Indian J.Pharm. Educ. Res.

44(4), Oct - Dec, 2010

Determination of In-vitro Sun Protection Factor (SPF) of Murraya Koenigii L. (Rutaceae) Essential oil Formulation
Rekha B Patil , Shantanu Kale *, Devanshi M Badiyani and A.V. Yadav
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MGV's Pharmacy College, University of Pune, Pune, Maharashtra, India.

Government Pharmacy College, Karad, Shivaji University, Kolhapur, Maharashtra, India. *Author for Correspondence: shantanu_kale@yahoo.com

Abstract The present study was designed to study the sunscreen activity of herbal formulation. There is no evidence regarding the sun protection factor studies on essential oil of Curry leaf oil (Murraya koenigii L. Spring., Rutaceae). This study investigates its in vitro sun protection factor (SPF) by COLIPA method of Curry leaf oil in a cream formulation. The sun protection factor were analysed by using Optometrics LLC, SPF- 290S is a recording ultraviolet visible (UVVIS) spectrophotometer using samples exposed to Xenon arc lamp. The sun protection factor of Curry leaf oil cream exhibited less activity (SPF= 2.040.02) suggesting it can be used to maintain the natural pigmentation of the skin or can be used as an adjuent in other formulations to enhance the activity. Key Words: Essential oil, Murraya koenigii, Curry leaf oil formulation, Sun Protection Factor, UV. Abbreviations: UV: Ultraviolet, 290-400 nm; UV-B, 280-320 nm; UV-A, 320-400 nm; BED, Biologically Efficient Dose; MED, Minimal Erythemal Dose; SPF: Sun Protection Factor. INTRODUCTION The purpose of sunscreen product is to prevent the skin from tanning and burning by screening out the UV-A and UV-B radiations in sunlight 1.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. Evaluation of SPF has been performed for a long time in vivo on human volunteers according to the COLIPA method 2. It is based on the minimal erythemal dose (MED) related mainly to the biological effect of UV-B irradiation . Murraya koenigii L. Spring. (Rutaceae) is an aromatic pubescent shrub or small tree commonly known as curry leaf in India. It often forms undergrowth in forests throughout India and in Andaman Island, growing up to an altitude of 1500m4. In traditional system of medicine, Murraya koenigii L. Spring. is used as antiemetic, antidiarrhoeal, in dysentery, febrifuge, blood purifier, tonic, stomachic, flavouring agent in curries and chutney. The oil is used externally for bruises, eruption 5-8. The essential oil from leaves of Murraya koenigii L.
Indian Journal of Pharmaceutical Education and Research Received on 15/12/2009; Modified on 14/4/2010 Accepted on 3/7/2010 APTI All rights reserved
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exhibit strong antibacterial as well as antifungal activity

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9-

. On commercial basis Curry leaf oil may find use as

fixative for a heavy type of soap and perfume industry 4. In aromatherapy, aromatherapists have used Curry leaf essential oil against diabetes, hair loss and a means of helping the skin maintain its natural pigmentation. The present study deals with the determination of sun protection factor (SPF) as cosmetic use of Murraya koenigii L. leaf essential oil. The carbapol base cream formulation containing isolated Curry leaf oil and its in vitro evaluation for sun protection factor (SPF) by using COLIPA method. The study was designed with an objective to in vitro determination of sun protection factor, using Optometrics LLC, SPF- 290S, of the investigational sunscreen cream sample. The SPF 290S is a recording UV-VIS spectrophotometer designed and optimized for the in vitro determination of SPF values, on a variety of sunscreen and cosmetic products. MATERIAL AND METHODS 2.1 Plant Material: The plant specimens for the proposed study were collected from in-house garden in Nashik; district Nashik, Maharashtra, India. The specimens were identified and authenticated by Botanical Survey of

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India, Pune, Maharashtra, India. (Ref. BSI/WC/TECH/2008/501 Date 15 October, 2008). The herbarium of the plant was deposited in the BSI against voucher no. RBMUK1 10/2008. 2.2 Isolation of essential oil: Fresh Curry leaves (voucher specimen deposited) were obtained from the in-house garden, were cleaned and washed thoroughly under running water. The excess water was drained out completely and the leaves were dried under shade for 7-8 days. Dried leaves of M. koenigii L. were coarsely powdered in grinder. Weighed (550 g) were macerated in alcohoh for 7 days with frequently shaking, distilled the solvent up to concentrate. That concentrated mass were subjected to hydrodistillation in a Clavenger-type apparatus for 8 h. The distillate was extracted with Diethyl ether, the ethereal layer was dried over anhydrous sodium sulphate and ether distilled of on gently heated on water bath. The yield of the oil obtained was found to be 0.95 %. 2.3 Physico-chemical properties of oil: After isolation of oil, the physico-chemical parameters for oil were tested for the identity of oil. Specific gravity: 0.8858 g/ml at 250C, relative density n25D: 0.993 g/ml, optical rotation: -5.3 , viscosity by Brookfield viscometer: 13 Cp, acid number: 1.15, saponification value: 122.8, ester value: 121.03. 2.4 Preparation of cream formulation: Sun protection formulation is a main issue for formulators nowadays. Considering the need of the market, formulations are more difficult to make than for standard care products. That is a challenge, which is achieved by preparing a stable formulation by considering the things: the difficulty to stabilize components, the interaction between the ingredients, water resistance, easy spreadibility, pleasant feeling during and after application, stable formulation and non irritant. Carbopol 940 has been selected the gel forming polymer for the preparation of the semi-solid formulation of curry leaf oil, because the rheological property of Carbopols have extensively been studied as a function of Concentration, pH and cross-linked density. Cream formulation of isolated Curry leaf oil from leaves of Murraya koenigii L. were prepared using formulae given in (Table No. 1). For the formulation to prepare plain gel Carbopol-940 was soaked in Water (80%). After
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that, it was homogenized and then heated to 800C. In another 5% quantity of Water was added Disodium EDTA, Sodium methyl paraben and kept aside. In another 5% quantity of Water was added Triethanolamine and kept aside. Other is oil phase, was prepared by collecting and heating Sodium propyl paraben, Cetostearyl alcohol, Stearic alcohol, Cetomacrogol-1000, along with Cetyl alcohol at 800C. Homogenized heated Carbapol and added Disodium EDTA, Sodium methyl paraben, in water and quickly added oil phase and homogenized for 15 minutes. The cream was kept at room temperature. Then Murraya koenigii L. oil was weighed (5%) and added in the formed emulsion under constant homogenization. The cream was once more neutralized with Triethanolamine under constant stirring. The final pH of the formulation was 6.0. Whole formulation was stored in well closed amber coloured glass bottle and was compounded fresh for all studies. 2.5 Physical parameter of cream tested: The finished product was evaluated for its safe use, by studying its sensory evaluation, pH, spreadibility, specific gravity, heavy metal testing for Lead (Pb), total aerobic microbial count and patch testing for irritancy: etc. as per official methods and results are mentioned in (Table No.2). 2.6 Determination of the in vitro sun protection factor: Approximately 100 mg of the investigational sample was applied and spread on 50 sq.cm area to obtain a sample film thickness of 2 l/cm2 on Transpore Tape to get an even film as suggested in the operation manual of Optometrics LLC for the sample preparation and application technique. The samples thus prepared were exposed to Xenon arc lamp for determining the sun protection factor. Scan of the sample were run from 290 nm to 400 nm. This study was performed by Transmittance Measurement of the sample. The Optometrics Model SPF290 Analyzer is a computercontrolled instrument that is designed to measure the sunscreen protection factor of sunscreen preparations. For US FDA standards the protection factor is calculated over the wavelength range from 290 to 400 nm. The SPF-290 software uses Trapezoidal Approx. calculation technique to approximate the integral for SPF and Erythemal UVA protection factor. These include UVA/UVB ratio, critical wavelength, cumulative

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Indian J.Pharm. Educ. Res. 44(4), Oct - Dec, 2010

absorbance, etc. The Average Absorbance method is used for calculating average protection factor; this method averages and computes the standard deviation based on the absorbance scan data. This method of calculation gives a better average value assuming that sample thickness is the largest variable in performing a protection factor measurement. For the calculation of standard deviation, Diffey's method is used, is based on B. L. Diffey's paper on using Transpore Tape as the substrate for SPF measurements . Diffey's equation applies weighting by recognizing that the MPF (Monochromatic protection factor) measurements for a set of scans have some distribution. Therefore, the standard deviations of the MPF measurements at each wavelength are factored in to the Diffey SPF standard deviation calculation. Win SPF has used the following equation for calculating SPF.
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RESULT The Formulation of essential oil of Murraya koenigii L. was studied for all parameters of cream as well as for in vitro sun protection factor. The results of cream and sun protection factor test summarized in (Table No. 2 and 3) showed that cream parameters complies as per official acceptance criteria's and SPF for Curry leaf oil cream formulation 2.040.02 shows minimum sun protection activity for sunlight and erythema.

DISCUSSION Determination of SPF in the Curry leaf oil cream formulation To initiate an analysis, a reference scan (which consists of data from the 23 wavelengths) was acquired with the blank substrate in the incident beam. The samples were then applied to the substrate and the first sample scan was made. Data was collected in the same manner as the reference data, ratioed to the reference and plotted as a MPF. Ratioing the sample signal to the reference signal negates any effect of wavelength dependent variables in the optical system (source, monochromator, and detector). Up to 6 sample scans were made to compensate for variables in the substrate and sample application. Three readings were taken consecutively and its average value is shown in Table No.2. There is no evidence regarding the sun protection factor studies on essential oil of Murraya koenigii L. from the literature survey it could be found that aromatherapists have used Curry leaf oil in helping the skin maintain its natural pigmentation. It is a discovery that Curry leaf oil shows SPF activity. CONCLUSION This study has shown that Curry leaf oil cream shows the low sun protection factor so the cream can be used in maintaining the natural skin pigmentation or it can be used as additives in other formulations to enhance the activity. ACKNOWLEDGEMENTS The author wish to thank the Dr. Mrs. Anupama Wagale, In charge, The Kelkar Education Trust's, Scientific Research Center, Mulund (E), Mumbai for technical guidance about Sun Protection Factor of Formulation.

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Indian J.Pharm. Educ. Res. 44(4), Oct - Dec, 2010

Table No. 1. Formulation content Sr. No 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Ingredients Cetostearyl Alcohol Stearic Acid Cetomacrogol Cetyl Alcohol Carbopol 940 Disodium EDTA Sodium Methyl Paraben Sodium Propyl Paraben Triethanolamine q.s. Demineralised Water Murraya koenigii L. Oil Weight (%w/w) 05 02 02 01 0.5 0.02 0.3 0.06 (to pH 5-6) Up to 100.00 05

Table No.2 Physical parameters of Murraya oil Cream formulation Sr. No. 1 2 3 4 5 6 7 8 9 Parameters Colour Odour Spreadibility pH Specific gravity Limit test for Lead Viscosity (26 0 C)S Total microbial count Patch test for irritancy Observations Off-White Spicy Good and uniform 6.5 0.97 Passes 251646.3 (2 rpm) Nil No Irritation Reaction Persist

Table No. 3. Result expressed as the average and S.D. of three determinations replicated of SPF values with critical wavelength. Test Curry Leaf Oil Cream Sample Parameters SPF Standerd Deviation Critical Wavelength Average value 2.04 0.02 38.37

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6. Drury HC. The Useful Plants of India. 2 nd Edition. London: Allen; 1978;78. 7. Peter KV. Curry leaf - A good ingredient for vegetable preparations. Indian Farmers Digest 1978;10:13-4. 8. Prajapati ND, Purohit SS, Sharma AK and Kumar T. A Handbook of Medicinal Plants. Jodhpur: Agro bios, 2003;352-3. 9. Goutam MP and Purohit RM. Antimicrobial activity of the essential oil of the leaves of Murraya koenigii (Linn) Spreng (Indian Curry leaf). Indian J. Pharm. 1974;36(1);1112. 10. Kishore N, Dubey NK, Tripathi RD and Singh SK. Fungitoxic activity of leaves of some higher plants. Natl. Acad. Sci. Lett. 1982;5(1):9. 11. Diffey BL, Robson J. J. Soc. Cosmet. Chem 1989;40:127-33.

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