WO2023161402A1

WO2023161402A1 - Composition for the treatment of inflammation

Info

Publication number: WO2023161402A1
Application number: PCT/EP2023/054652
Authority: WO
Inventors: Miguel Ángel BARES DOMÍNGUEZ; Lydia BARES LÓPEZ; Noureddine El Aouad; Hristina EMILOVA HRISTOVA
Original assignee: Bares Dominguez Miguel Angel; Bares Lopez Lydia
Priority date: 2022-02-24
Filing date: 2023-02-24
Publication date: 2023-08-31
Also published as: ES2949572A1

Abstract

The present invention relates to a composition for the treatment of an inflammatory disease or condition, such as fibromyalgia, comprising a wood extract of Ceratonia siliqua, which may be administrated by a transdermal patch, as well as a method for the preparation thereof.

Description

COMPOSITION FOR THE TREATMENT OF INFLAMMATION

DESCRIPTION

BACKGROUND

The beneficial health properties of garrob (Ceratonia siliqua) have been described in numerous publications. In 2014 Durazzo et al. (Food Chem. 2014, 153, 109) found antioxidant properties of carob seed meal, of special interest to the food industry. Benchikh et al. (Ind. Crops. Prod. 2014, 60, 98) showed an excellent source of natural antioxidants in extracts of unripe Ceratonia siliqua that could be used in the cosmetic, pharmaceutical and food industries. For example, Rached et al. used carob pulp extract for yogurt in an encapsulated and free form, the latter being the one that contains the greatest antioxidant power (Food Funct., 2016, 7, 1319). Amessis-Ouchemoukh et al. demonstrated that extracts of Ceratonia siliqua are very effective in neutralizing free radicals (Ind. Crops. Prod. 2017, 95, 6). In the cosmetic industry, plant extracts with antiaging properties are used (CN105640854; KR20150060004; KR101339915; KR201301 13729; KR10080155).

On the other hand, Meziani et al. found antimicrobial compounds in Ceratonia siliqua leaves and pods for the bacterium Pectobacterium atrosepticum, which causes potato rot (Microb. Pathogenesis 2015, 78, 95). Simultaneously, Hsouna et al. identified antimicrobial activity in essential oil of Ceratonia siliqua pods against Listeria monocytogenes in minced beef (I nt. J. of Food Microbiol., 2011 , 148, 66).

Likewise, it has been described that the pulp of the carob contains polyphenols that serve for the treatment of diabetes in humans (S. Gruendel, A.L. Garcia, B. Otto, K. Wagner, M. Bidlingmaier, L. Burget, et al., Br. J. Nutr. 2007, 98, 1170), as antitumor therapy (Custodio et aL, Rec. Nat. Prod., 2013, 7, 225), with hepatoprotective and nephroprotective effect (Hsouna et aL, Food Chem. Toxicol., 201 1 , 49, 3183), or for heart disease (Nasar-Abbas et al., Compr. Rev. Food Sci. Food Sat, 2016, 15, 63).

Asgari et al. (The Natural Products Journal, 2012, 2, 1 ) described that carob has antidiarrheal properties, not only acting for detoxification and constipation, but also providing a rich source of calories. WO201 1089012 describes a method for producing a medicinal product for diarrhea through the extract of the fruit of Ceratonia siliqua. ES2736014 describes the use of an extract of Ceratonia siliqua for the prevention and/or treatment of hemorrhoids. Fibromyalgia is a chronic disease characterized by generalized musculoskeletal pain, with exaggerated hypersensitivity (allodynia and hyperalgesia) in multiple body areas and predefined points (tender points), without demonstrable organic alterations. It is associated with a wide variety of symptoms, including persistent fatigue and nonrestorative sleep. In addition, it often coexists with other rheumatological and psychiatric disorders. Fibromyalgia affects approximately 2 to 5% of the general population in different countries and affectswomen 10 times more than men.

Currently there is still no universally accepted cure for fibromyalgia, there are treatments that have been shown during controlled clinical trials to be effective in reducing symptoms such as patient education, exercise, behavioral therapies and some drugs, such as tricyclic antidepressants, selective serotonin reuptake inhibitors, antidepressants dual serotonin reuptake inhibitors and noradrenaline, reversible monoamine oxidase inhibitors, nonsteroidal anti-inflammatory drugs, antiepileptics, GABA agonists, hypnotics, muscle relaxants or hormone therapy.

Collado Mateo et al. (Nourishment Hospitalaria. 2015;32(5):2126-2135) studied the effects on physical condition in women with fibromyalgia of a six-week treatment with Ganoderma lucidum and compared them with those of a treatment with carob fruit flour (Ceratonia siliqua).

Therefore, there is a need for a remedy for the treatment of fibromyalgia of comfortable and painless application.

DESCRIPTION OF THE INVENTION

The present invention provides a symptomatic relief of inflammation based on natural ingredients which may be applied painlessly and extremely comfortably, directly on the skin and away from the affected place, thus avoiding suffering by contact in the application of the remedy. The inventors of the present invention have found that the transdermal application of a pharmaceutical composition comprising an extract of wood of carob (Ceratonia siliqua), is highly efficient in treating the symptoms of inflammation. Specifically, they have observed a clear improvement in patients diagnosed for fibromyalgia, chronic fatigue syndrome, Covid-19, multiple sclerosis, prostatitis, arthritis or psoriasis. In a first aspect, the present invention relates to a composition comprising an extract of wood of Ceratonia siliqua for use in the treatment of a disease or inflammatory condition. In a preferred embodiment, in the extract is made of Ceratonia siliqua wood, the wood is in the form of sawdust or wood shavings of Ceratonia siliqua. In a preferred embodiment, these extracts are ethanolic extracts which may be obtained by a mixture of ethanol and water in different proportions. The extract of the present invention can be obtained in multiple ways known to the expert in the art, such as pressing, maceration, absorption or distillation.

In a preferred embodiment of the first aspect of the invention, the inflammatory disease or condition is selected from fibromyalgia, Covid-19, chronic fatigue syndrome, multiple sclerosis, prostatitis, arthritis and psoriasis. Preferably, the inflammatory disease or condition is fibromyalgia.

In a preferred embodiment of the first aspect of the invention, the composition is administered orally, cutaneously, subcutaneously, intravenously or intramuscularly, preferably administered by cutaneous route. Preferably, this composition is in the form of tablets, hard capsules, softgels, granules or powders for suspension or solution, oral solutions, syrups, jacking, solution for injection, transdermal patch, cream, gel, ointment, emulsion, oil or solution. In a preferred embodiment of the first aspect of the invention, the composition is a transdermal patch. In another preferred embodiment, the composition is a solution, preferably, this solution is applied in spray. Preferably, the transdermal patch comprises an adhesive. Preferably, the transdermal patch comprises a porous membrane. In a preferred embodiment, the porous membrane comprises Ceratonia siliqua wood. In another preferred embodiment, the patch of the present invention comprises wood and bark of Ceratonia siliqua. The authors of the present invention have observed that a disc of 7 mm in diameter and 2 mm thick of Ceratonia siliqua wood is able to totally eliminate the symptoms of inflammation, specifically the symptoms of fibromyalgia when it comes into contact with the skin of an affected subject. The same happens when the form of administration is a lotion that is applied in spray.

The composition can also be administered orally, and be in the form of tablets, hard capsules, soft capsules, granules or powders for suspension or solution, oral solutions, syrups or lows.

In a preferred embodiment of the first aspect of the invention, the transdermal patch comprises at least one outer barrier layer and one inner layer comprising the extract. Preferably, the composition comprises water, ethanol, acetone, methanol or combinations thereof. In a preferred embodiment, the composition comprises acetone. In a preferred embodiment, the composition comprises methanol. In a preferred embodiment, the composition comprises ethanol.

In a preferred embodiment of the first aspect of the invention, the composition further comprises at least one plasticizer. Preferably, the plasticizer is selected from dibutylphthalate, triethylcitrate, polyethylene glycol, polypropylene glycol or mixtures thereof.

In a preferred embodiment of the first aspect of the invention, the composition further comprises at least one permeability enhancer.

The extract of the invention may comprise the following polyphenols in different proportions (% wt.): gallic acid, pyrogallol, 3-OH-tyrosol, 4-amino benzoic, protocatch nico, chlorogenic, catechol, catechin, caffeine, p-OH-benzoic, caffeic, vanilla, ferulic, iso-ferulic, e-vanillic, resveratrol, alpha-coumaric, benzoic, 3,4,5-methoxycinnamic, salicylic, coumarin, p- coumaric.

The extract of the invention may also comprise the following flavonoids in different proportions (% wt.): naringin, routine, hisperidine, rosmarinic acid, quercetrin, quercotin, hispertine, kaempferol, apegenin, 7-OH-flavone.

The ethanolic extract of the invention is obtained by the following process:

1 ) Pulverizing the Ceratonia siliqua wood preferably to a particle size of less than 1 mm.

2) Extracting the active components by a process of solid/liquid extraction with a mixture of ethanol/water at room temperature, preferably with a solid/liquid ratio of 1/10

3) Filtering and drying the extract.

The mixture of ethanol/water for the extraction step is preferably used in a relation of ethanol/water which may be selected from 0.90:0.10, 0.80:0.20, 0.70:0.30, 0.60:0.40, 0.50:0.50, 0.40:0.60, 0.30:0.70, 0.20:0.80, 0.10:0.90. More preferably 0.80:0.20 or 0.70:0.30. In a second aspect, the present invention relates to a process for the preparation of the patch of the first aspect comprising the following steps: a. obtaining an extract of wood of Ceratonia siliqua, b. diluting the extract obtained in step (a) in at least one acceptable solvent, and c. loading the diluted extract in step (b) into a substrate.

In a third aspect, the present invention relates to the use of a composition comprising a wood extract of Ceratonia siliqua for the treatment of a disease or inflammatory condition other than hemorrhoids, preferably for the treatment of fibromyalgia, Covid-19, chronic fatigue syndrome, multiple sclerosis, prostatitis, arthritis and psoriasis. Preferably, the composition is a transdermal patch.

This invention provides a method for supplying the extract of wood of Ceratonia siliqua through the skin of a subject comprising the administration of a transdermal patch to the skin of said subject.

This invention provides a method for treating a human subject afflicted with an inflammatory disease or condition other than hemorrhoids comprising periodic administration of a transdermal patch to such human subject.

This invention provides a transdermal patch as described herein for use in the prevention and/or treatment of a human subject afflicted with a disease or inflammatory condition other than hemorrhoids.

In a preferred embodiment, the transdermal patch also comprises a highly porous membrane. In another preferred embodiment, the pharmaceutical composition additionally comprises an adhesive. In another preferred embodiment, the pharmaceutical composition comprises at least one permeability enhancer. In one embodiment, the transdermal patch is a matrix patch. In another embodiment, the matrix patch comprises of an adhesive. In one embodiment, the transdermal patch is a reservoir patch. In another embodiment, the deposition patch also comprises a highly porous membrane. In another embodiment, the reservoir patch comprises at least one permeability enhancer. In another embodiment, the permeability enhancer is selected from a group consisting of fatty acid, an alcohol, diethylene glycol, monoethyl, alphatocopherol, a sulfoxide, an azone, a pyrrolidone or a derivative thereof, a terpene, a terpenoid, methyl acetate, butyl acetate and a cyclodextrin. In another embodiment, the permeability enhancer is oleic acid. In another embodiment, the permeability enhancer is isopropyl myristate. Still in another embodiment, the permeability enhancer is an azone.

In one embodiment, the transdermal patch has a total area of about 5-50 cm². In another embodiment, the transdermal patch has a total area of approximately 5-30 cm². In another embodiment, the transdermal patch has a total area of about 5-20 cm². In another embodiment, the transdermal patch has. a total area of about 5-10 cm². In another embodiment, the transdermal patch has a total area of 5 cm². In another embodiment, the transdermal patch has a total area of 10 cm². In another embodiment, the transdermal patch has a total area of 20 cm².

In one embodiment, the coating is a polyethylene terephthalate (PET) coating. In another embodiment, the PET coating is siliconized or has a fluoropolymer layer. And still in another embodiment, a backing layer comprises of a polymer selected from a group consisting of PET, polypropylene and polyurethane.

This invention also provides a method for supplying an extract of wood of Ceratonia siliqua through the skin of a subject comprising the administration of a transdermal patch to the skin of a subject as described herein.

A "transdermal patch" can include, for example, matrix patches and reservoir patches. The matrix patches contain the extract to be supplied in a semi-solid matrix comprising an extract and an adhesive. The deposit patch contains a layer, separate from the adhesive, containing the extract to be supplied.

A "needle patch" is a transdermal patch with small needles that micro-pierce the skin in order to increase the permeation or permeability of the extract that is administered through the barrier. In one embodiment, the transdermal patch described herein is a patch of needles.

A "highly porous membrane" is a membrane that has a high permeability of gas, air, and liquid. Membrane parameters that affect its permeability can be, for example, total weight per surface area, thickness, porosity, medium flow pore size, and air permeability Gurley number (a unit describing the number of seconds required for 100 cubic centimeters of air to pass through a given material of 6.45 cm2 (1.0 square inch) at a differential pressure of 0.013 kg/cm2 (4.88 inches of water (0.188 psi) (ISO 5636-5:2003)). The flow decreases with increasing Gurley number and the thickness of the membrane increases. Other factors that affect flow include membrane pore size and weight per surface area. In addition, lower Gurley numbers are associated with an increased risk of leakage from the deposit. Suitable membrane parameters should be selected such that 1 ) the flow through the membrane is not affected, and 2) provides an effective barrier to prevent reservoir fluid from leaking.

In one embodiment, the highly porous membrane has the following parameters: 1 -20 g/^m2 total weight per surface area, 8-120 microns thick, 40-99% porosity volume, optionally, 75-90% porosity volume, 1 -200 s/50 ml Gurley Number, and up to 1 .1 microns of medium flow pore size. In another embodiment, the highly porous membrane has the following parameters: 3.0-16 g/^m2 total weight per surface area, 20-120 microns thick, 80-90% porosity volume, 1 -5 s/50 ml Gurley number, and 0.3 - 1.1 microns of the medium flow pore size.

In another embodiment, the highly porous membrane has the following parameters: 40- 50% porosity volume, 8-35 microns thick, 4-20 g/^m2 base weight, and 20-200 s/50 ml Gurley Number, and < 0.1 microns pore size. And even in another embodiment, the highly porous membrane has the following parameters: 75-90% volume of porosity, I Q- 120 microns thick, 3-20 g / m² base weight, 1 -100 s / 50 ml of Gurley Number, and 0.05- 1 .0 microns pore size.

The term "composition" is understood as in a pharmaceutical composition, nutraceutical or as a medical device and is intended to encompass a product comprising of an extract of wood of Ceratonia siliqua and an inert ingredient(s) or vehicle or carrier.

"Permeation or permeability enhancers" are agents that increase the bioavailability of the extract. Permeation enhancers include, but are not limited to, fatty acids including oleic acid, propylene glycol, aloe vera oil, isopropyl myristate, heterocyclic nitrogen n- dodecyl heptane-2-ketone, soybean oil, diethylene glycol monoethyl, alpha-tocopherol, alcohol (e.g., ethanol or isopropanol), sulfoxide (e.g., dimethyl sulfoxide), azones (e.g., lauryl caprolactone), pyrrolidone (and derivatives thereof), terpenes, terpenoids, ethyl acetate, methyl acetate, butyl acetate and cyclodextrins.

In one embodiment, the permeation enhancer is an oleic acid. In another embodiment, the permeation enhancer is an isopropyl myristate. And even in another embodiment, the permeation enhancer is an azone. In one embodiment, permeation enhancers can be up to 15% by weight of the composition.

A "backing layer" is a layer of waterproof flexible cover that protects the patch from the outside environment. A backing layer can be composed of a material such as a polymer that includes, but is not limited to, PET, polypropylene and polyurethane.

"Managing the subject" or "administering to the (human) patient" means delivery of, dispensing of, or application of the composition of the present invention.

"Treatment" means, for example, inducing inhibition, regression, or stasis of a condition or disease, inhibiting, reducing, or decreasing the severity or substantially eliminating, or improving a symptom of the disease or disorder. In the present case, the composition of the invention has been shown to substantially alleviate the symptoms of people affected by fibromyalgia treated with said composition.

A transdermal patch usually consists of a small adhesive bandage containing the drug to be administered and these bandages can come in various forms. The simplest type is a one-piece adhesive comprising a drug-containing reservoir placed on a carrier. The reservoir is typically made up of a pharmaceutically acceptable pressure-sensitive adhesive, but in some cases may consist of a non-adhesive material, the surface of which in contact with the skin is provided with a thin layer of a suitable adhesive. Because transdermal patches release the drug by diffusion through the skin, the rate of drug release from the patch is governed by Fick's law and is proportional to the saturation concentration of the drug in the reservoir.

Therefore, the present invention focuses on a transdermal device for releasing compounds with antioxidant activity from particles of Ceratonia siliqua for the treatment of a disease or inflammatory condition. Specifically, for the treatment of an inflammatory disease or condition, it is selected from fibromyalgia, Covid-19, chronic fatigue syndrome, multiple sclerosis, prostatitis, arthritis and psoriasis. QReferibly, the disease is fibromyalgia.

The technical problem solved with this invention is the treatment for inflammation and associated symptoms of an inflammatory disease or condition is selected from fibromyalgia, Covid-19, chronic fatigue syndrome, multiple sclerosis, prostatitis, arthritis and psoriasis, preferably fibromyalgia, by treatment with Ceratonia siliqua, being non- invasive, since the product acts only in contact with the skin.

An example of transdermal patch object of the present invention consists of a disc of about 7 mm in diameter and 2 mm thick of Ceratonia siliqua wood that has been impregnated in an extract of the wood itself to improve its properties. Ceratonia siliqua wood has been shown to have anti-inflammatory properties by itself, and discs of it can be used in contact with the skin to obtain the desired effect. Another example of transdermal patch object of the present invention consists of a porous membrane to which an extract of wood of Ceratonia siliqua, preferably wood, is applied.

The inclusion of wood inside a patch and the increase in the concentration of active compounds involved in impregnating it with an extract of the same plant is an additional advantage, since it allows to reduce the size of the device, without losing the beneficial effect of it.

EXAMPLES

This invention will be better understood by reference to the examples below, but those skilled in the art will easily appreciate that the specific examples detailed are only illustrative of the invention.

Example 1 : obtaining the wood extract of Ceratonia siliqua.

Ceratonia siliqua wood was pulverized to a particle size of less than 1 mm and the active components were extracted with a 80/20 ethanol/water mixture at room temperature, for 24 h in the dark, in a 1/10 solid/liquid ratio. After that time, the extract was filtered through Whatman No. 1 paper and dried under reduced pressure to constant weight.

Example 2: Measurement of the antioxidant activity of the extract

The antioxidant activity of the extract obtained in example 1 was analyzed using the method described in Brand-Williams, W. et al. (1995) LWT - Food Science and Technology, 28(1 ), 25-30, adapted as described in Carmona-Jimenez, Y et al. (2014) Food Chemistry 165, 198-204. This activity was tested on dilutions of the extract in different solvents: dichloromethane (DCM), hexane, acetone and methanol and is shown in Figure 1. It was observed that the extract diluted in acetone or methanol has a high antioxidant activity, better than when the other solvents are used. These solvents are also more environmentally friendly. Example 3: preparation of a transdermal patch comprising the extract.

The extract obtained as explained in example 1 was redissolved in ethanol to form a solution of 500 mg of extract / ml and in it sheets of Ceratonia siliqua wood of 2 mm thickness were immersed to enrich them in the active components.

The wood was left in contact with the extract for 48 h at room temperature and in darkness. Once impregnated, the wooden sheets were allowed to dry and cut to the desired shape. The impregnated wood discs thus obtained were put in direct contact with the skin.

T ransdermal patches were prepared with a support layer containing an adhesive element on which a disc 7 mm in diameter and 2 mm thick of Ceratonia siliqua wood impregnated in the extract described above was placed. Using the adhesive support, the transdermal patch could be fixed to the skin comfortably so that the wooden disc impregnated with Ceratonia siliqua wood extract was in contact with the skin, allowing the absorption of the active compounds of the extract by the body.

Example 4: Transdermal patch effectiveness

Fibromyalgia

The composition of the present invention was tested on 36 women between 33x and 65 years. Among them, 30 showed improvement in symptoms of between 70 and 80%, especially in muscle and bone pain, chronic fatigue, in addition to a notable decrease in burning sensation in the palms of the hands and feet, as well as in anxiety and depression.

Chronic fatigue syndrome

The composition of the present invention was tested on 8 of 10 women aged between 28 and 43 years. An improvement of chronic fatigue disease was observed in less than 24 hours, which is within the central sensitivity syndrome, as is fibromyalgia. This improvement is progressively manifested up to 80%, in terms of muscle and cognitive weakness, irritability, sleep and mood, using the active ingredient of the wood of Ceratonia Siliqua on the skin, both in transdermal patches, lotion or cream.

Multiple sclerosis

The composition of the present invention was tested on 5 of 7 people, 3 of them female aged between 25 and 38 years. Improvement in initial MS disease was observed. The improvement was progressive, since up to 70-80% of patients were relieved between 24 and 48 hours, in the following symptoms: tingling, pain and weakness in extremities, balance, fatigue, memory and concentration, as well as blurred vision and pain in the eyes and mood. Patients describe that the time between relapses is prolonged, using the wood of Ceratonia Siliqua on the skin, both in transdermal patches, lotion or cream.

Prostatitis

The composition of the present invention was tested on 8 of 12 men aged between 53 and 66 years. An improvement was seen in the condition called prostatitis. This relief is manifested in 48 hours, progressively improving up to 70% in the symptomatology related to irritation and difficulty when urinating, as well as in its frequency, using the composition of the invention on the skin, both in transdermal patches, lotion or cream.

COVID-19

The composition of the present invention was tested on 4 people aged between 30 and 42 years. A progressive improvement of up to 60% in the symptoms of Covid-19 was observed, relieving joint and muscle pain, using the wood Ceratonia Siliqua on the skin, both in transdermal patches, lotion or cream.

Psoriasis

The composition of the present invention was tested on 12 people of different gender aged between 25 and 45 years. An improvement in psoriasis symptoms was observed in 9 of them in a few weeks: relief in itching, inflammation and peeling, which are shown progressively (depending on the amount of skin submitted to treatment), using a Ceratonia Siliqua ointment on the affected area.

Arthritis

The composition of the present invention was tested on 12 people of different gender, aged between 43 and 60 years. Progressive improvement in arthritis disease was observed. This relief manifests itself after 24 hours (depending on the severity), and progressively improving up to 80% pain, swelling and stiffness, using the wood of Ceratonia Siliqua on the skin, both in transdermal patches, lotion or cream.

Example 5: Analysis of the composition of the extract from example 1 .

The chemical composition of the extract in example 1 was analyzed by HPLC as explained in Torres, J. L. et al. (2002) Journal of Agricultural and Food Chemistry, 50(26), 7548-7555; Villaho, D. et al. (2005) Analytica Chimica Acta, 538(1-2), 391-398; Hanan A. Jambi (2015) Life Science Journal 12(12), 1 -5.

Table 1 shows the concentration in mg/kg of extract and the relative abundance of a series of compounds in the extract of example 1 . Table 2 shows the concentration and relative abundance of flavonoids in the extract in example 1. Table 3 shows the concentration and relative abundance of volatile compounds in the extract of example 1 . Finally, Table 4 shows the relative concentrations and abundances of some compounds in the patch obtained according to example 3, and how there is an increase in concentration of these compounds in the patch of example 3 compared to the extract.

Table 1 . Results of the analysis of polyphenols in the wood extract of Ceratonia siliqua prepared according to example 1 .

Table 2. Results of the analysis of flavonoids in the wood extract of Ceratonia siliqua prepared according to example 1 .

Table 3. Results of the analysis of volatile compounds in the wood extract of Ceratonia siliqua prepared according to example 1 .

Table 4. Concentration of some compounds in the patch described in Example 3.

Example 6: Evaluation of the antioxidant activity

A. DPPH Radical Scavenging Assay

The DPPH (2,2-diphenyl-1 -picrylhydrazyl) is a relatively stable free radical characterized by a violet color, the contact of this compound with an antioxidant result on its reduction inducing its transformation into a yellow compound the diphenyl picryl hydrazine. The intensity of the coloration is proportional to the antioxidant concentration. In this assays we used the protocol previously described by Mechqoq et al. 2022 (Cosmetics, 2022. 9(5): p. 94). Briefly, the extracts obtained as indicated in example 1 were diluted at various concentrations ranging between 50 and 1000 pg/mL. Then, 50 microliters of each concentration were added to 1950 pL of DPPH-methanol solution (20 mg/L). The solutions were then incubated in the dark for 30 min, and the absorbance was measured at 515 nm using a spectrophotometer. Ascorbic acid was used as a positive control, and all tests were carried out in triplicate. The radical-scavenging activity of the samples are expressed as inhibition percentage, the latter is calculated according to Equation (1 ) below:

DPPH inhibition

Where OB_biank corresponds is the control (containing all reagents except the tested compound), and OD_sampie is the samples. The inhibition values were plotted against concentration, and the obtained linear regression equation was used to calculate the IC₅o value. For all the inhibition assay a low IC50 value corresponds to a strong inhibitory activity.

B. Ferric Reducing Antioxidant Power (FRAP) Assay

In contrast with the DPPH scavenging assay, the FRAP method is based on the reduction of ferric ion (Fe³⁺) of the potassium ferricyanide (III) [K₃Fe(CN)₆] complex to ferrous ion (Fe²⁺) [4], The total antioxidant activity by ferric thiocyanate (FTC) was performed using the method described by Oyaizu 1986 (The Japanese journal of nutrition and dietetics, 1986. 44(6): p. 307-315) with slight modifications. Briefly, the extract was prepared at various concentrations ranging between 50 and 1000 pg/mL, a 0.1 mL volume of each extract dilution was mixed with 2.5 mL of 1% (w/v) potassium ferricyanide solution and PBS (0.04 M; pH 7.4). The latter were incubated for 20 min at 50°C. Then, 2.5 mL of a 10% (w/v) trichloroacetic acid (TCA) solution was added to each dilution. Then, 2.5 mL of the supernatant ware collected and mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% iron chloride (III) (FeCh). After a 5 min incubation at room temperature, the absorbance was measured at 700 nm, and ascorbic acid was used as a positive control. Total antioxidant activity was calculated as percentage of inhibition, using the same equation previously used for DPPH scavenging activity (Equation 1 ). An efficient concentration of 50% (EC50) was determined by plotting the inhibition values against different concentrations of the sample.

The results of the antioxidant assays are summarized in table 1 .

Ascorbic acid 0.921 0.07 23.067 2.124

Ceratonia siliqua

153.642 42.377 6.173 0.12

(ethanol-water 80:20)

Table 1 . Antioxidant activity of Ceratonia siliqua extract by DPPH and FRAP methods.

The Ceratonia siliqua (ethanol-water 80:20) wood hydroalcoholic extract showed very interesting antioxidant activities for both DPPH and FRAP. In fact, this extract exhibited a DPPH inhibition IC50 value of 153.642 ± 42.377 pg/mL in contrast with the positive control that showed a lower value of 0.921 ± 0.07 pg/mL, despite being higher than the positive control, Ceratonia siliqua IC50 value remains strong in comparison with other plant species such as Thymus vulgaris (aerial parts), Acacia nilotica (bark) or Adenocarpus artemisiifolius (leaves).

Moreover, the obtained EC50 value was much lower than the positive control (EC50= 23.067 ± 2.124 pg/ml), with a value of 6.173 ± 0.12 pg/ml. the low EC50 value suggest a stronger ferric ion reducing antioxidant power.

The antioxidant activity is closely related to fibromyalgia. Many researchers have shown that patients with fibromyalgia are subjected to oxidative stress caused by an abnormal proliferation of free radicals in their nervous systems. The antioxidant ability of the extract from Ceratonia siliqua supports its use for the treating of this pathology as well as other conditions in which a high level of ROS are produced. Example 7: Evaluation of the anti-inflammatory activity

A. Cvcloxygenase-2 inhibition assay

The cyclooxygenase-2 assay was carried out according to the protocol described by Jan et al. 2020 (European Journal of Medicinal Chemistry, 2020. 186: p. 1 11863). Prior to the experiment, 10 pl of COX-2 enzyme solution (350 unit/ml), was added to a 50 pl cofactor solution composed of glutathione (0.9 mM), N,N,N,N-tetramethyl-p- phenylenediamine dihydrochloride (0.24 mM) and hematin (1 mM) in TrisHCI buffer (0.1 M, pH 8.0). Ceratonia siliqua hydroalcoholic extract was prepared in solution with concentrations ranging between 15 and 240 pg/ml. In a 96-well microplate, 20 pl of each sample concentration were mixed with 60 pl of enzyme, the reaction medium was then incubated for 5 min at 25 °C, the enzymatic reaction was initiated by addition 20 pl of arachidonic acid (30 mM). After a 10 min incubation, the absorbance of the samples was measured at 570 nm. Rofecoxib (IC₅o= 0.242 pg/mL) was used as positive control. Experiments were performed in triplicates, and the inhibition percentage of COX-2 was calculated by the Equation (2):

COX-2 inhibition (%)= °^Dcon ' °^Dbl ' °^Dsam ' ^{0Dbl sam} _x 1₀₀ (2)

(0D_con - 0D_bl) where OD_con is the Control [without sample], OD_bi is the Blank (without sample and enzyme), OD_sam is the Sample, and OD_bi sam is the Blank Sample (without enzyme). Extracts were initially tested at 200 pg/mL. The final results are expressed as IC₅o values.

B. 5-Lipooxyqenase inhibition assay

The 5-lipooxygenase inhibition assay was performed according to the protocol previously described by Mahnashi et al. 2021 (Journal of ethnopharmacology, 2021. 273: p. 113976). Briefly, the plant sample was prepared in concentrations ranging between 15 and 240 pg/ml, the different sample concentrations were dissolved in 250pl of PBS (0.05 M; pH 6.3), then 250 pl of 5-LOX solution (10,000 unit/ml) were added. After 5 min incubation at room temperature, 1000 pl of the substrate solution (0.6 mM, linoleic acid) was added, followed by a 10 min at room temperature. The absorbance of the samples was measured at 234 nm. Zileuton (IC50= 2,457 pg/mL) was used as positive control. Experiments were performed in triplicates, and the inhibition percentage was determined following the method previously described for the cyclooxygenase assay.

The results of the anti-inflammation assays are summarized in table 2. Cyclooxygenase-2 IC50 (pg/mL) 5-Lipooxygenase IC50 (pg/mL)

Mean value Mean value

Rofecoxib 0.998 0.065 ND ND

Zileuton ND ND 2.564 1.432

Ceratonia siliqua

7.791 1.941 5.375 0.669

(ethanol-water 80:20)

Table 2. Anti-inflammatory activity of Ceratonia siliqua extract evaluated by inhibition COX-2 and 5-LOX inhibition assays.

The observation of the obtained results showed that for the cyclooxygenase-2 inhibitory assay, C. siliqua wood hydroalcoholic extract have an IC50 value of 7.791 ± 1 .941 pg/mL in comparison with the positive control (Rofecoxib, z COX-2-inhibitor) that showed an IC50 value of 0.998 ± 0.065 pg/mL. When for the 5-Lipooxygenase, C. siliqua extract exhibited an IC50 value of 5.375 ± 0.669 pg/ml, in contrast with the positive control (Zileuton IC50= 2.564 ± 1.432 pg/mL). Despite being higher than the positive controls, the C. siliqua extract have a strong COX-2 and 5-LOX inhibitory activity with good IC50 values. Those results shows that C. siliqua wood hydroalcoholic extract have a strong anti-inflammatory activity with a double action against both cyclooxygenase-2 and 5- lipooxygenase. Thus, these results support the use of the extract from Ceratonia siliqua of the invention for the modulation of inflammation process in which these enzymes are involved.

Claims

CLAIMS A composition comprising an extract from wood of Ceratonia siliqua for use in the treatment of an inflammatory disease or condition. The composition for use according to claim 1 , wherein the wood is in the form of sawdust or wood shavings of Ceratonia siliqua. The composition for use according to any one of claims 1 or 2, wherein the extract is an ethanolic extract. The composition for use according to any of claims 1 to 3, wherein the inflammatory disease or condition is selected from fibromyalgia, Covid-19, chronic fatigue syndrome, multiple sclerosis, prostatitis, arthritis and psoriasis. The composition for use according to any of claims 1 to 4, wherein the disease or inflammatory condition is fibromyalgia The composition for use according to any of claims 1 to 5, wherein said composition is administered orally, cutaneously, subcutaneously, intravenously or intramuscularly, preferably administered by cutaneous route. The composition for use according to claim 6, wherein said composition is administered included in tablets, hard capsules, softgels, granules or powders for suspension or solution, oral solutions, syrups, jacking, solution for injection, transdermal patch, cream, gel, ointment, emulsion, oil or solution. The composition for use according to claim 7, wherein the composition is administered included in a transdermal patch, cream, gel, ointment, emulsion, oil or solution. The composition according to any of claims 1 to 8, wherein said composition comprises a pharmaceutically acceptable solvent, preferably comprises water, ethanol, acetone, methanol or combinations thereof. The composition according to claim 9, wherein comprises acetone.

11 . The composition according to claim 9, wherein comprises methanol.

12. The composition according to claim 9, wherein comprises ethanol.

13. The composition according to any of claims 1 to 12, which further comprises at least one plasticizer.

14. The composition according to claim 13, wherein the plasticizer is selected from dibutylphthalate, triethylcitrate, polyethylene glycol, polypropylene glycol or mixtures thereof.

15. The composition according to any of claims 1 to 14, which further comprises at least one permeability enhancer.

16. A transdermal patch comprising a porous membrane, wherein said porous membrane comprises Ceratonia siliqua wood or an extract thereof as defined in claims 1 -15.

17. The transdermal patch according to claim 16 which comprises at least one outer barrier layer and one inner layer comprising the extract.

18. Procedure for the preparation of the transdermal patch according to any of claims 17 or 18 comprising the following steps: a. obtaining an extract of wood of Ceratonia siliqua as defined in claims 1 - 15; b. diluting the extract obtained in step (a) in at least one acceptable solvent, and c. loading the diluted extract in step (b) onto a substrate.

19. Process according to claim 18, wherein the extraction in step (a) is carried out with a mixture of ethanol/water.