An hydroxytyrosol-rich composition from olive vegetation water and method of use thereof

<b>Field</b> of <b>the Invention</b>

0001This invention relates to a hydroxytyrosol-rich composition for use in protecting skin against adverse effects of exposure to ultraviolet radiation, an injectible formulation comprising the composition and the use of a treatment agent for the preparation of a pharmaceutical composition for protecting skin against adverse effects of exposure to ultraviolet radiation.

<u>References</u>

0002<ul id="ul0001" list-style="none" compact="compact"><li><nplcit id="ncit0001" npl-type="s"><text>Armstrong, B.K. and Doll, R., International. J. Cancer 15:617-631 (1975</text></nplcit>).</li><li><nplcit id="ncit0002" npl-type="s"><text>Bartsch, H., et al., Carcinogenesis 20:2209-2218 (1999</text></nplcit>).</li><li><nplcit id="ncit0003" npl-type="s"><text>Braga, C., et al., Cancer 82:448-453 (1998</text></nplcit>).</li><li><nplcit id="ncit0004" npl-type="s"><text>Chan, J.M., et al., Seminars in Cancer Biology 8:263-273 (1998</text></nplcit>). <nplcit id="ncit0005" npl-type="b"><text>d'Amicis, A. and Farchi, S., in: Advances in Nutrition and Cancer 2 (Zappia, V., et al., Eds.) 67-72, Kluwer Academic/Plenum Publishers, New York (1999</text></nplcit>).</li><li><nplcit id="ncit0006" npl-type="s"><text>Deiana, M., et al., Free Radic. Biol. Med. 26:762-769 (1999</text></nplcit>). <nplcit id="ncit0007" npl-type="s"><text>de la Puerta, R., et al., Biochem. Pharmacol. 57:445-449 (1999</text></nplcit>).</li><li><nplcit id="ncit0008" npl-type="s"><text>Ficarra, P., et al., Farmaco 46:803-815 (1991</text></nplcit>).</li><li><nplcit id="ncit0009" npl-type="b"><text>Gerber, M., Epidemiology of Diet and Cancer, ed. M.J. Hill, 263-275 (1994</text></nplcit>).</li><li><nplcit id="ncit0010" npl-type="s"><text>Kohyama, N., et al., Biosci. Biotechnol. Biochem. 61:347-350 (1997</text></nplcit>).</li><li><nplcit id="ncit0011" npl-type="s"><text>Kuller, L.H., Journal of the American Dietetic Association 97:S9-S15 (1997</text></nplcit>).</li><li><nplcit id="ncit0012" npl-type="s"><text>La Vecchia, C., et al., European Journal of Cancer Prevention 7:461-464 (1998</text></nplcit>).</li><li><nplcit id="ncit0013" npl-type="s"><text>Manna, C., et al., FEBS Letters 470:341-344 (2000</text></nplcit>).</li><li><nplcit id="ncit0014" npl-type="s"><text>Martin-Moreno, J.M., et al., Int. J. Cancer 58:774-780 (1994</text></nplcit>).</li><li><nplcit id="ncit0015" npl-type="s"><text>Mattson, F.H. and Grundy, S.M., J. Lipid Res. 26:194-202 (1985</text></nplcit>).</li><li><nplcit id="ncit0016" npl-type="s"><text>Owen, R.W., et al., J. Can. Res. Clin. Onc. 125:S31 (2000a</text></nplcit>).</li><li><nplcit id="ncit0017" npl-type="s"><text>Owen, R.W., et al., Eur. J. Cancer 36:1235-1247 (2000b</text></nplcit>).</li><li><nplcit id="ncit0018" npl-type="s"><text>Owen, R.W., et al., Food Chem. Toxic. 38:647-659 (2000c</text></nplcit>).</li><li><nplcit id="ncit0019" npl-type="s"><text>Parthasarathy, S., et al., Proc. Natl. Acad. Sci. USA 87:3894-3898 (1990</text></nplcit>).</li><li><nplcit id="ncit0020" npl-type="s"><text>Petroni, A., et al., Thromb. Res. 78:151-160 (1995</text></nplcit>).</li><li><nplcit id="ncit0021" npl-type="s"><text>Risch, H.A., et al., Journal of the National Cancer Institute 86:1409-1415 (1994</text></nplcit>).</li><li><nplcit id="ncit0022" npl-type="s"><text>Romani, A., et al., J. Agric. Food Chem. 47:964-967 (1999</text></nplcit>).</li><li><nplcit id="ncit0023" npl-type="s"><text>Tsimidou, M., et al., Food Chem. 44:53-60 (1992</text></nplcit>).</li><li><nplcit id="ncit0024" npl-type="s"><text>Visioli, F., et al., FEBS Letters 468:159-160 (2000</text></nplcit>).</li><li><nplcit id="ncit0025" npl-type="s"><text>Visioli, F. and Galli, C., Nutr. Rev. 56:142-147 (1998</text></nplcit>).</li></ul>

<u>Background of the Invention</u>

0003A high amount of dietary fat has been implicated in the development of several diseases (Owen <i>et al.,</i> 2000c). Atherosclerosis (Kuller, 1997) and coronary heart disease (Gerber, 1994), as well as cancer of the breast (La Vecchia <i>et al.,</i> 1998), prostate (Chan <i>et al.,</i> 1998), ovary (Risch <i>et al.,</i> 1994), and colon (Armstrong and Doll, 1975) have each been associated with elevated dietary fat. However, evidence indicates that it is not only the amount, but also the type of dietary fat that is important in the etiology of some cancers (Bartsch <i>et al.,</i> 1999).

0004Olive oil, the principal fat component of the Mediterranean diet, has been associated with a lower incidence of coronary heart disease (Owen <i>et al.,</i> 2000b; Parthasarathy <i>et al.,</i> 1990; Mattson and Grundy, 1985) and certain cancers (d'Amicis and Farchi, 1999; Braga <i>et al.,</i> 1998; Martin-Moreno <i>et al.,</i> 1994). Several laboratories have reported that the hydrolysis of the olive oil phenolics oleuropin and other family members lead to small phenolic components with strong chemoprotective activity (Owen <i>et al.,</i> 2000a; Manna <i>et al.,</i> 2000). In particular, the olive oil phenolic hydroxytyrosol prevents low density lipoprotein (LDL) oxidation (Visioli and Galli, 1998), platelet aggregation (Petroni <i>et al.,</i> 1995), and inhibits 5- and 12-lipoxygenases (de la Puerta <i>et al.,</i> 1999; Kohyama <i>et al.,</i> 1997). Hydroxytyrosol has also been found to exert an inhibitory effect on peroxynitrite dependent DNA base modification and tyrosine nitration (Deiana <i>et al.,</i> 1999), and it counteracts cytotoxicity induced by reactive oxygen species in various human cellular systems (Manna <i>et al.,</i> 2000). Finally, studies have shown that hydroxytyrosol is dose-dependently absorbed in humans following ingestion, indicating its bioavailability (Visioli <i>et al.,</i> 2000).

0005Conventionally, olive oil production involves crushing olives, including the pits, to produce a thick paste. During this procedure, the crushed olives are continuously washed with water, a process known as "malaxation." The paste is then mechanically pressed to squeeze out the oil content. In addition to providing olive oil, the pressing also squeezes out the paste's water content. Such washing and pressing steps yield a considerable amount of water, referred to as "vegetation water."

0006Both the pit and the pulp of olives are rich in water-soluble, phenolic compounds. Such compounds are extracted from olives during malaxation, according to their partition coefficients, and end up in the vegetation water. This explains why various phenolic compounds, such as oleuropein and its derivatives, produced in olive pulp, can be found in abundance in vegetation waters. Similarly, a number of monophenolic compounds, such as tyrosol and its derivatives, produced in olive pits, are also abundant in vegetation waters.

0007Because of the strong chemoprotective activity of hydroxytyrosol, it is desirable to develop a method which produces an aqueous olive extract with a high percentage of hydroxytyrosol.

0008<patcit id="pcit0001" dnum="WO0218310A1"><text>WO 02/18310 A1</text></patcit> describes a method of obtaining a hydroxytyrosol-rich composition from vegetation water comprising the steps of producing vegetation water from olives, adding acid to the vegetation water, incubating the acidified vegetation water, particularly, until the vegetation water has weight ratio of hydroxytyrosol to oleoeuropein of between 5:1 and 200:1. Hydroxytyrosol obtained by the method may be used: (i) as a natural anti-bacterial, anti-viral and/or fungicidal product for agricultural and/or pest control applications, and (ii) as a therapeutic and/or an anti-oxidant for a variety of health purposes.

Summary of the Invention

0009The invention includes a hydroxytyrosol-rich composition for use in protecting skin against adverse effects of exposure to ultraviolet radiation, wherein the composition has a weight ratio of hydroxytyrosol to oleuropein of between 1:1 and 4:1. In one embodiment, the composition is to be orally and/or topically administered.

0010In another aspect, the invention includes an injectible formulation comprising the hydroxytyrosol-rich composition

0011In another aspect, the invention includes the use of a treatment agent having a weight ratio of hydroxytyrosol to oleuropein of between 1:1 and 4:1 for the preparation of a pharmaceutical composition for protecting skin against adverse effects of exposure to ultraviolet radiation. In one embodiment, the composition is to be orally and/or topically administered to a subject in need of such protection. The subject may be a human.

0012In one embodiment, the treatment agent further comprises a sunscreen for topical applications.

0013A suitable method of producing a hydroxytyrosol-rich composition has the steps of <ol id="ol0001" compact="compact"><li>(a) producing vegetation water from olives, preferably from the meat (or pulp) of depitted olives,</li><li>(b) adding acid to the vegetation water, preferably, in an amount to produce a pH between 1 and 5, and more preferably between 2 and 4, and (c) incubating the acidified vegetation water until at least 50%, preferably at least 75%, and more preferably at least 90% of the oleuropein originally present in the vegetation water has been converted to hydroxytyrosol. The acidified vegetation water is incubated for a period of at least two months, and even more preferably, the acidified vegetation water is incubated up to a period of approximately between 6-12 months.</li></ol>

0014The incubating is carried out until the vegetation water has a weight ratio of hydroxytyrosol to oleuropein of between 1:1 and 4:1.

0015The incubating may be carried out until the vegetation water has a weight ratio of hydroxytyrosol and tyrosol of between 3:1.

0016The method may further include fractionating the incubated, vegetation water to separate hydroxytyrosol from other components, and/or drying the vegetation water rich in hydroxytyrosol to produce a dried extract.

0017The incubated vegetation water may be extracted with an organic solvent to produce a 20%, or preferably 95% or more rich fraction in hydroxytyrosol.

0018The injectable composition that includes a hydroxytyrosol-rich composition may be prepared by one or more of the methods described above.

0019A further suitable method of producing a hydroxytyrosol-rich composition includes the steps of (a) producing vegetation water from olives; (b) hydrolyzing the oleuropein and other large phenolic molecules by addition of acid (c) optionally, drying the vegetation water; (d) contacting the optionally dried vegetation water with a supercritical fluid; and (e) recovering the hydroxytyrosol-rich composition from the contacted vegetation

0020The hydroxytyrosol-rich composition may include at least about 95 percent by weight hydroxytyrosol. The hydroxytyrosol-rich composition may include at least about 97 percent by weight hydroxytyrosol. The hydroxytyrosol-rich composition may include at least about 99 percent by weight hydroxytyrosol.

0021A further suitable method of producing a hydroxytyrosol-rich composition includes the steps of (a) producing vegetation water from olives; (b) hydrolyzing the oleuropein and other large phenolic molecules by addition of acid (c) optionally, drying the vegetation water; (d) extracting the vegetation water with a suitable organic solvent, such as Ethyl Acetate (EtAc); and (e) recovering a fraction that contains hydroxytyrosol in a purity equal or higher than 95% of the total phenolic fraction. The hydroxytyrosol-rich composition may include at least 20% of a phenolic fraction containing about 95 percent by weight hydroxytyrosol. The EtAc fraction may be purified by silica gel chromatography or other gel chromatography to obtain an hydroxytyrosol fraction containing 95% or more by weight hydroxytyrosol.

0022The recovering step described above may include the steps of (a) recovering the supercritical fluid, where the supercritical fluid contains the hydroxytyrosol; and (b) vaporizing the supercritical fluid to extract the hydroxytyrosol-rich composition. The contacting step described above may comprise the steps of (a) providing a porous membrane having opposite sides in a module under pressure with the membrane serving as a barrier interface between a fluid and a dense gas, the membrane being nonselective for said hydroxytyrosol; (b) providing the supercritical fluid into the module on one side of the membrane and the vegetation water on the opposite side of the membrane; (c) and extracting the hydroxytyrosol across the membrane as driven by a concentration gradient of the hydroxytyrosol between the vegetation water and the supercritical fluid. The porous membrane may be a hollow fiber membrane. The supercritical fluid may be carbon dioxide.

0023A further suitable method of producing a hydroxytyrosol-rich composition includes the steps of (a) producing vegetation water from olives; (b) hydrolyzing the oleuropein and other large phenolic molecules by addition of acid; and (c) spray drying, i.e., evaporating the acidified vegetation water thereby resulting in a powder containing hydroxytyrosol. The evaporation step described above may be performed by the addition of maltodextrins to the acidified vegetation water to preferably result in a powder containing approximately 1 to 5% hydroxytyrosol, and more preferably, a powder containing approximately 2% hydroxytyrosol.

0024The use of protecting skin against adverse effects of exposure to ultaviolet radiation (UVR) may comprise administering to a subject in need of such protection a pharmaceutically effective amount of a treatment agent having a weight ratio of hydroxytyrosol to oleuropein of between 1:1 and 4:1.

0025The agent may also include a sunscreen for topical applications. In one embodiment, the agent is administered topically. Preferably, the agent is administered orally.

0026These and other aspects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying figures and tables.

<u>Brief Description of Figures</u>

0027<ul id="ul0002" list-style="none" compact="compact"><li><figref idref="f0001">Figure 1</figref> shows the structures of phenolic compounds and their precursors detected in olive oil: ligstroside (I); oleuropein glucoside (II); aglycone of ligstroside (III); aglycone of oleuropein glucoside (IV); dialdehydic form of ligstroside aglycone laking a carboxymethyl group (V); dialdehydic form of oleuropein glucoside aglycone lacking a carboxymethyl group (VI); tyrosol (VII); hydroxytyrosol (VIII).</li><li><figref idref="f0002">Figure 2</figref> shows the HPLC analysis of a hydroxytyrosol-rich composition of the invention after supercritical carbon dioxide extraction from vegetation water obtained from the meat of depitted olives.</li><li><figref idref="f0003">Figure 3</figref> shows the HPLC analysis of a hydroxytyrosol-rich composition of the invention following supercritical carbon dioxide extraction, with synthetic hydroxytyrosol.</li><li><figref idref="f0004">Figure 4</figref> shows the HPLC analysis of a hydroxytyrosol-rich composition of the invention after acidic hydrolysis of vegetation water obtained from the meat of depitted olives.</li><li><figref idref="f0005">Figure 5</figref> shows the HPLC analysis of a hydroxytyrosol-rich composition of the invention following ethyl acetate extraction of hydroxytyrosol from vegetation water obtained from depitted olives and hydrolyzed by acid addition.</li><li><figref idref="f0006">Figure 6</figref> shows the HPLC analysis of pure (95% or more) hydroxytyrosol obtained after purification by gel chromatography on silica gel.</li><li><figref idref="f0007">Figure 7</figref> shows the mass spectrum of a hydroxytyrosol-rich composition of the invention.</li><li><figref idref="f0008">Figure 8</figref> illustrates the fragmentation pathway of hydroxytyrosol.</li></ul>

<u>Detailed Description of the Invention</u>

I.

Definitions

0028Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

0029By "oleuropein" is intended secoiridoid glucoside oleuropein (Structure II in <figref idref="f0001">Figure 1</figref>).

0030By "tyrosol" is intended 4-hydroxyphenethyl alcohol (Structure VII in <figref idref="f0001">Figure 1</figref>).

0031By "hydroxytyrosol" is intended 3, 4-dihydroxyphenethyl alcohol (Structure VIII in the <figref idref="f0001">Figure 1</figref>).

II.

Suitable Methods of producing a hydroxytyrosol-rich composition

0032The invention provides, in one aspect, a hydroxytyrosol-rich composition . It has been discovered that under specific conditions, as described below, hydroxytyrosol may be obtained from the vegetation water of olives.

A.

Producing Vegetation Water

0033A suitable method i employs olives that may be obtained from conventional and commercially available sources such as growers. Preferably, the vegetation water is obtained from pitted olives. The olives processed according to the method disclosed herein may be pitted by any suitable means. Pits in the olives contain tyrosol which is an undesired component in the vegetation water and which may not be appreciably broken down by the acid treatment described below. The pits may be separated from the pulp manually or in an automated manner as described below. Preferably, such means should be capable of segregating the pits without breaking them, which might otherwise cause higher concentrations of tyrosol in the vegetation water. Furthermore, hydroxytyrosol may be extracted from vegetation water obtained from olives that have not been pitted.

0034To produce vegetation water, olive pulp from the olives is first pressed to obtain a liquid-phase mixture including olive oil, vegetation water, and solid byproducts. Thereafter, the vegetation water is separated from the rest of the liquid phase mixture and collected. Exemplary methods of obtaining vegetation water are described in co-owned U.S. Patent Application Nos. <patcit id="pcit0002" dnum="US6165475A"><text>6,165,475</text></patcit> and <patcit id="pcit0003" dnum="US6197308B" dnum-type="L"><text>6,197,308, both to R. Crea</text></patcit>.

0035For purposes of commercial production, it may be desirable to automate various aspects of the invention. In this regard, exemplary methods contemplates the use of an apparatus as disclosed in U.S. Patent Nos. <patcit id="pcit0004" dnum="US4452744A"><text>4,452,744</text></patcit>, <patcit id="pcit0005" dnum="US4522119A"><text>4,522,119</text></patcit> and <patcit id="pcit0006" dnum="US4370274A"><text>4,370,274, each to Finch et al.</text></patcit>

0036Briefly, Finch <i>et al.</i> teach an apparatus for recovering olive oil from olives. Initially, olives are fed to a pulper that separates the olive pits from the olives to obtain a pitless olive meat. The meat is then taken up by an extraction screw that subjects the meat to an extraction pressure sufficient to withdraw a liquid phase, comprising oil, water and a minor proportion of olive pulp. The liquid phase is collected in a bin and then sent to a clarifying centrifuge that separates the pulp from the liquid phase to obtain a mixture comprising olive oil and vegetation water. A purifying centrifuge then separates the vegetation water and a small proportion of solid matter from the mixture to obtain an olive oil, substantially free of vegetation water, that is collected in a tank. According to Finch <i>et al.,</i> the water is put to a disposal means such as a sewer. The above mentioned method, in sharp contrast, provides for the collection, saving and use of the vegetation water to extract hydroxytyrosol.

0037Additional devices that may be used in practicing the mentioned method are disclosed in Italian Patent Nos. <patcit id="pcit0007" dnum="IT1276576"><text>1276576</text></patcit> and <patcit id="pcit0008" dnum="IT1278025"><text>1278025</text></patcit>.

0038As above, these devices can be used to separate the pulp from the pits prior to processing of the crushed olive pulp into oil, water, and solid residues.

B.

Conversion of Oleuropein to Hydroxytyrosol

0039The oleuropein contained in the vegetation water is converted to hydroxytyrosol. The pH of the vegetation water may be decreased by the addition of acid, and the vegetation water allowed to incubate under conditions which, according to the discovery of the invention, promote acid hydrolysis of oleuropein to hydroxytyrosol. The sample may then be fractionated or extracted to separate hydroxytyrosol from other compounds.

0040Preferably, the added acid is citric acid. The acid is added to the vegetation water, preferably to adjust the pH to 1-5, and more preferably, to a pH of 2-4. Solid citric acid can be added while continuously stirring in an amount of preferable 11,34 to 22,68 kg (25 to 50 pounds) of acid per about 3785 l (1000 gallons) of vegetation water. The pH of the resulting solution can be monitored, and further addition of acid may be necessary to achieve the desired pH. Exemplary methods showing the conversion of oleuropein to hydroxytyrosol following the addition of citric acid are given in Examples 1 and 2.

0041The acid may also be an organic or inorganic acid other than citric acid. Exemplary acids which may be used in the present invention include the inorganic substances known as the mineral acids -- sulfuric, nitric, hydrochloric, and phosphoric acids -- and the organic compounds belonging to the carboxylic acid, sulfonic acid, and phenol (benzyl) groups. The addition of acid to the vegetation water serves several purposes: (i) it stabilizes the vegetation water from air (oxygen) polymerization of phenolic molecules; (ii) it prevents fermentation of the vegetation water by endogenous and/or exogenous bacteria and yeast; and (iii) it provides for the hydrolysis of oleuropein and other large phenolic molecules containing hydroxytyrosol, converting them into hydroxytyrosol, as shown in Examples 1 and 2. Tables 1 and 2, in Examples 1 and 2, respectively, contain data from two samples of vegetation water and the respective percent composition of various components in the samples over time following the addition of citric acid.

0042The mixture may be allowed to incubate until hydroxytyrosol is 75-90% of the total combination of oleuropein and hydroxytyrosol. Furthermore, substantially none of the oleuropein in the original mixture may remain.

C.

Purification of Hydroxytyrosol

0043Following the conversion of oleuropein to hydroxytyrosol, preferably by acid addition, the incubated vegetation water may be fractionated by a number of methods known in the art. Exemplary methods of fractionation include partitioning with an organic solvent, such as Ethyl Acetate, chromatographic methods, including gel chromatography and high pressure liquid chromatography (HPLC), or supercritical fluids.

0044Alternatively, vegetation water obtained as described above after acidification, provides a solution which is rich in low molecular weight polyphenols, particularly hydroxytyrosol and a small amount of tyrosol and oleuropein. The concentration of hydroxytyrosol in the processed water may range from 4 - 5 grams per liter to 10 - 15 grams per liter depending upon the degree of dilution by addition of water during the olive oil extraction. A suitable method of extraktion or purification may selectively enrich the content of hydroxytyrosol without the addition of contaminants. Thus, the major polyphenolic component, hydroxytyrosol, may be isolated from other members of the polyphenolic family, impurities, suspended solids, tannins, and other molecules contained in the vegetation water. Hydroxytyrosol may therefore be produced in a purity and quantity not readily available by current synthetic or natural extraction methods.

0045A supercritical fluid is a gas that becomes very dense above its critical temperature and pressure. Its properties are between those of a gas and liquid, resulting in increased ability to dissolve compounds. Its relatively high density, high diffusivity, and low viscosity allow it to extract compounds faster than conventional liquid solvents. Carbon dioxide is the gas used most widely for supercritical fluid processing of foods and food ingredients because it is natural, nontoxic, non-flammable, and relatively inert and leaves no residue in the extracted product. Typical liquid extraction with supercritical carbon dioxide is usually done by dispersing one phase in the other in large contacting columns or towers, where the solute containing fluid, such as juices, flows downward by gravity, and the supercritical carbon dioxide flows upward. Mass transfer occurs at the interface between the two phases.

0046Alternatively, continuous extraction of liquids and suspensions can be achieved using supercritical fluids, such as carbon dioxide, and porous membranes instead of contacting columns. Instead of dispersing the phases, the liquid is fed continuously through porous polypropylene membranes configured as hollow fiber bundles or spiral wound sheets. The liquid passes through the porous membranes within a pressurized module, while supercritical carbon dioxide flows countercurrently on the other side of the membrane. The pressure in the module is essentially the same, so that the extraction is driven by the concentration gradient between the fluid and the supercritical carbon dioxide. The extract may be recovered by vaporizing the carbon dioxide for recycling. An exemplary method of extraction using supercritical carbon dioxide and porous membranes is described in <patcit id="pcit0009" dnum="US5490884A"><text>U.S. Patent No 5,490,884</text></patcit>.

0047Other supercritical fluids, instead of, or in combination with, carbon dioxide. These fluids include methane, ethane, propane, butane, isobutane, ethene, propene, hydrofluorocarbons, tetrafluoromethane, chlorodifluoromethane, carbon dioxide, dinitrogen monoxide, sulphur hexafluoride, ammonia, and methyl chloride.

0048Example 3 describes a small scale experiment in support of the invention, wherein hydroxytyrosol was isolated from vegetation water using supercritical carbon dioxide and porous membranes. HPLC and mass spectrometry analysis of the isolated hydroxytyrosol shows the sample to be 97-99% pure hydroxytyrosol. Thus, the invention provides a hydroxytyrosol-rich composition may contain at least about 80% hydroxytyrosol, preferably at least about 90% hydroxytyrosol, more preferably at least about 95% hydroxytyrosol, even more preferably at least about 97% hydroxytyrosol, and yet, even more preferably at least about 99% hydroxytyrosol.

0049Prior to extraction with a supercritical fluid the vegetation water may have carriers, which are known to those of skill in the art, such as maltodextran and/or polypropylene beads, added to the solution; and/or the solution may be dried. The drying step preferably removes at least about 90%, more preferably at least about 95%, and even more preferably at least about 98% of the water from the vegetation water.

0050An important feature of membrane reactors is the fact that contact surface interfacial area can be added independently of fluid velocities. Accordingly, a suitable method contemplates a large scale unit where the surface membrane area of the membrane used for extraction is at least about 100 square yards, preferably at least about 300 square yards, and even more preferably at least about 600 square yards to allow separation of hydroxytyrosol from large volumes of vegetation water. Thus, the membrane system would, in one aspect, be able to accommodate a flow rate of between 1 - 20 liters per minute, preferably between 5-10 liters per minute.

0051Additional purification methods may also be used in accordance with the invention as mentioned above. HPLC isolation of hydroxytyrosol is described in: Ficarra <i>et al.</i>, 1991; Romani <i>et al.,</i> 1999; and Tsimidou, 1992.

III.

Hydroxytyrosol-Rich Dietary Supplement

0052The claimed hydroxytyrosyl-rich composition may be used for a variety of applications. For example, hydroxytyrosol can be used: (i) as a natural anti-bacterial, anti-viral and/or fungicidal product for agricultural and/or pest control applications (not according to the invention), and (ii) as a therapeutic and/or an anti-oxidant for a variety of health purposes. The hydroxytyrosol may be administered to a mammalian subject, such as a person desirous of one or more of the benefits associated with hydroxytyrosol.

0053Accordingly, provided herein are compositions for the protection of skin damage resulting from exposure to ultraviolet radiation (UVR). The hydroxytyrosol can be administered orally or parenterally. Oral dosage forms can be in a solid or liquid form. Such dosage forms can be formulated from purified hydroxytyrosol or they can be formulated from aqueous or aqueous-alcoholic extracts. Regarding the latter, aqueous or aqueous-alcoholic (e.g., water or water-ethanol) extracts can be spraydried to provide a dry powder that can be formulated into oral dosage forms with other pharmaceutically acceptable carriers. The aqueous or aqueous-alcoholic extracts can be formulated to contain various weight ratios of hydroxytyrosol to oleuropein of 4:1.

0054The extracts may also be formulated to contain various weight ratios of hydroxytysol and tyrosol of between about 2:1 and about 50:1, preferably between about 5:1 and about 30:1.

0055Preferably, the composition is orally administered to a patient in need of protection against skin damage resulting from exposure to UVR. The solid oral dosage form compositions in accordance with this invention are prepared in a manner well known in the pharmaceutical arts, and comprise hydroxytyrosol in combination with at least one pharmaceutically acceptable carrier. In making such compositions, a hydroxytyrosol-rich composition, either in substantially pure form or as a component of a raw distillate or extract, is usually mixed, diluted or enclosed with a carrier. The carrier can be in a solid form, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Alternatively, the carrier can be in the form of a capsule or other container to facilitate oral administration. Thus, the solid oral dosage forms for administration in accordance with the present invention can be in the form of tablets, pills, powders or soft or hard gelatin capsules.

0056Alternatively, the hydroxytyrosol for oral administration can be in liquid form wherein the pharmaceutically acceptable carrier is water or an aqueous-alcoholic medium.

0057The compositions for administration in the present invention can also be formulated with other common pharmaceutically acceptable excipients, including lactose, dextrose, sucrose, sorbitol, mannitol, starches, gums, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, methylcellulose, water, alcohol and the like. The formulations can additionally include lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxybenzoates, sweetening agents or flavoring agents. Further, the compositions of the present invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a subject.

0058Parenteral formulations for use in accordance with the present invention are prepared using standard techniques in the art. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques. Such formulations are commonly prepared as sterile injectable solutions, using a parenterally acceptable carrier such as isotonic saline solution or as a sterile packaged powder prepared for reconstitution with sterile buffer or isotonic saline prior to administration to a subject. In one preferred embodiment the parenteral formulation is an injectible formulation which comprises between 1 and 500 mg/ml of the hydroxytyrosol rich composition of the present invention. More preferably, the injectible formulation comprises between 1 to 100 mg/ml of the hydroxytyrosol rich composition, even more preferably, between 10 to 100 mg/ml of the hydroxytyrosol rich composition, and most preferably about 10 mg/ml of the hydroxytyrosol rich composition.

0059From the foregoing, it can be seen how various objects and features of the invention are met. Those skilled in the art can now appreciate from the foregoing description that the broad teachings of the present invention can be implemented in a variety of forms. Therefore, while this invention has been described in connection with particular embodiments and examples thereof, the true scope of the invention should not be so limited. Various changes and modification may be made without departing from the scope of the invention, as defined by the appended claims.

0060The following examples illustrate methods of producing hydroxytyrosol-rich compositions in accordance with the invention. The examples are intended to illustrate, but in no way limit, the scope of the invention.

<u>EXAMPLES</u>

EXAMPLE 1

Conversion from Oleuropein to Hydroxytyrosol Following the Addition of About 11,34 kg (25 Pounds) of Citric Acid / 3785 l (1000 Gallons)

0061Table 1 shows the conversion of oleuropein to hydroxytyrosol over time following the addition of about 11,34 kg (25 pounds) of citric acid per 3785 l (1000 gallons) of vegetation water. The percentages in Table 1 are shown as weight percentages of the total phenolic compounds in the solution. As demonstrated in Table 1, hydroxytyrosol comprises over 80% of the phenolic compounds in the solution after 12 months. <tables id="tabl0001" num="0001"><table frame="all"><title><u>Table 1</u></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="29mm" /><colspec colnum="2" colname="col2" colwidth="34mm" /><colspec colnum="3" colname="col3" colwidth="34mm" /><colspec colnum="4" colname="col4" colwidth="36mm" /><colspec colnum="5" colname="col5" colwidth="36mm" /><thead><row><entry namest="col1" nameend="col5" align="center" valign="top"><u>Conversion from Oleuropein to Hydroxytyrosol Following the Addition of About 11,34 kg (25 Pounds) of Citric Acid / 3785 l (1000 Gallons)</u></entry></row><row><entry valign="top"><b>Component</b></entry><entry valign="top"><b>Composition at T=2mo.</b></entry><entry valign="top"><b>Composition at T=3mo.</b></entry><entry valign="top"><b>Composition at T=4.5mo.</b></entry><entry valign="top"><b>Composition at T=12mo.</b></entry></row></thead><tbody><row><entry>Hyd roxytyrosol</entry><entry>30.4%</entry><entry>32%</entry><entry>48.4%</entry><entry>80.2%</entry></row><row><entry>Tyrosol</entry><entry>2.5%</entry><entry>5%</entry><entry>2.2%</entry><entry>3.6%</entry></row><row><entry>Oleuropein</entry><entry>41%</entry><entry>36.6%</entry><entry>25.1%</entry><entry>1.2%</entry></row><row><entry>Oleuropeinagly cone</entry><entry>4.2%</entry><entry>4.6%</entry><entry>2.7%</entry><entry>3.7%</entry></row></tbody></tgroup><tgroup cols="5" rowsep="0"><colspec colnum="1" colname="col1" colwidth="29mm" /><colspec colnum="2" colname="col2" colwidth="34mm" /><colspec colnum="3" colname="col3" colwidth="34mm" /><colspec colnum="4" colname="col4" colwidth="36mm" /><colspec colnum="5" colname="col5" colwidth="36mm" /><tbody><row><entry namest="col1" nameend="col5" align="justify">not according to the invention not according to the invention not according to the invention</entry></row></tbody></tgroup></table></tables>

EXAMPLE 2

Conversion from Oleuropein to Hydroxytyrosol Following the Addition of About 22,68 kg (50 Pounds) of Citric Acid / 3785 l (1000 Gallons)

0062Table 2 shows the conversion of oleuropein to hydroxytyrosol over time following the addition of about 22,68 kg (50 pounds) of citric acid per 3785 l (1000 gallons) of vegetation water. The percentages in Table 2 are shown as weight percentages of the total phenolic compounds in the solution. Significantly, as demonstrated in Table 2, hydroxytyrosol comprises over 45% of the phenolic compounds in the solution after 2 months. <tables id="tabl0002" num="0002"><table frame="all"><title><u>Table 2</u></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="57mm" /><colspec colnum="2" colname="col2" colwidth="55mm" /><colspec colnum="3" colname="col3" colwidth="55mm" /><thead><row><entry namest="col1" nameend="col3" align="center" valign="top"><u>Conversion from Oleuropein to</u><u>Hydroxytyrosol Following</u><u>the Addition of About</u><u>22,68</u><u>kg</u><u>(50</u><u>Pounds)</u><u>of Citric Acid / 3785 l (1000 Gallons)</u></entry></row><row><entry valign="top"><b>Component</b></entry><entry valign="top"><b>Composition at T=2mo.</b></entry><entry valign="top"><b>Composition at T=12mo.</b></entry></row></thead><tbody><row><entry>Hydroxytyrosol</entry><entry>45.7%</entry><entry>78.5%</entry></row><row><entry>Tyrosol</entry><entry>2.9%</entry><entry>3.3%</entry></row><row><entry>Oleuropein</entry><entry>28.7%</entry><entry>1.5%</entry></row><row><entry>Oleuropeinaglycone</entry><entry>4.1%</entry><entry>3.5%</entry></row></tbody></tgroup><tgroup cols="3" rowsep="0"><colspec colnum="1" colname="col1" colwidth="57mm" /><colspec colnum="2" colname="col2" colwidth="55mm" /><colspec colnum="3" colname="col3" colwidth="55mm" /><tbody><row><entry namest="col1" nameend="col3" align="justify">not according to the invention</entry></row></tbody></tgroup></table></tables>

EXAMPLE 3

Extraction of Hydroxytyrosol from Vegetation Water

0063An aliquot (0.5 ml) of vegetation water containing about 40 mg of dry solid (maltodextran) was mixed with polypropylene porous beads and dried. The dry mix was used for extraction with supercritical carbon dioxide (PoroCrit, LLC, Berkeley, CA). The collected sample (about 2.0 mg) was analyzed by HPLC. The profile of the sample is shown in <figref idref="f0002">Figure 2</figref> and Table 3 shows the area under the major peak to be 97%. When synthetic hydroxytyrosol was added to the sample and analyzed by HPLC, one major peak appeared, as shown in <figref idref="f0003">Figure 3</figref>, indicating that the major product of the sample is hydroxytyrosol (Table 4). <tables id="tabl0003" num="0003"><table frame="all"><title><u>Table 3</u></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="22mm" /><colspec colnum="4" colname="col4" colwidth="25mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><thead><row><entry namest="col1" nameend="col5" align="center" valign="top"><u>Peak Analysis of</u><figref idref="f0002"><u>Figure 2</u></figref><u>HPLC Results</u></entry></row><row><entry valign="top"><b>Peak No.</b></entry><entry valign="top"><b>Time</b></entry><entry valign="top"><b>Height (µV)</b></entry><entry valign="top"><b>Area (µV-sec)</b></entry><entry valign="top"><b>Area (%)</b></entry></row></thead><tbody><row><entry>1</entry><entry>5.935</entry><entry>215542</entry><entry>6687705</entry><entry>97.476</entry></row><row><entry>2</entry><entry>11.433</entry><entry>5686</entry><entry>173104</entry><entry>2.523</entry></row></tbody></tgroup></table></tables><tables id="tabl0004" num="0004"><table frame="all"><title><u>Table 4</u></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="22mm" /><colspec colnum="4" colname="col4" colwidth="25mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><thead><row><entry namest="col1" nameend="col5" align="center" valign="top"><u>Peak</u><u>Analysis of</u><figref idref="f0003"><u>Figure</u><u>3</u></figref><u>HPLC Results</u></entry></row><row><entry valign="top"><b>Peak No.</b></entry><entry valign="top"><b>Time</b></entry><entry valign="top"><b>Height (µV)</b></entry><entry valign="top"><b>Area (µV-sec)</b></entry><entry valign="top"><b>Area (%)</b></entry></row></thead><tbody><row><entry>1</entry><entry>2.875</entry><entry>1345</entry><entry>13895</entry><entry>0.26</entry></row><row><entry>2</entry><entry>3.278</entry><entry>1076</entry><entry>14140</entry><entry>0.265</entry></row><row><entry>3</entry><entry>6.641</entry><entry>211204</entry><entry>5241105</entry><entry>98.240</entry></row><row><entry>4</entry><entry>11.961</entry><entry>2587</entry><entry>65811</entry><entry>1.233</entry></row></tbody></tgroup></table></tables>

EXAMPLE 4

Extraction of Hydroxytyrosol from Acidified Vegetation Water

0064An aliquot (1 liter) of vegetation water after acidic hydrolysis was vigorously shaken with ethyl acetate in a shaking flask. The organic solvent was separated from the aqueous solution and evaporated off by rotory evaporator. The resulting thick oil (about 20 g.) was collected and analyzed by HPLC. The profile of this sample is shown in <figref idref="f0005">Figure 5</figref>, and Table 5 shows the area of the major peak to be 97.457% indicating that hydroxytyrosol represents about or more than 95% of the total polyphenolic fraction in the water. Total phenolic determination by standard colorimetric assay shows that the hydroxytyrosol is contained in the oil at approximately 20% in weight. <tables id="tabl0005" num="0005"><table frame="all"><title><u>Table 5</u></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="23mm" /><colspec colnum="4" colname="col4" colwidth="25mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><thead><row><entry namest="col1" nameend="col5" align="center" valign="top"><u>Peak</u><u>Analysis of</u><figref idref="f0005"><u>Figure</u><u>5</u></figref><u>HPLC Results</u></entry></row><row><entry valign="top"><b>Peak No.</b></entry><entry valign="top"><b>Time</b></entry><entry valign="top"><b>Height (µV)</b></entry><entry valign="top"><b>Area (µV-sec)</b></entry><entry valign="top"><b>Area (%)</b></entry></row></thead><tbody><row><entry>1</entry><entry>3.873</entry><entry>7620</entry><entry>46501</entry><entry>2.542</entry></row><row><entry>2</entry><entry>13.575</entry><entry>95112</entry><entry>1782793</entry><entry>97.457</entry></row></tbody></tgroup></table></tables>

0065This fraction was used for further purification of hydroxytyrosol by gel chromatography. Dry silica (150 g) was suspended in ethyl acetate (300 ml) to obtain a thick slurry. The slurry was poured into a glass column and the silica was allowed to stand for 15 minutes to sediment. The thick oil containing about 20% (4 g) hydroxytyrosol was dissolved in 25 ml of ethyl acetate and slowly poured over the silica gel. The purification of the hydroxytyrosol was obtained by gravity elution of the product and by the addition of ethyl acetate as the solvent. The fractions containing the pure hydroxytyrosol were collected and pooled together. The solvent was evaporated until a yellow oil was produced. As shown in <figref idref="f0006">Figure 6</figref> and in Table 6, this oil is essentially pure hydroxytyrosol (97-99%) as verified by HPLC and mass spectroscopy. The yield of this purification is about 2.8-3.0 g. Hydroxytyrosol or ca. 65%. <tables id="tabl0006" num="0006"><table frame="all"><title><u>Table 6</u></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="22mm" /><colspec colnum="4" colname="col4" colwidth="25mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><thead><row><entry namest="col1" nameend="col5" align="center" valign="top"><u>Peak</u><u>Analysis of</u><figref idref="f0006"><u>Figure</u><u>6</u></figref><u>HPLC Results</u></entry></row><row><entry valign="top"><b>Peak No.</b></entry><entry valign="top"><b>Time</b></entry><entry valign="top"><b>Height (µV)</b></entry><entry valign="top"><b>Area (µV-sec)</b></entry><entry valign="top"><b>Area (%)</b></entry></row></thead><tbody><row><entry>1</entry><entry>2.875</entry><entry>1345</entry><entry>13895</entry><entry>0.26</entry></row><row><entry>2</entry><entry>3.278</entry><entry>1076</entry><entry>14140</entry><entry>0.265</entry></row><row><entry>3</entry><entry>6.641</entry><entry>211204</entry><entry>5241105</entry><entry>98.240</entry></row><row><entry>4</entry><entry>11.961</entry><entry>2587</entry><entry>65811</entry><entry>1.233</entry></row></tbody></tgroup></table></tables>

0066Mass spectrometry analysis of the samples obtained as described by the two procedures in Examples 3 and 4, as shown in <figref idref="f0007">Figure 7</figref>, confirmed that the major product is hydroxytyrosol. The sample was diluted to a final concentration of 26 micrograms per milliliter with methanol and analyzed in negative ionization mode on a Finnigan LCQ fitted with an ESI probe. The infusion was at 3 microliters per minute using an integrated syringe pump. The temperature was 270C, needle voltage +4.2 V, sheath gas 45 units, and auxiliary gas 10 units. The fragmentation pathway of hydroxytyrosol is shown in <figref idref="f0008">Figure 8</figref>. As can be seen in <figref idref="f0007">Figure 7</figref>, hydroxytyrosol (mass / charge 153.1) and its fragmentation products (123.1 and 105.1 mass / charge) account for the majority of the product abundance in the multi-stage spectrum.

EXAMPLE 5

Protection Against Skin Damage from UltraViolet Radiation

Olivenol Compositions

0067The test article was Olivenol (Lot#1A-1B). Olivenol is the crude water preparation obtained by acidic hydrolysis of vegetation water (500 ml) evaporated to dryness by rotory evaporator and subsequent lyophilization. The test article vehicles were aqueous 0.5% w/v methylcellulose (oral administration) and methyl alcohol, 99.9% A.C.S. spectrophotometric grade (topical administration). Formulations were prepared once during the study and the test article was considered 75% active for the purpose of dosage calculations.

Mice

0068One hundred male Crl:SKH1-<i>hr</i>BR hairless mice (Source: Charles River Laboratories, Inc., Portage, Michigan USA) were randomly assigned to ten dosage groups (Groups 1 through 10), ten mice per group as indicated in Table 1. The body weights of male mice ranged from 17 to 28 grams. <tables id="tabl0007" num="0007"><table frame="all"><title><b>Table 5: Experimental Design</b></title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="34mm" /><colspec colnum="3" colname="col3" colwidth="30mm" /><colspec colnum="4" colname="col4" colwidth="30mm" /><colspec colnum="5" colname="col5" colwidth="22mm" /><colspec colnum="6" colname="col6" colwidth="38mm" /><thead><row><entry align="center">Group</entry><entry align="center">Formulation and Dosage (mg/kg/day)*</entry><entry align="center">Concentrati on (mg/mL)*</entry><entry align="center">Route of Administrati on</entry><entry align="center">Number of Male Mice</entry><entry align="center">Frequency of Formulation Administrati on (Days)</entry></row></thead><tbody><row><entry align="center" valign="bottom">1</entry><entry align="center" valign="bottom">0 (Vehicle)</entry><entry align="center" valign="bottom">0 (Vehicle)</entry><entry align="center" valign="bottom">Oral</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">31</entry></row><row><entry align="center" valign="bottom">2</entry><entry align="center" valign="bottom">Olivenol (1)</entry><entry align="center" valign="bottom">0.1</entry><entry align="center" valign="bottom">Oral</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">31</entry></row><row><entry align="center" valign="bottom">3</entry><entry align="center" valign="bottom">Olivenol (10)</entry><entry align="center" valign="bottom">1.0</entry><entry align="center" valign="bottom">Oral</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">31</entry></row><row><entry align="center" valign="bottom">4</entry><entry align="center" valign="bottom">Olivenol (100)</entry><entry align="center" valign="bottom">10.0</entry><entry align="center" valign="bottom">Oral</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">31</entry></row><row><entry align="center" valign="bottom">5</entry><entry align="center" valign="bottom">Olivenol (100)</entry><entry align="center" valign="bottom">10.0</entry><entry align="center" valign="bottom">Oral</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">10</entry></row><row><entry align="center" valign="bottom">6</entry><entry align="center" valign="bottom">0 (Vehicle)</entry><entry align="center" valign="bottom">0 (Vehicle)</entry><entry align="center" valign="bottom">Topical</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">31</entry></row><row><entry align="center" valign="bottom">7</entry><entry align="center" valign="bottom">Olivenol (1)</entry><entry align="center" valign="bottom">0.25</entry><entry align="center" valign="bottom">Topical</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">31</entry></row><row><entry align="center" valign="bottom">8</entry><entry align="center" valign="bottom">Olivenol (10)</entry><entry align="center" valign="bottom">2.50</entry><entry align="center" valign="bottom">Topical</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">31</entry></row><row><entry align="center" valign="bottom">9</entry><entry align="center" valign="bottom">Olivenol (100)</entry><entry align="center" valign="bottom">25.00</entry><entry align="center" valign="bottom">Topical</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">31</entry></row><row><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">Olivenol (100)</entry><entry align="center" valign="bottom">25.00</entry><entry align="center" valign="bottom">Topical</entry><entry align="center" valign="bottom">10</entry><entry align="center" valign="bottom">10</entry></row></tbody></tgroup><tgroup cols="6" rowsep="0"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="34mm" /><colspec colnum="3" colname="col3" colwidth="30mm" /><colspec colnum="4" colname="col4" colwidth="30mm" /><colspec colnum="5" colname="col5" colwidth="22mm" /><colspec colnum="6" colname="col6" colwidth="38mm" /><tbody><row><entry namest="col1" nameend="col6" align="justify">* Groups 1-5: dosage volume = 10mL/kg. Groups 6-10: dosages and concentrations assume a mouse body weight of 25 grams and an administration volume of 0.1 mL/mouse (i.e., 100 mcL/mouse).</entry></row></tbody></tgroup></table></tables>

Administration of Olivenol Compositions and UVR Exposure

0069Formulations were orally administered (via gavage) to appropriate mice once daily for either 31 (Groups 1 through 4) or 10 (Group 5) consecutive days. Formulations were topically administered (100 mcL/mouse) to appropriate mice once daily for either 31 (Groups 6 through 9) or 10 (Group 10) consecutive days.

0070On the 28^th day (Groups 1 through 4 and 6 through 9) or the 7^th day (Groups 5 and 10) of formulation administration, mice in Groups 1 through 10 were exposed to UVR (i.e., wavelengths in the UVB and UVA portions of the electromagnetic spectrum). The source of irradiation was a Berger Compact Arc high intensity solar simulator (Solar Light Company, Philadelphia, PA) with a WG320 Schott glass filter (1 mm) coupled to an Oriel light pipe. The radiant intensity of the source was monitored continuously with a PMA 2100 meter (Solar Light Company, Philadelphia, PA) or comparable device. On day 28 or 7, the interval between the formulation administration and the start of UVR exposure was less than 15 minutes for most mice and slightly more than 15 minutes for a small number of mice.

0071Checks for viability were made twice daily. Clinical observations were recorded at least weekly, including once before the first formulation administration and once immediately before UVR exposure. Clinical observations were also recorded at approximately 24, 48 and 72 hours after irradiation. Body weights were recorded once weekly during the administration period and at terminal sacrifice.

Sacrifice of Mice

0072All mice survived to schedule sacrifice. Scheduled sacrifice occurred after the final examination, approximately 72 hours after completion of the UVR exposures (CO₂ asphyxiation). Dorsal skin, including the UVR exposure sites, were removed and retained in neutral buffered 10% formalin for possible histopathological examination.

0073Calculated mean UVR dose values (MED) and standard deviations were determined for appropriate groups as follows. The lowest instrumental UVR dose to cause any cutaneous response at a site of exposure was determined for each mouse. The mean calculated UVR dose for each group for each observational time point was determined. If administration of the test article has no influence on the UVR dose required to elicit cutaneous responses, based on this method of calculation, a mean calculated UVR dose value equivalent to 1.0 MED would be expected at 48 hours after irradiation. A mean calculated UVR dose value greater than 1.0 would indicate a protective effect of the test article. For any mouse that had no skin reactions in any of the six UVR exposure sites, an imputed value of 2.8 was assigned for the purpose of calculation and the > symbol was included as a prefix to the group mean calculated UVR dose value. Additionally, ratios (clinical observations) and averages with standard deviations (body weights) were calculated.

0074Group means and standard deviations were calculated and tabulated for body weights and body weight changes.

Results

0075Skin reactions that occurred in the UVR exposure sites included erythema, edema and flaking and the severity of the skin reactions tended to be dependent on the UVR exposure dose.

0076There was an indication of a mild dosage-dependent protective effect against UVR-induced cutaneous inflammation in hairless mice orally administered Olivenol for 31 days and a moderate dosage-dependent protective effect in mice topically administered Olivenol for 31 days. In this type of study, it was anticipated that the mean calculated UVR dose value would be approximately equal to 1.0 at 48 hours after UVR exposure in naïve mice.

0077In mice orally administered Olivenol for 31 days (Groups 1 through 4), the mean calculated UVR dose values at 48 hours after UVR exposure were 1.2, 1.3, 1.4 and 1.5 in the 0 (Vehicle), 1, 10 and 100 mg/kg/day dosage groups, respectively. In mice topically administered Olivenol for 31 days (Groups 6 through 9), the mean calculated UVR dose values at 48 hours after UVR exposure were 1.5, 1.5, >1.9 and >2.2 in the 0 (Vehicle), 1, 10 and 100 mg/kg/day dosage groups, respectively. The > symbol was included as a prefix to the mean calculated UVR dose values in Groups 8 and 9 because no cutaneous reactions occurred in any of the UVR exposure sites for two mice in each of those groups. For those four mice an imputed value of 2.8 was assigned for the purpose of calculation.

0078At 72 hours after UVR exposure, the protective effect of the test article was less definitive. However, in mice topically administered 100 mg/kg/day Olivenol for 31 days (Group 9) the mean calculated UVR dose was 1.6 at 72 hours after UVR exposure, as compared with a value of 1.2 for the appropriate control group (Group 6).

0079The 1.5 mean calculated UVR dose value in Group 6 [0 (Vehicle), topical administration] at 48 hours after UVR exposure was unanticipated. Since the value was substantially greater than the anticipated value of approximately 1.0, the vehicle may have had some impact on cutaneous susceptibility to UVR exposure. However, there was a clear increase in the mean calculated UVR dose values in the mice topically administered 10 and 100 mg/kg/day Olivenol dosages for 31 days, as compared with mice topically administered 0 (Vehicle) mg/kg/day Olivenol.

0080There was no indication of a protective effect against UVR-induced cutaneous inflammation in hairless mice administered the 100 mg/kg/day Olivenol dosage for 10 days vial the oral (Group 5) or topical (Group 10) route, as compared with the appropriate control groups. In mice orally or topically administered the 100 mg/kg/day Olivenol dosage for 10 days, the mean calculated UVR dose values at 48 hours after UVR exposure were 1.0 and 1.3, respectively. These values were comparable to the values that occurred in the appropriate 0 (Vehicle) mg/kg/day dosage groups (i.e., Groups 1 and 6, respectively).

0081The skin reactions that occurred at 24 hours after UVR exposure were not considered useful in making a determination on the protective potential of the test article because these reactions tend to be less reproducible than those that occur later.

0082Two mice in each of Groups 1 and 3 developed urogenital ulcerations. One mouse in each of Groups 6 and 7 developed lump(s). These are common findings in male hairless mice and were not considered test article-related. Necropsy revealed that all tissues appeared normal. Body weight and body weight changes observed throughout the experimental protocol were unremarkable.

0083There was an indication of a mild dosage-dependent protective effect against UVR- induced cutaneous inflammation in male hairless mice orally administered Olivenol for 31 days and a moderate dosage-dependent protective effect in mice topically administered Olivenol for 31 days. The high Olivenol dosage, 100 mg/kg/day, afforded cutaneous protection via the oral and topical administration routes.

0084There was no indication of a protective effect against UVR-induced cutaneous inflammation in hairless mice administered the 100 mg/kg/day Olivenol dosage for 10 days via the oral or topical route.