j. Cosmet. sci., 50, 221-229 (July/August 1999) Comparison of antioxidant activity of extract from seeds of white pepper (Piper nigrurn, Linn.) to commercial antioxidants in 2% hydroquinone cream ARANYA MANOSROI, MASAHIKO ABE, and JIRADEJ MANOSROI, Pharmaceutical-Cosmetic Raw Materials and Natural Products Research and Development Center (PCRNC), Natural Products for Thai Traditional Medicines Research Unit, Department of Pharmaceutical Technology, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand (A.M., J.M.) The Cosmetic Quality Control Subcommittee, FDA, Ministry of Health, Nontaburi, Thailand (A.M.) Faculty of Science and Technology, Science University of Tokyo, Yamazaki 2641, Noda, Chiba 278, Japan (M.A.) and Institute of Colloid and Interface Science, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku, Tokyo 162, Japan (M.A.). Accepted for publication June 30, 1999. Synopsis Powdered dry fruits of white pepper (Piper nigrum, Linn.) were extracted with dichloromethane and frac- tionareal into various portions. The powder obtained from the aqueous weakly acidic fraction, which gave a yield of 1.09% w/w of crude pepper, was tested for antioxidative activity in comparison with commercial antioxidants (sodium metabisulfite and BHT) at 0.1%, 0.5%, and 1.0% w/w in 2% w/w hydroquinone cream. The systems were incubated in a dark room at 24 ø + 1.0øC for two weeks and at 45 ø + 0.5øC for three months. The physical stability and the percentages of hydroquinone remaining after two weeks and one, two, and three months were determined by UV spectrophotometry at 294 nm according to official standard procedures. The experiment revealed that oxidation degradation of hydroquinone was accelerated by heat even with the existence of antioxidants. The higher percentages of remaining hydroquinone were observed for higher antioxidant concentration but showed lower physical stability of the formulation, especially in the cases of 1% BHT and 1% extract. In two months at 45 ø + 0.5øC, the extract demonstrated the same activity as the other two commercial antioxidants at all concentrations, with about 20-30% more hydroquinone remaining than in the control system. The creams containing 0.1% and 0.5% extract gave good physical formulation stability, with about 66% and 72% of hydroquinone remaining. This suggested the possibility of using an extract from white pepper at 0.1% and 0.5% as a water-soluble antioxidant for substances that are oxidation-susceptible. INTRODUCTION One of the most important characteristics of many cosmetic products is stability. Hy- droquinone, a hypopigmenting agent employed percutaneously to lighten localized 221

222 JOURNAL OF COSMETIC SCIENCE areas of hyperpigmented skin such as blemishes, lentigo, melasma, chloasma, and freck- les, is known for its high oxidative reactivity. It is one of the chemicals that are difficult to stabilize. It becomes brown in air due to oxidation. One gram of hydroquinone is soluble in 17 ml of water and freely soluble in alcohol (1,2). In Thailand, hydroquinone is classified in a specially controlled cosmetic category according to the FDA-Cosmetic Act BE 1992. In antifreckle and hair dyeing cosmetic products, its content is limited by law to 2% w/w. Most hydroquinone products used in creams, gels, lotions, and oint- ments are locally manufactured in Thailand. Being located in a tropical area, most Thai people suffer from solar effects on their skin. Freckles are one of these effects. Hydroquinone is one of the cheapest hypopigmenting agents available in the Thai market that most middle and lower class Thai people have preferred to use along with sunscreening products. However, with low stability and the side effects of allergy and irritation by hydroquinone for many consumers, the Cosmetic Control Division, FDA, of Thailand has special concerns for these products. In 1995, the FDA and the Toxicology Division of the Department of Medical Science, Ministry of Health, in Thailand reported that six out of 20 samples collected from the market contained more hydroquinone than the allowed amount (3). This was excused because of the high instability of hydroquinone, and most manufacturers had put the excess hydroquinone in their products in order to keep the constant amount of 2.0% during storage. Recently, many researchers have been involved in finding means to prevent or delay deterioration by oxidative reactions in cosmetic preparations. A variety of antioxidants both from natural sources and synthetic processes are available in the market. In Thai markets, antioxidants that are usually incorporated in hydroquinone formulations are sodium metabisulfite, BHT (butylated hydroxy toluene), BHA (butylated hydroxy an- isole), ascorbic acid, vitamin E, citric acid, and/or combinations of these chemicals. Their shelf-life is about one year. Hydroquinone itself has been used as an antioxidant in combination with others in the concentration of 0.05-0.1% (4). Various disadvantages of some of these antioxidants have caused concern. For example, an application of BHA and BHT is now restricted in many countries, since undesirable effects of these additives on the enzymes of the liver and lungs can occur (5). Occasionally, the antioxidant ability of vitamin E is less active (6). More recently, research has focused on developing safer and more effective antioxidants from natural sources, such as Rosmarinus officinalis L. (7,8) and Piper spp. (8-10). Chemical constituents of white pepper (Piper nigrum, Linn.) and their application to food preservation have been evaluated (6). However, a study of the antioxidative activity of the extract from white pepper has never been performed for cosmetic preparations. Hydroquinone, which is known for its high sensitivity to oxi- dation, has been chosen as an indicator for comparison of the antioxidative activity of white pepper extract to commercial antioxidants in the form of 2% w/w hydroquinone cream. MATERIALS AND METHODS EXTRACTION AND FRACTIONATION OF PEPPER SEEDS An amount of 729.56 g of the powdered dry fruits of white pepper (P. nigrum, Linn.)