INTRODUCTION Research into the use of naturally-occurring chemical compounds for application as topical medication is motivated in part, by growing

public concern over the possible health risks associated with products of this type that contain synthetic active agents. Consequently, efforts continue toward the development of safe and effective therapeutic agents based on natural compounds. Research at Poseidon involves a discovery program to: (1) identify existing natural compounds currently in human use that may have alternative biological actions suitable for industrial or pharmaceutical applications; (2) identify natural products derived from marine and terrestrial flora and fauna. I. MENTHOL PROPYLENE GLYCOL CARBONATE Menthol is a natural product extracted from peppermint oil. Menthol and its various isomers and analogs, such as isopulegol, N-ethyl-p-menthane-3-carboxyamide and pmenthane-3, 8-diol, are used in commerce as cooling agents. These compounds impart a cooling sensation to a variety of products such as cosmetics, perfumes, personal care products, oral hygiene products, confectionery, cigarettes, cough drops, nasal inhalants and the like. Although it is known to have some emollient and antiinflammatory action, the effects are generally mild or require very high concentrations. The strong odor of menthol has made it difficult to use this material for these applications. For this reason, there had been much effort and significant successes in developing analogs that have minimal mint odor, yet high cooling effect. One of these compounds is menthol propyleneglycol carbonate (MPC). Recent research by Poseidon scientists on MPC demonstrated significant inhibition of TPA-induced inflammatory response and inhibition of angiogenesis. By comparison menthol showed no appreciable anti-inflammatory effect under the same test conditions.

Fig. 1. Comparison of the structure of menthol (left) and MPC (right)

MPC is a GRAS (generally regarded as safe by US FDA) material known by number 3806 in the U.S. FEMA (Flavor and Manufacturers Association) list and as number 444 on the JECFA (Joint FAO/WHO Expert Committee on Food Additives) list. The latest JECFA assessment conducted in 1999 of menthol and its derivatives including MPC reaffirmed this compounds safety as an ingredient in food and cosmetics. MPC as an Anti-Inflammatory Agent

Fig. 2. Inhibition of TPA induced edema (ear thickening) by topical application of MPC on the mouse ear

The mouse ear edema model is a standard animal test procedure to document the antiinflammatory effect of an agent. In this test, edema was induced in mice through the topical application of 10l of TPA (Tetradecnoylphorbol acetate) in acetone (2.5g/ear) to both the inner and outer surface of one ear of each mouse. Each test compound, diluted with acetone to a concentration of 10% was applied topically to the inflamed mouse ear immediately after TPA application, so as to deliver 2.5 mg/ear. The reference drug, indomethacin (0.5mg/ear), was administered as a positive control. The thickness of each ear was measured before treatment and 4 hours after induction of inflammation, using a micrometer (Mitutoyo Co.). Anti-inflammatory effect was expressed as the reduction in ear thickness with respect to the control group. When menthol and its isomers were tested for anti-inflammatory effects using the mouse edema assay, we observed no significant effects. However, when MPC was tested in the same assay, a dramatic inhibition of TPA-induced mouse ear edema was found. This effect was further tested by the topical application of MPC (referred in this study as HR-008) at 2% and 10%. The data are presented in Figure 2. The study showed approximately a 30% reduction of the ear thickness by the application of a 2% solution of MPC in ethanol, and a 50% reduction using a 10% solution of MPC. MPC as an Anti-angiogenic Agent The effect of MPC was studied by culturing aortic explants in three-dimensional matrix gels according to the procedure of Kruger and Figg (Kruger E.A. and Figg, W.D. Protein binding alters the activity of suramin, carboxyamidotriazole, and UCN01 in an ex Vivo Rat Aortic Ring Angiogenesis Assay. Clinical Cancer Research, 7:1867-1872, 2001).

The photographs shown in Figures 3 and 4 show that MPC exerted significant anti-

angiogenic activity in a standard rat aortic ring assay. SUMMARY The findings described in this report indicate that menthol propyleneglycol carbonate (MPC), a GRAS food ingredient, is an effective nontoxic biochemical that may be used clinically as an anti-inflammatory and anti-androgenic agent. These observations open new market opportunities to use MPC in diverse medical conditions that involve angiogenesis and inflammation. For more details about these studies, please download this brief description of the biological effects of MPC. Please click here. II. Extract of Inula viscosa Inula viscosa General description of the plant and its uses Inula viscosa (Ait.) L. (Compositae; syn. Dittrichia viscosa), is a sturdy perennial shrub that grows in the wild around the Mediterranean basin. It is an aromatic plant that disperses a smell of camphor. The leaves and stems of the plant are coated with a sticky resin secreted from glandular hairs growing on the surface of the leaves. The local variant of Inula viscosa that grows in Israel, being located in the most southern latitude of its normal range, is believed to possess higher biological activity compared to its European relatives. In Israel, the species is exposed to warmer climate, longer sunshine exposure days and lack of rainfall that can wash off some of the resin. The longer period of exposure to pathogenic attacks during the hot summer is postulated to selectively enhance the local plants production of defensive phytochemicals. The increased UV protection of the leaf is made possible by the release of the resin on the leafs surface.

Traditional medicinal uses of Inula viscosa Hailed as the King of Medicinal Plants, Arab villagers in Israel, Palestine and Jordan have used Inula viscosa to alleviate or cure various ailments. The Arab physician El Tamimi, who practiced in Jerusalem during the 10th century, wrote that the drink Raesen, made from Inula viscosa and added to honey, was the drink of the kings. It was effective in alleviating rheumatism, colds, and even acted as an aphrodisiac. The traditional medicine practices are documented in Herbs Pouch1, a book written in Hebrew, which includes interviews and first-hand evidence of Inula viscosas many uses. Since ancient times, Inula viscosa has been widely used as treatment for the following: Wounds The application of fresh leaves, or a powder made of dry

leaves on open or bleeding wounds or burns results in the stoppage of bleeding, and serves as an effective antiseptic and anti-inflammatory agent. Treatment using Inula viscosa is preferred by villagers over modern medications, since it promotes faster wound healing. The powder from the dried Inula viscosa leaf is mixed with oil and applied externally to affected parts of the Dry and rough skin body. About 200 grams of Inula viscosa leaves are boiled in water for 5 minutes; filtered extract is poured into a bath full of warm water in which the arthritic person immerses Arthritis and rheumatism himself for 30 minutes. For rheumatism, a powder made from Inula viscosas dried leaves is mixed with olive oil and applied on the affected area. Treatment for hemorrhoids consists of steaming Inula viscosa leaves and sitting over the vapors. A paste is also Hemorrhoids prepared from ground leaves, mixed with olive oil and applied as a topical ointment. Bronchitis and respiratory The extract is added to boiling water and the tonic consumed. infections Inula viscosa leaves are boiled in water and the tonic is High blood pressure consumed. and diabetes The biochemical basisof Inula viscosas medicinal properties The wide range of biological activity of Inula viscosa extracts has been described in the scientific literature. Among these are Inula viscosas properties as: a microbial inhibitor. Inula viscosa has potential applications in the protection of food crops, in the protection of coatings against mold and mildew, in wood preservation against fungal decay, in the prevention of fouling on submerged marine structures, and in the protection of humans against growth dermatophytes and Candida albicans; an anti-inflammatory agent; an anti-oxidant; and an anti-ulcer agent Some of these effects are hypothesized to occur because of synergy among the diverse constituents of the biochemicals produced by this unusual plant. Various chromatographic techniques show that extracts of Inula viscosa possess a high content of the following: Bioflavonoids, a powerful anti oxidant; Tomentosin (sesqui terpenic lactones), a powerful antiseptic, microbial and fungi inhibitor; Saponins, a naturally-occurring foamy substance known to possess anti-cancer effects; Sterols, a cholesterol-lowering group of naturally-occurring chemicals; and

 Carotenoids

The anti-inflammatory properties of Inula viscosa Unlike most well known herbal medicinal plants, Inula viscosa was unheard of in medical literature until a decade ago. Since then, over 60 scientific papers have been published about the broad-spectrum medicinal activity of Inula viscosa and its extracts. Several patents related to Inula viscosa have been filed in recent years, and start-up biotechnology companies focusing on the plants potential and wide-ranging therapeutic properties have carried out laboratory testing of Inula viscosas extracts. The anti-inflammatory and wound-healing properties of Inula viscosa are described in a few publications and summarized below: A study published in 2001 by scientists at the Department of Pharmacology at the University of Valencia in Spain showed that inuviscolide, one of the sesquiterpene lactones found in Inula viscosa acted as the plants main antiinflammatory agent.3 The study was designed to examine the anti-inflammatory effects of the sesquiterpene acid ilicic acid, and inuviscolide on cell degranulation, leukotriene biosyntheses, neurogenic drive and glucocorticoid-like interactions. Inuviscolide acted by interfering with leukotriene synthesis and PLA(2)-induced mastocyte release of inflammatory mediators. In the 2001 study, Swiss female mice were used to measure the ear edema induced by phorbol esters or ethyl phenylpropionate (EPP), and the paw edema induced by phospholipase A(2) (PLA(2)) or serotonin. One topically applied dose was applied in the ear models and a subcutaneous or intraperitoneal injection on the paws. Quantitative analysis of leukotriene B(4) (LTB(4)) formation was performed on rat peritoneal neutrophils by high performance liquid chromatography (HPLC). The lactone inuviscolide reduced the PLA(2)-induced edema (ID(50): 98 uM/kg). Modifiers of the glucocorticoid response did not change the effect on serotonininduced edema. Ilicic acid showed minor in vivo effects, but was slightly more potent than inuviscolide on the 12-O-tetradecanoylphorbol 13-acetate (TPA). Acute edema test (ID(50): 0.650 micromol per ear). Inuviscolide reduced LTB(4) generation in intact cells, with an IC(50) value of 94 microM. Earlier research conducted by the University of Valencia in 1999, sought to establish a strong basis for the documented use of Inula viscosa as a remedy for various skin diseases.4 Flavonoids (rhamnocitrin (1), 7-O-methylaromadendrin (3), and 3-O-acetylpadmatin (4)), inuviscolide, ilicic acid, and a digalactosyldiacylglycerol, inugalactolipid A (6), were isolated from the CH2Cl2 extract,

identified and characterized as the topical anti-inflammatory properties of Inula viscosa. All these compounds proved to be effective against 12-Otetradecanoylphorbol-13-acetate-induced ear edema in mice. However, it lacked activity against against arachidonic acid-induced edema. Ilicic acid and inugalactolipid showed significant effects on a multiple-dose murine chronic dermatitis model. Inula viscosas effectivity as an antipyretic and antiseptic was studied by scientists at the Faculty of Medicine and Surgery at the University of Genoa.5 Pharmacological test on rabbits, which were made hyperpyretic in laboratory conditions, gave satisfactory antipyretic results. Gas chromatographic separation from a high-boiling fraction of seven azulenes, two of them identified as 1,4dimethyl-azulene (about 50% and chamazulene (32%), confirmed the antiphlogistic action ascribed to the plant. Through methods such as solvent extraction, thin layer chromatography and gas chromatography, eucalyptol was identified in a fraction of essential oils obtained from fresh leaves of the plant, supporting the balsamic and antiseptic properties of Inula viscosa. The anti-inflammatory action of the Inula viscosa extract In order to verify the biological activity of Inula viscosa extract as an antiinflammatory agent, a study was conducted using the mouse ear edema assay. In this method, 10 l of TPA (tetradecanoylphorbol acetate) was applied in acetone (2.5 g/ear) to both the inner and outer surface of the mouse ear to induce inflammation. The thickness of each ear was measured with a micrometer before treatment and four hours after induction of edema. The effect of the topical application of Inula viscosa extract at various concentrations in preventing inflammation was also measured (see Fig. 4).

Fig. 4. The reduction of the ear thickness by the application of nula viscosa extract at various concentration

In the graph below (Fig. 5) the data shows that with a concentration as low as 2%, Inula viscosa extract is sufficient in reducing inflammation by as much as 60%. By comparison, indomethacin as the standard reference, produces as much as 95% inhibition. However, indomethacin is not suitable for dermatologic use.

Fig. 5. The anti-inflammatory effect as percentage of control group

REFERENCES: 1. Nissim Krisspil, Herbs Pouch (translated from the original Hebrew), Jerusalem: Kana publishers (1982). 2. Stephen Spiteri, The Pharmacognosy of the local plant Inula viscosa [abstract online] (Malta: University of Malta, Dept. of Pharmacy, 1998, accessed 20 March 2004); available from http://www.cis.um.edu.mt/~phcy/symp98/StephenSpiteri.html 3. Victoriano Hernandez, Maria del Carmen Recio, Salvador Manez, Jose Maria Prieto, Rosa Maria Giner, Jose Luis Rios, A mechanistic approach to the in vivo anti-inflammatory activity of sesquiterpenoid compounds isolated from Inula viscosa, Planta Medica (November 2001) : 67 (8), 726-731. 4. S. Manez, M.C. Recio, I. Gil, C. Gomez, R.M. Giner, P.G. Waterman, J.L. Rios, A glycosyl analogue of diacylglycerol and other anti-inflammatory constituents from Inula viscosa, J Nat Prod. (April 1999) : 62(4), 601-604. 5. L. Lauro, C. Rolih, Observations and research on an extract of Inula viscosa, (article in Italian), Boll Soc Ital Biol Sper. (September 1990): 66(9), 829-834.