The Investigation of the Rabbit and Human Skin Irritation of Herbal Anti-wrinkle Cream

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1 Original Article The Investigation of the Rabbit and Human Skin Irritation of Herbal Anti-wrinkle Cream Narisa Kamkaen*, Wimon Phuntuwate, Weerasak Samee, Artitaya Boonrod and Charoen Treesak Faculty of Pharmacy, Srinakharinwirot University, Nakhonnayok, 26120, Thailand * Corresponding author: narisa@swu.ac.th ABSTRACT Flower extract of Caesalpinia pulcherrima possesses an impressive anti-oxidant activity. In this study, we examined safety of the anti-wrinkle cream containing the flower extract by means of skin irritation tests on rabbit skin (N = 7) and human skin (N = 30). This formulated cream was oil-in-water type containing 0.5% Caesalpenia flower extract. Safety was evaluated based on the Primary Irritation Index (PII). The results showed that in rabbit the cream was safe as the erythema and edema reactions at 24 hr observations were relatively low in rabbit, and somewhat negligible in human. All reactions disappeared at 72-hr observations. In human we found that our 0.5% Caesalpenia cream either with or without Aloe vera extract was safe as only 2 subjects reported slight itching on their skin sites applied with the herbal cream without Aloe vera extract. No rash was observed on any formulation. Since the cream with Aloe vera extract exhibited a somewhat lower irritancy than did the one without Aloe vera, the 0.5% Caesalpenia cream with Aloe vera extract may have a potential for further development for cosmetic products. Thai Pharm Health Sci J 2007;2(1):20-25 Introduction Anti-wrinkle product has been increasingly popular especially those with herbal or natural ingredients. The flower of Caesalpinia pulcherrima Swartz. (Leguminosae) (Peacock flower, Pride of Barbados, and Peacock's crest) was found to have antimicrobial and antiinflammation properties. 1,2 Therefore it has been added as an herbal ingredient in an anti-wrinkle cream (by the authors: N. Kamkaen and W. Samee). It is thus necessary to examine skin irritation of the cream formulation. In cosmetic industry, evaluation of irritancy potential to human skin of any chemicals or formulations is a necessity. This must be done by means of in vivo and in vitro tests to determine the risk of irritation due to the contact between these compounds and human skin. The most commonly used test is the rabbit skin irritation test 12 th year of Srinakharinwirot Journal of Pharmaceutical Sciences described in the OECD test guideline 404 and in the European Chemicals Bureau Annex V part B.4 ( which was initially described by Draize et al (1944). 3 In this animal test, the test substances, either raw materials or finished formulated products, are to apply on the rabbit s shaved skin. A score of skin reactions is based on physiological observations on the animals. Caesalpinia pulcherrima Swartz. is an ornamental plant with its flowers appearing in a variety of colors including yellow, pink, off-white, and red with yellow margins. 4 It is a common medicinal plant in India, Taiwan, and South-East Asian countries including Thailand. In alternative medicine, different parts of this plant have been used for treatment of inflammation, bronchitis and malarial infection, diarrhea and dysentery, and also used as abortifacient and emmenagogue. 1,5 20 Thai Pharmaceutical and Health Science Journal, Vol. 2 No. 1, Jan. Apr. 2007

2 Decoction of the plant is used to treat various infections. 6 Earlier works reported the isolation of diterpenoids 6-9, peltogynoids and flavanoids in this plant. 8,10,11 Some constituents were found to possess antitumor 7,9, antimicrobial 6 and anti-inflammatory properties 2. In our laboratory, we found that crude flower extract of Caesalpinia pulcherrima possesses an impressive antioxidant activity using DPPH radical scavenging assay with an EC 50 of 0.05 mg/ml. We also formulated an antiwrinkle cream containing crude extract of Caesalpinia pulcherrima flower in oil-in-water (O/W) emulsion. It was found that antioxidant activity of the cream was adequately stable under accelerated condition (%AA = 71.8 ± 1.8) (unpublished research report of a pharmacy undergraduate project, under supervision of two N. Kamkaen and W. Samee). In order to assure safety of the anti-wrinkle cream, we conducted skin irritation tests on rabbit and human in this present study. Materials and Methods Preparation of herbal anti-wrinkle cream Air-dried flower (100 g) of Caesalpinia pulcherrima was powdered and macerated with ethanol (2 L) for 7 days. The extract was filtered and concentrated by rotary evaporator. The thick red brown residue (6.55 g) was kept in the dessiccator. This crude extract of Caesalpinia pulcherrima flower was then mixed in oil-in-water emulsion for the formulation of herbal anti-wrinkle cream using Aloe vera extract as vehicle instead of pure water. The ingredient contents in the formula (% w/w) are listed in Table 1. The procedure to prepare herbal cream was as follows. Salicylic acid was dissolved in propylene glycol and glycerin in a beaker. Salicylic acid, as a keratolyic agent, was used in an acceptable means as suggested by Thai FDA that salicylic acid in cosmetics with concentrations of 1 to 1.5% (ph 2.8) was allowed. 12 The oil phase (as shown in Table 1) was melted by heating a beaker to 65 C using a water bath. Water phase (as shown in Table 1) was heated in a separate beaker to 70 C. Once the desired temperature was reached, the oil phase was added into the water phase. When the mixture temperature decreased to 40 C, Germaben II was added and the mixture was homogenized for 30 min. This resulting smooth cream was then tested for its viscosity, particle size, ph, physical appearance and antioxidant activity. Table 1 Ingredients of cream base and herbal cream formulations Cream Herbal Ingredient (%w/w) base cream A. Oil phase Cetomacrogol Cetomacrogol Stearic acid Stearyl alcohol Cetyl alcohol Silicone oil Isopropyl myristate B. Water phase Sodium EDTA Sodium salicylate Aloe vera extract (50% w/w) Caesalpinia pulcherima flower extract C. Others Salicylic acid Propylene glycol Glycerine Germaben II Total Rabbit skin irritation test An evaluation was conducted using seven rabbits. The back of the animals was clipped free of fur with an electric clipper 24 hours before application of the sample. For the experiment, the clipped areas of skin of each rabbit were divided into four sites within the same area (25 25 mm). The herbal cream (500 mg) was mixed and immediately applied to one site with an approximate amount of 80 mg per cm 2. Another site was used as a control, i.e., applied with cream base. Both the treated and controlled sites were covered by gauze and the back of the rabbit was wrapped with a non-occlusive bandage. Thai Pharmaceutical and Health Science Journal, Vol. 2 No. 1, Jan. Apr

3 The animals were then returned to their cages. After 24 hours, the bandage and the test materials were removed and 1 hour later the sites were examined for skin irritation. Observation of the sites was done at 24 hours after application, and repeated at 48 and 72 hours thereafter. The reactions, defined as erythema and edema, were evaluated according to the scoring system for skin reactions (Table 2). Table 2 Classification system for skin reactions Reaction Score Erythema No erythema 0 Very slight erythema (barely perceptible) 1 Well-defined erythema 2 Moderate to severe erythema 3 Severe erythema (beet redness) to eschar formation 4 Edema No edema 0 Very slight edema (barely perceptible) 1 Well-defined edema (edges of the area well defined by 2 definite raising) Moderate edema (raising approximately 1 mm) 3 Severe edema (raised more than 1 mm and extending 4 beyond the area of exposure) Total possible score for primary irritation 8 The Score of Primary Irritation (SPI) was calculated for each rabbit as the following. Scores for erythema and edema at 24, 48 and 72 hours were summed and divided by the number of the observations for the treated sites. 13 The SPI for the control sites were calculated in the same fashion. The differences between the summary scores from the treated sites and the control sites were calculated. The Primary Irritation Index (PII) was calculated as the arithmetical mean of the SPI values of the six animals. The irritation degree was categorized as negligible, or slight, moderate or severe irritation based on the PII (Table 3). Human skin irritation test Two cream formulations consisting of 0.5% Caesalpenia crude extract with and without Aloe vera extract as vehicle were tested for human skin irritation. Thirty healthy volunteers (15 women and 15 men, aged years) were included in this test. The irritation reactions of the two formulations were observed 15 min after topical administration. Each subject was applied cream base (control) and herbal cream on separate but adjacent areas on the same lower forearm. Table 3 Response categories of irritation in rabbit Category Primary Irritation Index (PII) Negligible Slight irritation Moderate irritation Severe irritation 5 8 Results Rabbit skin irritation test At 24 hours, it was found that in seven rabbits, erythemas with score of 1-2 were found on the skin sites that were applied with cream base and with herbal cream (Table 4). On sites applied with cream base, erythemas with scores of 2 (well-defined erythema) were found in 5 out of 7, while those applied with herbal cream were found 6 out of 7. After 72 hours, most erythemas found on either sites applied with cream base or with herbal cream disappeared or reduced in severity. In terms of edema, the occurrences of edema on sites applied with cream base and sites with herbal cream at 24 hours were also comparable. Edema with score of 1 (very slight edema) was found in 4 out of 7 and 5 out of 7 on sites with cream base and with herbal cream respectively. At 72 hours, all edemas disappeared. Human skin irritation test The results of the irritation test on 30 volunteers suggested that 0.5% Caesalpenia cream either with or without Aloe vera extract was safe as only 2 subjects reported slight itching on their skin sites applied with the herbal cream without Aloe vera extract (Table 5). No rash was observed on any formulation. 22 Thai Pharmaceutical and Health Science Journal, Vol. 2 No. 1, Jan. Apr. 2007

4 Table 4 Score of erythema and edema after application of test materials Rabbit No. Reaction Score for skin reaction* Cream base (Control) Herbal cream 24 h 72 h 24 h 72 h Erythema Edema Erythema Edema Erythema Edema Erythema Edema Erythema Edema Erythema Edema Erythema Edema Primary Irritation Index (PII) 20/28 = /28 = 0.71 Category of irritation based on PII Slight irritation Slight irritation * Score for erythema: 0 = No erythema, 1 = very slight erythema (barely perceptible), 2 = well-defined erythema; Score for edema: 0 = No edema, 1 = very slight edema (barely perceptible) (see also Table 2) Table 5 The irritation reaction on human subjects applied with 0.5% Caesalpenia cream with and without Aloe vera extract Rash Itching Reaction Irritation category 0.5% Caesalpenia cream based on PII without with Aloe vera extract Aloe vera extract Negligible (0 0.4) Slight ( ) 0 0 Moderate (2 4.9) 0 0 Negligible (0 0.4) Slight ( ) 2 0 Moderate (2 4.9) 0 0 Discussions Assessment of irritation of pharmaceutical and cosmetic products with natural compounds is a significant step in the evaluation of their biocompatibility. Researchers and regulatory agencies recognize the important role of in vitro and animal tests play in the biologic evaluation of cosmetics products. Based on the results of this in vivo investigation, the irritant properties of the Caesalpenia cream after direct application to rabbit skin were somewhat evident, but less relevant to clinical significance. This is the case since we also found that the cream caused almost no skin reactions in human. Our findings were consistent with the arguments that the rabbit skin irritation test by Draize et al (1944) may have some disadvantages. 3 First, rabbit and human skins have Thai Pharmaceutical and Health Science Journal, Vol. 2 No. 1, Jan. Apr

5 different physiological properties and different responses to environmental and chemical agents However, biological basis for the variability of skin irritation among species remains unknown. 18 Despite the increasing evidence of differences of the skin responses between rabbit and human, data based on rabbit skin test have been taken as a reference to determine the irritant potential of chemicals with an exception of rare published studies comparing results obtained both from animals and humans. 14,16,19 Another reason for the shortcoming of skin irritation by Draize et al (1944) is the fact that some compounds are more toxic for rabbits than for humans and vice versa. 13,14,16 This confirms that results from animal and human tests are sometimes not interchangeable. Moreover, the Draize test sometimes lacks reproducibility. 20,21 It was also a concern about the animal s suffering and discomfort since eschar formation can be observed with severe irritants. The presence of salicylic acid in our preparation might have also contributed to the irritation as the substance is a keratolytic agent. Salicylic acid with the concentration in our study was probably not likely to cause significant irritation since overt keratolytic effect can be observed in a much higher concentration, e.g., 5%. 22 In this study, we investigated the human skin irritation study compared with the one on rabbit skin. The discrepancy of irritation reactions was found where the cream was much less potential to cause such reactions on human skin. We found that our 0.5% Caesalpenia cream without Aloe vera extract produced only mild irritation on human skin. In addition, 0.5% Caesalpenia cream with Aloe vera extract seemed to exhibit a somewhat lower irritancy potential than the one without Aloe vera extract. Thus, the 0.5% Caesalpenia cream with Aloe vera extract may have a potential for further development for cosmetic products. Acknowledgement The authors would like to express appreciation for the financial support by the Faculty of Pharmacy, Srinakharinwirot University. References 1. Srinivas KVNS, Rao YK, Mahender I, et al. Flavonoids from Caesalpinia pulcherrima. Phytochemistry 2003;63: Rao YK, Fang SH, Tzeng YM. Anti-inflammatory activities of flavonoids isolated from Caesalpinia pulcherrima. J Ethnopharmacol 2005;14: Draize JH, Woodard G, Calvery HO. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J Pharmacol Exp Ther 1944;82: Roach JS, McLean S, Reynolds WF, Tinto WF. Cassane diterpenoids of Caesalpinia pulcherrima. J Nat Prod 2003;66(10): Chiang LC, Chiang W, Liu MC, Lin CC. In vitro antivial activities of Caesalpinia pulcherrima and its related flavonoids. J Antimicrob Chemother 2003;52: Ragasa CY, Hofilena JG, Rideout JA. New furanoid diterpenes from Caesalpinia pulcherrima. J Nat Prod 2002;65: Che CT, McPherson DD, Cordell GA, Fong HHS. Pulcherralpin, a new diterpene ester from Caesalpinia pulcherrima. J Nat Prod 1986;49: McPherson DD, Che TT, Cordell GA, Soejarto DD, Pezzuto JM, Fong HHS. Diterpenoids from Caesalpinia pulcherrima. Phytochemistry 1986;25: Patel AD, Freyer AJ, Webb RL, et al. Pulcherrimins A-D, novel diterpene dibenzoates from Caelsalpinia pulcherrima with selective activity against DNA repairdeficient yeast mutants. Tetrahedron 1997;53: Namikoshi M, Nakata H, Saitoh T. Homoisoflavonoids from Caesalpinia sappan. Phytochemistry 1987;26: Passador EAP, Da-Silva MF, Das-Fo ER, Fernandes JB, Vieire PC, Pirani JR. A pyranochalcone and a flavanone from Neoraputia magnifica, Phytochemistry 1997;45: Thai Food and Drug Administration. BHA. (Accessed on Jul. 10, 2007, at hppt:// product/bha.shtml) 13. ECETOC. ECETOC Monograph 32: use of human data in hazard classification for irritation and sensitization. Brussels Thai Pharmaceutical and Health Science Journal, Vol. 2 No. 1, Jan. Apr. 2007

6 14. Phillips L-II, Steinberg M, Maibach HI, Akers WA. A comparison of rabbit and human skin response to certain irritants, Toxicol Appl Pharmacol 1972;21(3): Marzulli FN, Maibach HI. The rabbit as a model for evaluating skin irritants: a comparison of results obtained on animals and man using repeated skin exposures. Food Cosmet Toxicol 1975;13: Nixon GA, Tyson CA, Wertz WC. Interspecies comparisons of skin irritancy. Toxicol Appl Pharmacol 1975;31: Scott RC, Corrigan MA, Smith F, Mason H. The influence of skin structure on permeability: an intersite and interspecies comparison with hydrophilic penetrants. J Invest Dermatol 1991;96: Campbell RL, Bruce RD. Direct comparison of rabbit and human primary skin irritation responses to isopropylmyristate. Toxicol Appl Pharmacol 1981;59: Hoffmann S, Cole T, Hartung T. Skin irritation: prevalence, variability, and regulatory classification of existing in vivo data from industrial chemicals. Regul Toxicol Pharmacol 2005;41: Weil CS, Scala RA. Study of intra- and interlaboratory variability in the results of rabbit eye and skin irritation tests. Toxicol Appl Pharmacol 1971;19: Spielmann H. Aternativen in der toxikologie. In: Gruber FP, Spielmann H (eds.). Alternativen zu Tierexperimenten. Berlin. Spektrum Akadamisher Verlag, 1996: pp Nook TH. In vivo measurement of the keratolytic effect of salicylic acid in three ointment formulations. Br J Dermatol 1987;117(2): Thai Pharmaceutical and Health Science Journal, Vol. 2 No. 1, Jan. Apr