CA2377118C - Antipsoriatic compositions, method of making, and method of using - Google Patents

Abstract

A psoriasis treatment composition derived from the plant Asphodelus Microcarpus includes 3-methylanthralin, chrysophanol, aloe-emodin, aloe-emodin monoacetate, and/or derivatives thereof.
The composition is prepared by extracting a liquid from the Asphodelus Microcarpus root and mixing the liquid with acetic acid. A method of treatment includes applying the composition to an affected area of skin at a frequency sufficient to effect an alleviation of symptoms, typically once per day for 14-56 days.

Description

ANTIPSORIATIC COMPOSITIONS, = z METHOD OF MAKING, AND METHOD OF USING
BACKGROUND OF INVENTION
1. Field of the Invention The present invention relates to skin treating compositions, to a method of making the compositions, and to a method of using the compositions in the treatment of skin. In another aspect, the present invention relates to antipsoriasis compositions, to a method of making the compositions, and to a method of using the compositions to treat psoriasis. In even another aspect, the present invention relates to botanical-derived skin treating compositions, to a method of making the compositions, and to a method of using the compositions to treat skin.
This application is a division of Canadian Patent Application No. 2,249,458 filed March 19, 1997.

2. Description of the Related Art Psoriasis is a chronic skin condition characterized by itchy, flaky skin. It is estimated that two percent of the United States population, more than four million people, will suffer from psoriasis during their lives. Psoriasis conditions can range from mild to severe.
In the United States, between about 150, 000 and 250, 000 new cases of psoriasis occur each year, with about 40,000 of these cases classified as severe. Sufferers of psoriasis must endure not only the irritating disease itself, but also the embarrassment of skin disfigurement.
The total annual cost for treating psoriasis on an outpatient basis is estimated at more than $1.5 billion. It is estimated that psoriasis sufferers are spending an average of $500 per year on psoriasis treatment to achieve only temporary relief. Severe cases that require hospitalization may require an expenditure of up to $10,000.
The compound 3-methylanthralin has long been utilized in the treatment of psoriasis, and is listed in the Merck Index as an antipsoriatic. Chrysarobin is a mixture of compounds derived from Goa powder, and includes 3-methylanthralin. Goo powder itself is derived from the wood and bark of Andria Araroba Aguiar (Fam. Legum.inosae) . Literature references describing the isolation of and structure of Chrysarobin date back to the early 1.800s. A method of reducing Chrysarobin to obtain 3-methylanthralin was known as early as 1931.
Known psoriasis treatments include: antimetabolites such as methotrexate; corticosteroids such as triamcinolone creams or injection, clobeasol propinate cream, and hydrocortisone;
keratolytic/destructive agents such as anthralin or salicylic acid; lubricants such as hydrogenated vegetable oils and white petroleum; oral retinoids such as etretinate or isotretinoin tablets; photochemotherapy such as methoxsalen or trioxsalen capsules, and coal tar; and topical cholecalciferol analogs such as calcipotriene ointment, a topical vitamin D3; known commercially as Dovonex ~, (Squib, Huffalo, NY). , Numerous botanical protocols for the treatment of psoriasis are known, including the use of extracts of various herbs, roots, seeds, flowers, berries, and twigs. See Therapeutic Botanical Protocol for Psoriasis, Protocol Journal of Botanical Medicines, August 1994, pp. 1-38.
A composition for the treatment of burns and erythema has been described in CH-A-671336, the composition including Aspbodelus verus Albus'root extract and four other plant species.
However, the known psoriasis treatments suffer from one or more deficiencies, including potential toxic side effects and achieving only temporary relief. Thus-there is a need for improved psoriasis treatment.
SUI~lARY OF INVENTION
It is an object of the present invention to provide a composition and method of treating psoriasis.
It is another object of the present invention to provide ~S a method for making 3-methylanthralin.
It is even another object of the present invention to 2a provide a method of making chrysophanol.
It is still another object of the present invention to provide a method of making aloe-emodin.

It is a further object to provide a method of making aloe-emodin monoacetate.
It is an additional object to provide a composition extracted from a botanical specimen that has efficacy in treating psoriasis.
It is another object to provide such a composition having a plurality of polyphenols therein.
It is a further abject to provide a method of treating psoriasis with such a composition.
It is yet another object to provide a method of extracting such a composition from the botanical specimen.
These and other objects of the present invention are achieved by the composition and methods of the present invention.
According to even still another embodiment of the present invention there is provided a method of making a product comprising at least one of 3-methylanthralin, chrysophanol, aloe-emodin, and aloe-emodin monoacetate comprising contacting root of the plant Asphodelus Microcarpus with acetic acid to form the product. A further embodiment of the method includes recovering 3-methylanthralin, chrysophanol, aloe-emodin, or aloe-emodin monoacetate from the product. Any of the products may be further derivatized, According to yet even another embodiment of the present invention there is provided a method of obtaining either 3-methylanthralin, chrysophanol, or aloe-emodin by recovery of the desired compound from the root of the plant Asphodelus Microcarpus. The polyphenols may be further derivatized.
BRIEF DESCRIPTION OF THE DRAWINGS
3o FIG. 1 is a schematic representation of the analytical fractionation scheme utilized in Example 4.
FIG. 2 shown the peak assignments used to generate the data of Table 3 for the XAD-2 resin DCM eluate fraction 23.
FIGS. 3 and 4, show, respectively, the direct probe (DP) mass spectrometry data and liquid secondary ion mass spectrometry (LSIMS? data pertaining to the XAD-2 resin DCM
eluate fraction 23 of FIG. 1.
FIGS. 5, 6, and 7 show, respectively, a GC-MS
chromatogram, DP data, and LSIMS data for the chemical composition of XAD-2 resin MeOH eluate DCM-insoluble fraction 26 of FIG. 1.
FIGS. 8, 9, and 10 show, respectively, a GC-MS
chromatogram, DP data, and LSIMS data for the hydrolyzed XAD-2 resin MeOH eluate DCM-insoluble fraction 29 of FIG. 1.
FIGS. 11, 12, and 13 show, respectively, a GC-MS
chromatogram, DP data, and LSIMS data for carboxylic acid fraction 28.
FIG. ~ 14 shows GC-MS chromatogram data from the methylated carboxylic acids of Example 5.
FIGS. 15, 16, and 17 show, respectively, a GC-MS
chromatogram, DP data, and LSIMS data for the neutral/phenol fraction 36 of FIG. 1.
FIGS. 18-23 show, respectively, GC-MS chromatograms, DP data, and LSIMS data for the neutral/phenol fraction DCM
silica gel eluate and the neutral/phenol fraction MeOH silica gel eluate.
FIG. 24 is a plot showing the effects of the treatment solution of Example 2 on HNF in fibroblasts.
DETAILED DESCRIPTION OF THE INVENTION
In the practice of the present invention, a composition is made from the plant Asphodelus Microcarpus that comprises compounds useful in the treatment of mammalian skin disorders, especially psoriasis. Asphodelus Microcarpus is a plant that is native to the Middle East; specifically, it can be easily found in the Northern regions of Israel, as well as in regions from the Canary Islands to Asia Minor.
Tn the practice of the present invention, the root of Asphodelus Microcarpus is utilized. In a specific embodiment of the present invention, the root or portions thereof may be administered to treat mammalian skin disorders. Preferably, in such a treatment, the outer skin of the root is first removed, and then the inner portion of the root is applied to the afflicted skin region. Additionally, raw extract from the roots may also be applied~to the afflicted skin region.
It is to be understood in the practice of the present invention that the roots of Asphodelus M.icrocarpus may be harvested at any time. However, it is preferred that the roots be harvested at a time when they are full of liquid which for Asphode3us Microcarpus growing in Israel is generally from February to May.
Preparation of the Asphodelus Microcarpus roots is generally as follows. Excess dirt and other foreign matter should be removed from the roots, generally by shaking and water washing. After dirt and other foreign matter have been removed from the root, the next step is to remove the outer layer of the root. This can be accomplished by using a scraper, knife, a peeler such as a potato peeler, or the like.
The next step is to extract liquid from the roots.
Methods for obtaining liquid from a compressible liquid containing solid are well known to those of skill in the art, and any such method may be utilized. Simple methods include mashing, squeezing, pulverizing, liquefying, or compressing the roots.
Preferably, the roots are liquefied in a commercially available "juicer."
The liquid thus obtained is mixed with acetic acid.
Generally for this step, the volume ratio of Asphodelus Microcarpus root juice to acetic acid is in the range of about 1:20 to about 20:1. Preferably, the volume ratio is in the range of about 1:10 to about 10:1, more preferably in the range of about 1:5 to about 5:1, even more preferably approximately 4:1.
The Asphodelus Microcarpus root liquid is next mixed with acetic acid at any temperature suitable for creating a mixture of 3-methylanthralin, chrysophanol, aloe-emodin, and aloe-emodin monoacetate. It is preferable that the mixing occur _""'~

with both the Asphodelus Microcarpus root liquid and acetic acid in the liquid state. Thus the contacting temperature is above the freezing point but below the boiling point for the mixture:
Optionally, in the practice of the present invention, brimstone may be mixed in with the Asphodelus Microcarpus root and acetic acid. The brimstone may be in any suitable form, but is preferably ground, and more preferably ground to a flour-like consistency.
The Asphodelus Microcarpus root/acetic acid mixture comprises 3-methylanthralin, chrysophanol, aloe-emodin, and aloe emodin monoacetate and has been shown to be useful in the treatment of psoriasis. While this mixture is shown herein as being derivable from the Asphodelus Microcarpus root, it should be understood that the 3-methylanthralin, chrysophanol, and aloe emodin can be obtained by the methods that are known in the art.
Aloe-emodin monoacetate can be made by contacting aloe-emodin with acetic acid.
3-Methylanthralin, chrysophanol, aloe-emodin, and aloe emodin monoacetate can be individually recovered from the mixture using separation techniques as are well known in the art.
The treatment composition of the present invention may include a wide range of the polyphenol components and/or their derivatives. In the method of the present invention fox treating psoriasis, the composition may be administered to a mammalian organism by any route known in the art. Nonlimiting examples of suitable routes of administration include oral, parenteral, topical, and the like. Specific nonlimiting examples of carrying out such routes of administration include injection, IV administration, pills, tablets, capsules, liquids, gels, creams, soaps, shampoos, dermal patches, inhaled aerosols, sprays, suppositories, and the like. Topical administration is preferred for human psoriasis.
The frequency of administration varies with the strength of the composition, but an exemplary treatment schedule comprises a topical application once per day until symptoms are _ '7 eradicated. The course of treatment naturally is dependent upon the severity of the affliction, and may last from 14 days up to 56 days, although this is not intended as limiting.
The present invention is described as being suitable for the treatment of humans for psoriasis, which is to be understood to include, but not be limited to, exfoliative psoriatic dermatitis, pustular psoriasis, and guttatte variant psoriasis.
It is also believed that the compositions of the present invention axe useful in the treatment of other skin disorders and conditions, including eczema.
In addition to being used in the treatment of skin disorders, the compositions of the present invention are also believed to be useful in the treatment of psoriatic arthritis.
EXAMPLES
The following examples are provided 'merely to illustrate the invention and are not intended to limit the scope of the invention in any manner.
Example 1 Obtaining Raw Extract Raw extract was obtained from the Asphodelus Microcarpus plant as follows. Approximately 1 1b of roots of the plant were obtained from a location in Israel. Dirt and other foreign matter was removed from the roots by shaking, and then by washing with water, after which the outer skin of the roots was removed. Liquid was extracted from the peeled roots utilizing a commercially available "juicer". The 1 1b of roots yielded approximately 300 cc of raw extract.
Example 2 Pretaaration of Treatment Solution Approximately 650 cc (although 400-800 cc are usable, depending upon the severity of the affliction) of the raw extract obtained by the method of Example 1, approximately 350 cc (although 200-600 cc are usable) of acetic acid, and -~L
.8.
approximately 1 teaspoon of brimstone, ground to flour-like texture, were mixed together. This mixture does not require refrigeration, although the raw extract should be refrigerated until the acetic acid is introduced if kept in an unmixed state.
Example 3 Analysis of the Treatment Solution The treatment solution of Example 2 was subjected to various types of chemical and physical analysis, the results of which are presented in Tables 1 and 2.
to Table 1 Proximate Analysis Data*
Physical description of sample Dark brown liquid containing sediment and suspended particulate matter, acetic acid like odor Ph (as received) 1.95 Acetic acid content (by titration)5.05! wiw Insoluble matter 2.14! wlw Ash weight (mineral content) 0.746! wJw Specific gravity (filtrate) 1.103 glml Total dissolved solids (filtrate)12.94! wlw Chloroform extractables (filtrate)0.84% wlw (organic soluble components) *Analysis performed by Stillwell & Cladding Testing Laboratories, 3 o Inc., New York, N.Y.

_: L

Table 2 Elemental/Heavy Metal Composition*
Element ~ . Conc. ppm: Element Canc: ppm:.

Sulfur 4260 Bismuth NO < 3 ppm Potassium 1640 Lead ND < 3 ppm to Calcium 1075 Antimony _ ND < 3 ppm Sodium 216 Boron ND < 2 ppm Phosphorous 142 Indium ND < 2 ppm Magnesium 100 Molybdenum ND < 1 ppm is Silicon 7 Arsenic ND < 1 ppm Iron - 7 Selenium ND < 1 ppm Aluminum 5 Tellurium ND < 1 ppm Zinc 2 Thallium ND < 1 ppm Tin 1 Lithium ND < 1 ppm Strontium 0.9 Cobalt ND < 1 ppm Cop~aer 0.9 Niobium ND < 1 ppm Manganese 0.4 Gallium NO < 1 ppm Titanium 0.4 Germanium ND < 1 ppm Nickel 0.4 Silver ND < 1 ppm l Chromium 0.2 Cadmium ND < .5 ppm ~~

l Barium 0.1 Mercury NO < .5 ppm Vanadium 0.06 Be Ilium ND < .1 m l 3 s * Elemental/heavy metal analysis was conducted by Umpire and Control Services; lnc., West Babylon, N.Y.

Example 4 Fractionation of Treatment Solution The solution of Example 2 was also subjected to analysis . As the solution of Example 2 is too complex for direct analysis, it was fractionated according to the scheme in FiG. 1 using a combination of wet analytical chemistry, column chromatography and solvent extraction procedures. Throughout the analytical fractionation, isolates were subjected to cell culture bioassay, as described in Example 6. Fractions testing positive in the cell culture bioassay were then directed to additional to separation and/or chemical characterization until nearly pure isolates were obtained.
A 500 g sample 10 of the treatment solution of Example 2 was filtered through a Buchner funnel containing Whatman #1 filter paper and a 1 cm bed volume of Celite°
analytical filtering aid. The filtrate 12 (485 g, 97% of original sample) tested posiaive in the cell culture bioassay and was equivalent in potency to the unfiltered treatment solution of Example 2. The filter retentate 14 (14.6 g, 2.92%) was a dark reddish brown colored material with a clay-like consistency.
2o This substance tested negative in the cell culture bioassay.
Analysis of this sediment indicates it to be largely composed of sucrose, complex carbohydrates, cellulosic debris, lignins, inorganic minerals, and clay.
The filtrate was then passed through a preconditioned chromatographic column packed with a 20 x 350 mm bed volume of XAD-2 resin. (Supelcopak-2° absorbent) at a flow rate of 5 ml/minute. XAD-2 resin is a hydrophobic porous polymer absorbent based on styrene-divinylbenzene copolymer. This resin has a high affinity for absorbing nonpolar, organic-soluble components from aqueous solutions. The column was preconditioned by washing it with 1.0 L of dichloromethane (DCM), followed by 1.0 L of methanol (MeOH) and finally 1.0 L of distilled/deionized water.
All solvents were ultra-high-purity ~~capillary-analyzed~~ grade suitable for trace-level chemical analysis procedures. The distilled water used throughout the analysis was obtained from a Milli-Q~ purification system. It was double distilled in glass and then further purified by passing it through ion-exchange and activated carbon filters . After the filtrate was passed through the XAD-2 resin, the column was washed with 2 L of distilled water. The filtrate and wash 15 from the XAD-2 resin column tested negative in the call culture bioassay and were discarded.
This dark brown colored fraction contains the water-soluble compounds of the treatment solution, such as acetic acid, sugars, low-molecular-weight polar organic acids, tannins, and other l0 biologically inert components.
The XAD-2 resin column containing the treatment solution retentate 16 was blown dry to remove as much of the residual wash water as possible. The treatment solution organic-soluble components were then eluted from the column with 2.0 L
of MeOH followed by 2.0 L of DCM. The MeOH and DCM XAD-2 resin eluates were collected separately. The treatment solution XAD-2 resin MeOH eluate 21 yielded 4.6 g, which corresponds to 0.92%
of the original treatment solution on a weight basis. The eluate was amber colored. The treatment solution XAD-2 resin DCM eluate ltf 23 contained 0.51 g or O.lx of the original sample. This eluate was bright yellow colored. Both of these fractions tested positive in the cell culture bioassay. However, higher activity was observed in the treatment solution XAD-2 resin MeOH eluate.
Therefore, this isolate was subjected to additional fractionation.
The treatment solution XAD-2 resin MeOH eluate 21 was concentrated to dryness in a round bottom flask using a rotary evaporator at reduced temperature and pressure. The residue was a dark red crystalline substance. The residue was dissolved in 200 ml of DCM, producing an amber-colored solution. However, not all the residue was soluble in DCM. The DCM-insoluble materials were then dissolved in 100 m1 of MEOH. The methanol-soluble components had a dark red color. Thus two additional fractions were prepared. The treatment solution XAD-2 resin MEOH eluate s~.~ I)CM-soluble fraction 25 contained 1.7 g (0.34x) , and the -~i treatment solution XAD-2 resin MeOH eluate DCM-insoluble fraction 26 yielded 2.9 g (0.58%). Both of these fractions tested positive in the cell culture bioassay.
The treatment solution XAD-2 resin MEOH eluate DCM
soluble fraction 40 contains organic-soluble neutral, phenolic, and acidic components. It was extracted 6 times with 100 ml portions of saturated sodium bicarbonate solution to remove the carboxylic acid fraction. The saturated sodium bicarbonate extract 28 was acidified to Ph 2.0 using 1 N HCL and then back extracted 6 times with 50 ml portions of DCM to partition the carboxylic acids into the organic phase. The treatment solution carboxylic acid fraction 33 contained 166 mg (0.03%) and was weakly positive in the cell culture bioassay. The fraction had an amber-colored appearance.
The treatment solution XAD-2 resin MeOH eluate DCM-soluble fraction, after extraction of the carboxylic acids, contained 1.52 g or 0.30% of the original sample. This isolate 31 was named the treatment solution "neutral/phenol fraction"
based on its chemical composition. It yielded a strong positive response in the cell culture bioassay. Upon refrigeration this fraction was observed to form a yellow-orange-colored crystalline precipitate. The precipitate was isolated by filtration through a sintered glass type Gooch crucible. The precipitate 36 isolated from the neutral/phenol fraction after extraction of acids yielded 186 mg (0.04%) of bright yellow-orange crystals.
This isolate was found to be the most highly active fraction in the cell culture bioassay. The neutral/phenol fraction 38 after harvesting of the crystalline precipitate was found to contain 1.33 g (0.27%) . This sample also tested strongly positive in the cell culture bioassay.
Some additional subfractions were prepared from the isolates described above. A portion of the treatment solution carboxylic acid fraction was methylated with diazomethane reagent to produce the corresponding methyl esters of the sample. This was done to enhance the volatility of the acids to improve the - L
-i3-gas chromatographic separation. The treatment solution XAD-2 resin MeOH eluate DCM-insoluble fraction was hydrolyzed with 1 N Hcl for 4 hours at 100°C to break down glycosidically conjugated species. The treatment solution neutral/phenol fraction 29 after harvesting of precipitate was subjected to additional minicolumn fractionation using a silica gel solid-phase extraction column. These procedures are described in more detail Example 5.
Example 5 - Chemical Characterization of the Fractions of Example 4 The nonvolatile components of the fractions were analyzed by a combination of electron ionization direct probe mass spectrometry (DP data) and by a high-mass technique, liquid secondary ion mass spectrometry (LSIMS data).
The chemical composition of the XAD-2 resin DCM eluate fraction 23 is shown in Table 3, with peak assignments corresponding to the GC-MS chromatogram shown in FIG. 2. The DP
and T.~SIMS spectra pertaining to the XAD-2 resin DCM eluate fraction 23 are shown in FIGS. 3 and 4, respectively. A
homologous series of nonvolatile compounds with molecular weights 474, 490, 506, and 508 were detected in the DP data. Accurate mass measurements were performed on these peaks using high-resolution (R = 10,000) mass spectrometry in order to determine their elemental formulas. The empirical formulas for these 2 5 compounds were found to be C23H22~11 ( 4 74 MW ) , C23HZZOlx ( 4 90 MW ) , C13H22O,3 (506 MW) , and C23Hz401a (508 MW) . In other fractions related homologues with molecular weights 478 and 492 were also observed. Exact chemical structures for these compounds are unknown. However, their elemental formulas and mass spectral fragmentation patterns indicate that they are a class of polyphenolic chemical compounds called bisflavanoids.
The chemical composition of XAD-2 resin MeOH eluate DCM-insoluble fraction 26 is summarized in Table 4. The GC-MS
chromatogram, DP data, and LSIMS data pertaining to this fraction are presented in FIGS. 5, 6 and 7. Most of this fraction was nonvolatile, and so the GC-MS peaks described make up only a small portion of this sample. The majority of the mass in this fraction consists of high-molecular-weight nonvolatile compounds.
The DP data show relatively trace levels of several S bisflavanoids. The LSIMS data show a complex mixture of high-mass compounds in the range 300-1000 to be present in this fraction. This fraction largely consists of ~~bound~~ compounds such as glycosides (phenolic compounds bound to sugar molecules) and other polar high-molecular-weight conjugates. This fraction was digested with HC1 and heat in order to hydrolyze the conjugates down to small molecules, which could then be identified.
The data on the hydrolyzed XAD-2 resin MeOH eluate DCM-insoluble fraction 29 are summarized in Table 5. The GC-MS
chromatogram, DP data, and LSIMS data for this fraction are shown in FIGS. 8, 9, and 10, respectively. Please note that the compounds in Table 3 that appear following hydrolysis are all present as bound components in the original fraction.

-'..-i Table 3 Chemical Composition XAD-2 Resin DCM Eluate Fraction 23 of FIG. 1 M5 Compound (Synonyms, Common Names,CAS~# Peak Spec Comments, Ete.) Area%..
#

66 phenol 108-95-2 1.43 282 sulfur (elemental sulfur six memberedNA 0.90 ring) 372 dibutylphthaiate (plasticizer) 84-74-2 0.29 441 hexadecanoic acid (palmitic acid)57-10-3 0.11 445 sulfur (S-8, cyclic sulfur, orthothrombic10544-50-09.02 sulfur, molecular sulfur) 452 1-hexadecanol (hexadecyl alcohol)36653-82-40.81 485 linoleic acid 60-33-3 0.91 519 3-methyl-1,8,9-anthra~netriol 491-59-8 5.86 (3-methylanthralin, Chrysarobin) 527 1,8-dihydroxy-3-methyl-9,10-anthracenedione481-74-3 11.76 (Chrysvphanol, Chrysophanic acid, 2 0 1.8-dihydroxy-3-methyl-1,8-anthraquinone) 549 1,8-dihydroxy-3-(hydroxymethyl)-9,10-NA 0.14 anthracenedione .

(Aloe-Emodin) 574 2,4-bis(dimethylbenzyl)-6-t-butylphenolNA 0.27 583 di-2-ethylhexylphthalate (plasticizer)117-81-7 65.44 619- monoacetate derivative of 1,8-dihydroxy-3-NA 2.23 622 (hydroxymethyl)-9,10-anthracenedione (Aloe-Emodin monoa~tate) ~DP bis-flavanoids with m.w.'s 474 NA 1.73 (C2,HZZO"), 490 (est.

Data (C~,Hz=O,Z), 506 (C=,HziO") and total 508 (Cz,Hz~O") for exact chemicals structures unknown DP 8~

3 0 LSIMS) LSIMS unknown high mass compounds m.w. NA 1.73 608 8 (est.

Data 696 total for OP 8~ S
t_SI

data DP = Data is from Electron Ionization Direct Insertion Probe LSIMS = Liquid Secondary Ion Mass Spectrometry Table 4 Chemical Composition XAD-2 Resin MeOH Eluate DCM-Insoluble Fraction !Fraction 26 of FIG. 1) MS Compound {Synonyms, Common Names, CAS # Peak Spec Comments;; Etc.) Area I
#

423 palmitic acid 57-10-3 17.05 447 unk. 216 m.w. aromatic NA 2.41 468 linoleic acid 60-33-3 28.08 519 3-methyl-1,8,9-anthracenetriol 491-59-8 27.82 (3-rnethylanthralin, Chrysarobin) 527 1,8-dihydroxy-3-methyl-9,10-anthracenedione481-74-3 12.78 (Chrysophanol, Chrysophanic acid, 1,8-dihydroxy-3-methyl-1,8-anthraquinone) 583 di-2-ethylhexylphthalate (plasticizes) 117-81-7 11.87 DP bis-flavanoids with m.w.'s 492 (CZ,H24O,z)NA trace and Data 506 (C2,Hu0,~) exact chemicals structures unknown LSIMS complex mixture of unknown compounds NA major m.w.

Data range 300-1000 (bound components, glycosides, higfi m.w. polar conjugates etc.) OP = Data is from Electron Ionization Direct Insertion Probe raw data file LSIMS = liquid Secondary Ion Mass Spectrometry Data file VG2268 Table 5 Chemical Composition of Hydro~zed XAD-2 Resin MeOH
Eluate DCM-Insoluble (Fraction 29 of FIG. 1) MS Compound (Synonyms, CommonCAS Peak Spec Names, # Area # Comments, Etc.) /;

57 tumaric acid 110-17-84.14 97 2-ethyl-t-hexanol (ptaslicizer104-7871.B
degradate) 123 2-furancarboxylic acid 88-14-22.64 151 4-oxo-pentanoic acid (fevutinic123-76-25.36 acid) 183 a-hydroxyhexanoic acid NA 0.62 271 branched dodecanol isomerNA 1.56 278 1-chlorododecane (probably112-52-70.91 a hydrolysis art'rfact) 282 1-dodecatlol (lauryl alcohol)112-53-815.7 318 dodecanoic acid (lauric 143-07-72.54 acid) 345 tributylphosphate (plasticizer)126-7381.29 348 1-tetradecanol (myristyl 112-72-15.89 alcohol) 36t 2-ethoxy-1dodecanot 297184-37.09 365 2.8-dihydroxy-3-methyl-1,4-napthoquinoneNA 3.29 (Droserone) 1 S 378 tetradecanoic acid (myristic544-63-82.31 acid) 420 2-ethoxy-1-tetradecanol NA 3.01 422 methyl palmilate 112-39-00.48 428 7-hydroxy5-melhoxy-2-methyl-4-oxo-4H-1-7338-5t-41.15 benzopyran-6tarboxaldehyde 4a hexadecanoic acid (palmitic57-l0-37.54 t acid) 440 a-hydroxytauric acid NA 3.4 461 heptadecanoic acid (margaric508-12-70.32 acid) 449 oleic acid 112-80-10.69 ?

. 480 linotoic acid Gt1.33-34.ti3 511 a-hydroxymyrislic acid 2507-5530.42 488 odadecanoic acid (stearic57-1 1.35 acid) i-4 518 3-methyl-1,8.9-anthracenetriol49159-80.42 -(3-methytanthralin, Chrysarobin) 520 1,8-dihydroxy-3-methyl-9,10-anthracenedione48174-34.71 (Chrysophano4 Chrysophanic acid.
1,8-dihydroxy-3-methyl-1,8-anthraquinone) 574 di-2-ethy~exytphthalate 11781-74.48 25 (plasticizer) OP bis-llavanoids with m.w.'sNA 11.98 Data 478 (Ci=HyO,=). 492 (C"Hr,O,,) and 306 (C=,H1,0") exact chemicals structures unknown OP = Data is from Electron Ionization 0'uect Insertion Probe raw data file The carboxylic acid fraction 28 data are summarized in Table 6. The GC-MS chromatogram, DP data, and LSIMS data pertaining to this fraction 28 are presented in FIGS. 11, 12, and 13. Carboxylic acid fractions often contain nonvolatile species that do not readily pass through gas chromatography. These compounds can be chemically derivatized into more volatile forms using methylation or silylation reagents. Therefore, the carboxylic acid fraction 28 was methylated using freshly prepared diazomethane reagent. This procedure converts the carboxylic acids into their corresponding methyl esters. Phenols are converted into methyl ethers. The methyl esters and methyl ethers are more volatile and chromatograph better than the free acids.
The GC-MS chromatogram from the methylated carboxylic acids is shown in FIG. 14. No additional compounds were detected in the methylated sample. All the same compounds observed in the underivatized carboxylic acid fraction were found as their methylated counterparts.
Table 7 summarizes the chemical composition of the 2.O yellow precipitate which was isolated from the neutral/phenol fraction 36 after extraction of the carboxylic acids. This fraction, which contains relatively few components, possesses the highest degree of activity in the cell culture bioassay. The GC-MS chromatogram, DP data, and LSIMS data pertaining to this fraction are shown in FIGS. 15-17.

Table 6 Chemical Composition of Carboxylic Fraction (Fraction 28 FIG. 1) MS Compound (Synonyms.CAS Peek SpeeCommon Hamea, a Area al Commrnts, Ete.) X

37-102anetnylacrytitacid79-41-414.4 19 2-memylbulyrie acd 1185302.04 57 pentanore aed 109-52J2.2 1t8hexanas acid tt2-82-t3.52 t202.furancarboxyficaud118-t4-29.t8 t unknown 138 m.w, NA 2.7 s3 audw containing compound 1672atnymexanoicaud t49-575t t8tbenzoic add 658502.91 t97ttrtanoie acid t24-07.28.29 2t7Denzerleacetieaad 10382-2t.2 227rlonanoicacid 1t2-03-04.74 242decanoicaeid 734-4855.2 2772Jyaroxy:l.,~~~ 87-4090.77 ~p (uesotic sad.
homosaicylic add) 277yneenylproperwie 621-82-90.69 sad (unnamic sad) 3053.4dactrbrtt0ertzoicaeidStJ4-50.88 318dodersnoic sad (lauric147-07-70.82 add) 323tOwmdecenoic acid 112-18-90.72 328tuuunerfioic acid 123-38-90.54 - 329pemacnbrophend (wood87-86-SO.
preservative) t t 342I~meUaxy~-hydroxybentaldehyde(van4tin)12133-50.75 772p-coumaric acid. 39s3-97-31.23 methyl ester 378telradenrioic add 544-63-80.72 (mynsttc acid) 385methylfmuWla. 23080710.72 408methyl4ister o! 596-6450.71 3-4.dimethoxydnnamic acd 41ata-0enzene dicarboxylic10021-00.6T
acid IterePhthalic xd) 423Lt-dihydf0-4.648tydtouy-3.methyi-i:ocoumann30951172U,42 - i8-hYdroxymeeein) a279.Nexadecenaic acid209 0.55 (paknilokie acid) 1.29.1 434hexaaecaivoic sad 57.10-7t.9 Ipa4nitic acd) 4389.octadecerwrc add 1 0.49 (oleic acdl t2-80-1 4789.12octmlacadienoicadd(Nnokicacd)80-33-31.03 464oaaaec>noic scd s7-t 0.69 (atearic add) 1.4 4913-memyl-1.8.9snttuacenetrrol491-59-80.55 -(3-methylanttuatin.
Ctuysarobin) 518t,84ihydroxy-3-methyl9.t0-anthracened'rone481-74.31.3 (Chryaoptunol, Chrysophanie acid.
t.8 J4rydroxy.3.n;ethyl L.B~anthrequmonel 572dl2edryltiexytphlhatatelplaatrdur)ItT.Bt.70.8 833saualene 768384-90.74 OP 0is-Ilavanoids wrilhNA 27.22 Datam.w's 492 IC"H"O,~ eat.
and total 508 (C~H~,O,rj a:act chemicals atrutttuea for unknown OP
d ISIMS

LSIMSunknown hr~b mass 14A 27 Drleconytotmrts rn 22 w. 530. 854 eW
rrH174U total laDPB
LSIMS

OP ~ Data a Iron Eleeuon lonitatron Direct Insenwn Prone taw oats file FM

LSIMS ~ LMrmf SeconJary Ion MaaaSpectrane~ry Data tile Vti2267 Table 7 S
Chemical Composition Precipitate Neutral/Phenol Fraction after Extraction of Acids (Fraction 36 of FIG. 1) MS Compound (Synonyms, Common Names, CAS # Peak Spec Comments, Etc.) Area #

419 methyl palmitate 112-39-0 0.53 464 methyl linoleate 1 i 2-63-0 1.09 510 3-methyl-1.8,9-anthracenetriol 491-59-8 19.69 l o (3-methylanthralin, Chrysarobin) , 517 1,8-dihydroxy-3-methyl-9,10-anthracenedione481-74-3 57.41 (Chrysophanol, Chrysophanic acid, 1,8-dihydroxy-3-methyl-1,8-anthraquinone) 583 di-2-ethylhexylphthalate (plasticizes)117-81-7 18.35 DP bis-flavanoids with m.w.'s 474 (C23H220"),NA 2.93 (est.

Data (Cz,H2,0,2). 506 (C2~Hu0") and 508 total (C23H2,O") for 1 s exact chemicals structures unknown DP

LSIMS) LSIMS unknown high mass compounds m.w. 618, NA 2.93 (est.
662, Data 696, 718, 736 and 758 total for DP &

LSIMS

data DP'= Data is from Electron Ionization Direct Insertion Probe raw data file LSIMS = Liquid Secondary fon Mass Spectrometry Data file VG2270 The neutral/phenol fraction following extraction of carboxylic acids and harvesting of the yellow precipitate was still too complex for direct analysis. Therefore, it was subjected to additional fractionation using a silica gel solid phase extraction minicolumn procedure. The fraction was passed through the silica gel column and was washed with DCM. A second more polar fraction was then eluted from the column using methanol. Out of the original 1.32 g of neutral/phenol fraction S were recovered 0.73 g (0.15% of original crude extract? in the DCM eluate and 0.59 g (0.12%) in the MeOH eluate. Therefore, the neutral/phenol fraction following extraction of carboxylic acids and harvesting of yellow precipitate was split into two additional subfractions for analysis. These were named the neutral/phenol fraction DCM silica gel eluate and the neutral/phenol fraction MEOH silica gel eluate. The chemical compositions of these two fractions are summarized in Tables 8 and 9. The GC-MS chromatograms, DP data, and LSIMS data pertaining to these two fractions are shown in FIGS. 17 through 22.

Table 8 Chemical Comeosition Neutral/Phenol Fraction Silica GeI MEOH Eluate MS Compound (5ynonyma. CAS Peak Sp~cCommon Nama. s Area a Commrrrtts, Ete.) Y.

134 3.5,5-trimed~yl-2ctrdchexenet-oneT859t 0.83 ((soptuxone) 205-5-hydroxymothyUurturyl1055 6.24 225 acetate (HMf acetate)t-58-3 231 gykxrytmonoaeetate 26sa6-35-51.03 1- ~ 244 glyCeryWiacetate 2539531-70.6 246 gtycerydriacetate 102-7811 (triaeetin) 254 deesrwic aad 334.48-S0.18 295 t-4.butanadiold(acetate628-6T.t0.57 311 unknovm aromatic NA 0.53 acetate 318 unknown arana8c acetateNA 0-65 328 dodecanok acid (tawlc113-07.72.
acid) t 334 t-benzettediol monoaeetateNA 2.03 (hydroquinone monos 353 unkraowm aromatic NA 0.29 acetate 1 S 359 unknown aromatic NA 0.13 acetate 3n myri:tir:rda ss4.s3-a2.ss 399 unknown aromatic NA 0.5 acetate 405 pentadecanoicaeid 100284-2t.6 421 t.d-0eruenedfearbo:yie100-2100.58 acid perephlha6e acid) a28 hexadecanoie add 57.10-3t (palmitic acid) l.se 436 hepfadecanoicadd 508-1271.67 474 dnokicacid 8033-32.,88 500 oraddecaraic add 57-t 39-02 (stearic acid) t-4 520 3-mettryl-t:8:9anthraeenetriot~9t-5982.57 2 0 1n: CttrysarobYr) 529 t.8-dfiydroxy:3metby49,t0anthraeenedionea81-7435.7 (Clttysolthtatd.
Chysophanic acid.
1.S:dfiydroay3methyt.t,8-anlhraquinorte) 539-rntxitne of brp chainNA 4 575 allphatle sutates 583 dl-2-athylhexytphthalate(PIasIkIzer)1178170.55 818 t.8-A9rydroay-3-(hyd<oxymedtyp-9.10-NA 3.82 -anthraeened-rone (Aioe.Enrodkt) 622 monoacelate derivativeNA 1.34 of l,8dihydroxyJ-(hydroxymethyl)-9.10-anMracenedione (Aloe-Emadinmonoaeetate) 2 5 6~ tmlawwn 378 m.w. NA 0.77 aranalie aceute 644 aQualene T683-8d-90.2T

OP bb-Ilavandds with NA 4.3 Oatsm.w.'s 474 (Cr,HnO"), (cal 478 total (CafipO,~. 490 (CrrH~On). lot 492 (C~hla,Op). OP
506 d (CHrrO") snd 5011 ~SIMS) (C~H"O,~ exact chemicals stnretures unknown lSIMSunknown high masa NA 4 Oalacompounds m.w. 608, 3 880 (est.
and 898 total br OP
b ~SIMS
data DP . Oats is from Eacarwr lonizadon Okeet lnseribn Probe raw dau Gk FM 10850 t.SIMS ~ liquid Secondary Ion Mass Spsdromelry Oata (1e vG2265 :() Table 9 Chemical Composition Neutral/Phenol Fraction Silica Gel DCM Eluate MS Compound (Synonyms, CAS Psak Spo Common Namss; a Ar~x At t Comments. Etc.) 283 unknown 154 m.w. NA 0.29 rnmpound 268 2.3Eihydro-2.54imethyl~H-tbenzopyran-4-696878720.88 one 278 S.8 hexasulfide (sixNA t.35 membered ring structure) 283 2,6~it-butyl-p-hydroxyanisoleNA 2,12 (antioxidant) 295 butylated hydroxy 128-3703.65 toluene (8H1- antioxdant) 301 methyl lsurate t 1 1.47 t-82.0 323 diethytphthalate 846-2 1.08 (plasticizer) 329 dodecyi acetate t 12-66-31.82 1 5 358 unknown bauoata NA 0.65 785 methylleidecanoate 173188-01.35 369 oaxrs)Adruramic aldehyde101-8600.65 373 bePtylbenzaate 120-51-40.71 388 meewnoate 713254.10.82 390 telradecyl acetate 638-59-50 40a txnryl sa&cylate 11858-11.35 417 acetylterulic acid 2596-47.82.12 425 methyl patmitate 112J9~04.53 433 dibutylphthalate Sd74.22,00 141 sulfur (S-8, eyUie 10544-50-09.59 suNur; orthothrbmbie sullur, molecular sulfw) 447 hexadecylacetate 829-70-91.88 151 rtathylheptadecanoate17319260.94 471 methyl finobaee 112-63-015:A2 478 methylslesrate 112-61-80.76 515 3-meMy1-1,8.9anthracenettiol49159-85.06 (3erKdrylaMfualin, Chrysarobin) 319 1.8-dihydroxy3-melhyl.g,l0.anthracenedione481-74.315.00 533 (Ctwysophar>al, CMysophanic acid.
t.8-dihydroxy-3-methyl-1.8-anthraquinane) 543 di-2-ethylhexylpMhalate117-81-712.53 (plasticizer) 817 monoacetate derivativeNA BZ4 of l,8dihydroxy.3-(hydroxyrtwthyp:9,10-anthracenedione (aloe-Emodin monoacetate) 3 ~ 839 methyl tamesate 10485-JO-81-08 OP bb-8avanoida with NA 9.71 Oatsm.w.'s 474 (CnH,rO, (eat.
) 490 total (Cr,11,r0"). 492 1w (C~fl,.0"). 508 OP
(C"ItrrO.,) b and 508 (CnHNO,~ lSIM$) exaC chemicals sbudures unknown; also unknown peaks at m.w. 570 and ISIMSunknawm h'gh mass NA 9.71 Oatseorr>pounda m.w. t (esl 540. 818. otal 648 and 682 for OP

IStMS
data OP ~ Oata is Iron Electron Ionization Oked Insertion Probe raw data file ISIMS = liquid Secondary ton Masa Spewomeby Oata tits VG2266 The data obtained from all of the fractions of Example 4 were pooled together and normalized to produce a comprehensive summary. These data are summarized in Tables l0A-F, which describe the chemical composition of the organic soluble S fraction, which constitutes approximately 1% of the treatment solution on a w/w basis but contains 100% of the 'biological activity. The compounds in this table are grouped together by chemical class. It should be noted that the quantitative date (% w/w) provided in this and the other tables are not exact but l0 rather are semiquantitative. The data were derived from a combination of GC area % integrations and from gravimetric determinations made throughout the fractionation. Highly accurate quantitation data would only be possible if analytical reference standards were available for all compounds detected so 15 that detector response factors could be determined and the data adjusted.

Data Summary: Composition of OrQani.c Soluble Fraction Grouped by Chemical Class Table 10A
Aiphatic Carboxylic Acids CAS # ! wlw 2-methylacryiic acid 79-41-4 0.27 2-methylbutyric acid 116-53-0 0.04 fumaric acid 110-17-8 1.50 I

pentanoic acid 109-52-4 0.04 hexanoic acid 142-&2-1 0.07 2-furancarboxyiic acid 88-14-2 1.13 2-ethylhexanoic acid 149-57-5 0.02 4-oxo-pentanoic acid ttevulinic acid)123-76-2 '1.94 a-hydroxyhexanoic acid NA 0.23 octanoic acid 124-07-2 0.118 nonanoic acid 112-05-0 0.09 decanoic acid 334-48-5 0.11 2 0 dodecanoic acid (iauric acid) 143-07-7 1.09 10-undecenoic acid 112-38-9 0.01 nonanedioic acid 123-3B-9 0.01 tetradecanoic acid (myristic acidj 544-63-8 1.05 pentadecanoic acid 1002084-20.12 2 5 hexadecanoic acid (palmitic acid) 57-10-3 9.82 a-hydroxyfauric acid NA 1.232 a-hydroxymyristic acid 2507-55-30.15 heptadecanoic acid 506-12-7 0.24 oleic acid 112-80-1 0.259 tinoteic acid 60-33-3 12.26 stearic acid 57-11-4 3:43 paimitoleic acid 2091-29-40:01 Table 10B
Aromatic Carboxylic Acids ...__ benzoic acid 65-85-0 0.06 benzeneacetic acid 103-82-2 0.02 3,4-dichlorobenzoic acid 51-44-5 0.02 1,4-benzenedicarboxylic acid ~terephthaiic acid)100-21-0 0.06 cinnamic acid 621-82-9 0.01 acetyl ferulic acid , 2596-47-6 0.20 Table lOC
Esters glyceryl monoacetate 26446-35-50.08 glyceryl diacetate 25395-3i-70.04 gtyceryl triacetate (triacetin) 102-76-1 0.08 1,4-butanediol diacetate 628-67-1 0.04 2-hydroxy-5-methytfurfuryt acetate (HMF 10551-58-30.47 acetate) diethytphthalate (ptasticizer) 84-66-2 0.10 dibutylphthalate (plasticizer) 84-74-2 - 0.21 di-2-ethylhexylphthalate (ptasticizer) 117-81-7 11.70 tributylphosphate (ptasticizer) 126-73-8 0.47 methyl taurate 111-82-0 0.14 methyl tridecanoate 1731-88-0 0.13 methyl pentadecanoate 7132-64-1 0.08 methyl palmitate 112-39-0 0.60 2 0 methyl heptadecanoate 1731-92-6 0.09 methyl linoleate 112-63-0 1.51 methyl stearate 112-61-8 0.07 methyl farnesate 10485-70-80.10 2 5 dodecyl acetate 112-66-3 0.17 tetradecyt acetate 638-59-5 0.06 hexadecyt acetate 629-70-9 0.18 benzyl benzoate 120-51-4 0.54 benzyl salicytate 118-58-1 0.13 methyl ester of 3,4-dimethoxy cinnamic acid5396-64-5 0.01 unknown aromatic acetates NA 0.16 mixture of long chain aliphatic acetates NA 0.30 unknown benzoate NA 0.06 3 5 unknown 378 m.w. aromatic acetate NA 0.06 -Table lOD
Phenolic Compounds phenol 108-95-2 0.09 I

2-hydroxy-3-methylbenZOic acid (cresotic 83-40-9 0.01 acid, .
homosalicylic acid) ferulic acid methyl ester 2309-07-1 0.01 benzyl salicylate 118-58-1 0.13 3,4-dihydro-4,8-dihydroxy-3-methylisocoumarin309-5i-1120.01 (6-hydroxymellein) 7-hydroxy-5-methoxy-2-me#hyt-4-axo-4H-1-benzopyran-6-7338-51-4 0.42 carboxaldehyde 2,6-di-t-butyl-4~-methylphenoi {BHT, antioxidant)128-37-0 0.34 2,6-di-t-butyl-p-methyla~isole {antioxidant)NA 0.20 , 2,4-bis-(dimethylbenzyt)-6-t-bvtylphenol NA 0.02 (antioxidant) hydroquinone monoacetate NA 0.15 4-hydroxy-3-methoxybenzaldehyde (vanillin)121-33-5 0.01 p-coumaric acid methyl ester 3943-97-3 0.02 pentachlorophenol (PCP, wood preservative)87-86-5 0.002 3-methyl-1,8,9-anthracenetriol 491-59-8 11.77 (3-methylanthralin; Chrysarobin) 2 5 1,8-dihydroxy-3-methyl-9,10-anthracenedione481-74-3 10.37 (Chrysophanol, Chrysophanic acid, 1,8-dihydroxy-3-methyl-1,8-anthraquinone) 1,t3-dihydroxy-3-(hydroxymethyl)-9,10-anthracenedione481-72-1 0.30 (Aloe-Emodin) monoacelate derivative of 1.,8-dit~ydroxy-3-NA 0.83 3 0 (hydroxymethyl)-9,10-anthracenedione I
(Aloe-Emodin monoacetate) 2,8-dihydroxy-3-methyl-1,4-naphihoquinone NA 1.7 (Droserone) 9 bis-flavanoids with m.w.'s 474 (Cz,HzzO"),NA 6.27 478 (CzzHzzO,z), ' est.
5 490 (Cz,HzzO,z), 492 (Cz,Hz,O,Z), 506 (CZ,HzzO,z) total and 508 far (Cz,Hz,O") exact chemical structures unknown DP &
LSIIvIS

Table 10E
Atcohols 2-ethyl-1-hexanol 104-76-7 0.65 branched dodecanol isomer NA 0.57 1-dodecanol (dodecyi alcohol) 112-53-8 5.69 _-1-tetradecano! (tetradecyl alcohol] 112-72-1 2.14 i 2-ethoxy-1-dodecanol 29718-44-3 2.a,7 1-hexadecanoi (hexadecyi alcohol) 36653-82-4 0.05 2-ethoxy-1-tetradecanol NA 1.09 Table lOF
Misceltaneo~ts Carnpounds 3,5,5-trimethyl-2-cyclohexene-1-one (isophorone)78-59-1 0.06 2,3-dihydro-2,5-dimethyl-4H-1-ber~zopyran-4-one69687-87-2 0.08 2 o a-hexylcinnamic aldehyde 101-86-0 0.06 squalene 7683-64-9 0.03 1-chloro-dodecane (dodecyl chloride) 112-52-7 0.33 ' sulfur (elemental sulfur six membered ring) NA 0.18 2 5 sulfur (S-8, cyclic sulfur, ortho#hrombic sulfur,10544-50-0 0.90 molecular sulfur) unknown 138 m.w. sulfur-containing compound NA 0.05 unknown 216 m.w. aromatic compound NA 0.87 3 o unknown 154 m.w. aromatic compound NA D.03 complex mixture of high molecular weight unknownNA 6.27 compounds in the range 400-1000, many of these est.
compounds are conjugates of the compounds identified total in for this study such as glycosides, polar conjugates, DP &
high m.w. LSIMS
esters etc. I

Exam",ple 6 - In vivo Study. Application of the Treatment Solution to a Flaky Skin Mouse The flaky skin mouse (fsn) is a genetically engineered mouse with an autosomal recessive mutation causing the skin to resemble that exhibited in human psoriasis (see Sundberg et al., J. Vet. D.iagn. Invest. 4:312-17, 1992) In the homozygous affected flaky skin mouse (fsn/fsn), where the mutation is on both chromosomes, histological features such as marked acanthosis, hyperkeratosis with focal LU parakeratosis, subcorneal pustules, dermal capillary dilation, and dermal infiltration of inflammatory cells are seen.
Ten affected mice (fsn/fsn) and 10 normal littermate controls (fsn/-) were obtained from The Jackson Laboratory in Bar Harbor, ME. The animals were maintained.using standard diet and housing:
The dorsal surface of each animal was shaved. One-half of the dorsal surface received weekly topical treatments of the solution of Example 2, while the other half served as an untreated control. The solution of Example 2 was applied to the skin using a sterile cotton swab. Treatments continued for up to seven weeks. Weekly biopsies of treated and untreated areas were also removed for histological examination. Following the seventh week of treatment, the animals were necropsied for gross pathological changes.
The solution of Example 2 was found to induce dramatic gross and microscopic changes only for the hyperproliferative skin of the affected (fsn/fsn) mice with minimal to no effects noted for the (fsn/-) control mice. Observations by a veterinary dermatopathologist described the skin response of the affected animals as being similar to a "chemical burn." Histologic findings indicated that the keratinocyte growth of the affected mouse skin was markedly reduced, and that the treated skin began to slough off a bit but remained fully attached as a biological bandage allowing healing of the underlying skin. This effect was not observed in the control animals or untreated skin in the affected animals. Results are presented in Table 11.

Table 11 Summary of Flaky Skin Mouse Study I.D. NUMBERPhenotype Histological Gross PathologicComments Assessment Changes _ 1 (fsnlfsn)Flaky skin Reduction None observed in proliferation of keratinocytes 2 (fsnlfsn)Fiaky skin Reduction None observed in proliferation of 0 keratinocytes 3 - (fsnlfsn)Flaky skin Reduction None observed in proliferation of keratinocytes (fsnlfsn) Flaky skin Reduction None observed in proliferation of keratinocytes (fsn/fsnjFlaky skin Reduction None observed , in proliferation of keratinocytes 6 (fsnlfsn)Flaky skin Reduction None observed in proliferation of keratinocytes 7 (fsnlfsn)Flaky skin Reduction None observed in proliferation of ' 0 keratinocytes 8 (fsnlfsn)Flaky skin Reduction None observed in proliferation of keratinocytes 9 (fsnlfsn)Flaky skin Reduction None observed in proliferation of keratinocytes >_ S 10 (fsn/fsn)Confusing Marginal changeNone observedJackson tabs phenotype suggest mistyping of animal 11 (+!-) Normal skin No change None observed 12 (+!-) Normal skin No change None observed 13 (+I-) Normal skin No change None observed 1 4 (+I-) Normal skin No change None observed 3 0 15 (+/-) Normal skin No change None observed 16 (+I-) Normal skin No change tVone observed 17 (+I-) Normal skin No change None observed 18 (+p) Normal skin No change None observed 19 (+I-) Normal skin No change None observed 20 (+/-) Normal skin No change None observed Example 7 - Bioassay - In vivo Study Bioassay The in vivo study of Example 6 using the flaky skin mouse model was indicative of keratinocyte cell specifically with respect to the activity of the treatment solution compound.
S In vitro cell testing is necessary to detect activity with respect to cell type for purposes of developing a bioassay for treatment solution activity, and second to further study results obtained using the flaky skin mice of Example 6. Because human skin contains both keratinocytes and fibroblasts, either of which may be involved in the psoriasis disease process, pure cultures of human fibroblasts were grown from normal adult human skin, keloid scars (hyperproliferative fibroblasts), and commercially available certified pure cultures of normal human adult epidermal keratinocytes. These cell types were tested :S separately for their growth responses to treatment with the treatment solution of Example 2.
Protocol Cell growth is measured by examining the amounts of DNA
synthesis, for as cells grow and divide, more DNA is produced.
>o Radioactive thymidine is added to the cell culture media. The assay is conducted as follows: Cultured fibroblasts are grown in complete minimal essential medium (CMEM) containing 10% fetal bovine serum as a growth factor source. Epidermal keratinocytes were cultured in the presence of complete keratinocyte growth medium (CKGM) supplemented with bovine pituitary extract, hydrocortisone, and epidermal growth factor (EGF) as a growth factor source.
Healthy growing cells were seeded into Corning 24 well tissue culture plates at a density of 1.0 X 10' cells per well in 1.0 ml of either CMEM or CKGM depending on cell type. The cells were incubated for 24-36 hours or until they reached 60-70%
confluency at 37°C in the presence of 5% CO2. The medium was then changed to either MEM without the 10% serum or KGM without 35 hydrocortisone or ECF, but leaving the BPE in the media. This allows starvation of the cells, or their regression to a nongrowth phase where although they are not dividing, they remain biochemically active. The cells were allowed to incubate under these conditions for 24 hours. The starving media were then removed and replaced with 1.0 ml/well of complete growth media CMEM or CKGM containing no treatment, treatment solution of v Example 2, or a subfraction from Example 4 at a dilution of 1:5000, or solvent alone such as acetic acid or DMSO.
Statistically significant numbers of repetitions were performed for each treatment. Additionally 1 ~Ci tritiated thymidine (3H) was also added per well. Following these treatments, the cells o were allowed to incubate for 24 hours using identical conditions as above. Following the end of the incubation period, the wells were washed 3 times with phosphate buffered saline (PBS) and fixed with 12.50 trichloroacetic acid (TCA) for 10 minutes followed by methanol for 10 minutes. The plates were air dried S and the cells solubilized in 1.0 ml of 0.2 N NaOH at 37°C for 1 hour. Growth was determined by measuring the level of radioactivity present. This was accomplished by counting 0.9 ml of the solubilized cells in a scintillation counter.
0 Results Fibroblasts Fibroblasts from normal adult skin and keloid scar were cultured and assayed for effects of the treatment solution on growth as described above. Neither normal nor keloid fibroblasts were inhibited by the treatment solution. A typical graph for these experiments is shown in FIG. 24.
Keratinocytes The treatment solution of Example 2 was fractioned as in Example 4, with the chemical compositions of those fractions 30 analyzed in Example 5 and results shown in Tables 3-9 above.
Tables 12-14 summarize the percentages of the 4 main active ingredients in each of those fractions, and mean percentages of inhibition in the keratinocyte bioassay for each fraction. Finally, mean percentages of inhibition are 35 normalized to the percentages of each active ingredient in the fractions mentioned above.

Table 12 1?ercentaaes of 4 Main Active Ingredients in the Treatment Solution of Example 2, Compiled from Tables 3-10 Fraction FIG.1 3-methyl chrysuobialoe. aloe-emodin l~F. N0. .ttuhralin cmodin morxucetate XAD-2 Resin DCM 23 ~ 5.8dSd I 1.76;E0.1496 2.2396 Eluate XAD-2 26 27:8256 12.7896 09E Os6 1. 0 Rcsin MeOH Eiuate~DCht-lnsol.

Hydrolyzed Z-92 29 0.4296 4 7t 091, 096 Resin-MeOH Eluate DCM

huol.

Z-92 Carboxylic 28 ' O. S5 ~6 1. 3 0 c 05>:
'~~

Acid Fx 15 Precipitate 36 19.(9 57.41 05F; 096 ~

From Neutral!

Phenol Fx Ncucral/ 31 2.5T56 5.79E 3-8y'.Sb1.3496 Phenol Silica Gel MeOH

8luatt Neutral! 31 5.06 15.0096 096 6.2496 ~~I Sihca Gel DCM

Eluete Ctude Z-92 10 11. 77 10. 37 0. 30960. 83 96 9<: St Table 13 Fraction Analysis Summary Results -r5 % Growth Inhibition of Keratinoc~tes by Fraction Fraction Ra! Meant 7/12 '.1217128 8!3 8!28 9/1;9115 10/6 10!13 No. Inhibit.
From FIG. 1 23 6~~ X96 26 50 9E ~ 44 56 _ j 'b 96 ~

29 - 7 sr; 7 ~

.3b 635 5095 7 31 305'6 30~ 3996 31 9; 9:; 9a 99 98 80 94 89 56 ~ 9E 56 56 ~

?7 zip 1756 2a~

?? 17'~ 1596 18'b 7? 1b5:; 1656 775b 7756 7956 84~ 6996 3 5 ?? 94 ~ 94 5~

Table 14 Percent Inhibition per Percentage of Ingredient in Fraction for 4 Main Active Ingredients in the Treatment Solution of Examble 2 (Calculated From Mean %
Inhibition from Table 13?
Fraecion DescriptionFraction Ref. 3-cnethy(chrysarobiBloc- aloe-emodin From No.

FIG. 1 From FIG. 1 aiuhralin emodinmonoaceute XAD-Z Resin DCM 23 10.58'x5.27~'x 4439E 2896 ~

Eluate ..

~D-2 26 1.?9~;!-3.9156 096 0~

Resin MeOH Eluatc-DCM-Insol.

Hydrolyzed Z-92 29 16.66 1.48 09b 096 XAD

Resin-MeOH Eluate DCM

lnsoi.

Z-92 Carboxylic 23 72.725'E30.769b 0x 096 ~

Acid Fx Precipitate 36 3.19 1.1596 05E 096 From Neutral!

Phenol Fx Neutral! 31 11.67 S. ?696 7. 22. 38 ~ 85 96 ~

Phenol Silica Gcl McOli Eluate Neutral! 3I ! 8.18 6. I 09'0 14. 74 ~. 3 96 56 Pherbl Silica Gcl DCM

Eluate Ctude Z-92 10 6.545 7.4296 256.6592.7796 Example 8 - Neutralizata.on Study 'S As the pH of the treatment solution of Example 2 is very acidic, studies were undertaken to neutralize the Ph to 7.0 in order to examine inhibitory properties in the neutral state.
Crude treatment solution was adjusted to pH 7.O with 1.0 N NaOH.
Eferatinocytes were assayed for growth in the presence of the 3o neutralized extract using bioassay methodology as described above, except that neutralized treatment solution was added as the test compound at a dilution of 1:5000.
Results of this analysis indicated that neutralization removes activity of the treatment solution. While not wishing 3s to be limited by theory, it is hypothesized that the addition of acetic acid during the preparation sequence for the treatment solution may acetylate reactive molecules, conferring additional biological activity: This may confer increased abilities to enter cells, etc. Neutralization of such entities by raising the pH to 7.0 may render the active moieties inactive, as reflected by the dramatically decreased activity observed with these studies.
S
Example 9'- Protein Study This Example examines whether or not any proteins were present in the treatment solution of Example 2. Toward this end, treatment solution was denatured at 65°C in the presence of sodium dodecyl sulfate (SDS) and electrophoresed on 12%
polyacrylamide gels (PAGE) in the presence of SDS.
Following electrophoretic analysis and staining with Coomassie blue, no proteins were evident on visual inspection of the gel.
t5 While the illustrative embodiments of the invention have been described with particularity, it will be understood that various other modifications will be apparent to and can be readily made by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is not intended that the scope of the claims appended hereto be limited to the examples and descriptions set forth herein but rather that the claims be construed as encompassing all the features of patentable novelty that reside in the present invention, including all features that would be treated as equivalents 25 thereof by those skilled in the art to which this invention pertains.

Claims (48)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition comprising an extract from Asphodelus microcarpus and a pharmaceutical carrier for topical administration.

2. The composition of claim 1, wherein said pharmaceutical carrier is selected from the group consisting of gels, sprays, creams, and dermal patches.

3. The composition of claim 1, wherein said pharmaceutical carrier is a cream.

4. The composition of claim 1, wherein said pharmaceutical carrier is a gel.

5. The composition of claim 1, wherein said extract is included in said pharmaceutical carrier in an amount effective to reduce the symptoms of psoriasis.

6. The composition of claim 1, wherein said extract comprises effective amounts of 3-methylanthralin, chrysophanol, aloe-emodin, and aloe-emodin acetate.

7. For use in the topical treatment of psoriasis, a composition comprising an extract from Asphodelus microcarpus and a pharmaceutical carrier.

8. The composition of claim 7, wherein said pharmaceutical carrier is selected from the group consisting of liquids, gels, sprays, creams, and dermal patches.

9. The composition of claim 7, wherein said pharmaceutical carrier is a cream.

10. The composition of claim 7, wherein said pharmaceutical carrier is a gel.

11. The composition of claim 7, wherein said extract is included in said pharmaceutical carrier in an amount effective to reduce the symptoms of psoriasis.

12. The composition of claim 7, wherein said extract comprises effective amounts of 3-methylanthralin, chrysophanol, aloe-emodin, and aloe-emodin acetate.

13. A method for preparing a pharmaceutical preparation comprising:
a) providing:
i) an extract from Asphodelus microcarpus; and ii) a pharmaceutical carrier for topical administration; and b) preparing a pharmaceutical preparation from said extract and said pharmaceutical carrier.

14. The method of claim 13, wherein said pharmaceutical carrier is selected from the group consisting of liquids, gels, sprays, creams, and dermal patches.

15. The method of claim 13, wherein said pharmaceutical carrier is a cream.

16. The method of claim 13, wherein said pharmaceutical carrier is a gel.

17. The method of claim 13, wherein said extract is included in said pharmaceutical carrier in an amount effective to reduce the symptoms of psoriasis.

18. The method of claim 13, wherein said extract comprises effective amounts of 3-methylanthralin, chrysophanol, aloe-emodin, and aloe-emodin acetate.

19. A pharmaceutical preparation prepared according to claim 13.

20. A composition comprising an effective amount of purified aloe-emodin monoacetate in a pharmaceutical carrier.

21. The composition of claim 20, wherein said effective amount is an amount sufficient to relieve the symptoms of psoriasis in a patient.

22. The composition of claim 20, further comprising at least one compound selected from the group consisting of 3-methylanthralin, chrysophanol, and aloe-emodin.

23. The composition of claim 20, wherein said composition further comprises 3-methylanthralin and chrysophanol.

24. The composition of claim 20, wherein said pharmaceutical carrier is selected from the group consisting of pills, tablets, capsules, liquids, gels, sprays, creams, dermal patches, and suppositories.

25. The composition of claim 20, wherein said pharmaceutical carrier is a cream.

26. The composition of claim 20, wherein said pharmaceutical carrier is a gel.

27. A composition comprising a cream including an effective amount of aloe-emodin monoacetate.

28. A composition comprising a gel including an effective amount of aloe-emodin monoacetate.

29. A method for preparing a pharmaceutical preparation comprising:
a) providing purified aloe-emodin monoacetate; and b) formulating said pharmaceutical preparation, wherein said pharmaceutical preparation comprises an effective amount of said substantially purified aloe-emodin monoacetate.

30. The method of claim 29, wherein said effective amount is an amount sufficient to relieve the symptoms of psoriasis in a patient.

31. The method of claim 29, further comprising providing at least one compound selected from the group consisting of 3-methylanthralin, chrysophanol, and aloe-emodin and combining said at least one compound with said aloe-emodin monoacetate to provide said preparation.

32. The method of claim 29, wherein said pharmaceutical carrier is selected from the group consisting of pills, tablets, capsules, liquids, gels, sprays, creams, dermal patches, and suppositories.

33. The method of claim 29, wherein said pharmaceutical carrier is a cream.

34. The method of claim 29, wherein said pharmaceutical carrier is a gel.

35. A method for preparing a pharmaceutical preparation comprising:

a) providing purified aloe-emodin monoacetate and at least one compound selected from the group consisting of 3-methylanthralin, chrysophanol, and aloe-emodin; and b) formulating said pharmaceutical preparation, wherein said pharmaceutical preparation comprises an effective amount of said purified aloe-emodin monoacetate and said at least one compound selected from the group consisting of 3-methylanthralin, chrysophanol, and aloe-emodin.

36. The method of claim 35, wherein said effective amount is an amount sufficient to relieve the symptoms of psoriasis in a patient.

37. The method of claim 35, wherein said pharmaceutical preparation is selected from the group consisting of pills, tablets, capsules, liquids, gels, sprays, creams, dermal patches, and suppositories.

38. A method for preparing a pharmaceutical gel or cream comprising:
a) providing purified aloe-emodin monoacetate; and b) preparing a pharmaceutical gel or cream comprising an effective amount of said aloe-emodin monoacetate.

39. The method of claim 38, further comprising providing at least one compound selected from the group consisting of 3-methylanthralin, chrysophanol, and aloe-emodin and combining said at least one compound with said aloe-emodin monoacetate to provide said preparation.

40. For use in inhibiting keratinocyte proliferation, a composition comprising an effective amount of aloe-emodin monoacetate.

41. The composition of claim 40, wherein said composition further comprises at least one compound selected from the group consisting of 3-methylanthralin, chrysophanol, and aloe-emodin.

42. The composition of claim 40, wherein said composition is a cream.

43. The composition of claim 40, wherein said composition is a gel.

44. For use in treating psoriasis, a composition comprising an effective amount of aloe-emodin monoacetate.

45. For use in treating eczema, a composition comprising an effective amount of aloe-emodin monoacetate.

46. A commercial package comprising a container containing therein a composition according to claim 1, 2, 3, 4, 5 or 6 and written matter which states that the composition can or should be used for treating psoriasis.

47. A commercial package comprising a container containing therein a composition according to claim 20, 22, 23, 24, 25, 26, 27 or 28 and written matter which states that the composition can or should be used for treating psoriasis.

48. A commercial package comprising a container containing therein a composition according to claim 20, 22, 23, 24, 25, 26, 27 or 28 and written matter which states that the composition can or should be used for treating eczema.