% extract was found to be 0.36±0.022, 1.8±0.0099, 8.27±0.014 and 10.05±0.027 for 200ppm, 1000ppm, 10,000ppm and concentration of cream equivalent to 200ppm extract respectively. Invitro sunscreen activity by transmission spectroscopy In-vitro sunscreen activity of all developed cream containing leaf extract were checked by transmission spectoscopy. In this method transmission spectrum of all developed formulations were obtained by UV-vis spectro- -phototomerter from 290nm-400nm and shown in Table 3 and Figure 2. The transmission values further calculated to find out % protection against UV-A and UV-B and to calculate average UVA protection factor and depicted in Table 4. Percent blocking of UVA rays of increasing concentration of cream containing extract was found to be 73.84, 83.416, 87.035 and UVB rays is found to be 82.672, 86.4766, 92.088 respectively. Average UVA protection factor was found to be 4.36, 6.67 and 8.87 for increasing concentration of cream containing LEBM. Wavelength 290 295 300 305 310 315 320 SPF EE (λ)x(λ) 0.015 0.0817 0.2874 0.3278 0.1864 0.0839 0.018 (A) Negligible Negligible Negligible Negligible Negligible Negligible Negligible EE (λ)x(λ)x(a) (A) 0.0372±0.0037 0.0222±0.0015 0.0193±0.0023 0.0182±0.0023 0.0167±0.0029 0.0174±0.0024 0.018±0.0015 0.187±0.024 EE (λ)x(λ)x(a) 0.00056 0.00181 0.0055 0.0060 0.0031 0.0015 0.0003 (A) 0.305±0.002 0.2778±0.001 0.263±0.001 0.255±0.002 0.2507±0.002 0.2561±0.001 0.262±0.002 2.59±0.014 EE (λ)x(λ)x(a) 0.004575 0.022696 0.075586 0.083589 0.04673 0.021491 0.004716 (A) 0.971±0.01 0.891±0.01 0.842±0.002 0.816±0.0004 0.804±0.0004 0.819±0.003 0.836±0.0001 8.3±0.019 EE (λ)x(λ)x(a) 0.014565 0.072795 0.24212 0.267518 0.149791 0.068718 0.015046 (A) 0.043±0.0035 0.0295±0.0009 0.0254±0.001 0.0224±0.0012 0.0192±0.0007 0.0214±0.0009 0.0225±0.0003 0.234±0.011 EE (λ)x(λ)x(a) 0.00065 0.00241 0.00728 0.00733 0.00358 0.00179 0.00041 (A) 0.132±0.0226 0.0874±0.0012 0.0688±0.0018 0.054±0.0009 0.0499±0.0002 0.0524±0.0011 0.0582±0.0023 0.61±0.014 EE (λ)x(λ)x(a) 0.00198 0.0071 0.0198 0.0177 0.0093 0.0044 0.00104 (A) 0.585±0.0005 0.534±0.0006 0.505±0.0006 0.488±0.0004 0.478±0.00 0.487±0.0002 0.501±0.00028 4.96±0.0045 EE (λ)x(λ)x(a) 0.00878 0.043648 0.145151 0.159852 0.089164 0.040851 0.009016 (A) 1.039±0.0014 0.953±0.001 0.908±0.003 0.879±0.0004 0.867±0.0025 0.882±0.002 0.902±0.0043 8.94±0.017 EE (λ)x(λ)x(a) 0.015581 0.07784 0.260945 0.288153 0.161599 0.074012 0.01623 (A) 0.0564±0.0029 0.0424±0.0025 0.0399±0.0026 0.0335±0.0027 0.0291±0.0005 0.0317±0.0030 0.039±0.001 0.36±0.022 EE (λ)x(λ)x(a) 0.00085 0.00346 0.01147 0.01098 0.0054 0.00265 0.00070 (A) 0.212±0.0008 0.1924±0.0006 0.183±0.001 0.177±0.001 0.175±0.0004 0.177±0.002 0.183±0.0005 1.8±0.0099 EE (λ)x(λ)x(a) 0.003184 0.015719 0.052594 0.05807 0.032573 0.014888 0.003299 (A) 0.958±0.001 0.885±0.002 0.840±0.001 0.813±0.001 0.803±0.002 0.811±0.002 0.826±0.001 8.27±0.014 EE (λ)x(λ)x(a) 0.014363 0.072288 0.241545 0.266649 0.149707 0.068018 0.014869 (A) 1.149±0.030 1.070±0.012 1.025±0.0021 0.990±0.0013 0.973±0.0004 0.985±0.0007 1.002±0.0016 10.05±0.027 EE (λ)x(λ)x(a) 0.017228 0.087399 0.29447 0.324473 0.18133 0.08265 0.018042 *All the values are reported in the mean of two readings ± SD. Discussion Measurement of SPF is ultimate way to determine effectiveness of sunscreen formulation. The higher the SPF, the more protection a sunscreen offers against UV-light. Sunscreens are used to aid the body's natural defense mechanisms to protect against harmful UV radiation from the sun. Its function is based on its ability to absorb, reflect or scatter the sun's rays. To be an effective sunscreen, a product should have a wide range of absorbance between 290 and 400 nm [2]. Different organic and inorganic sunscreen ingredients are approved by Australian Govt. Department of Health and Ageing, Therapeutic Goods Administration (TGA) for use in Australia whereas only 16 ingredients are listed in US- FDA monograph [18]. However, inorganic sunscreens are cosmetically unacceptable because of their opaque quality and occlusiveness [19]. These sunscreen ingredients have also been increasingly reported for allergic and contact dermatitis, phototoxic and photo-allergic reactions, contact urticaria and even solitary cases of severe anaphylactic reactions [4]. A number of people with sensitive skin, such as those suffering from skin hypersensitivity don t want to use chemical sunscreens due to concern about skin exposure to unknown chemicals. Although a variety of hypoallergenic cosmetic products have been introduced for customers with sensitive skin, there are still limited options in sunscreen agents. Therefore, the researchers have turned their attention towards developing herbal sunscreen agents which are effective with less or no side. Topical cosmetic formulations are the most preferred treatments asked by patients and are also often most prescribed by family physicians and dermatologists for sun burn. Patients feel more comfortable using topical therapies because they have milder side effects, are easier to use, are generally less expensive and are more readily available [20]. In this study topical formulations containing leaves extract of Butea monosperma were evaluated for sunscreen activity by in-vitro method transmission spectroscopy and spectroscopic methods [11, 13]. 3

Figure 1: Absorbance Spectrum of Formulated Creams of Leaves extract of Butea monosperma for detrmination of SPF Table 3: In-vitro sunscreen activity of Cream of Leaves extract of Butea monosperma by Transmission spectrophotography Wavelength 0.5% cream 1% cream 1.5% cream % T±SD MPF % T±SD MPF % T±SD MPF 290 2.56±0.99 39.06 1.60±0.71 62.50 1.30±0.71 77.22 295 10.43±1.39 9.59 6.01±0.47 16.64 4.78±0.99 20.92 300 15.42±1.59 6.49 9.01±0.59 11.10 6.99±1.05 14.32 305 17.43±1.75 5.74 10.21±0.45 9.80 7.72±0.81 12.96 310 18.56±1.58 5.39 11.26±0.56 8.88 8.36±0.69 11.97 315 19.34±1.29 5.17 12.03±0.66 8.32 8.97±0.78 11.15 320 20.24±1.34 4.94 12.66±0.66 7.90 9.38±0.68 10.66 325 21.09±1.53 4.74 13.24±0.76 7.56 9.86±0.77 10.15 330 21.38±0.88 4.68 13.65±0.62 7.33 10.14±0.66 9.86 335 22.26±1.35 4.49 14.27±0.91 7.01 10.29±0.91 9.72 340 22.83±1.28 4.38 14.78±1 6.77 10.84±0.75 9.23 345 22.95±0.65 4.36 15.14±0.95 6.61 11.25±0.81 8.89 350 23.46±0.71 4.26 15.70±1.17 6.37 11.73±1.03 8.53 355 24.23±0.76 4.13 16.24±1.17 6.16 12.00±0.92 8.33 360 24.61±0.59 4.06 16.71±1.28 5.99 12.39±0.92 8.07 365 25.06±0.84 3.99 16.81±1.27 5.95 12.78±1 7.83 370 25.69±0.98 3.89 17.27±1.66 5.79 12.96±0.98 7.72 375 26.17±0.95 3.82 16.75±1.31 5.97 13.29±0.86 7.52 380 26.70±0.83 3.75 17.55±0.89 5.70 13.59±0.81 7.36 385 27.20±0.83 3.68 17.97±0.59 5.56 13.92±0.76 7.18 390 27.77±1.07 3.60 18.26±0.40 5.48 14.11±0.69 7.09 395 28.23±1.26 3.54 18.63±0.59 5.37 14.28±0.71 7.01 400 28.75±1.61 3.48 18.67±0.62 5.36 14.67±1.06 6.82 All the values are reported in the mean of two readings ± SD. 4

Figure 2: Transmittance Spectrum of Formulated Creams of Leaves extract of Butea monosperma for detrmination of SPF Table 4 : Determination of percent protection for Cream of Butea monospemra against UV rays and UVA analysis Fomulation %Protection against UVA % protection against UVB Average UVA protection factor 0.5% cream 73.84 82.672 4.363 1% cream 83.416875 86.476667 6.679 1.5% cream 87.035 92.088 8.8727 Table 5: Sun protection factor (SPF) rating, by the way, applies only to UVB radiation Skin Type Details Ideal SPF I Always burns easily, never tans (Sensitive) 8 or more II Always burns easily, tans minimally (Sensitive) 6-7 III Burns moderately, tans gradually (Light brown, Normal) 4-5 IV Burns minimally, always tans well (Moderate brown, Normal) 2-3 V Barely burns, tans profusely (Dark brown, Insensitive) 2 VI Never burns, deeply pigmented (Insensitive) Not indicated Though, in-vitro methods present some limits; it gives accurate and precise result and avoid the exposure of human subjects to harmful ultraviolet radiation. Excipients and other active ingredients can also produce UV absorption bands, thus interfering with those of UVA and UVB sunscreen. This effect is reflected in a finished formulation. There was no indication of an influence of excipients on the absorbance of product however very low effect was realized at high concentration. The proof of sunscreen products efficacy is of high importance for the protection of public health as the UV-B fraction of solar radiation is the main contributor to skin sunburn, immunosuppression and skin cancer [21]. The UVB causes most of the skin problems related to sun exposure: like aging, wrinkles and cancer [22]. The potential of UV radiation to cause skin damage rises exponentially with decreasing wavelength. UV light at 280 nm is 1000 times more damaging than light at 340 nm, therefore, a sunscreen s ability to block UV-B is more important to prevent the negative effects of sun exposure [23]. Hence in the present study the spectroscopic method emphasized on protection against UVB by considering the absorbance in the UVB range i.e. from 290nm-320nm. From the result of the present study it demonstrated that cream show protection against UVB radiation and indicated as SPF 0.18 to 8.3 for 0.5% cream, 0.23 to 8.9 for 1% cream and 0.36 to 10.05 for 1.5% cream in different concentration. Each formulation evaluated for different concentration and the found SPF value revealed that different concentration of each formulation can be applicable for different skin type. Different skin type, I-VI requires ideal SPF as shown in Table 5 [22]. As per our findings and ideal SPF required for different skin type, 0.5% cream, 1000pppm can be applicable for skin type VI; 10,000ppm can be applicable for skin type IV and 40,000ppm for Type I. 1% cream, 200 and 1000pppm can be applicable for skin type VI; 10,000ppm can be applicable for skin type III and 20,000ppm for Type I. 1.5% cream, 200ppm is applicable for skin type VI and 1000pppm is applicable for skin type V; 10,000ppm can be applicable for skin type III and 13,000ppm for Type I. In addition to the SPF applicable to UV-B, protection, UVA analysis, % protection against UV- A and UV-B rays were also calculated by Transmission Spectroscopy. In the present study, Transmittance spectroscopy using thin film technique was adopted to evaluate sunscreen activity in order to produce a value which describes the amount of UVA and UVB protection and average SPF against UVA by taking transmittance spectrum of a sunscreen in either region. The result shows that as the concentration of the extract in formulation increases, UV- rays protection and average UVA protection factor also increases which 5

reflected in Table 4. Conclusion Thus the results of the present study conclude that the formulated cream has potency to protect against UVA and UVB rays indicating sunscreen activity as well the formulations produced by incorporating different concentration of extracts can be applicable for different type of Skin respectively as per SPF value. References 1) Gaikwad M and Kale S. Formulation and in vitro evaluation for sun protection factor of Moringa oleifera lam (family-moringaceae) oil sunscreen cream. International Journal of Pharmacy and Pharmaceutical Sciences 2011; 3(4):371. 2) Dutra EA, Oliveira DAGC, Kedor-Hackmann ERM, Santoro MIRM. Determination of sun protection factor (SPF) of sunscreens by ultraviolet Spectrophotometry. Brazilian Journal of Pharmaceutical Sciences 2004; 40(3): 381-385 3) Center for Global Environmental Research, National Institute for Environmental Studies Japan. The Ozone hole. (http://www.theozonehole.com/uvrays) 4) Gasparro FP, Mitchnick M, Nash JF. A Review of Sunscreen and Efficacy. Photochemistry and Photobiology. 1998. 68(3). 243-56. 5) Lien APH, Hua H, Chuong PH. Free Radicals, Antioxidants in Disease and Health. International Journal of Biomed Sci. 2008, 4(2): 89-96. 6) Mehta JP, Pandya CV, Parmar PH, Golakiya BA. Isolation, characterization and antimicrobial activity of Buteamonosperma (Lam.) Iran J Pharmacol and Therap. 2011, 10(2): 76-81 7) Shahavi VM and Desai SK. Anti-inflammatory activity of Butea monosperma flowers. Fitoterapia. 2008, 79: 82-85. 8) Sumitra M, Manikandan P and Suguna L. Efficacy of Butea monosperma on dermal wound healing in rats. Int J of Biochem and Cell Biol. 2005, 37: 566 573. 9) Yadava RN and Tiwari L. New antifungal flavone glycoside from Butea monosperma O. Kuntze. J Enzyme Inhib Med Chem. 2007, 22: 497-500. 10) More BH, Sakharwade SN, Tembhurne SV, Sakarkar DM. Evaluation of formulated herbal cream containing leaf extract of Butea monosperma. FS J Pharm Res 2012; 1 (4): 21-24. 11) Mansur JS, Breder MNR, Manusur MCA, Azulay RD. Determinacao do fato de potecao sola po espectrofotometrica. An. Bras. Dermatol. 1986; 61: 121-124. 12) Ashwat MS, Shailendra S and Swarnlata S. Invitro Antioxidant activity of ethanolic exatrcts of Centella asiatica, Punica granatum, Glycerrhiza glabra and Areca catechu. Res. J. Med. Plant 2007; 1: 13-16 13) Kaur CD and Saraf S. Photoprotective activity of alcoholic extract of Camellia sinensis, Int. J. Pharmacol. 2011;7(3):400-404. 14) Sayre RM, Agin PP, Levee GJ, Marlowe E. A Comparison of in vivo and in vitro testing of sun screening formulas. Photochemistry and Photobiology 1979; 29(3): 559-566. 15) DIN-German Industry Standard 67502. Characterization of UVA protection effect of dermal sun protection agents by transmission measurements under consideration of the light protection factor (Jan 2004). 16) Labsphere UV-1000s Ultraviolet Transmittance Analyzer. SPF Analysis of Sunscreens. Technical Note: A Discussion of Optical Geometry and Measurement Effects. 17) Oliveira1 SL, Mansanares AM, Da-Silva EC and Barja PR. In vitro determination of the sun protection factor of sunscreens through photoacoustic spectroscopy: A new approach. Eur. Phys. J. Special Topics 2008; 153: 475 478. 18) Federal Register 43/166, 38206-69. U.S.A for overthe-counter human use; proposed safety, effective and labeling conditions. 19) Physical UVA+UVB sunscreen/sunblock: Titanium Dioxide [cited on 2011 Mar 29] available from URL: http://www.smartskincare.com/skinprotection/sunbloc ks /sunblocktitanium-dioxide.html 20) Vender RB. Topical acne therapies: Optimizing patient compliance. Skin Therapy Letter - Family Practice Edi., 2008; 4: 1-4. 21) Patil RB, Kale S, Badiyani DM and Yadav AV. Determination of In-vitro Sun Protection Factor (SPF) of Murraya Koenigii L. (Rutaceae) Essential oil Formulation. Indian J. Pharm. Educ. Res. 2010; 44(4): 11-12. 22) Kolhapure SA. Clinical Trial Report: Evaluation of the efficacy and safety of Sun Screen Lotion in sunburns, The Antiseptic 2004; 101(10), 420-421 23) Allen MW, Bain G. Measuring the Sun Protection Factor (SPF) of Sunscreens. Thermo Fisher Scientific, Madison, WI, USA. (http://www.analiticaweb.com.br/ newsletter/07/51463) Source of support: Nil; Conflict of interest: None declared 6