JOURNAL OF COSMETIC SCIENCE 122 Ac2, Ac3), Curcuma (Kc1, Kc2, Kc3), Tamarindus (Tc1, Tc2, Tc3) and Centella creams (Hc1, Hc2, Hc3). They contained 1, 3, and 5% w/w of herbal extract, respectively. STABILITY STUDIES Stability studies were carried out for all the formulations at different temperature condi- tions (4°, 25° and 37° ±2°C) and a relative humidity of 60% for three months. All the evaluation parameters, i.e., color, odor, pH, viscosity, spreadability, and phase separation, were studied at different time intervals, i.e., 15, 30, 60 and 90 days (30). STUDY DESIGN Healthy human volunteers who were willing to give their informed consent were in- cluded in the subjective study. Volunteers having known hypersensitivity reactions to any of the formulation ingredients, or with skin wounds or scratches on the volar forearm, were not selected. The biophysical study was done according to Table II. The volar fore- arm was used as the site for the application of the formulations because of the need for uniformity in assessment of all the skin parameters at a time and because this site was more convenient and volunteer-acceptable. EVALUATION STUDIES Physicochemical parameters and microbial examination. Several physicochemical parameters were measured for each prepared cream formulation (18,20). These physicochemical pa- rameters provided information regarding formula stability and skin compatibility. Vis- cosity was measured using a Brookfi eld viscometer. All the evaluations were carried out Table II Protocol and Design of the In Vivo Study Parameters Protocol and design Place This prospective study was conducted at the University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India. Number of Total: 60 (30 males and 30 females) (ten groups having six volunteers each) volunteers Site 1: Baseline values to observe initial compliance and safety to the skin (CC0) Site 2: Base cream (BC) Site 3: Coded formulations (Cc1, Cc2, Ac1, Ac2, Kc1, Kc2, Tc1, Tc2, Hc1, Hc2) Age of volunteers 22–45 years Application dose 5 mg/cm2 to 2 cm2 area of skin. Twice a day on the volar side of the forearm during four weeks. Readings taken every morning Evaluation Skin hydration determination by corneometer Skin viscoelasticity determination by cutometer Sebum determination by sebumeter Melanin determination by mexameter

HERBAL FORMULATIONS FOR SKIN IMPROVEMENT 123 in triplicate, presented as ± SD. Microbial contamination of the prepared formulations was tested according to Indian Standards methods. The total viable counts, i.e., total bacterial, yeast, and mold counts, were recorded by using a colony counter (21). Determination of sun protection factors (SPFs). The in vitro SPFs were determined according to the method described by Mansur et al. (22) and Sayre et al. (23), utilizing UV spectro- photometry and the following equation: 320 290 spectrophotometric SPF CF I Abs u O u O u O ¦EE where CF = the correction factor (10) EE (λ) = the erythmogenic effect of radiation with wavelength λ I(λ) = the intensity of solar light of wavelength λ and Abs (λ) = the spec- trophotometric absorbance values at wavelength λ. The values of EE × I are constants (24). Safety test. A patch test design was conducted to make sure that the prepared formulations did not cause any skin irritation. Cream formulations were applied on the volar forearm with the help of surgical gauze (0.5 g/cm2), and the score was determined using the scale defi ned in the Indian Standards (25). The positive comments of all volunteers helped us to begin the biophysical studies. The fi ve formulations with 5% w/w extract that caused irritation were not used in further study. Psychometric evaluations. The products were compared based on sensory evaluation, and ranking was done as per score obtained according to the hedonic scale. The cream formu- lations were applied twice a day, once in morning and once in evening at the same time, on the volunteers for up to six weeks, and observations are made by ranking method. The volunteers were asked various questions, and according to their answers ranking was done between 0–9 of the hedonic scale: 8–9 (extreme liking), 5–7 (medium), 1–3 (dislike), 6 (between extreme liking and medium), and 4 (between medium and dislike). Ranking was perfect verbally for appearance, fragrance, lathery feel, softness, irritation, stickiness, smoothness, and after effect on the skin. Overall ranking was done on the basis of an aver- age score for each product, determined as follows: E1 = average score of each product V1 = total score of product given by volunteer No.1 V2 = total score of product given by volunteer No.2 Vn = total score of product given by volunteer No. n N = number of volunteers Biophysical evaluation. Three test areas were marked on the forearms of the volunteers. One test area was treated twice daily with the test formulation, the second was treated with the base cream, and the third was left untreated and used as a control. At the baseline visit and after one, two, and four weeks of regular use, skin viscoelasticity, fi rmness, hydration level, sebum content, and melanin content were determined. Skin hydration was deter- mined by a Corneometer® CM 825, which is based on capacitance measurement (26). Sebum determination was done by a Sebumeter® SM 815, which is based on grease-spot photometry, whereby a special tape becomes transparent in contact with the sebum on the skin surface (27). Melanin and erythema determination is based on absorption/refl ection and was done by a Mexameter® MX 18 (28). Skin viscoelasticity was determined by a