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.
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0. 34. A composition comprising a gel including an effective amount of aloe-emodin monoacetate.
0. 33. A composition comprising a cream including an effective amount of aloe-emodin monoacetate.
0. 26. A composition comprising an effective amount of purified aloe-emodin monoacetate in a pharmaceutical carrier.
0. 7. A composition comprising an extract from Asphodelus microcarpus and a pharmaceutical carrier for topical administration.
5. A method for making an antipsoriatic composition comprising the step of mixing together 3-methylanthralin, chrysophanol, aloe-emodin, and aloe-emodin monoacetate.
1. A composition for the treatment of psoriasis produced by the step comprising mixing together effective amounts of 3-methylanthralin, chrysophanol, aloe-emodin and aloe-emodin monoacetate.
0. 44. A method for preparing a pharmaceutical gel or cream comprising:
a) providing purified aloe-emodin monoacetae; and
b) preparing a pharmaceutical gel or cream comprising an effective amount of said aloe-emodin monoacetate.
3. A method for treating psoriasis comprising the steps of:
mixing together 3-methylanthralin, chrysophanol, aloe-emodin, and aloe-emodin monoacetate; and
applying the mixture to an area of skin affected by psoriasis with sufficient frequency to effect an alleviation of psoriatic symptoms.
0. 19. 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.
0. 35. 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.
0. 46. A method for inhibiting keratinocyte proliferation comprising
a) providing
i) keratinocytes and
ii) a composition comprising an effective amount of aloe-emodin monoacetate; and
b) applying said composition to said keratinocytes, wherein said effective amount is sufficient to inhibit keratinocyte proliferation as compared to control samples.
0. 13. A method for treating psoriasis comprising
a) providing:
i) a composition comprising an extract from Asphodelus microcarpus and a pharmaceutical carrier for topical administration; and
ii) a patient having symptoms psoriasis; and
b) topically applying said composition to said patient under conditions such that said symptoms of psoriasis are reduced.
0. 41. 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.
2. The composition recited in
4. The method recited in
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0. 25. A pharmaceutical preparation prepared according to
0. 27. The composition of
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0. 29. The composition of
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This is a divisional of pending application Ser. No. 08/621,043, filed Mar. 22, 1996.
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.
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. Goa powder itself is derived from the wood and bark of Andria Araroba Aguiar (Fam. Leguminosae). Literature references describing the isolation of and structure of Chrysarobin date back to the early 1800s. 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, Buffalo, N.Y.).
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, Aug. 1994, pp. 1-38.
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.
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 a method for making 3-methylanthralin.
It is even another object of the present invention to 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 object 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.
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.
In 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 Microcarpus 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 Asphodelus 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 for treating psoriasis, the composition may be administered to a mammalian organism by any route known in the art. Non-limiting examples of suitable routes of administration include oral, parenteral, topical, and the like. Specific non-limiting 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 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 psonasis.
It is also believed that the compositions of the present invention are 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.
The following examples are provided merely to illustrate the invention and are not intended to limit the scope of the invention in any manner.
Raw extract was obtained from the Asphodelus Microcarpus plant as follows. Approximately 1 lb 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 lb of roots yielded approximately 300 cc of raw extract.
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 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.
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.
TABLE 1
Proximate Analysis Data*
Dark brown liquid containing
sediment and suspended
particulate matter, acetic
Physical description of sample
acid like odor
Ph (as received)
1.95
Acetic acid content (by
5.05% w/w
titration)
Insoluble matter
2.14% w/w
Ash weight (mineral content)
0.746% w/w
Specific gravity (filtrate)
1.103 g/ml
Total dissolved solids
12.94% w/w
(filtrate)
Chloroform extractables
0.84% w/w
(filtrate) (organic soluble
components)
*Analysis performed by Stillwell & Gladding Testing Laboratories, Inc., New York, N.Y.
TABLE 2
Elemental/Heavy Metal Composition*
Element
Conc. ppm
Element
Conc. ppm
Sulfur
4260
Bismuth
ND < 3 ppm
Potassium
1640
Lead
ND < 3 ppm
Calcium
1075
Antimony
ND < 3 ppm
Sodium
216
Boron
ND < 2 ppm
Phosphorous
142
Indium
ND < 2 ppm
Magnesium
100
Molybdenum
ND < 1 ppm
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
Copper
0.9
Niobium
ND < 1 ppm
Manganese
0.4
Gallium
ND < 1 ppm
Titanium
0.4
Germanium
ND < 1 ppm
Nickel
0.4
Silver
ND < 1 ppm
Chromium
0.2
Cadmium
ND < .5 ppm
Barium
0.1
Mercury
ND < .5 ppm
Vanadium
0.06
Beryllium
ND < .1 ppm
*Elemental/heavy metal analysis was conducted by Umpire and Control Services, Inc., West Babylon, N.Y.
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
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 positive 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. 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 20×350 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 biologically inert components.
The XAD-2 resin column containing the treatment solution retentate 18 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 23 contained 0.51 g or 0.1% 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 ml 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 DCM-soluble fraction 25 contained 1.7 g (0.34%), and the 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 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.
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 LSIMS spectra pertaining to the XAD-2 resin DCM eluate fraction 23 are shown in
TABLE 3
Chemical Composition XAD-2 Resin DCM Eluate
Fraction 23 of
MS
Compound (Synonyms, Common Names,
Peak
Spec #
Comments, Etc.)
CAS #
Area %
66
phenol
108-95-2
1.43
282
sulfur (elemental sulfur six membered ring)
NA
0.90
372
dibutylphthalate (plasticizer)
84-74-2
0.29
441
hexadecanoic acid (palmitic acid)
57-10-3
0.11
445
sulfur (S-8, cyclic sulfur, orthothrombic sulfur,
10544-50-0
9.02
molecular sulfur)
452
1-hexadecanol(hexadecyl alcohol)
36653-82-4
0.81
485
linoleic acid
60-33-3
0.91
519
3-methyl-1,8,9-anthracenetriol
491-59-8
5.86
(3-methylanthralin, Chrysarobin)
527
1,8-dihydroxy-3-methyl-9,10-anthracenedione
481-74-3
11.76
(Chrysophanol, Chrysophanic acid,
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-butylphenol
NA
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 monoacetate)
DP
bis-flavanoids with m.w.'s 474 (C23H22O11), 490
NA
1.73 (est.
Data
(C23H22O12), 506 (C23H22O13) and 508 (C23H24O13)
total for
exact chemicals structures unknown
DP &
LSIMS)
LSIMS
unknown high mass compounds m.s. 608 &
NA
1.73 (est.
Data
696
total for
DP & LSIMS
data
DP = Data is from Electron Ionization Direct Insertion Probe
LSIMS = Liquid Secondary Ion Mass Spectrometry
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
TABLE 4
Chemical Composition XAD-2 Resin MeOH Eluate
DCM-Insoluble Fraction (Fraction 26 of FIG. 1)
MS
Compound (Synonyms, Commons Names,
Peak
Spec #
Comments, Etc.)
CAS #
Area %
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-methylanthralin, Chrysarobin)
527
1,8-dihydroxy-3-methyl-9,10-
481-74-3
12.78
anthracenedione
(Chrysophanol, Chrysophanic acid, 1,8-
dihydroxy-3-methyl-1,8-anthraquinone)
583
di-2-ethylhexylphthalate (plasticizer)
117-81-7
11.87
DP
bis-flavanoids with m.w.'s 492
NA
trace
Data
(C23H24O12) and 506
(C23H22O13) exact chemicals structures
unknown
LSIMS
complex mixture of unknown compounds
NA
major
Data
m.w. range 300-1000 (bound components,
glycosides, high m.w. polar conjugates
etc.)
DP = Data is from Electron Ionization Direct Insertion Probe raw data file FM10647
LSIMS = Liquid Secondary Ion Mass Spectrometry Data file VG2268
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
TABLE 5
Chemical Composition of Hydrolyzed XAD-2 Resin MeOH
Eluate DCM-Insoluble (Fraction 29 of FIG. 1)
MS
Spec
Compound (Synonyms, Common Names
Peak
#
Comments, Etc.)
CAS #
Area %
57
fumaric acid
110-17-8
4.14
97
2-ethyl-1-hexanol (plasticizer degradate)
104-76-7
1.8
123
2-furancarboxylic acid
88-14-2
2.64
151
4-oxo-pentanoic acid (levulinic acid)
123-76-2
5.36
183
α-hydroxyhexanoic acid
NA
0.62
271
branched dodecanol isomer
NA
1.56
278
1-chlorododecane (probably a hydrolysis
112-52-7
0.91
artifact)
282
1-dodecanol (lauryl alcohol)
112-53-8
15.7
318
dodecanoic acid (lauric acid)
143-07-7
2.54
345
tributylphosphate (plasticizer)
126-73-8
1.29
348
1-tetradecanol (myristyl alcohol)
112-72-1
5.89
361
2-ethoxy-1-dodecanol
29718-44-3
7.09
365
2,8-dihydroxy-3-methyl-1,4-
NA
3.29
napthoquinone (Droserone)
378
tetradecanoic acid (myristic acid)
544-63-8
2.31
420
2-ethoxy-1-tetradecanol
NA
3.01
422
methyl palmitate
112-39-0
0.46
428
7-hydroxy-5-methoxy-2-methyl-4-oxo-4H-
7338-51-4
1.15
1-benzopyran-6-carboxaldehyde
441
hexadecanoic acid (palmitic acid)
57-10-3
7.54
440
α-hydroxylauric acid
NA
3.4
461
heptadecanoic acid (margaric acid)
506-12-7
0.32
449
oleic acid
112-80-1
0.69
480
linoleic acid
60-33-3
4.93
511
α-hydroxymyristic acid
2507-55-3
0.42
488
octadecanoic acid (stearic acid)
57-11-4
1.35
516
3-methyl-1,8,9-anthracenetriol
491-59-8
0.42
(3-methylanthralin, Chrysarobin)
520
1,8-dihydroxy-3-methyl-9,10-
481-74-3
4.71
anthracenedione (Chrysophanol,
Chrysophanic acid, 1,8-
dihydroxy-3-methyl-1,8-anthraquinone)
574
di-2-ethylhexylphthalate (plasticizer)
117-81-7
4.48
DP
bis-flavanoids with m.w.'s 478
NA
11.98
Data
(C22H22O12), 492 (C23H24O12) and
506 (C23H22O13) extract chemicals
structures unknown
DP = Data is from Electron Ionization Direct Insertion Probe raw data file FM10827
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
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 6
Chemical Composition of Carboxylic Fraction
(Fraction 28 FIG. 1)
MS
Compound (Synonyms, Common Names,
Peak
Spec #
Comments, Etc.)
CAS #
Area %
37-40
2-methylacrylic acid
79-41-4
14.4
49
2-methylbutyric acid
116-53-0
2.04
57
pentanoic acid
109-52-4
2.2
118
hexanoic acid
142-62-1
3.52
120
2-furancarboxylic acid
88-14-2
9.18
153
unknown 138 m.w. sulfur containing compound
NA
2.7
167
2-ethylhexanoic acid
149-57-5
1
181
benzoic acid
65-85-0
2.91
197
octanoic acid
123-07-2
6.29
217
benzeneacetic acid
103-82-2
1.2
227
nonanoic acid
112-05-0
4.74
242
decanoic acid
334-48-5
5.2
273
2-hydroxy-3-methylbenzoic acid (cresotic acid
83-40-9
0.77
homosalicylic acid)
277
phenylpropenoic acid (cinnamic acid)
621-82-9
0.69
305
3,4-dichlorobenzoic acid
51-44-5
0.88
318
dodecanoic acid (lauric acid)
143-07-7
0.62
323
10-undecenoic acid
112-38-9
0.72
328
nonanedioic acid
123-38-9
0.54
329
pentachlorophenol (wood preservative)
87-86-5
0.11
342
3-methoxy-4-hydroxybenzaldehyde (vanillin)
121-33-5
0.75
372
p-coumanic acid, methyl ester
3943-97-3
1.23
378
tetradecanoic acid (myristic acid)
544-63-8
0.72
395
methyl ferulate
2309-07-1
0.72
408
methyl ester of 3,4-dimethoxycinnamic acid
5396-64-5
0.71
414
1,4-benzene dicarboxylic acid (terephthalic acid)
100-21-0
0.67
423
3,4-dihydro-4,8-dihydroxy-3-methyl-isococoumarin
309-51-112
0.42
(6-hydroxymellain)
427
9-hexadecnoic acid (palmitoteic acid
2091-29-4
0.55
434
hexadecanoic acid (palmitic acid)
57-10-3
1.9
438
9-octadecenoic acid (oleic acid)
112-80-1
0.49
478
9,12-octadecadienoic acid (linoleic acid)
60-33-3
1.03
484
octadecanoic acid (stearic acid)
57-11-4
0.69
491
3-methyl-1,8,9-anthracenetriol
491-59-8
0.55
(3-methylanthralin, Chrysarobin)
516
1,8-dihydroxy-3-methyl-9,10-anthracenedione
481-74-3
1.3
(Chrysophanol, Chrysophanic acid,
1,8-dihydroxy-3-methyl-1,8-anthraquinone)
572
di-2-ethylhexylphthalate (plasticizer)
117-81-7
0.6
633
squalene
7683-64-9
0.74
DP
bis-flavanoids with m.w.'s 492 (C23H24O12) and
NA
27.22
Data
506 (C23H22O13) extract chemicals structures
est. total
unknown
for OP &
LSIMS
LSIMS
unknown high mass compounds m.w. 530, 654
NA
27.22
Data
and 740
est. total
for DP &
LSIMS
DP = Data is from Electron Ionization Direct Insertion Probe raw data file FM10648
LSIMS = Liquid Secondary Mass Spectrometry Data file VG2267
Table 7 summarizes the chemical composition of the 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
TABLE 7
Chemical Composition Precipitate Neutral/Phenol
Fraction after Extraction of Acids (Fraction 36 of FIG. 1)
MS
Compound (Synonyms, Common Names
Peak
Spec #
Comments, Etc.)
CAS #
Area %
419
methyl palmitate
112-39-0
0.53
464
methyl linoleate
112-63-0
1.09
510
3-methyl-1,8,9-anthracenetriol
491-59-8
19.69
(3-methylanthralin, Chrysarobin)
517
1,8-dihydroxy-3-methyl-9,10-
481-74-3
57.41
anthracenedione (Chrysophanol,
Chrysophanic acid, 1,8-
dihydroxy-3-methyl-1,8-anthraquinone)
583
d-2-ethylhexylphthalate (plasticizer)
117-81-7
18.35
DP
bis-flavanoids with m.w.'s 474
NA
2.93
Data
(C23H22O22), 492 (C23H24O12),
(est.
506 (C23H22O13) and 508 (C23H24O13)
total for
exact chemicals structures unknown
DP &
LSIMS)
LSIMS
unknown high mass compounds m.w. 618,
NA
2.93
Data
662, 696, 718, 736, and 758
(est.
total for
DP &
LSIMS
data
DP = Data is from Electron Ionization Direct Insertion Probe raw data file FM10645
LSIMS = Liquid Secondary Ion 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 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
TABLE 8
Chemical Composition Neutral/Phenol Fraction Silica Gel MEOH Eluate
MS
Compound (Synonyms, Common Names,
Peak
Spec #
Comments, Etc.)
CAS #
Area %
134
3,5,5-trimethyl-2-cyclohexene-1-one
78-59-1
0.83
(isophorone)
205-
5-hydroxymethylfurfuryl acetate (HMF acetate)
10551-58-3
6.24
225
231
glycerylmonoacetate
26446-35-5
1.03
244
glyceryldiacetate
25395-31-7
0.6
246
glyceryltriacetate (triacatin)
102-76-1
1
254
decanoic acid
334-48-5
0.18
295
1,4-butanediol diacetate
628-87-1
0.57
311
unknown aromatic acetate
NA
0.53
319
unknown aromatic acetate
NA
0.65
326
dodecanoic acid (lauric acid)
143-07-7
2.11
334
1,4-benzenediol monoacetate (hydroquinone
NA
2.03
monoacetate)
353
unknown aromatic acetate
NA
0.29
359
unknown aromatic acetate
NA
0.13
372
myristic acid
544-63-8
2.66
399
unknown aromatic acetate
NA
0.5
405
pentadecanoic acid
1002-84-2
1.6
421
1,4-benzenedicarboxylic acid (terephthalic acid)
100-21-0
0.58
428
hexadecanoic acid (palmitic acid)
57-10-3
11.58
456
heptadecanoic acid
506-12-7
1.67
474
linoleic acid
60-33-3
2.88
500
octadecanoic acid (stearic acid)
57-11-4
39.02
520
3-methyl-1,8,9-anthracenetriol
491-59-8
2.57
(3-methylanthralin, Chrysarobin)
529
1,8-dihydroxy-3-methyl-9,10-anthracenedione
481-74-3
5.7
(Chrysophanol, Chrysophanic acid,
1,8-dihydroxy-3-methyl-1,8-anthraquinone)
539-
mixture of long chain aliphatic acetates
NA
4
575
583
di-2-ethylhexylphthalate (plasticizer)
117-81-7
0.55
616
1,8-dihydroxy-3-(hydroxymethyl)-9,10-
NA
3.82
anthracenedione
(Aloe-Emodin)
622
monoacetate derivative of 1,8-dihydroxy-3-
NA
1.34
(hydroxymethyl)-9,10-anthracenedione
(Aloe-Emodin monoacetate)
636
unknown 378 m.w. aromatic acetate
NA
0.77
644
squalene
7683-64-9
0.27
DP
bis-flavanoids with m.w.'s 474 (C23H22O11), 478
NA
4.3 (est.
Data
(C22H22O12), 490 (C23H22O12), 492 (C22H24O12), 506
total for
(C23H22O13) and 508 (C23H24O13) exact chemicals
DP &
structures unknown
LSIMS)
LSIMS
unknown high mass compounds m.s. 608, 680
NA
4.3 (est.
Data
and 696
total for
DP &
LSIMS
data
DP = Data is from Electron Ionization Direct Insertion Probe raw data file FM10650
LSIMS = Liquid Secondary Ion Mass Spectrometry Data file VG2265
TABLE 9
Chemical Composition Neutral/Phenol Fraction Silica Gel DCM Eluate
MS
Compound (Synonyms, Common Names,
Peak
Spec #
Comments, Etc.)
CAS #
Area %
263
unknown 154 m.w. compound
NA
0.29
268
2,3-dihydro-2,5-dimethyl-4H-1-benzopyran-4-
69687-87-2
0.88
one
278
S-6 hexasulfide (six membered ring structure)
NA
1.35
283
2,6-di-t-butyl-p-hydroxyanisole (antioxidant)
NA
2.12
295
butylated hydroxy toluene (BHT, antioxidant)
128-37-9
3.65
301
methyl laurate
111-82-0
1.47
323
diethylphthalate (plasticizer)
84-66-2
1.06
329
dodecyl acetate
112-66-3
1.82
358
unknown benzoate
NA
0.65
365
methyl tridecanoate
1731-88-0
1.35
369
α-hexylcinnamic aldehyde
101-86-0
0.65
373
benzyl benzoate
120-51-4
0.71
386
methylpentadecanoate
7132-64-1
0.82
390
tetradecyl acetate
638-59-5
0.59
404
benzyl salicylate
118-58-1
1.35
417
acetylferulic acid
2596-47-6
2.12
425
methyl plamitate
112-39-0
4.53
433
dibutylphthalate
84-74-2
2.00
441
sulfur (S-8, cyclic sulfur, orthothrombic sulfur,
10544-50-0
3.59
molecular sulfur)
447
hexadecyl acetate
629-70-9
1.88
451
methyl heptadecanoate
1731-92-6
0.94
471
methyl linoleate
112-62-0
15.82
478
methyl stearate
112-61-8
0.76
515
3-methyl-1,8,9-anthracenetriol
491-59-8
5.06
(3-methylanthralin, Chrysarobin)
519-
1,8-dihydroxy-3-methyl-9,10-anthracenedione
481-74-3
15.00
533
(Chrysophanol, chrysophanic acid,
1,8-dihydroxy-3-methyl-1,8-anthrquinone)
543
di-2-ethylhexylphthalate (plasticizer)
117-81-7
12.53
617
monoacetate derivative of 1,8-dihydroxy-3-
NA
6.24
(hydroxymethyl)-9,10-anthracenedione
(Aloe-Emodin monoacetate)
639
methyl famesate
10485-70-8
1.06
DP
bis-flavanoids with m.w.'s 474 (C23H22O11), 490
NA
9.71 (est.
Data
(C23H22O12), 492 (C23H24O12), 508 (C23H22O13)
total for
and 508 (C23H24O13) exact chemicals structures
DP &
unknown, also unknown peaks at m.w. 570 and
LSIMS)
602
LSIMS
unknown high mass compounds m.w. 540, 616,
NA
9.71 (est.
Data
646 and 662
total for
DP &
LSIMS
data
DP = Data is from Electron Ionization Direct Insertion Probe raw data file FM10649
LSIMS = Liquid Secondary Ion Mass Spectrometry Data file 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 10A-F, which describe the chemical composition of the organic soluble 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 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 that detector response factors could be determined and the data adjusted.
TABLE 10A
Data Summary: Composition of Organic Soluble
Fraction Grouped by Chemical Class
Aliphatic Carboxylic Acids
CAS #
% w/w
2-methylacrylic acid
79-41-4
0.27
2-methylbutyric acid
116-53-0
0.04
fumaric acid
110-17-8
1.50
pentanoic acid
109-524
0.04
hexanoic acid
142-62-1
0.07
2-furancarboxylic acid
88-14-2
1.13
2-ethylhexanoic acid
149-57-5
0.02
4-oxo-pentanoic acid (levulinic acid)
123-76-2
1.94
α-hydroxyhexanoic acid
NA
0.23
octanoic acid
124-07-2
0.118
nonanoic acid
112-05-0
0.09
decanoic acid
33448-5
0.11
dodecanoic acid (lauric acid)
143-07-7
1.09
10-undecenoic acid
112-38-9
0.01
nonanedioic acid
123-38-9
0.01
tetradecanoic acid (myristic acid)
544-63-8
1.05
pentadecanoic acid
1002084-2
0.12
hexadecanoic acid (palmitic acid)
57-10-3
9.82
α-hydroxylauric acid
NA
1.232
α-hydroxymyristic acid
2507-55-3
0.15
heptadecanoic acid
506-12-7
0.24
oleic acid
112-80-1
0.259
linoleic acid
60-33-3
12.26
stearic acid
57-11-4
3.43
palmitoleic acid
2091-29-4
0.01
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 (terephthalic acid)
100-21-0
0.06
cinnamic acid
621-82-9
0.01
acetyl ferulic acid
2596-47-6
0.20
TABLE 10C
Esters
glyceryl monoacetate
26446-35-5
0.08
glyceryl diacetate
25395-31-7
0.04
glyceryl triacetate (triacetin)
102-76-1
0.08
1,4-butanediol diacetate
628-67-1
0.04
2-hydroxy-5-methylfurfuryl acetate (HMF acetate)
10551-58-3
0.47
diethylphthalate (plasticizer)
84-66-2
0.10
dibutylphthalate (plasticizer)
84-74-2
0.21
di-2-ethylhexylphthalate (plasticizer)
117-81-7
11.70
tributylphosphate (plasticizer)
126-73-8
0.47
methyl laurate
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
methyl heptadecanoate
1731-92-6
0.09
methyl linoleate
112-62-0
1.51
methyl stearate
112-61-8
0.07
methyl farnesate
10485-70-8
0.10
dodecyl acetate
112-66-3
0.17
tetradecyl acetate
638-59-5
0.06
hexadecylacetate
629-70-9
0.18
benzyl benzoate
120-51-4
0.54
benzyl salicylate
118-58-1
0.13
methyl ester of 3,4-dimethoxy cinnamic acid
5396-64-5
0.01
unknown aromatic acetates
NA
0.16
mixture of long chain aliphatic acetates
NA
0.30
unknown benzoate
NA
0.06
unknown 378 m.w. aromatic acetate
NA
0.06
TABLE 10D
Phenolic Compounds
phenol
108-95-2
0.09
2-hydroxy-3-methylbenzoic acid (cresotic acid,
83-40-9
0.01
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-
309-51-112
0.01
methylisocoumarin (6-hydroxymellein)
7-hydroxy-5-methoxy-2-methyl-4-oxo-4H-1-
7338-51-4
0.42
benzopyran-6-carboxaldehyde
2,6-di-t-butyl-4-methylphenol (BHT,
128-37-0
0.34
antioxidant)
2,6-di-t-butyl-p-methylanisole (antioxidant)
NA
0.20
2,4-bis-(dimethylbenzyl)-6-t-butylphenol
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)
18-dihydroxy-3-methyl-9,10-anthracenedione
481-74-3
10.37
(Chrysophanol, Chrysophanic acid,
1,8-dihydroxy-3-methyl-1,8-anthraquinone)
1,8-dihydroxy-3-(hydroxymethyl)-9,10-
481-72-1
0.30
anthracenedione (Aloe-Emodin)
monoacetate derivative of 1,8-dihydroxy-3-
NA
0.83
(hydroxymethyl)-9,10-anthracenedione
(Aloe-Emodin monoacetate)
2,8-dihydroxy-3-methyl-1,4-naphthoquinone
NA
1.19
(Droserone)
bis-flavanoids with m.w.'s 474 (C23H22O11),
NA
6.27 est.
478 (C22H22O12), 490 (C23H22O12), 492
total for
(C23H24O12), 506 (C23H22O13) and 508
DP &
(C23H24O13) exact chemical structures
LSIMS
unknown
TABLE 10E
Alcohols
2-ethyl-1-hexanol
104-76-7
0.65
branched dodecanol isomer
NA
0.57
1-dodecanol (dodecylalcohol)
112-53-8
5.69
1-tetradecanol (tetradecyl alcohol)
112-72-1
2.14
2-ethoxy-1-dodecanol
29718-44-3
2.57
1-hexadecanol (hexadecyl alcohol)
36653-82-4
0.05
2-ethoxy-1-tetradecanol
NA
1.09
TABLE 10F
Miscellaneous Compounds
3,5,5-trimethyl-2-cyclohexene-1-one
78-59-1
0.06
(isophorone)
2,3-dihydro-2,5-dimethyl-4H-1-benzopyran-4-
69687-87-2
0.08
one
α-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
sulfur (S-8, cyclic sulfur, orthothrombic sulfur,
10544-50-0
0.90
molecular sulfur)
unknown 138 m.w. sulfur-containing
NA
0.05
compound
unknown 216 m.w. aromatic compound
NA
0.87
unknown 154 m.w. aromatic compound
NA
0.03
complex mixture of high molecular weight
NA
6.27 est.
unknown compounds in the range 400-1000,
total for
many of these compounds are conjugates of the
DP &
compounds identified in this study such as
LSIMS
glycosides, polar conjugates, high m.w. esters
etc.
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. Diagn. 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 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
Histological
Gross Pathologic
I.D. NUMBER
Phenotype
Assessment
Changes
Comments
1 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
2 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
3 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
4 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
5 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
6 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
7 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
8 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
9 (fsn/fsn)
Flaky skin
Reduction in
None observed
proliferation of
keratinocytes
10 (fsn/fsn)
Confusing
Marginal change
None observed
Jackson Labs
phenotype
suggest mistyping
of animal
11 (+/−)
Normal skin
No change
None observed
12 (+/−)
Normal skin
No change
None observed
13 (+/−)
Normal skin
No change
None observed
14 (+/−)
Normal skin
No change
None observed
15 (+/−)
Normal skin
No change
None observed
16 (+/−)
Normal skin
No change
None observed
17 (+/−)
Normal skin
No change
None observed
18 (+/−)
Normal skin
No change
None observed
19 (+/−)
Normal skin
No change
None observed
20 (+/−)
Normal skin
No change
None observed
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.
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 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. 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 1.0×104 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 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 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 μCi tritiated thymidine (3H) was also added per well. Following these treatments, the cells 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 12.5% trichloroacetic acid (TCA) for 10 minutes followed by methanol for 10 minutes. The plates were air dried 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.
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 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 normalized to the percentages of each active ingredient in the fractions mentioned above.
TABLE 12
Percentages of 4 Main Active Ingredients
in the Treatment Solution of Example 2,
Compiled from Tables 3-10
REF.
3-methyl
aloe-
aloe-emodin
Fraction
NO.
anthralin
chrysarobi
emodin
monoacetate
XAD-2 Resin
23
5.86%
11.76%
0.14%
2.23%
DCM Eluate
XAD-2
26
27.82%
12.78%
0%
0
Resin MeOH
Eluate-DCM-
Insol.
Hydrolyzed
29
0.42%
4.71%
0%
0%
Z-92 XAD
Resin-MeOH
Eluate
DCM Insol.
Z-92
28
0.55%
1.3%
0%
0%
Carboxylic
Acid Fx
Precipitate
36
19.69%
57.41%
0%
0%
From Neutral/
Phenol Fx
Neutral/
31
2.57%
5.7%
3.82%
1.34%
Phenol Silica
Gel MeOH
Eluate
Neutral/
31
5.06%
15.00%
0%
6.24%
Phenol
Silica Gel
DCM Eluate
Crude Z-92
10
11.77%
10.37
0.30%
0.83%
TABLE 13
Fraction Analysis Summary Results -
% Growth Inhibition of Keratinocytes by Fraction
Fraction Ref. No.
Mean %
From
Inhibit.
7/12
7/21
7/28
8/3
8/28
9/14
9/15
10/6
10/13
23
62%
60%
64%
26
50%
51%
44%
56%
29
7%
7%
28
40%
51%
35%
35%
36
63%
50%
76%
31
30%
20%
39%
31
92%
94%
99%
98%
80%
94%
89%
??
21%
17%
24%
??
17%
15%
18%
??
16%
16%
10
77%
77%
79%
84%
69%
??
94%
94%
TABLE 14
Percent Inhibition per Percentage of Ingredient
in Fraction for 4 Main Active Ingredients in the Treatment
Solution of Example 2 (Calculated From Mean %
Inhibition from Table 13)
Fraction Description From
Fraction Ref. No.
3-methyl
aloe-
aloe-emodin
from
anthralin
chrysarobi
emodin
monoacetate
XAD-2 Resin DCM
23
10.58%
5.27%
443%
28%
Eluate
XAD-2
26
1.79%
3.91%
0%
0%
Resin MeOH Eluate-DCM-
Insol.
Hydrolyzed Z-92 XAD
29
16.66%
1.48%
0%
0%
Resin-MeOH Eluate DCM
Insol.
Z-92 Carboxylic
28
72.72%
30.76%
0%
0%
Acid Fx
Precipitate
36
3.19%
1.15%
0%
0%
From Neutral/
Phenol Fx
Neutral/
31
11.67%
5.26%
7.85%
22.38%
Pherol Silica Gel MeOH
Eluate
Neutral/
31
18.18%
6.13%
0%
14.74%
Phenol Silica Gel DCM
Eluate
Crude Z-92
10
6.54%
7.42%
256.6%
92.77%
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 7.0 with 1.0 N NaOH. Keratinocytes were assayed for growth in the presence of the 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 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.
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.
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 thereof by those skilled in the art to which this invention pertains.
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