WO2023187598A1

WO2023187598A1 - Compositions and methods

Info

Publication number: WO2023187598A1
Application number: PCT/IB2023/052985
Authority: WO
Inventors: Yehoshua KORCHIA-MAOR
Original assignee: Biblical Lands Ltd
Current assignee: Biblical Lands Ltd
Priority date: 2022-03-27
Filing date: 2023-03-26
Publication date: 2023-10-05
Anticipated expiration: 2024-09-27
Also published as: IL307496A

Abstract

There is provided a method of treating psoriasis, comprising administering to a patient in need of such treatment a composition comprising a terpene-enriched olive oil. Other embodiments are also disclosed.

Description

COMPOSITIONS AND METHODS

This application claims Paris Convention priority from, and the US benefit of, US Provisional Application No. 63324094, filed March 27, 2022, and entitled "Compositions and Methods". The contents of said application are incorporated herein by reference in their entirety.

Background

Olive oil is a liquid fat obtained from olives (the fruit of Olea europaea; family Oleaceae), a traditional tree crop of the Mediterranean Basin. Olive oil has been known for thousands of years, being mentioned inter alia in the Bible. It is produced by pressing whole olives and extracting the oil.

While the quality and exact composition of olive oil depends on factors such as cultivar, the region of growth, altitude of growth, time of harvest (i.e. degree of ripeness at which the olives were harvested), the growing conditions (including amount of precipitation and temperature), and the production conditions, all olive oil is composed primarily of the mixed triglyceride esters of the saturated fatty acids palmitic acid and stearic acid, and the unsaturated fatty acids oleic acid, linoleic acid, and in some cases linolenic acid; other fatty acids may also be present. Also present are often small amounts of the triterpene squalene (up to 0.7%) and sterols (about 0.2% phytosterol and tocosterols). In general, edible olive oil has not more than 2% by weight free fatty acids.

Terpenes are a class of compounds consisting of connected isoprene units:

viz. units of the formula (CsHs , where n is an integer of value 2 or greater. Most often the isoprene units are connected head-to-tail, although in some cases they are connected head- to-head. Terpenes of the formula CioHie are often referred to as monoterpenes, terpenes of the formula C15H24 are often referred to as sesquiterpene, terpenes of the formula C20H32 are often referred to as diterpenes, and terpenes of the formula C30H48 are often referred to as triterpenes. In nature, terpenes are produces primarily by plants. Terpenoids are terpenes that contain additional functional groups, most commonly in the form of oxygen atoms that appear between what would otherwise be carbon-hydrogen bonds or carboncarbon bonds. Olive oil has few to no monoterpenes, sesquiterpenes or diterpenes.

Psoriasis is a long-lasting, noncontagious autoimmune disease characterized by raised areas of abnormal skin. These areas are red, or purple on some people with darker skin, dry, itchy, and scaly. Psoriasis varies in severity from small, localized patches to complete body coverage. Injury to the skin can trigger psoriatic skin changes at that spot, which is known as the Koebner phenomenon. The five main types of psoriasis are plaque, guttate, inverse, pustular, and erythrodermic, although there are other types of psoriasis that together constitute about 10% of all cases. The most common current topical treatment for psoriasis involves the application of corticosteroid preparations.

Brief Description

The presently claimed invention is based on the observation that olive oil which has been used as an extractant for certain parts of certain plants can be used to treat psoriasis.

A method for preparing such olive oil is to mix (a) edible olive oil, (b) Boswelia sacra resin, and (c) fresh fruits of Boswelia sacra, Commiphora gileadensis, and Commiphora myrrha, wherein the amount of olive oil is at least 10 times by weight greater than each of the other ingredients, up to 70 times by weight greater than each of the other ingredients. The ingredients are heated and continuously mixed at 60-65°C. The mixture is then allowed to cool to room temperature and filtered to remove solids. The mass of the product collected after filtering is about the same as was the mass of the olive oil prior to the extraction process.

The resulting olive oil has a significantly higher content of mono-, sesqui- and diterpenes, and related terpenoids, than does naturally-occurring olive oils. In particular, whereas the content of mono-, sesqui- and diterpenes and related terpenoids (which, for purposes of this patent application, may include some compounds that have the formula selected from C10H14, C10H14O, CioHisO, C10H20O, C11H20O2, C12H20O2, C14H22O, C15H24O, C15H26O, and C15H28O, as will be specified below), taken together (hereinafter: "total terpenes"), in naturally-occurring olive oil is generally not higher than 1.2 wt.%, and usually lower, in the terpene-enriched olive oil used in accordance with embodiments of the present invention the total terpenes content is between about 4 wt.% and 8 wt.%; in some cases it is about 6 wt.% The primary components of the total terpenes are a-pinene, sabinene, myrcene, limonene, and caryophyllene E, which constitute respectively about 39-45%, about 6.0-12%, about 10.4-11.5%, about 12-12.7% and about 1-2% by weight of the total terpenes. Additional mono-, sesqui- and diterpenes or related terpenoids that may be present in an amount of 0.1-1.5 percent by weight each of the total terpenes, and collectively in an amount of 10-16 wt.% of the total terpenes, are a-phellandrene, 3-6-carene, p-cymene, Z-[3- ocimeme, E-[3-ocimeme, y-terpinene, a-campholenal, trans-pinocarveol, trans verbenol, pinocarvone, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, borneal acetate, a-copaene, p-bourbonene, |3-elemene, a-humulene, 9-epi-E-caryophyllene, y- muurolene, germacrene D, |3-selinene, y-cadinene, 6-cadinene, and p-eudesmol. Additional compounds included in the total terpenes that may or may not be present in small amounts include a-thujene, camphene, verbenene, 0-pinene, a-terpinene, 1,8-cineole, cis-sabinene hydrate, a-terpinyl acetate, curzerene, shyobunol, 2E-decenal, 2E,4Z-decdienal, 2E,4E- decadienal, 0-copaene, 6,9-guiaiadiene, trans-muurola-4(14),5-diene, a-selinene, a- muurolene, E,E- a-farnesene, caryophyllene oxide, t-cadinol, terpinolene, citronellol, carvone, citronellyl formate, 6-elemene, trans-muurola-3,5-diene, liguloxide, and selin-11- en-4-a-ol.

The terpene-enriched olive oil, such as that prepared as described herein, may be applied directly to the skin, or it may be formulated in ways known in the art, for example as a shampoo, solution, suspension, emulsion, cream, ointment, powder, liniment, salve, etc., which may be, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The terpene-enriched olive oil may additionally or alternatively be ingested directly, or it may be formulated into a pill, tablet, caplet, gelcap etc., and/or in discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The material may also be presented as a bolus, electuary or paste.

To treat psoriasis, the terpene-enriched olive oil may be applied to the affected skin, for example two or three times daily for a minimum of two weeks, but longer as necessary. Alternatively or additionally, the terpene-enriched olive oil may be ingested e.g. twice daily each time in an amount of up to 5 ml.

There is thus provided, in accordance with an embodiment of the invention, a method of treating psoriasis, comprising administering to a patient in need of such treatment a composition comprising a terpene-enriched olive oil having a total free fatty acid content of not more than 2 wt.% and a total content of mono-, sesqui- and diterpenes and related terpenoids in the range of 4 to 8 wt.%, the total mono-, sesqui- and diterpenes and related terpenoids comprising 70-85 percent by weight a-pinene, sabinene, myrcene, limonene, and caryophyllene E. In some embodiments, the total terpene content is about 6 wt.%. In some embodiments, the total mono-, sesqui- and diterpenes and related terpenoids comprise 10-16% by weight of a mixture of additional terpenes and/or terpenoids selected from the following: a-phellandrene, 3-6-carene, p-cymene, Z-p-ocimeme, E-p-ocimeme, y- terpinene, a-campholenal, trans-pinocarveol, trans verbenol, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, 2E-decenal, borneal acetate, a-copaene, -bourbenone, P-elemene, a-humulene, 9-epi-E-caryophyllene, y-muurolene, germacrene D, P-selinene, y- cadinene, 6-cadinene, and p-eudesmol. In some embodiments, the composition is administered topically. In some embodiments, the composition is administered orally.

There is also provided, in accordance with an embodiment of the invention, a method of treating psoriasis, comprising administering to a patient in need of such treatment a composition comprising olive oil which has been used as an extractant for a mixture of Boswelia sacra resin, Boswelia sacra fruits, Commiphora gileadensis fruits, and Commiphora myrrha fruits. In some embodiments, the composition is administered topically. In some embodiments, the composition is administered orally. In some embodiments, the olive oil which has been used as an extractant has a total free fatty acid content of not more than 2 wt.% and a total content of mono-, sesqui- and diterpenes and related terpenoids in the range of 4 to 8 wt.%, the total mono-, sesqui- and diterpenes and related terpenoids comprising 70-85 percent by weight a-pinene, sabinene, myrcene, limonene, and caryophyllene E. In some embodiments, the total terpene content of the olive oil which has been used as an extractant is about 6 wt.%. In some embodiments, the total mono-, sesqui- and diterpenes and related terpenoids comprise 10-16% by weight of a mixture of additional terpenes and/or terpenoids selected from the following: a-phellandrene, 3-6- carene, p-cymene, Z-p-ocimeme, E-p-ocimeme, y-terpinene, a-campholenal, trans- pinocarveol, trans verbenol, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans- carveol, 2E-decenal, borneal acetate, a-copaene, p-bourbenone, p-elemene, a-humulene, 9- epi-E-caryophyllene, y-muurolene, germacrene D, p-selinene, y-cadinene, 6-cadinene, and P-eudesmol.

There is also provided, in accordance with an embodiment of the invention, a composition comprising a terpene-enriched olive oil having a total free fatty acid content of not more than 2 wt.% and a total content of mono-, sesqui- and diterpenes and related terpenoids in the range of 4 to 8 wt.%, the total mono-, sesqui- and diterpenes and related terpenoids comprising 70-85 percent by weight a-pinene, sabinene, myrcene, limonene, and caryophyllene E, for use in the treatment of psoriasis. In some embodiments, the total terpene content is about 6 wt.%. In some embodiments, the total mono-, sesqui- and diterpenes and related terpenoids comprise 10-16% by weight of a mixture of additional terpenes and/or terpenoids selected from the following: a-phellandrene, 3-5-carene, p- cymene, Z-p-ocimeme, E-p-ocimeme, y-terpinene, a-campholenal, trans-pinocarveol, trans verbenol, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, 2E-decenal, borneal acetate, a-copaene, P-bourbenone, P-elemene, a-humulene, 9-epi-E-caryophyllene, y-muurolene, germacrene D, p-selinene, y-cadinene, 5-cadinene, and p-eudesmol. In some embodiments, the composition is administered topically. In some embodiments, the composition is administered orally. There is also provided, in accordance with an embodiment of the invention, olive oil which has been used as an extractant for a mixture of Boswelia sacra resin, Boswelia sacra fruits. Commiphora gileadensis fruits, and Commiphora myrrha fruits, for use in the treatment of psoriasis. In some embodiments, the composition is administered topically. In some embodiments, the composition is administered orally. In some embodiments, the olive oil which has been used as an extractant has a total free fatty acid content of not more than 2 wt.% and a total content of mono-, sesqui- and diterpenes and related terpenoids in the range of 4 to 8 wt.%, the total mono-, sesqui- and diterpenes and related terpenoids comprising 70-85 percent by weight a-pinene, sabinene, myrcene, limonene, and caryophyllene E. In some embodiments, the total terpene content of the olive oil which has been used as an extractant is about 6 wt.%. In some embodiments, the total mono-, sesqui- and diterpenes and related terpenoids comprise 10-16% by weight of a mixture of additional terpenes and/or terpenoids selected from the following: a-phellandrene, 3-5- carene, p-cymene, Z-0-ocimeme, E-0-ocimeme, y-terpinene, a-campholenal, trans- pinocarveol, trans verbenol, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans- carveol, 2E-decenal, borneal acetate, a-copaene, p-bourbenone, 0-elemene, a-humulene, 9- epi-E-caryophyllene, y-muurolene, germacrene D, p-selinene, y-cadinene, 6-cadinene, and |3-eudesmol.

Brief Description of the Figures

Embodiments of the invention will be better understood with the aid of the figures, in which:

Figure 1 shows qualitatively the enhanced proliferation of cells stimulated with terpene- enriched olive oil, compared to a control;

Figure 2 presents graphs showing the viability levels of HaCaT levels after stimulation with psoriasis-inducing cytokines, viz. IL17A and TN Fa, in different concentrations and combinations (A) or in different time points (B). The results are shown as percentage of the control. The data are mean ± SE (n=4);

Figure 3 shows the levels of reactive oxygen species (ROS) in HaCaT cells stimulated with IL17A and TNFa in different incubation periods;

Figure 4 shows the gene expression levels of four known biomarkers for psoriasis, after different incubation times;

Figure 5 shows qRT-PCR analysis for Psoriasin gene (SA007) expression after 48 hours treatment with different samples (CBC 10 micromolar, terpene-enriched olive oil 80 microgram/ml); Figure 6 shows qRT-PCR analysis for TRB3 gene expression after 48 hours treatment with different samples (same dosages as in Fig. 5); and

Figure 7 shows qRT-PCR analysis for VEGF gene expression after 48 hours treatment with different samples (same dosages as in Fig. 5).

Examples

Example 1

A batch of terpene-enriched olive olive oil was prepared as follows. Five kg of edible olive oil, 500 g of Boswelia sacra resin, 350 g of fresh Boswelia sacra fruits, 350 g fresh Commiphora gileadensis fruits, and 80 g of fresh Commiphora myrrha fruits were heated and continuously mixed at 58-65°C for 120-180 minutes. The mixture was then allowed to cool to room temperature and filtered to remove solids. Approximately 5 kg of terpene- enriched olive oil was recovered.

Both before and after this process, the olive oil had less than 2 wt.% free fatty acids. Prior to the process, the olive oil contained less than 1.1 wt.% (and olive oil usually contains not more than 0.2 wt.%) total mono-, sesqui- and diterpenes and related terpenoids. After the process, the total mono-, sesqui- and diterpenes and related terpenoids constituted approximately 6 wt.% of the total product.

Samples were analyzed as follows: samples were diluted in methanol (1%) for GCMS analysis. A Hewlett Packard G 1800B GCD system with a HP-5971 gas chromatograph with electron ionization detector running GCD Plus Chemstation with SPB-5 (30 m x 0.25 mm x 0.25 urn film thickness) column were used. Experimental conditions: inlet, 250°C; detector, 280°C; splitless injection/purge time, 1.0 min; initial temperature, 40°C; initial time, 5.0 min; rate, 5°C/min; final temperature, 270°C, helium flow rate 1 ml/min. The Wiley Mass Spectral library (2017) was used for the identification of compounds. Analysis of the total mono-, sesqui- and diterpenes and related terpenoids components in this way by gas chromatography/mass spectrometry (GC/MS) showed the principal components to be a- pinene, sabinene, myrcene, limonene, and caryophyllene E, which collectively constituted 72.7 wt% of the total terpenes. Additional components present were a-phellandrene, 3-6- carene, p-cymene, Z-0-ocimeme, E-P-ocimeme, y-terpinene, a-campholenal, trans- pinocarveol, trans verbenol, pinocarvone, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, borneal acetate, a-copaene, p-bourbonene, -elemene, a-humulene, 9-epi-E- caryophyllene, y-muurolene, germacrene D, p-selinene, y-cadinene, 6-cadinene, and p- eudesmol, which collectively constituted 15.4 wt.% of the total terpenes. Example 2

Example 1 was repeated with a different batch of olive oil, resin and fruits.

Both before and after this process, the olive oil had less than 2 wt.% free fatty acids. Prior to the process, the olive oil contained less than 1.1 wt.% total mono-, sesqui- and diterpenes and related terpenoids (viz. total terpenes). After the process, the total terpenes constituted approximately 6 wt.% of the total product.

Analysis of the total terpenes components by gas chromatography (GC) and/or gas chromatography/mass spectrometry (GC/MS) showed the principal components to be a- pinene, sabinene, myrcene, limonene, and caryophyllene E, which collectively constituted 71.1 wt% of the total terpenes. Additional components present were a-phellandrene, 3-6- carene, p-cymene, Z-p-ocimeme, E-P-ocimeme, y-terpinene, a-campholenal, trans- pinocarveol, trans verbenol, pinocarvone, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, borneal acetate, a-copaene, p-bourbonene, p-elemene, a-humulene, 9-epi-E- caryophyllene, y-muurolene, germacrene D, p-selinene, y-cadinene, 6-cadinene, and p- eudesmol, which collectively constituted 10.9 wt.% of the total terpenes.

Example 3

Example 1 was repeated with a different batch of olive oil, resin and fruits.

Analysis of the total terpenes components by gas chromatography (GC) and/or gas chromatography/mass spectrometry (GC/MS) showed the principal components to be a- pinene, sabinene, myrcene, limonene, and caryophyllene E, which collectively constituted 76.5 wt% of the total terpenes. Additional components present were a-phellandrene, 3-6- carene, p-cymene, Z-p-ocimeme, E-p-ocimeme, y-terpinene, a-campholenal, trans- pinocarveol, trans verbenol, pinocarvone, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, borneal acetate, a-copaene, p-bourbonene, p-elemene, a-humulene, 9-epi-E- caryophyllene, y-muurolene, germacrene D, p-selinene, y-cadinene, 6-cadinene, and p- eudesmol, which collectively constituted 13.0 wt.% of the total terpenes.

Example 4 The oil of example 1 was applied to the skin of patients diagnosed with psoriasis two times daily for 60 days. Improvement in the condition was reported by users.

Example 5 - Treatment of Psoriasis in in vitro model

For the purpose of mimicking the pathological features of psoriasis in an in vitro model, HaCaT cells, spontaneously immortalized human keratinocytes, were used. In order to determine the optimal conditions for the disease in an in vitro model, the cells were exposed to psoriasis-inducing cytokines (e.g interleukin 17A (IL17A) and tumor necrosis factor alpha (TNF a)) at various concentrations, alone or in combination, for different times of incubation. The cells were then analyzed for different psoriasis-related features.

Cells were cultured according to standard mammalian tissue culture protocols and sterile technique. Human Keratinocytes, HaCaT, were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM). All media was supplemented with 10% fetal bovine serum, streptomycin (100 mg/ml), penicillin (100 U/ml) and Nystatin (12.5 U/ml). Cells were incubated in 5% CO2 at 37°C. All tissue culture cells were maintained in 75 cm² cell culture treated flask (Eppendorf) and all the media and supplements were obtained from Biological Industries.

The cells' viability following treatment was determined using a commercially available MTT assay kit (Abeam, abl46345) and performed according to manufacturer's instructions. Briefly, cells were seeded in a 96-well plate (Eppendorf) at a density of lxlO⁴ cells/well (n=4). After 24h, the media was replaced with "Stimulation media". This media is supplemented with known psoriasis-inducing cytokines, IL17-A (50 ng/ml) and TN Fa (10 ng/ml). The cytokines were purchased from Abeam. Different conditions were evaluated for optimal validation of the model. According to the MTT standard protocol, after the desired incubation time, the media was removed, and the cells were incubated with serum-free media containing 0.5 mg/ml MTT reagent for 4 hours at the incubator. The MTT purple crystals formed by the viable cells were dissolved using isopropanol containing 0.04 mol/L HCI. Quantification was determined by measuring the optical density at 570 nm in a spectrophotometer reader (Spark, Tecan). Results were presented as proportional viability (%) by comparing between treated and untreated groups.

Cellular Reactive Oxygen Species (ROS) production was measured using a DCFDA assay kit (Abeam, abll3851)) according to the manufacturer's protocol. DCFDA (2',7'- dichlorofluorescein diacetate), a cell-permeable fluorogenic dye, is deacetylated to a non- fluorescent compound by cellular esterases and later oxidized into highly fluorescent DCF (2',7'-dichlorofluorescein) by ROS, allowing measurement of hydroxyl, peroxyl and other ROS activity within the cell. Here, HaCaT cells were seeded in 96-well plates at a density of lxlO⁴ for different time periods. After 24h, "stimulation" media was added and the cells were incubated for different periods of time. After incubation, the cells were washed twice with IX buffer assay and then incubated with 25 pM DCFDA in IX buffer assay in a 37°C incubator for 45 minutes and washed twice with IX buffer assay. Cell fluorescence in the 96- well plates was measured at 488 nm excitation and 525 nm emission using a Tecan spark plate reader (Tecan US, Inc. Morrisville, NC). The results are shown as percentage of the control.

To conduct RNA purification and quantitative reverse transcriptase polymerase chain reaction (qRTPCR) analysis of gene expression, cells were seeded in T25 flasks at a density of 5xl0⁵ cel Is/flask in. After 24h the media was replaced with "stimulation" media for different incubation times. After incubation with the stimulation media, the cells were harvested for RNA purification.

RNA was purified from cells' pellets using RNeasy mini kit (QIAGEN), according to manufacturer instructions. RNA concentration and purity was determined by using the UV- Vis micro volume Nanodrop lite (Thermo scientific). cDNA was synthesized from the purified RNA using the High-Capacity reverse transcription kit (Thermo scientific, USA). qRTPCR reaction was preformed using a Fast SYBR™ Green Master Mix (Thermofisher, USA) in The StepOnePlus™ Real-Time PCR System (Applied Biosystems™). The cycle conditions for realtime PCR were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 1 min. The experiment was performed by three independent experiments with triplicate. The following table summarizes the primers used in this study:

GAPDH = glyceraldehyde-3-phosphate dehydrogenase; TRB3 = tribbles homolog 3, IL8 = interleukin 8, VEGF = vascular endothelial growth factor

Figure 1 shows qualitatively the enhanced proliferation of the stimulated cells, compared to the control. A, B: HaCaT cells' pellet after 4 and 5 days of cultivation, respectively. Larger cells pellet can be observed in the stimulated cells (see red arrows). C: HaCaT cells' growing media after 4 days of cultivation. Stimulated cells' media switched to yellow, indication higher proliferation. Figure 2 shows HaCaT cells' viability levels after stimulation with psoriasis-inducing cytokines: HaCaT cells' viability levels are shown after stimulation with IL17A and TNFa, in different concentrations and combinations (A) or at different time points (B). The results are shown as percentage of the control. The data are mean ± SE (n-4). Part A in Figure 2 shows that a combination of both IL17A (at 50 ng/ml) and TNFa (at 10 ng/ml) produced the highest viability levels on day 4 after the stimulation, compared to the control. Part B in Figure 2 shows that this effect was reduced at day 5 after the stimulation. Therefore, it was established that the optimal time point for the evaluation of psoriasis-related features is at day 4 after the induction of psoriasis. The same pattern can be observed in Figure 1 (A+B) when comparing the larger cells' pellet after stimulation and in (C) in Figure 1 by the change in the color of the growing media, which is in direct correlation with the number of cells.

Figure 3 shows the levels of ROS in HaCaT cells stimulated with the psoriasis-inducing cytokines IL17A and TNFa after different incubation periods. The results are shown as percentage of the control. The data are mean ± SE (n=4). It is seen that stimulated HaCaT cells accumulated the highest levels of free radicals on day 4 after the stimulation, compared to unstimulated cells.

Figure 4 shows the gene expression levels of four known biomarkers for psoriasis, after different incubation times: Psoriasin (A), TRB3 (B), IL8 (C) and VEGF (D); "CTL" - control. The results are shown as fold-change of the control. The data are mean ± SE (n=3). It is seen that the gene encoding for IL8 protein, a key component in the inflammatory process, was upregulated (4.6 fold-change) in the stimulated cells, on day 4 (C), and that the VEGF gene, a major biomarker for the formation of new blood vessels (angiogenesis) also showed elevated expression levels (7.5 fold-change) in the stimulated cells (D). It is also seen that the expression levels of Psoriasin and TRB3 genes, two well-described psoriasis-related genes, were evaluated. The expression of psoriasin and TRB3 were significantly upregulated in stimulated cells only, on day 4 (63.5 and 18.6 fold-change, respectively, (A) and (B) respectively in Figure 4). These results serve as validation of the model of qRT-PCR analysis for specific biomarkers' gene expression after disease-inducing cytokines treatment.

Figures 5, 6 and 7 show the gene expression levels of, respectively, Psoriasin (a known biomarker for psoriasis), TRB3 gene, and VEGF gene, after 48 hours treatment with cannabichromene (CBC), which is a known down-regulator of expression of VEGF, or with terpene-enriched olive oil. The results are shown as fold-change of the control. The data are mean ± SE (n=3). The result show that terpene-enriched olive oil at 80 pg/ml significantly reduced (~50%) the expression level of the two psoriasis related genes (psoriasin and TRB3).

Claims

Claims:

1. A method of treating psoriasis, comprising administering to a patient in need of such treatment a composition comprising a terpene-enriched olive oil having a total free fatty acid content of not more than 2 wt.% and a total content of mono-, sesqui- and diterpenes and related terpenoids in the range of 4 to 8 wt.%, the total mono-, sesqui- and diterpenes and related terpenoids comprising 70-85 percent by weight a-pinene, sabinene, myrcene, limonene, and caryophyllene E.

2. The method of claim 1, wherein the total terpene content is about 6 wt.%.

3. The method of claim 1 or claim 2, wherein the total mono-, sesqui- and diterpenes and related terpenoids comprise 10-16% by weight of a mixture of additional terpenes and/or terpenoids selected from the following: a-phellandrene, 3-5-carene, p-cymene, Z-0- ocimeme, E-|3-ocimeme, y-terpinene, a-campholenal, trans-pinocarveol, trans verbenol, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, 2E-decenal, borneal acetate, a-copaene, 0-bourbenone, |3-elemene, a-humulene, 9-epi-E-caryophyllene, y- muurolene, germacrene D, 0-selinene, y-cadinene, 6-cadinene, and P-eudesmol.

4. The method of any one of claims 1 to 3, wherein the composition is administered topically.

5. The method of any one of claims 1 to 4, wherein the composition is administered orally.

6. A method of treating psoriasis, comprising administering to a patient in need of such treatment a composition comprising olive oil which has been used as an extractant for a mixture of Boswelia sacra resin, Boswelia sacra fruits, Commiphora gileadensis fruits, and Commiphora myrrha fruits.

7. The method claim 6, wherein the composition is administered topically.

8. The method of claim 6 or claim 7, wherein the composition is administered orally.

9. The method of any one of claims 6 to 8, wherein the olive oil which has been used as an extractant has a total free fatty acid content of not more than 2 wt.% and a total content of mono-, sesqui- and diterpenes and related terpenoids in the range of 4 to 8 wt.%, the total mono-, sesqui- and diterpenes and related terpenoids comprising 70-85 percent by weight a-pinene, sabinene, myrcene, limonene, and caryophyllene E.

10. The method of claim 9, wherein the total terpene content of the olive oil which has been used as an extractant is about 6 wt.%.

11. The method of claim 8 or claim 9, wherein the total mono-, sesqui- and diterpenes and related terpenoids comprise 10-16% by weight of a mixture of additional terpenes and/or terpenoids selected from the following: a-phellandrene, 3-5-carene, p-cymene, Z-|3- ocimeme, E-P-ocimeme, y-terpinene, a-campholenal, trans-pinocarveol, trans verbenol, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, 2E-decenal, borneal acetate, a-copaene, p-bourbenone, p-elemene, a-humiilene, 9-epi-E-caryophyllene, y- muurolene, germacrene D, p-selinene, y-cadinene, 6-cadinene, and p-eudesmol.

12. A composition comprising a terpene-enriched olive oil having a total free fatty acid content of not more than 2 wt.% and a total content of mono-, sesqui- and diterpenes and related terpenoids in the range of 4 to 8 wt.%, the total mono-, sesqui- and diterpenes and related terpenoids comprising 70-85 percent by weight a-pinene, sabinene, myrcene, limonene, and caryophyllene E, for use in the treatment of psoriasis.

13. The composition for use of claim 12, wherein the total terpene content is about 6 wt.%.

14. The composition for use of claim 12 or claim 13, wherein the total mono-, sesqui- and diterpenes and related terpenoids comprise 10-16% by weight of a mixture of additional terpenes and/or terpenoids selected from the following: a-phellandrene, 3-6-carene, p- cymene, Z-p-ocimeme, E-p-ocimeme, y-terpinene, a-campholenal, trans-pinocarveol, trans verbenol, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, 2E-decenal, borneal acetate, a-copaene, p-bourbenone, p-elemene, a-humulene, 9-epi-E-caryophyllene, y-muurolene, germacrene D, p-selinene, y-cadinene, 5-cadinene, and p-eudesmol.

15. The composition for use of any one of claims 12 to 14, wherein the composition is administered topically.

16. The composition for use of any one of claims 12 to 15, wherein the composition is administered orally.

17. A composition comprising olive oil which has been used as an extractant for a mixture of Boswelia sacra resin, Boswelia sacra fruits, Commiphora gileadensis fruits, and Commiphora myrrha fruits, for use in the treatment of psoriasis.

18. The composition for use of claim 17, wherein the composition is administered topically.

19. The composition for use of claim 17 or claim 18, wherein the composition is administered orally.

20. The composition for use of any one of claims 17 to 19, wherein the olive oil which has been used as an extractant has a total free fatty acid content of not more than 2 wt.% and a total content of mono-, sesqui- and diterpenes and related terpenoids in the range of 4 to 8 wt.%, the total mono-, sesqui- and diterpenes and related terpenoids comprising 70-85 percent by weight a-pinene, sabinene, myrcene, limonene, and caryophyllene E.

21. The composition for use of claim 20, wherein the total terpene content of the olive oil which has been used as an extractant is about 6 wt.%.

22. The composition for use of claim 20 or claim 21, wherein the total mono-, sesqui- and diterpenes and related terpenoids comprise 10-16% by weight of a mixture of additional terpenes and/or terpenoids selected from the following: a-phellandrene, 3-6-carene, p- cymene, Z-p-ocimeme, E-p-ocimeme, y-terpinene, a-campholenal, trans-pinocarveol, trans verbenol, terpinene-4-ol, a-terpineol, myrtenal, verbenone, trans-carveol, 2E-decenal, borneal acetate, a-copaene, p-bourbenone, p-elemene, a-humulene, 9-epi-E-caryophyllene, y-muurolene, germacrene D, P-selinene, y-cadinene, 6-cadinene, and P-eudesmoL