MXPA98000697A - Use of calendulic glycosides for the treatment of psoria - Google Patents

Abstract

Use of glycosidic compounds as pharmaceutical agents against psoriasis, compounds therefor and pharmaceutical formulations thereof.

Description

USE OF CALENDULIC GLYCOSIDES FOR THE • TREATMENT OF PSORIASIS Field of the invention The present invention relates to compounds and extracts of plants containing compounds which are indicated to have an inhibitory effect on cell proliferation. More specifically, the invention relates to glycosidic compounds that are derived from Calendula species, the glycosides of plants that have a cytostatic effect on cells and their use as cytostatic agents, in particular in the treatment of psoriasis. Background of the Invention Calendula plant extracts have been used in the culture of medicine for the treatment of various ailments for centuries. Said extracts for example, have been used as, or in, anti-inflammatory drugs and the like. International patent application WO 91/15218 teaches a therapeutic composition against psoriasis comprising as active ingredient a solvent extract of at least six different herbs. This application teaches that marvelous decoctions can be used against gastric and intestinal ulcers externally as well as to wrap wounds of slow healing. It is now established that marigold extract, in fact, is used by itself as the active component in a therapeutic composition against psoriasis. Pizza C, and Tommasi N., Phytochemistry, vol. 27, number 7, p. 2205-2208 (1988) teaches the isolation and structure of sesquiterpene glycoside for Calendula arvensis. It is established that Calendula arvensis, L. (Mixed Compound) is an herbaceous plant used in the medicine of the Italian people as an anti-inflammatory and antipyretic remedy. It is not suggested that the obtained sesquiterpene glycosides have cytostatic activity or can be used in the treatment of psoriasis. Mascó N. et al., Phytotherapy Research, Vol. 1, p. 28-31 (1987) teaches that marigold extract is known for anti-inflammatory activity. It is not mentioned that marigold extract is used as an anti-psoriatic agent. Gracza L. Planta Medica 53, page 227 (1987) describes several terpene derivatives containing oxygen from Calendula officinalis.

The uses of these are indicated for leukorrhea and trichomonacidal activity. The use of terpene derivatives as anti-psoriatic agents is not mentioned. Fazakas B, and Rácz, G. Pharmacia Vol. XIII, number 2, page 91 (1965) also teaches that the extracts of the flowers of Calendula officinalis are used in traditional herbal medicine for leucorrhoea (excessive fluoride alba) and showed good trichomonacidal activity. Gracza L. and Szász K. Acta Pharm. Hung. 38, pgs. 118-125 (1968) reports on a chemical analysis of marigold petals (Calendula officinalis) with the help of separation and identifying the solution or solutions that are responsible for the effect trichomonacid reported by Frazakas and Rácz (before). The most active liquid compounds isolated were described as terpene alcohols and terpene lactones according to spectroscopy data. Jakupovic and others. Planta Medica 54 (3) p. 254-256 (1988) teaches the extraction and isolation of five sesquiterpene glycosides from Calendula pérsica. The potential or real uses for the extracted and isolated molecules are not mentioned. Ahmed A Ahmed et al., Journal of Natural Products Vol. 56, number 10, p. 1821-1824 (1993) refers to the extraction of Calendula arvensis products. The products are described as four new sesquiterpene glycosides and three known ones. There is no reference to possible uses for them. EP 364442 B1 describes a therapeutic composition against psoriasis comprising an oily extract of at least three herbs selected from a scale of herbs, the scale of which may include Calendula. However, it is established that the separated extracts of herbs do not provide a curative effect against psoriasis when used alone. In addition, it is established that the calendula decoctions by themselves are used for, among others, gastric and intestinal ulcer treatment. Calendula decoctions are not described as being beneficial for the treatment of skin diseases involving abnormal skin cell proliferation regimens (e.g., hyperproliferation), in diseases such as psoriasis.

DE 3836519 C2 alleges that a pharmaceutical preparation containing mixed cut inflorescences of Calendula officinalis, with milk fat as a balsam base, is useful for treating psoriasis. However, the composition is described as being capable of causing allergy, which can lead to to the interruption of the treatment and the active component of the composition is not indicated or identified. Cytostatic activity is not indicated and it is also not evident which component or mixture of competent in the pharmaceutical preparation based on Calendula officinalis is / are the component (s) (s) act? ve (s) In addition, there does not appear to be any real evidence to show that the composition was used in the treatment of psoriasis. There is a need for the development of new pharmacocitostats that are effective in combating initiation, maintenance and / or development. of disease involving hyperproliferation of cells of the dermis, in particular in the treatment of psoriasis SUMMARY OF THE INVENTION It is an object of the present invention to provide the use of active compounds or purified plant extracts comprising at least one active compound in the preparation of a medicament for the treatment of disease involving hyperproliferation of dermal cells, particularly in the treatment of psoriasis A second objective of the invention is to provide compounds Active and / or purified Calendula for use in the treatment of disease involving hyperproliferation of cells of the dermis, in particular in the treatment of psoriasis. A third objective of the invention is to provide isolated compounds for use in the treatment of disease involving hyperproliferation of dermal cells, in particular in the treatment of psoriasis. This and other objects of the invention will be apparent from the following description and examples. DETAILED DESCRIPTION OF THE INVENTION In accordance with one aspect of the present invention, the use of a compound of the general formula (I) is provided. wherein R1 and R2 are independently selected for H, OH, H3C CH3 H3C COO "\ / C / C = C C = C / \ / \ H COO" H CH- and esters related thereto; R3 is selected from -OH, O O O II II li CH3CH2CH (CH3) CO-, CH3CH2C (CH3) = CHCO- CH3CO- H3C CH3 H3C COO- O \ / \ / II C = CC = C and CH3C (CH3) = CHCO- / \ / \ H COO "H CH3 and esters related thereto; R4 is selected from aliphatic, monocyclic ring systems or polycyclic, saturated or unsaturated C6-C? 2 optionally substituted by alkyl d-Ce, H, OH, = CH3 or alkyl carboxyloxy C? -C4 or R4 represents a straight or branched chain alkane group of C1-C5 substituted with said ring system; R5 is selected from = CH3, -CHO, -COOH and -CH2OH and related esters and ethers derived therefrom; R6 is selected from -OH, O O O II II II CH3CO-, (CH3) 2CHCO-, CH3CH2CH (CH3) CO-, H3C CH3 H3C COO "\ / \ / C = C and C = C / \ / \ H COO- H CH3 In the preparation of a drug for the treatment of disease involving hyperproliferation of dermal cells. For the purposes of the present invention "related esters and ethers" refers to all defined esters of R groups mentioned herein and, in general, to straight or branched chain C-C20 carboxyalkyl esterification acids saturated or unsaturated Suitable examples include acid esterification comprising methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and all isomers of alkyl groups of pentanyl, pentenyl, hexanyl and exeyl. Also included with the term "related esters" are aromatic acids such as benzoic acid and cinnamic acid. C 1 -C 20 alkyl esters comprising straight or branched chain alkyl groups as defined for "related esters" above, also related herein. In a preference there is provided a compound of the general formula (I) wherein R1 and R2 are independently selected from H, -OH H3C CH3 H3C COO "\ / \ / C = C and C = C; / \ / \ H COO "H CH3 R is selected from -OH, O O O II CH3C (CH3) CO-, CH3CH2C (CH3) = CHCO-, CH3CH2CH (CH3) CO- H3C CH3 H3C COO- O \ / \ / || C = C, C = C and CH3CO- / \ / \ H COO- H CH3 R4 is selected from the group Rb is selected from the group CH3, -CHO, -COOH and -CH2OH; and O O R6 is selected from OH, (CH3) 2CHCO-, CH3CH2CH (CH3) CO-, O H3C COO- H3C CH3 II \ / \ / CH3CO- C = C and C = C / \ / \ H CH3 H COO "In a further preference the use of a compound that can be extracted from Calendula species is provided. General Formula (I) wherein R 1 and R 2 are independently selected from HyOH (/? OH or aOH); OO II II R3 is selected from OH, CH3C (CH3) = CHCO-, CH3CO-, O O II II CH3CH2C (CH3) = CHCO- and CH3CH2CH (CH3) CO-; R is selected from R5 is CH3; and R6 is selected from OH, O O O II CH3CO-, (CH3) = CHCO- and CH3CH2CH (CH3) CO- More preferably, the use of a compound of the Formula (I) extracted from Calendula species is provided wherein R1 and R2 are independently selected from H and OH (αOH or αOH); R3 is selected from OH and (E) -3-methylpent-2-enoate, that is, ? R is selected from R5 is CH3; and R6 is OH. The skilled person will also appreciate that the groups R1, R2, R6 and R3 can be located in both the axial and equatorial positions. Naturally, the skilled person will appreciate that the functional isomers are physiologically functional of the Formula (I) found both in Calendula species and isomers synthetically derived therefrom, including conformational and constitutional isomers as well as the D and L forms of the Formula (I) HE encompass the present invention. Examples of conformational isomers in the invention are encompassed in Formulas (la) and (Ib) as follows: (Ia) (Ib) wherein R1, R2, R3, R4. R5 and R6 are as defined for Formula (1). Naturally, the skilled person will appreciate that other constitutional isomeric variants that refer to "chair" (Formula (la)) and "can" (Formula (Ib)) forms having physiological functionality are permissible within the scope of the invention. The compounds of Formula (I) can be isolated from Calendula species using conventional organic solvent extraction technology. Generally, the compounds of Formula (I) can be extracted from any plant tissue such as leaves, stems, flower parts, roots, shoots and the like. In a further embodiment of the invention, novel compounds of the invention are provided according to the general Formula (I) as presented above wherein R1 and R2 are independently selected from -OH, H, H3C CH3 H3C COO "\ / \ / C = C and C = C / \ / \ H COO" H CH3 R3 is selected from (E) -3-2-methylpent-enoate, and H3C C H3 \ / C = C / \ H COO " R 4 is selected from monocyclic or polycyclic saturated or unsaturated C 6 -C 2 aliphatic ring systems optionally substituted by C 1 -C 6 alkyl, H, -OH, = CH 2 or carboxyloxy C 1 -C 4 alkyl; R5 is selected from C-C4 alkyl or -CHO, -COOH and -CH3OH; R6 is selected from \ / \ / C = C C = C / \ / \ H COO- H CH3 physiologically functional isomers thereof. In a preference, novel compounds are provided according to formula (I) wherein R1 and R2 are independently selected from -OH, H H3C CH3 H3C COO- \ / \ / C = C and C = C; / \ / \ H COO- H CH3 R3 is selected from (E) -3-methylpent-2-enoate and H3C C H3 \ / C = C / \ H COO " Rs is selected from -CH3, -CHO, -CH2OH and -COOH; R6 is selected from C = C and C = C / \ / \ H COO "H CH; and physiologically functional isomers thereof. In a further preference there are provided compounds of Formula (I) wherein R1 and R2 are independently selected from H and -OH; R3 is (E) -3-methylpent-2-enoate; R4 is selected from R5 is CH3; and R6 is OH; and physiologically functional isomers thereof The skilled person will also appreciate that the groups R1, R2. Rs and R3 can be located in the axial or equatorial position. "Physiologically functional isomers" for the purposes of the present invention mean those isomers that are capable of substantially decreasing or stopping the hyperproliferation of the cells of the dermis. Therefore, it should be noted that such isomers have a substantially cytostatic effect on the cells of the dermis and as such are indicated to be useful in the treatment of skin diseases such as psoriasis. Examples of novel compounds according to the invention include: (i) (rel) -1aa, 4a ?, 7a ?, 7ba-Decahydro-1, 1, 4 ?, 7a-tetramethyl-1H-cycloprop (e) azulene 4? -O- (2-E- (3-met.lpent-2-enoyl) -? -quinovopyranoside (Van-1 (ii) (re /) - 5 ?, 7 ?, 14? -eudesimo-4 (15) -in-11-O- (2-E- { 3-methyl.}. pent-2-enoyl) -? -fucopyranoside (Van- 10-2). (iii) (re /) - 1aa, 4a ?, laß, 7ba-Decahydro-1, 1, 4 ?, 7a-tetramethyl-1H-cycloprop (e) azulen-4? -O- (2-E-. { 3-methyl.}. Pent-2-enoyl) -? - fucopyranoside (Van-10-4).

The preferred novel compounds of the invention on the basis of their biological activity are (ii) and (iii) above and physiologically functional derivatives and analogs thereof. The most preferred compound based on its biological activity is compound (iii) above. As an embodiment of the invention, the use of compounds of the Formula (I) is included in a composition for the treatment of disease that involves hyperproliferation of the cells of the dermis, in particular for the treatment of psoriasis. Naturally, the skilled person will appreciate that such use may include the use of known compounds isolated from Calendula species such as, (iv) (rel) -1aa, 4a ?, laß, 7ba-Decahydro-1, 1, 4 ?, 7a- tetramethyl-1H-cycloprop [e] azulene-4? -0-? -fucopyranoside (Van-15A).

A preferred compound used in the preparation of a medicament for the treatment of a skin disorder such as psoriasis, is compound (iii) above. The skilled person will appreciate that the analogs of the compounds (i) to (iv) above can be synthesized therefrom in situ. For example, where the groups R1 and R6 are both OH or R2 and R6 are both -OH can be esterified to the angelato, tiglato or other ester by reaction of the appropriate acid chloride or acid anhydride with the appropriate starting compound, for example compound (iii) in the presence of a suitable base such as pyridine. Both alcohol functions will be esterified by this technique. Derivatives with different ester groups on R1 or R6 v.gr., tiglato and angelato, can also be prepared by reacting suitable compounds such as compound (iii) with a mixture of two acid chlorides or two anhydrides. This will produce the four possible isomers that can be separated using standard techniques such as chromatography on silica gel or the like. The branching of ethyl crotonate (R3) on the compounds (i) to (i1i) can be replaced by other carboxylic acid esters using the following general procedure. The hydroxyl functions of R1 and R6 can be protected by the formation of the acetonide by reaction of the compound with acetone in the presence of toluene sulfonic acid by similar techniques known in the art such as those described in Organic Synthesis, Second Edition, TW Greene, PGM Wuts (ISBN 0 471 62301 6) (e.g., Chapter 2 pp. 123-127) incorporated herein by reference, such as by producing, for example, a following compound A or similar compounds.

The ethyl crotonate branching can be removed by hydrolysis of acid or base of for example, compound (iii), to give the product with R3 = OH. The resulting compound can then be reacted with an appropriate acid chloride or acid anhydride in the presence of a base such as pyridine to give an ester such as tiglato or angelato on R3 Alternatively said ester can be formed by transestepfication of compound A (above) with an excess organic acid such as tiglic or angelic acids in the presence of a suitable acid or basic catalyst Finally the protective acetonide group can be separated by suitable separation reagents such as iodine in methanol as described in Greene TW (supra). As a preferred embodiment of the invention, the use of at least one compound isolated from a species of Calendula is provided., such as Calendula officinalis, for the manufacture of a medicament for the treatment of psoriasis. The compound is usually a plant glycoside, such as a sesquiterpene glycoside. A therapeutical composition against psoriasis is also encompassed within the scope of the invention. at least one plant glycoside isolated from a Calendula species, such as Calendula off icinalis. Generally, the therapeutic composition contains at least one glycoside of purified plant, such as a sesquiterpene glycoside of Formula (I). Naturally, the expert will appreciate that said compositions may comprise two or more plant glycosides at any concentration that is capable of causing a therapeutic cytostatic effect. Therefore, the therapeutic compositions may comprise extracts from Calendula Plants substantially without undesirable contaminant compounds. Plant extracts, for example, can den undergo a certain number of steps of extract of solvents substantially to remove undesirable components from the desirable components such as those encompassed by Formula (I) Naturally, plant extracts that have been subjected to said solvent extracting steps may contain more than one plant glycoside and may contain Various plant ghcosides Said plant extracts may be subjected to additional solvent extraction procedures to isolate the glycoside compounds of individual active plants of Formula (I) A description of a general method for obtaining purified extracts of Calendula species is given below. containing active components capable of producing a cytostatic effect on cells of the dermis, the refinement of said extracts and the isolation of active components of extracts and / or subsequent purification thereof. The flowers of a Calendula species are pulverized in a mill and the resulting powder extracted with "Soxhlet" with petroleum ether until exhaustion The extract can concentrate, for example, under reduced pressure using an evaporator, such as a rotary evaporator to provide an extract residue, the crude extract The crude extract can then be fractionated via, for example, sebaceous liquid chromatography (CLS) using suitable solvents and solvent systems of increased polarity Suitable solvents include solvents such as polar and non-polar organic solvents and mixtures of same. Examples of solvents that can be used in a suitable extraction method for the purposes of the present invention include the following (volume ratios): Petroleum - EtOAc (EtOA O-10%) Petroleum - EtOAc (EtOA 12-18%) Petroleum - EtOAc (20-24% EtOAc) Oil - EtOAc (35-40% EtOAc) Petroleum - EtOAc (45% EtOAc) Petroleum - EtOAc (50-55% EtOAc) Oil - EtOAc (60% EtOAc) Petroleum - EtOAc (EtOAc 65-75%) Oil - EtOAc (EtOA 80-85%) Oil - EtOAc (EtOA 90-95%) Oil - EtOAc (EtOA 95%) EtOA - MeOH (0-5% MeOH) EtOA - MeOH (10-100% MeOH) mixtures of hexane, hexane: chloroform on volume ratios such as: Hexane Chloroform 100 0 95 5 9 1 8 2 6 4 4 6 0 100 mixtures of chloroform, and chloroform: methanol (e.g., 5: 1, 1: 1) and the like. Therefore, the crude extract can be fractionated several times using different mixtures of organic solvents at preselected ratios of, for example, increasing polarity. Under CLV, silica gel (Merck 7749) is usually packed in a receptacle, for example a funnel formed under vacuum applied, to give a column. The crude extract may be ready for introduction to the CLV column for example, by adsorption onto silica (eg, 1: 1 w / w) and placed on top of the column as described above, diluted at less once with a solvent or solvent system of increasing polarity, such as those mentioned herein in previously made relationships. The eluents from the CLV column are collected and can be subjected to further fractionation steps via column chromatography or other suitable chromatographic media. After fractionation of the crude extract, the collected eluents can be subjected to preparative thin layer chromatography (CD) for Analysis of active constituents contained therein For example, in a method a fraction of eluents designated Van-5-10, obtained from column chromatography using a chloroform-methanol mixture ratio of 955, was subjected to CCD analysis using an initial solvent system of hexane chloroform ethyl acetate in a volume ratio of 366, giving five fractions, designated Van-9-1, Van-9-2, Van-9-3, Van-9-4 and Van- 9-5 It can be considered that said fractions comprise purified mixtures of different glycosidic molecules encompassed by Formula (I) and therefore are able to be seen as substantially mixed. e free of contaminating components such as plant proteins, plant hormones and the like Said fractions of eluents having cytostatic activity form an embodiment of the present invention In a further step, the fractions Van-9-1 to Van-9-5 as mentioned earlier, they can be subjected to additional preparative thin layer chromatography (CCD prep) silica gel analysis using additional organic solvent preparations For example, the Van-9-2 eluent fraction subjected to CCD prep using as a solvent the hexane preparation chloroform ethyl acetate at a ratio of 364, giving the sub-fractions of Van-9- 2, designated Van-9-2B and Van-9-2C, the sub-fractions resulting from prep CCD step can be subjected to additional chromatographic steps such as high pressure liquid chromatography (CLAP). For example, the isolated fractions of CCD of Van-9-2-B and Van-9-2-C are subjected to CLAP using an octadecyl-silanol (analytical) column (ODS) with a solvent of 75% acetonitop in water containing 0.1% Tpfluoroacetic acid (TFA) The individual fractions obtained from CLAP techniques can then be evaluated for the active component using NMR techniques and analyzing said fractions for cytostatic activity m vitro For larger batches of raw materials such as dried calendula petals off i cinalis The following general extraction method can be used. The material of dried plants, for example, calendula off icinalis petals, can be ground to a coarser powder and extracted in a suitable organic solvent. Generally, the extraction method can be any extraction method. suitable as is known in the art, such as "Soxhlet" extraction, batch extraction and continuous extraction. For example, anti-psopathic compounds of Formula (I) are provided as a further aspect of the invention. which can be obtained from a Calendula species particularly in the manner of an extraction process by "Soxhlet", in continuous batch. Such extraction processes are known in the art. Preferably the Calendula species are Calendula officinalis and the preferred extraction method is a continuous extraction method. Suitable organic solvents for use in a continuous extraction method include polar and non-polar organic solvents such as methane, ethane, dichloromethane, toluene, hexane, ethyl acetate, isopropyl, and the like. Preferably, the solvent is a non-polar organic solvent such as heptane. The resulting extract is concentrated under vacuum until an appropriate weight of extract / total volume of percent solution ratio is obtained (% w / v) Solution solution ratio of adequate% p / v falls on the scale 10% w / w 60% w / v, more preferably between 15 w / v and 30% w / v and even more preferably around 20% w / v The resulting further concentrate is extracted at a solvent / water solution at an appropriate ratio of parts by weight: parts by weight such as 100 to 7 3 Preferably the ratio is from 100 to 8: 2 More preferably the ratio is 9 1 A suitable polar organic solvent can be selected from acetonitop, methanol, ethanol, ethyl acetate and the like A preferred polar organic solvent is acetonttplo The heptane fraction is discarded and the polar organic solvent fractions containing VAN (such as acetonitoplo fractions) can be combined Then, if the polar organic solvent is removed under vacuum The resulting solid it can be redissolved in a suitable polar or non-polar organic solvent such as ethyl acetate, methanol, acetonitrile, diethyl ether, dichloromethane, toluene, and the like, and then washed successively with a base such as sodium bicarbonate in a ratio of 10. : 30% w / v, water, dilute inorganic acid such as hydrochloric acid at a concentration of 0.05 to 0.1 M or diluted organic acid, citric acid at a concentration of 0.2 M to 1.0 M, and finally water. The organic solvent solution is then evaporated under vacuum. The resulting crude preparation can further be purified to an appropriate VAN-10 compound, such as VAN-10-4. The crude preparation can be redissolved in a suitable volume of organic solvent such as a polar organic solvent or an adequate volume of a mixture of at least one polar organic solvent: at least one non-polar organic solvent. Preferably, the organic solvent consists of a polar organic solvent mixed with a non-polar organic solvent. When a suitable mixture of organic solvents is used they can be miscible with each other. The% (v / v) ratio of polar organic solvent: non-polar organic solvent can be between 80:20 and 20: 80% (v / v) preferably 30: 70 and 70: 30% (v / v). For example, an amount of the crude extract can be redissolved in an adequate volume of polar organic solvent: non-polar organic solvent at a 50% percent polar organic solvent: 50% non-polar organic solvent. A preferred solvent mixture is 50% ethyl acetate: 50% Hexane Additional suitable solvent may be selected from polar and non-polar organic solvents as described above. The dissolved material may be loaded in a Flash chromatography system such as a Flash 75 column and eluted at a suitable flow rate using a mixture of suitable solvents. as the mobile phase of v.gr, 50% ethyl acetate: 50% hexane. A suitable flow rate of a flash chromatography column can be 50-500 ml / min depending on the size of column used, preferably 100-250 ml / mm, more preferably about 250 ml / min using, for example, a mixture of 50% ethyl acetate 50% hexane as the mobile phase The polar non-polar solvent solvent ratio for the mobile phase can be within the scales used to redissolve the crude extract as described above The actual ratio It will depend on the solvent mixtures employed Using instantaneous system described in the mixture, which of VAN can be isolated in a number of fractions These fractions can then be combined and evaporated to dryness After evaporation, the combined fractions (ie material semi-solid), can be redissolved in a suitable polar organic solvent, for example, such as those suitable polar solvents mentioned above and after the dissolved fractions purified by CLAP using a reverse phase column such as "Sphepsorb" column or similar A stock solution of the resulting (semi-crude) combined material obtained after the instant chromatography but before CLAP it can be redissolved in a solvent similar to that used as the mobile phase and divided into aliquots. The aliquots can then be processed by standard CLAP techniques known in the art, such as isocratic separation or by gradient elution . Naturally, the person skilled in the art will appreciate that the maximum concentration of semi-crude re-dissolved in the mobile phase can depend on the solvent employed. For example, up to 25 mg / ml of combined extract (semi-crude) can be re-dissolved in acetonitoplo which can then be divided into suitable volumes to be run in a CLAP column. Each sample runs for a time depending on the mobile phase used and the VAN-10 compounds can be eluted at appropriate time intervals. work by doing preliminary runs on an analytical column using different solvent systems such as the mobile phase and selecting a solvent system that results in an optimal elution rate and good resolution of the sample. In the mobile phase the% polar organic solvent% non-polar organic solvent can be in the range of 90 10, A 1090, preferably 70-80 3020, even more preferable Between 75-80 25-20 Therefore the purification by CLAP, a mobile phase consisting of water acetonitoplo can be 75% acetonitplo 25% water and flow rate can be established according to the size of column used, such as 5 ml / per min. Suitable polar organic solvents include those mentioned before. The preferred organic solvents are acetonitrile or methanol. It will be evident to the expert that the selection of solvent or solvent mixtures for each step in the isolation of active principles can be guided by the results of bioanalysis of finally separated fractions, the identification of active components found in the separated fractions, in the different steps of the extraction procedure. The compounds of the present invention can be found to have cytostatic activity in mouse embryonic fribroblasts cells in vitro. Therefore, the cytostatic activity of the compounds of the general Formula (I) has been demonstrated in a number of in vitro cytostatic tests. In addition, the cytostatic activity of compounds of the general Formula (I) has been demonstrated in vivo. It is noted that the compounds of the present invention are useful for the treatment of skin diseases wherein abnormally high cell proliferation is observed in the disease process. They can be used to treat skin disease disorders; as psoriasis, where the abnormally high cell proliferation is a recognized symptom of such skin disorders. The invention therefore also provides a method for the treatment of skin disorders such as psoriasis in mammals, including humans, which comprises the use of a clinically useful amount of the compound of the Formula (I) or physiologically functional derivative in a pharmaceutically useful form, once or several times a day or in any other appropriate program, for example, orally or applied topically. Further provided is a further or alternative aspect of the invention, a compound of Formula (I) or physiologically functional derivative thereof for use in therapy, for example; psoriasis and the like. The amount of the compound of the Formula (I) required to be effective as a cytostatic agent will, of course, vary and will ultimately be at the discretion of a veterinarian or practitioner. Factors that will be considered include the condition being treated, the route of administration and naturalization of the formulation, the body weight of the mammal, surface area, age and general condition and the particular compound that will be administered. An adequate effective dose of cytostatic compounds of the invention generally falls on the scale from about 0.01 to about 120 mg / kg of body weight, e.g. 0.1 to 120 mg / kg of body weight, preferably in the range of about 0.1 to 50 mg / kg, for example 0.5 to 50 mg / kg. The total daily dose can be given as a single dose, multiple doses, e.g. from two to six applications per day. For example, for a 75 kg mammal (v.gr, a human) the dose scale could be about 8 to 9000 mg per day and a normal dose could be about 50 mg per day. If discrete multiple doses are indicated, the treatment it can normally be 15 mg of a compound of the formula (I) given up to four times per day. While it is possible to administer only the active compound, it is preferable to present the active compound in a pharmaceutical formulation. The formulations of the present invention, for use medical, comprise a compound of the Formula (I) together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients The carrier (s) should be pharmaceutically acceptable in the sense of being compatible. (s) with the other ingredients of the formulation and substantially harmless (s) for the success thereof. Therefore, the present invention further provides a pharmaceutical formulation comprising a compound of the Formula (I) or physiologically functional thereof together with a pharmaceutically acceptable carrier thereof. Naturally, the skilled artisan will appreciate that any pharmaceutical formulation comprising The active compound of Formula (I) can include at least one active compound isolated and / or purified from an extract derived from a Calendula species. Preferably, the Calendula species is selected from at least one of Calendula officinahs, Calendula arvensis. , and Persian Calendula More preferably the active compound isolated from a Calendula species is isolated and / or purified from Calendula off icinalis Therefore, a pharmaceutical formulation may contain an active component isolated and / or purified from two or more species of Calendula whose active component can be formed of two or more active compounds encompassed by Formula (I) above In a further embodiment a pharmaceutical formulation can comprise a mixture of at least one active compound isolated and / or purified from an extract derived from Calendula officinalis and at least one active compound isolated and / or purified from Calendula arvensis or Persistent Calendula from Formula (I) A method is also provided for the preparation of a pharmaceutical formulation comprising associating a compound of Formula (I) or a physiologically derived compound. Functional thereof and a pharmaceutically acceptable carrier thereof Formulations according to the present invention include those suitable for oral or topical administration. Preferred formulations are those suitable for topical administration, such as to the skin. For external tissue infections., v. g, skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0 075 to 20% w / v, preferably 02 to 15% w / v and more preferably 05 at 10% w / v When Formula in an ointment, the active ingredients can be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredients can be formulated in a cream with an oil-in-water cream base. The aqueous phase of the cream may include, for example, 3% w / w of a polyhydric alcohol, ie an alcohol which has two or more hydroxyl groups such as propylene glycol, butanol-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. Topical formulations may conveniently include a compound that increases the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of said dermal penetration enhancers include dimethyl sulfoxide and related analogues. The oil phase of the emulsions of this invention may be constituted of known ingredients in a known manner. While the phase may merely comprise an emulsifier (known in some manner as an emulsifier), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or both a fat and an oil. Preferably a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together the emulsifier (s) with or without stabilizer (s) form the so-called emulsifying wax and the wax together with the oil and / or fat form the so-called emulsifying ointment base which forms the dispersed oily phase of the formulations of cream. Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glycerol mono-stearate and sodium lauryl sulfate.

The choice of oils or greases suitable for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in the majority of the oils that are likely to be used in pharmaceutical emulsion formulations is low, therefore the cream should preferably be a non-greasy, non-staining and washable product with a suitable consistency to prevent the leakage of tubes or other containers. Branched straight chain mono or dibasic alkyl esters, such as di-isoadipate, isocetyl sterate, and propylene glycol ester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a mixture of branched chain esters known as Crodamol CAP can be used, the last three being the preferred esters. These can be used alone or in combination depending on the required properties. Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils may be used. The formulations can conveniently be present in unit dosage form and can be prepared by any of the methods well known in the pharmacy art. All methods include the step of associating the active compound (s) with a vehicle that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately associating the active compound (s) with a liquid carrier or a finely divided solid carrier or both and, if necessary, shaping the product later into desired formulations. Formulations of the present invention suitable for oral administration may be present as discrete units such as capsules, tablets, troches, comprising the active ingredient in a sabotaged base usually sucrose and acacia or tragacanth, tablets comprising the active ingredient in an inert base such as gelatin and ghicken, or sucrose and acacia, and mouthwashes comprising the active ingredient in a suitable liquid carrier. Each formulation generally contains a predetermined amount of the active compound, such as a powder or granules, or a solution or suspension in aqueous or non-aqueous liquid such as syrup, an elixir an emulsion or a potion and the like A tablet can be made by compression or molding optionally with one or more accessory ingredients The tablets compressed can be prepared compr imaging in a suitable machine the active compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, (v. g, povidone, hydroxypropylmethyl cellulose gelatin), inert diluent, preservative, disintegrant lubricant (v. g, sodium starch glycolate, interlaced povidone, interlaced sodium carboxymethylcellulose) , surface active agent or dispersant The molded tablets can be made by molding in a suitable machine a mixture of the wetted powder compound with an inert liquid diluent. The tablets can optionally be coated or delineated and can be formulated in such a way as to provide slow or controlled release of the active ingredient therein using for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile. A syrup can be formed by adding the active compound to an aqueous concentrated solution. of a sugar, for example, sucrose, to which any necessary ingredient can also be added D? cho (s)? ancillary ingredient (s) can (include) sabopzantes, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol, for example ghcerol or sorbitol In addition to the ingredients mentioned above, the formulations of this invention may also include one or more accessory ingredients selected from diluents, solutions pH regulating agents, sabopzantes, binders, surface active agents, thick Gras, lubricants, preservatives (including antioxidants) and the like In a further aspect, the present invention provides the use of a compound of the Formula (I) or a physiologically functional derivative for the manufacture of a medicament for the treatment of psoriasis Figure 1 Diagram schematic showing the extraction steps to isolate the cytostatic compounds from Calendula off icinalis flours.

Figure 2: Results of the treatment of patients suffering from psoriasis with extract of Calendula officinalis. The invention will now be illustrated by the following non-limiting examples Example 1: Isolation of Van-6-1 Extract Reference is made to Figure 1. Extract and Fractionation of plant material: 400g of plant material powder was extracted by "Soxhlet" "with petroleum ether (60-80 °) until exhaustion. The extract was concentrated under reduced pressure using a rotary evaporator to yield 49g of extract residue. The plant material was further extracted with chloroform under conditions similar to those described above to give an additional extract. These crude extracts were then fractionated using vacuum liquid chromatography (CLV). The silica gel (Merck 7749) was packed in a cone funnel under vacuum applied to give a column approximately 10cm in diameter and 5cm in height. The extract (20g at a time) absorbed the silica for (1: 1, w / w) was then placed on top of the column and eluted with solvents of increasing polarity; that is, hexane, hexane containing increased concentrations of chloroform, chloroform and chloroform containing increased amounts of methanol. The CLD eluents were collected and the fractions analyzed for CCD for its constituents. Based on the CCD analysis, the CLD eluents were poured into wells giving thirteen fractions, (Table 1). These were tested for their cell growth inhibitory activity, following the 3T3 bioassay procedure (example 5). An eluent, designated Van-2-36 (extracted using a 1: 1 mixture of chloroform and methanol) was found to be the most active. TABLE 1 Fraction Solvent System Van-2-1 Hexane Van-2-2 Hexane Van-2-6 Hexane: Chloroform (95: 5) Van-2-12 Hexane: Chloroform (8: 2) Van-2-15 Hexane: Chloroform (6: 4) Van-2-21 Hexane: Chloroform (6: 4) Van-2-23 Hexane: Chloroform (6: 4) Van-2-25 Hexane: Chloroform (4: 6) Van-2-27 Hexane: Chloroform (4: 6) Van-2-31 Chloroform Van-2-35 Chloroform: Methanol (5: 1) Van-2-36 Chloroform: Methanol (1.1) Van-2-37 Chloroform.Metanol (1.1) The eluent Van-2-36 was subjected to silica gel column chromatography with mixtures of hexane: chloroform and chloroform: methanol of increasing polarity. The fractions diluted with 10- chloroform 60% (in hexane) gave Van-5-4 while those fractions diluted with 5% methanol (in chloroform (in hexane) gave Van-5-4 while those fractions diluted with 5% methanol (in chloroform) gave Van- 5-9 Both of these fractions appeared to be mixtures of fatty acids for (GC spectrum and NMR) and showed no inhibition of cell growth, Van-5-10 was located in a 5% methanol eluate while Van-5 was located in a 5% methanol eluate. -11, Van-5-12 and Van-5-15 were fractions obtained from the 10% methanol eluates.An additional attempt was made to purify Van-5-12 using CCD prep (silica gel, solvent- chloroform: methanol; 95.5). This gave Van-6-1 was found to be very active using cell growth bioanalysis (example 5). Example 2: Isolation of Van-9-2 The procedure of Example 1 (above) was continued to include CLV and CCD analysis. Van-5-10 was subjected to preparative CCD (silica gel; solvent; hexane; chloroform; ethyl acetate, 3: 6: 6) to give five fractions, Van-9-1 to Van-9-5, of which Van-9-2 was found to be biologically active using the bioanalysis of 3T3 cells (example 5). Example 3: Isolation of Van-10-2, Van-10-3 and Van-10-4 The protocols of examples 1 and 2 above were followed to reach Van-9-2.

Van-9-02 was separated in Van-9-2B and Van-9-2C by preparative CCD preparation (solvent: hexane: chloroform: ethyl acetate, 3: 6: 4). Van-9-2B and Van-9-2C were separated by CLAP in an ODS analytical column using 75% acetonitrile in water (plus 0.1% trifluoroacetic acid). This gave four fractions, Van-10-1, Van-10-2, Van-10-3 and Van-10-4. The NMR data for Van-10-2, Van-10-3 and Van-10-4 are provided below. It is thought that the stereochemistry of the Van compounds in the aromadendranol portion is the following structure: However, the skilled artisan would also appreciate that additional stereoisomeric variants of the aromadendranol portion having the following structures are also encompassed by the present invention. 2 Proton dH (J in Hz) 10 0.75 s 11 1.26 s 12 1.39 s 13a 4.6 br s 13b 4.8 br sr 4.9 d (7.9) 2 '5.85 d (7.8) 3' 4.25 dd (7.8, 3.1) 4 '1.55 d (6.5) 5 '6' 1"2" 5.90 br s 3"4" 2.10 m 5"1.9 (7) 6" 2.15 br s Chemical change data 'H-NMR (Pyridine-Ds) for Van-10-2 Carbon / Proton H dH (J in Hz) Multiplicities dc 1 C 19.4 1a 0.65 / 77 CH 29.7 2 CH2 19.4 3 CH2 39.3 4 C 81.7 4a CH 55.2 CH2 26.2 6 CH2 29.7 7 CH 39.1 7a 1.85 m CH 40.4 7b 0.10 t (9.4) CH 23.1 8 0.95 s Me 17.1 9 1.00 s Me 29.3 1.35 s Me 27.0 11 0.90 s Me 17.2 1"5.0 esc. CH 96.0 2 '5.55 t (8.3) CH 77.6 3 '4.25 dd (8.3) CH 77.1 3.7 t (d) 4' CH 75.0 '3.75 t (d) CH 73.0 6' 1.56 d (7) Me 19.0 1"C 166.4 2" 5.85 br s CH 115.8 3"C 162.0 4" 2.05 m CH2 34.3 5"0.95 c (7) Me 12.2 6" 2.25 dr s Me 17.0"Obtained from the J-mod experiment Data of chemical changes 1H and 13C-NMR (Pyridine-D5) for Van-10-3 Coal / Proton? DH (J in Hz) Multiplicity is * dc 1 C 19.4 1a 0.62 m CH 29.7 2 1.55, 1.8 m CH2 19.2 3 CH2 39.3 4 C 81.5 4a CH 55.2 5 CH2 26.3 6 CH2 29.7 7 1.85 m CH 39.3 7a 0.10 t (9.4) CH 40.4 b 1.10 s CH 23.1 8 1.15 s Me 17.1 9 1.35 s Me 29.8 10 0.90 d (7) Me 27.1 11 4.9 d (7.9) Me 17.2 1 '5.85 t (7.9) CH 96.3 2' 4.25 dd (7.9, 3.5) CH 74.4 3 '3.7 d (3.5; CH 73.5 4' 3.85 m CH 73.3 5 '1.60 d (6) CH 71.4 6' Me 17.9 1"5.85 br s C 166.6 2" CH 115.9 3"2.05 m C 161.7 4" 0.95 c (7) CH2 34.3 5"2.22 br s Me 12.2 6" Me 19.3 * Obtained from the experiments of J-mod and HC-COB1 Data of chemical changes 1H and 13C-NMR (Pyridine-D5) for Van-10-4 Example 4: Van-15A isolation Reference is made to Figure 1. The protocols of examples 1 and 2 were followed to obtain Van-6-1.

Van-6-1 was fractionated by column chromatography (Sephadex LH20: solvent: chloroform: methanol, 1: 1) in two fractions: Van-15-1 (heavier fraction containing fatty acids) and Van-15-2 (fraction lighter containing terpenes). Van-15-2, was subjected to preparative CCD (silica gel; solvent: chloroform: methanol 95: 5) to give Van-15A and a minor component Van-15B. The NMR data for the active component, Van-15A, is provided below. ___ Carbon / Proton dH (J in Hz) Multiplicities '' 1 C 19.4 1a 0.6 br q O) CH 29.7 2 CH2 19.2 3 CH2 39.3 4 C 81.7 4a CH 55.2 5 CH2 26.3 6 CH2 29.7 7 CH 39.3 7a 1.85 m CH 40.4 7b 0.10 t (9.4) CH 23.1 8 095 s Me 17.1 9 1.00 s Me 29.8 10 1.35 s Me 27.1 11 0.90 d (7) Me 17.2 V 4.4 d (7.3) CH 96.3 2 '3.56 CH 74.4 3' 3.56 CH 73.5 4 '3.69 d (2.6) CH 73.3 5' 3.56 CH 71.4 6 '1.25 d (6.07) Me 17.9' Obtained from the experiments of J-mod and HC-COB1 Data of chemical changes 1H and 13C-NMR (CDCL3) for Van-15A. Example 5: Reversible inhibition of Proliferation of Fibroblast-3T3 cell line. Mouse embryonic fibroblast cells (3T3-available from Flow) were plated on a 96-well tissue culture plate. The cells were suspended in Medium (Modification of Dulbeco DEA Medium Eagle (DMEM), Gibco) containing 10% fetal calf serum for (Gibco), 1% Glutamine (Gibco); 1% penicillin / streptomycin (Gibco); 1% non-essential amino acids (Gibco), at a concentration of 50,000 cells per ml. 100μl of the cell suspension was pipetted into each well to give a concentration of 5000 cells per well. The cells were incubated for 24 hours at 37 ° C under a 5% CO2 atmosphere. The medium was removed from the plate and variant dilutions of the compounds (0, 30, 45, 60, 100μl / ml), internally designated Van-10-4, Van-10-2, Van-10-3 and Van were prepared. -15A dissolving the compounds in 100% DMSO (Sigma). This was diluted with DMEM to give a DMSO concentration of 0.5%. The compounds were then diluted to the concentrations indicated (above) with DMEM containing 0.5% DMSO (Sigma) and added to the plate (100μl / well). Each dilution was performed in triplicate; the control was DMEM containing 0.5% DMSO. 50μl of DMEM containing 0.5μCi 1H-thymidine was added to each well. Next, 59μl of DMEM containing 40% fetal calf serum (Gibco); 4% penicillin / streptomycin (Gibco); 4% glutamine (Gibco); 4% non-essential amino acids (Gibco) were added to each well. Plates were incubated for 24 hours at 37 ° C in a 5% CO2 atmosphere. The medium including compounds under test was then removed and the plates were washed twice (2x) with saline with pH regulated phosphate (SRF) (pH 7.3). 100μl of 5% trypsin (Gibco) in Versene, (in EDTA chelating agent available from Gibco) were added to each well. The plate was further incubated for 15 minutes at 37 ° C in a 5% CO2 atmosphere, then the cells were collected from the well using a semi-automatic cell collector (Skatron). The collected cells were placed in a scintillation vial containing 4ml of "safe" Optiphase (LKB) and the radioactivity was counted using a liquid scintillation counter (LKB WALLAC 1217 Rack beta). Results are shown in table 2. ro n TABLE 2 0 μg / ml 15 μg / ml 22 5 μg / ml 30 μg / ml 50 jrg / ml Mean SD SD Average SD Average SD Average SD VAN 6-1 5517.1 186.1 3947.6 454.6 3370.5 772.8 2667.5 475.4 614.1 122.2 VAN 9-2B 4512.1 125 856.5 168 254.5 134 84.5 8 40.0 8 VAN 9-2C 4579.5 155 2759.8 301.9 1475.6 24 687.5 39 188.6 27 00 VAN 10-4 4475.1 393 633.6 45 111.1 19.2 88.6 17 34.5 16 VAN 10-2 2939.3 444 925.3 651 247.0 119 60.6 11 21.8 10 VAN 10-3 2742.8 175 1887.3 432 388.1 354 405.5 228 37.5 12 VAN 15-A 3052.0 274 1773.3 182 1154.8 73 1145.0 226.6 364.8 59 Example 6: Extraction and Fractionalization of Van-10-4 Powder Calendula Plant Description a) "Soxhlet" 500g of powder were extracted using preferred ether (60-80) or chloroform. The extraction period was usually around five days without considering the solvent used. The volume of solvent was about 3 liters and the crude yield was about 60g (preferred ether) or about 80g (chloroform). The crude was combined with an equal amount of silica and a powder form was allowed to dry for another period of five days. The total powder obtained for each batch was therefore approximately 120g (preferred ether) or 160g (chloroform). b) CLV 30g of the crude / silica powder was formed in layers on top of a silica CLV column (60Hgel) approximately 5cm deep by 10cm wide. This was covered with a layer of fine sand and fractionated using the following solvent system. 5 x 200ml 50% chloroform: preferred ether (60-80) 5 x 200ml 70% chloroform: preferred ether 5 x 200ml 100% chloroform 5 x 200ml 10% chloroform This last step gives the fractions containing Van-10-4. The CCD confirmed that all Van-10-4 has been removed from the CLV column at this stage. The relevant fractions were evaporated by rotation to dryness. The weight of the sample obtained is approximately 2-3 grams. c) "Sephadex" The inclusion of the "Sephadex" column at this stage allowed a much faster method to purify the CLV sample. The sample was redissolved in chloroform and loaded onto the column. The column used was 20cm high by 2cm wide and contained 13gm. of "Sephadex" (LH-20-100 lipophilic). The sample was eluted using 10% preferred ether: chloroform using flow rate 2-3ml / min and the samples were collected at 1min intervals. The presence of Van-10-4 was confirmed by CCD and the relevant fractions were measured and evaporated by rotation. The weight of the sample obtained was around 500 mg. d) Silica A small column of silica was used. A 50ml burette was filled with 20g of silica prepared as a slurry in preferred ether (60-80). The sample was redissolved in chloroform and loaded onto the column. The column was 40cm high by 10cm wide.

The sample was eluted using the following solvent system: 10ml volumes used: preferred ether (60-80) 10% chloroform: ether 20% chloroform: ether 40% chloroform: ether 50% chloroform: ether (sample initiates separation) 60% chloroform, ether 70% chloroform: ether 80% chloroform : ether 90% chloroform: ether 100% chloroform: ether 100% chloroform 10% ethyl acetate: chloroform 20% ethyl acetate: chloroform 30% ethyl acetate: chloroform 40% ethyl acetate: chloroform 50% ethyl acetate: chloroform 50% ethyl acetate: chloroform continued until all Van-10-4 eluted from the column. This was confirmed by CLD. Van-10-4 is eluted from the column. This was confirmed by CCD. This method gives samples that contain Van-10-4 plus several impurities (semi-purified samples). Example 7: Treatment of Psoriasis with extract of Calendula officinalis Materials 1. Extract of plant material. The preparation consisted of an extract based on chloroform (5% v / v of chloroform in absolute ethanol) of the plant material in the ratio of 20g of dead plant material extracted with 100ml of solvent. 2. Preparation of cream. The prepared extract, as mentioned before, was evaporated to dryness and weight. The weight of the "dry" extract was mixed with enough aqueous cream B.P. to form 100g of cream which has a yellow / orange color. 3. Preparation of the Placebo Cream. The Placebo cream was prepared by adding enough beta-carotene to the aqueous cream B.P. to give a product of approximately the same color as that containing natural drug. It was found that an extract of plant material obtained from Calendula officinalis (SSSB) is clinically useful to treat cases of psoriasis. After a long period of empirical observation of patients, a double blind clinical analysis was performed on a group of seven patients with psoriasis. The patients (4 men, 3 women) were 53 to 63 years old, except one who was 26 years old. They had had the disease from 8.5 to 25 years. The treatment was monitored by a series of pre and post-trial photographs and using a "psoriasis symptom graduation" before and after treatment. The treatment was applied twice a day as a cream for four weeks and there were three SSSB, Betnovate, and placebo treatments.

The results of the trial are shown in Figure 2. The average pretreatment graduation 11.1 and this was reduced to 3.5 after SSSB treatment, whereas it was 5.5 and 7.4 after Betnovate and placebo, respectively. Example 8: Isolation and Purification of Van-10-4 15 to 30g of crude extracted by "Soxhlet" as performed in Example 6, were resolved in 15ml of 50% ethyl acetate: 50% hexane. This material was loaded on a Flash 75 column (Biotage Limited) eluted at a flow rate of 150-200 ml / min using 50% ethyl acetate: 50% hexane, as the mobile phase. The time to elute the VAN compounds from the Flash column was calculated by CCD. A diluted solution of the crude mixture (redissolved in ethyl acetate) was applied on a CCD plate (silica gel 60). The VAN compound mixture was visualized using a vanillin developer: normal concentrated sulfuric acid (1 gram of vanillin: 100 ml of concentrated sulfuric acid) and the Rf value was calculated. The mobile CCD phase was adjusted by manipulating the polar: non-polar solvent ratio until the Rf value was around 3.0. The equivalent column volume was worked from the Rf value according to the manufacturer's instructions (Biotage Limited). The column volume for the Flash 75 system is about one liter and the mobile phase giving adequate Rf values was 40% ethyl acetate: 50% hexane. When this method was tested using the Flash 75 column, the VAN compounds eluted in fractions of 15-19 (equivalent to 4-5 column volumes). Each fraction collected was 250ml. In a repeated operation, under similar conditions, the VAN compounds were eluted in fractions of 14-18. These fractions were combined and evaporated to dryness giving an approximate yield of 200-400mg of semi-liquid material. This semi-liquid material was purified by CLAP using a reverse phase Spherisorb column. A stock solution of the semi-liquid material was prepared as 25 mg / ml in acetonitrile and aliquots of 200 μl were processed socratically. The mobile phase used was 75% acetonitrile, 25% water and the flow rate was 5 ml / min. Each sample run was 17 minutes and VAN-10-4 eluted approximately 14 minutes. NMR spectroscopy confirmed that the eluate was VAN-10-4. Example 9: Extraction of Calendula officinalis - Scale of 1Kq The dried petals of Calendula officinalis (1Kg) were ground to a coarse powder and extracted continuously with heptane (1-4 liters) ie the heptane was continuously distilled from extract and was percolated to through the mass of petals. The extract was concentrated under vacuum still volume of 400ml. The resulting concentrate was extracted three times with 500 ml. Of acetonitrile / water solution, 9: 1 (v / v). The acetonitrile fractions containing NPV were combined and the acetonitrile was removed by vacuum distillation. The resulting orange gum was redissolved in 500 ml of ethyl acetate and washed successively with 500 ml (0.1 M) of saturated sodium bicarbonate, 500 ml of water, 500 ml (0.1M) hydrochloric acid and finally 500 ml of water . The ethyl acetate solution was then distilled under vacuum to give 10 g of an orange oil.

Claims (40)

1. CLAIMS 1. Use of a compound of Formula (I):

   wherein R1 and R2 are independently selected for H, OH, H3C C H3 H3C COO- \ / c / c = c C = C / \ / \ H coo- H CH;

   and esters related to them; R3 is selected from -OH, O O O II

   CH3CH2CH (CH3) CO-, CH3CH2C (CH3) = CHCO-, CH3CO-, H3C C H3 H3C COO- O \ / \ / II C = CC = C and CH3C (CH3) = CHCO- / \ / \ H COO- H CH3 and esters related thereto; R 4 is selected from aliphatic, monocyclic or polycyclic, saturated or unsaturated C 6 -C 12 ring systems optionally substituted by C 1 -C 6 alkyl, H, OH, = CH 3 or carboxyloxy of d-C 4 alkyl or R 4 represents a chain alkane group straight or branched C? -C3 substituted with said ring system;

   R5 is selected from = CH3, -CHO, -COOH and -CH2OH and related esters and ethers derived therefrom; R6 is selected from -OH, O O O

   II II II CH3CO-, (CH3) 2CHCO-, CH3CH2CH (CH3) CO-,

   H3C CH3 H3C COO "\ / \ / C = C and C = C / \ / \ H COO- H CH3

   in the preparation of a medicament for the treatment of disease involving hyperproliferation of dermal cells.
2. 2. Use according to claim 1 of a compound of the formula (I) wherein: R and R2 are independently selected from H, -OH H3C CH3 H3C COO- \ / C = C and C = C / \ / \ H COO 'H CH3
3. 3. Use according to claim 1 or 2 of a compound of the Formula (I) wherein R3 is selected from -OH, O

   II

   CH3CH2C (CH3) = CHCO-, CH3CH2CH (CH3) CO-, H3C CH3 H3C COO "OO \ / \ / II II C = CC = C, CH3CO- and CH3C (CH3) CO- / \ / \ H coo- H CH;
4. 4. Use according to any of claims 1 to 3, of a compound of the Formula (I), wherein R4 is selected from the group
5. 5. Use according to any of claims 1 to 4, of a compound of the Formula (I) wherein R4 is selected from the group
6. 6. Use according to any of claims 1 to

   5, of a compound of Formula (I) wherein R 5 is selected from the group of CH 3, -CHO, COOH and -CH 2 OH.
7. 7. Use according to any of claims 1 to

   6, of a compound of Formula (I) wherein R6 is selected from OH,

   O O O II

   CH3CO-, (CH3) 2CHCO-, CH3CH2CH (CH3) CO-,

   H3C COO- H3C CH3 \ / \ / C = C and C = C / \ / \ H CH3 H COO
8. 8. Use according to claims 1 to 7, of a compound of the Formula (I) wherein R 1 and R 2 are independently selected from H and OH (αOH or αOH); O O

   II II R3 is selected from OH, CH3C (CH3) = CHCO-, CH3CO-, O O II

   CH3CH2C (CH3) = CHCO- and CH3CH2CH (CH3) CO- R4 is selected from

   R5 is CH3; and R6 is selected from OH, O O O II

   CH3CO-, (CH3) = CHCO- and CH3CH2CH (CH3) CO-.
9. 9. Use according to claim 8, of a compound of the Formula (I) wherein R 1 and R 2 are independently selected from H and OH (βOH or aOH); R3 is selected from OH and (E) -3-methylpent-2-enoate, that is,
10. R is selected from
11. Rs is CH3; and R6 is OH. 10. Use according to any of claims 1 to 9 of a compound of the Formula (I) in the preparation of a medicament for the treatment of psoriasis. 11. Use according to claim 10 of the compound:
12. 12. Use according to claim 10 of the compound: (Van-10-2).
13. 13. Use according to claim 10 of the compound:
14. 14. Use according to claim 10 of the compound:
15. 15. Use of a compound of Formula (I):

    wherein R1 and R2 are independently selected from -OH, H,

    H3C CH3 H3C COO "\ / \ / - C = C and C = C; / \ / \ H COO" H CH3

    R3 is selected from (E) -3-methylpent-2-enoate and

    H3C CH3 \ / C = C / \ H COO "

    R4 is selected from saturated or unsaturated C6-C ?2 monocyclic or polycyclic aliphatic ring systems optionally substituted by C?-C6 alkyl, H, -OH, = CH2 or carboxyloxyalkyl of C?-C4 or R4 represents an alkylene group straight or branched chain of C? -C6 substituted with said ring system; Rs is selected from C? -C alkyl or -CHO, -COOH and -CH3OH; R6 is selected from

    H3C CH3 H3C COO "\ / \ / C = C C = C / \ / \ H COO" H CH3 physiologically functional isomers thereof.
16. 16. A compound of the Formula (I) according to claim 15, wherein R1 R 'are independently selected from -OH, H, H3C, H3, H3C, COO, "/ c / c = c C = C / \ / \ H COO- H CH3
17. 1 7. A compound of the Formula (I) according to claim 1 or claim 16, wherein R3 is selected from

    (E) -3-2-methylpent-enoate, and H3C C H3 \ / c = c / \ H COO "
18. 18. A compound of the Formula (I) according to any of claims 1-5 to 17, in where R4 is selected from

    5
19. 19. A compound of Formula (I) according to any of claims 15 to 18, wherein R 4 is selected from the group
20. 20. A compound of the Formula (I) according to any of claims 15 to 19, wherein Rs is selected from the group of CH3, -CHO, -CH2OH and -COOH. 21. A compound of Formula (I) according to any of claims 17 to 19, wherein R6 is selected from
21. H3C CH3 H3C COO "\ / \ / C = C and C = C / \ / \ H COO- H CH3
22. 22. A compound of the Formula (I) according to any of claims 17 to 21, wherein R1 and R2 are independently selected from H and -OH; R 4 is (E) -3-methylpent-2-enoate;

    R4 is selected from

    R5 is CH3; and R6 is OH; and physiologically functional isomers thereof.
23. 23. A compound according to any of claims 15 to 22, which is:
24. 24. A compound according to any of claims 17 to 23, which is:
25. 25. Use of a compound isolated from a Calendula species according to any of claims 15 to 24, for the manufacture of a medicament for the treatment of psoriasis.
26. 26. Use according to claim 26, wherein the compound is isolated from Calendula officinalis, Calendula arvensis and / or Calendula persica.
27. 27. Use according to claim 25 or claim 26, wherein the compound is isolated from Calendula officinalis.
28. 28. A pharmaceutical formulation for the treatment of psoriasis comprising at least one compound isolated from at least one species of Calendula according to Formula (I) mixed with a pharmaceutically acceptable carrier.
29. 29. A formulation according to claim 28, wherein at least one compound according to Formula (I) is isolated and / or purified from an extract derived from at least one species of Calendula.
30. 30. A formulation according to claim 28 or 29, wherein at least one compound is isolated and / or purified from an extract derived from at least one of Calendula arvensis, Calendula persica and Calendula officinalis.
31. 31. A formulation according to any of claims 28 to 30, wherein at least one compound is isolated and / or purified from an extract of Calendula officinalis.
32. 32. A formulation according to any of claims 28 to 31, wherein at least one compound according to Formula (I) is:
33. 33.

    n 32, wherein at least one compound is
34. 34. A method for treating a skin disease that involves hyperproliferation of the dermis cells in a mammal comprising administering to the mammal an effective amount not
35. Toxic of a compound of Formula (I) according to claim 15. 35. A method according to claim 34, wherein the compound of Formula (I) is:
36. 36. A method according to claim 34 or 35, wherein the compound is selected from
37. 37. A method according to any of claims 34 to 36, wherein the skin disease is psoriasis.
38. 38. The compound

    to be used as a medication for psoriasis.
39. 39. The compound:

    to be used as a psoriasis medication.
40. 40. The compound:

    to be used as a psoriasis medication.