WO2016120241A1

WO2016120241A1 - Process for the preparation of a helichrysum-based product

Info

Publication number: WO2016120241A1
Application number: PCT/EP2016/051517
Authority: WO
Inventors: Daniele PIETRA; Alice BORGHINI; Guido Puricelli
Original assignee: Purytra Farmaceutici S.P.A.
Priority date: 2015-01-28
Filing date: 2016-01-26
Publication date: 2016-08-04

Abstract

The invention relates to a process for the preparation of an extract of Helichrysum italicum comprising the steps of: 1) boiling an aqueous solution of the drug of Helichrysum italicum for a time period in the range from 30 minutes to 2.5 hours; 2) optionally separating the boiled Helichrysum italicum product from the boiling decoction; 3) optionally drying the boiled Helichrysum italicum product so as to obtain a dry product of Helichrysum italicum; and 4) extracting the boiled product of Helichrysum italicum or the dry product of Helichrysum italicum with a solvent selected from the group consisting of water, ethanol, propylene glycol, glycerin, jojoba wax and derivatives thereof, a vegetable oil and mixtures thereof. The extract of the invention has a high ratio of polyphenols/phenols with respect to the allergens limonene, eugenol and linalool.

Description

"PROCESS FOR THE PREPARATION OF A HELICHRYSUM-BASED PRODUCT"

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DESCRIPTION FIELD OF THE INVENTION

The present invention relates to a process for obtaining a product based on the drug Helichrysum italicum, and the product thus obtained. The product of the invention is used in formulations for use in cosmetics, in dermatology and in the food industry.

PRIOR ART

The drug Helichrysum italicum is well known and is found in some commercially available preparations. Among them, we may mention infusion, decoction, essential oil, fluid extract, dry extract, lipophilic dry extract and macerated oil. The Helichrysum genus belongs to the Asteraceae family and comprises about six hundred species of flowering plants. The various species of Helichrysum are known to contain substances that may be useful for treating skin diseases, mainly because they have a certain antioxidant power. Among these species, Helichrysum italicum or angustifolium are used, which grow spontaneously in Southern Europe. Helichrysum italicum contains several substances including flavonoids, terpenes, phenols and polyphenols. Other species of the genus Helichrysum that contain substances potentially useful for treating skin diseases are, for instance, H. frigidum, H. lithoreum, H. melitense, H. montelinasanum, H. nebrodense, H. rupestre, H. saxatile, H. siculum and H. stoechas. The different species vary for the kinds of substances and the amounts contained therein. Many products on the market, however, have the drawback of having a low content of phenols and polyphenols and of having poor stability over time.

Document WO2007/083190 describes a process for preparing an extract of helichrysum which comprises a step of extraction of the drug helichrysum with a mixture of an organic solvent and water. The drug thus obtained has high amounts of substances, such as flavonoids, which allow the pharmacological application thereof as well as the dosage standardization. Document WO2013/050902 describes a method for making an extract of helichrysum in jojoba oil which comprises a step of putting the drug helichrysum in contact with a specific jojoba wax that allows obtaining an extract with a high flavonoid content to stimulate the epidermal keratinocytes. In particular, the extract is characterized by a high content of chrysin.

All formulations available on the market have a content of fragrance allergens which has been problematic in some subjects in both the cosmetic and the pharmaceutical fields.

Although the skin compatibility of cosmetic products is fully tested, it is inevitable that a number of individuals may experience a skin reaction (redness or irritation) to substances to which they are allergic.

A case report is found in the literature which reports the onset of dermatitis in a subject who used a formulation containing the liposoluble extract of helichrysum of Aboca [Foti C, Guida S, Antelmi A, Romita P, Corazza M. Allergic contact dermatitis caused by Helichrysum italicum contained in an emollient cream. Contact Dermatitis. 2013;69(1):62-3].

As a precaution, the current legislation provides for obligations to report the content of allergenic substances on the label in order to protect consumers in the field of cosmetics.

Among all allergens listed in legislation, Helichrysum italicum only contains limonene, linalool and eugenol.

The object of the present invention is therefore to provide an extract of helichrysum which has a high content of phenols/polyphenols and a low content of allergenic substances.

SUMMARY OF THE INVENTION

The above object is achieved by a process for the preparation of an extract of Helichrysum italicum comprising the steps of:

1 ) boiling an aqueous solution of the drug Helichrysum italicum for a time period in the range from 30 minutes to 2.5 hours;

2) optionally separating the boiled Helichrysum italicum product from the boiling decoction; 3) optionally drying the boiled Helichrysum italicum product so as to obtain a dry product of Helichrysum italicum; and

4) extracting the boiled Helichrysum italicum product or the dry Helichrysum italicum product with a solvent selected from the group consisting of water, ethanol, propylene glycol, glycerin, jojoba wax and derivatives thereof, a vegetable oil and mixtures thereof.

The inventors of the present invention have surprisingly discovered that boiling the specific drug Helichrysum italicum in water for a specific time period, i.e. from 30 minutes to 2.5 hours, leads to the deterpenization of the drug Helichrysum italicum, which allows eliminating the allergenic substances and at the same time having a high concentration of antioxidant polyphenols. In other words, the inventors found out that boiling the specific species Helichrysum italicum for the claimed time period allows to obtain a very advantageous optimal amount ratio of the antioxidant species with respect to the allergens. Furthermore a time period of 30 minutes to 2.5 hours is also convenient from an industrial point of view since the boiling time is short so as to favour the final process yield, but equally guaranteeing the lowering of the amount of the allergens as desired for hypoallergenic cosmetic products. According to what discovered by the inventors, boiling times lower than 30 minutes were not enough to remove allergens, while boiling times higher than 2.5 hours are capable to disrupt the desired phenol/polyphenol molecules thus reducing their content.

In the present invention, the term:

"drug Helichrysum italicum" means the Helichrysum italicum plant or parts thereof, either whole, chopped or crumbled, for example cut for infusion;

"boiled Helichrysum italicum product" means the solid phase remaining after treatment by boiling of the drug Helichrysum italicum substantially deprived of the presence of the allergens limonene, eugenol and linalool;

"substantially deprived of the presence of the allergens limonene, eugenol and linalool" and "does not substantially comprise the allergens limonene, eugenol and linalool", when referred to the product of step 3) or step 4), means a product having a negligible amount of allergens, strongly reduced compared to known products;

"boiling decoction" means any liquid phase remaining after treatment by boiling of the preparation of the drug Helichrysum italicum in water.

According to another aspect, the invention relates to an extract of Helichrysum italicum obtainable from the process of claim 1 . The extract of Helichrysum italicum of the invention surprisingly has a high ratio between the amount of polyphenols/phenols and the amount of eugenol, limonene and linalool with respect to known products.

In particular, the extract of the invention has a ratio between the amount of total polyphenols/phenols with respect to the amount of limonene higher than 10,000, a ratio between the amount of total polyphenols/phenols with respect to the amount of linalool higher than 10,000 and a ratio between the amount of total polyphenols/phenols with respect to the amount of eugenol higher than 10,000. More preferably, the extract of the invention has a ratio between the amount of total polyphenols/phenols with respect to the amount of limonene higher than 50,000, a ratio between the amount of total polyphenols/phenols with respect to the amount of linalool higher than 50,000 and a ratio between the amount of total polyphenols/phenols with respect to the amount of eugenol higher than 50,000. The extract of the invention is used in the cosmetics, pharmaceutical and food industry for its antioxidant properties.

In another aspect, therefore, the invention relates to a cosmetic composition comprising the extract of Helichrysum italicum described above and cosmetically acceptable carriers.

In another aspect, therefore, the invention relates to a pharmaceutical composition comprising the extract of Helichrysum italicum described above and pharmaceutically acceptable carriers.

In another aspect, therefore, the invention relates to a food or a food supplement comprising the extract of Helichrysum italicum described above and optionally food-grade carriers.

In yet a further aspect, the invention relates to the extract of Helichrysum italicum for use in the prevention and treatment of atopic dermatitis. Preferably, therefore, the invention relates to the extract of Helichrysum italicum for use in the prevention and treatment of irritations and chapping of the hand and face skin due to atmospheric agents, irritations and chapping of the nose in the presence of colds, body skin irritations due to chemicals, such as chlorine, vasculitis and frostbite of the feet and hands, hemorrhoids, venous insufficiency, sore throat and mouth, aphtae, gingivitis, psoriasis.

Surprisingly, the dry Helichrysum italicum product obtainable from step 3) of the invention does not substantially comprise the allergens limonene, eugenol and linalool and may find use as a product for use in food preparations. Among such food preparations we may mention preparations for infusion, such as herbal teas. DETAILED DESCRIPTION OF THE INVENTION

The invention therefore relates to a process for the preparation of an extract of Helichrysum italicum comprising the steps of:

2) optionally separating the boiled Helichrysum italicum product from the boiling decoction;

3) optionally drying the boiled Helichrysum italicum product so as to obtain a dry product of Helichrysum italicum; and

Step 1 ) of the process of the invention consists in bring an aqueous preparation of the drug Helichrysum italicum to boiling. Helichrysum italicum has a busty look, a silver-green colour and it flowers from the end of the spring to the beginning of the autumn.

Boiling step is carried out for a time period from 30 minutes to 2.5 hours, preferably from 1 hour to 1 .5 hours. Boiling step a) is preferably carried out by hydrodistillation or by boiling in an open container. More preferably, the boiling step is carried out in acidic environment, preferably in the range of pH from 2 to The separation step 2) of the aqueous solution from the boiled Helichrysum italicum product is preferably carried out through filtration and pressing.

The drying step 3) is preferably carried out in a suitable oven.

The separation step 2) and the drying step 3) are optional since the procedure may conveniently provide for carrying out a boiling step which leads to an increasing concentration of the solid product with removal of the water. For example, by continuing the boiling in an open container to a desired concentration to dryness of the boiled Helichrysum italicum product.

The extraction step 4) of the dry Helichrysum italicum product of step 3) or of the boiled product of step 1 ) is carried out with a solvent selected from the group consisting of water, ethanol, propylene glycol, glycerin, jojoba wax and derivatives thereof, a vegetable oil and mixtures thereof. Preferably, the solvent or the solvent mixture is selected from the group consisting of water/ethanol, ethanol, water/propylene glycol, propylene glycol, water/glycerin, glycerin, jojoba wax and a vegetable oil. Among the derivatives of jojoba wax we may mention the hydrogenated jojoba wax.

If the extraction is carried out with the water/ethanol mixture, a hydroalcoholic Helichrysum italicum extract is obtained.

If the extraction is carried out with ethanol, an alcoholic Helichrysum italicum extract is obtained.

If the extraction is carried out with the water/propylene glycol mixture, a hydroglycolic Helichrysum italicum extract is obtained.

If the extraction is carried out with propylene glycol, a glycolic Helichrysum italicum extract is obtained.

If the extraction is carried out with the water/glycerin mixture, a hydroglycerinated Helichrysum italicum extract is obtained.

If the extraction is carried out with glycerin, a glycerinated Helichrysum italicum extract is obtained.

If the extraction is carried out with jojoba wax or derivatives thereof, a Helichrysum italicum extract in jojoba is obtained. If the extraction is carried out with a vegetable oil, it can be preferably selected from corn germ oil, peanut oil, extra virgin olive oil, almond oil, hemp oil, sunflower seeds oil, sesame oil. The extract thus obtained will be a macerated oil Helichrysum italicum extract.

The extracts obtainable from the process may be subjected to a further concentration or drying step 5), for example by means of partial or total evaporation of the extraction solvent.

All the extracts obtainable by the process of the invention are substantially deprived of allergens, such as eugenol, limonene and linalool.

Eugenol, i.e. 2-methoxy-4-(propen-2-yl)-phenol, is a hydroxylated aromatic compound, a chain-modified guaiacol. Eugenol is a member of the chemical class of allylbenzenes.

Linalool is a monoterpene abundantly present in the essence of rosewood and linaloe.

Limonene is a hydrocarbon, a cycloolefin classified as cyclic monoterpene. Specifically, limonene is naturally found as R enantiomer, R-(+)-4-isopropenyl-1 - methyl-1 -cyclohexene. It is also known as S enantiomer, S-(-)-4-isopropenyl-1 - methyl-1 -cyclohexene. Limonene is colorless at room temperature and has a strong smell of oranges, lemons or turpentine depending on the chiral composition.

Since limonene, eugenol and linalool belong to the family of terpenes and terpenoids, the boiling step 1 ) of the process of the invention is a deterpenization step, i.e. a step that allows eliminating the three terpenes/terpenoids responsible for the allergenic effects of Helichrysum italicum extracts.

According to another aspect, the invention relates to an extract of Helichrysum italicum obtainable from the process of claim 1 . The extract of Helichrysum italicum of the invention surprisingly has a high ratio between the amount of polyphenols/phenols with respect to the amount of eugenol, limonene and linalool compared to known Helichrysum italicum products.

In particular, the extract of the invention or the Helichrysum italicum product of step 1 ) show a ratio of the amount of total polyphenols/phenols with respect to the amount of limonene higher than 10,000, a ratio of the amount of total polyphenols/phenols with respect to the amount of linalool higher than 10,000 and a ratio of the amount of total polyphenols/phenols with respect to the amount of eugenol higher than 10,000.

Preferably, the ratio between the amount of total polyphenols/phenols with respect to the amount of limonene is higher than 50,000, the ratio between the amount of total polyphenols/phenols with respect to the amount of linalool is higher than 50,000 and the ratio between the amount of total polyphenols/phenols with respect to the amount of eugenol is higher than 50,000. The extract of the invention is used in the cosmetics, pharmaceutical and food industry for its antioxidant properties.

In yet a further aspect, the invention relates to the extract of Helichrysum italicum for use in the prevention and treatment of atopic dermatitis.

Preferably, the invention relates to the extract of Helichrysum italicum for use in the prevention and treatment of irritations and chapping of the hand and face skin due to atmospheric agents, irritations and chapping of the nose in the presence of colds, body skin irritations due to chemicals, such as chlorine, vasculitis and frostbite of the feet and hands, hemorrhoids, venous insufficiency, sore throat and mouth, aphtae, gingivitis, psoriasis.

Surprisingly, the dry Helichrysum italicum product obtainable from step 3) of the invention does not substantially comprise the allergens limonene, eugenol and linalool and may find use as a product for use in food preparations. Among such food preparations we may mention preparations for infusion, such as herbal teas. As will be apparent from the following experimental part, the Helichrysum italicum products of the invention have been found to have an antioxidant activity comparable to, and in some cases even higher than, compounds with a known antioxidant activity such as chrysin, galangin, caffeic acid and chlorogenic acid, known to be contained in Helichrysum italicum itself. Without being bound to any theory, the inventors believe that this may indicate a possible presence of synergism between the various antioxidants components present in the extracts of the invention, when present simultaneously.

The inventors of the present invention have also shown that the antioxidant activity of the products of the invention remained stable over time up to 6 months from the preparation of the extracts.

The products of the invention were also found to have a softer smell intensity compared to the reference products; moreover, each product of the invention has its own specific olfactory note that is pleasant.

Experimental part

Example 1 : preparation or procurement of substances, of the drug Helichrysum italicum and of the reference helichrysum extracts (reference products)

1 a. Preparation of the drug Helichrysum italicum cut for infusion

The product drug Helichrysum italicum cut for infusion is a commercial helichrysum product sold by the farm Erboristeria Officinale di Daniele Patrizia, Olbiatempio, Sardinia, Italy, and is the Helichrysum italicum plant dried and crumbled cut for infusion, i.e. fragments of between 2 and 15 mm in size.

1 b. Preparation of the Helichrysum italicum decoction

The reference product 1 b was prepared according to the directions contained in Santini L. Rassegna clinico-statistica sulle proprieta terapeutiche dell'elicriso. Minerva medica, 1952, 714-719.

The drug Helichrysum italicum cut for infusion consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was placed in a beaker containing boiling distilled water, at a drug/water ratio of 20% w/v. Boiling was continued for 10 minutes, after which the drug was removed by filtration through filter paper. In order to evaluate the concentration of the dry residue in the decoction, three 100 μΙ portions of decoction were independently concentrated and dried under reduced pressure in a rotary evaporator at a temperature of 60 °C. By averaging the value of the concentration of the three independent measurements, it was obtained that the concentration of the decoction in relation to its dry residue was 5 mg/ml.

1 c. Preparation of the macerated oil in reference jojoba wax

The reference product 1 c was prepared according to the directions contained in Del Corso S, Pietra D, Borghini A, Bianucci AM. Jojoba oil Helichrysum extract and composition thereof, in particular, for treating a skin condition, and in the international patent application PCT/IB2012/055081 , Example 1 , page 21 .

The drug helichrysum cut for infusion (2.5 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was placed in a beaker and allowed to macerate in 10 ml of jojoba wax for 28 days. The extract was then decanted and pressed.

1 d. Product "Oleolito composto RD" (Macerated oil compound RD) of Del Corso Simone

The product Oleolito composto RD" (Macerated oil compound RD) of Del Corso Simone is a commercial herbal product sold in Italy, so it was bought at the pharmacy.

1 e. OTI Helichrysum italicum mother tincture

The OTI Helichrysum italicum mother tincture is the commercial product "Elicriso T.M." of OTI Sri and, being a commercial herbal product sold in Italy, it was bought at the pharmacy. In order to evaluate the concentration of the dry residue in the mother tincture, three 100 μΙ portions of mother tincture were independently concentrated and dried under reduced pressure in a rotary evaporator at a temperature of 60 °C. By averaging the value of the concentration of the three independent measurements, it was obtained that the concentration of the mother tincture in relation to its dry residue was 18 mg/ml. Example 2: preparation, optional separation and optional drying of the boiled products of the invention and of the extract of the invention 2aa. Preparation of the boiled Helichrysum italicum product

The drug Helichrysum italicum cut for infusion (250 g) consisting of dry Helichrysum italicum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was subjected to hydrodistillation in a Clevenger apparatus by boiling for 2.5 hours with 2 liters of water, with a drug:water ratio of 1 :8.

2ab. Preparation of the boiled Helichrysum italicum product

The drug Helichrysum italicum cut for infusion (250 g) consisting of dry Helichrysum italicum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was subjected to hydrodistillation in a Clevenger apparatus by boiling for 1 .0 hours with 2 liters of water, with a drug:water ratio of 1 :8.

2ac. Preparation of the boiled Helichrysum italicum product

The drug Helichrysum italicum cut for infusion (250 g) consisting of dry Helichrysum italicum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was subjected to hydrodistillation in a Clevenger apparatus by boiling for 1 .5 hours with 2 liters of water, with a drug:water ratio of 1 :8.

2ba. The drug Helichrysum italicum cut for infusion (250 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was boiled in an open container with 2 liters of water, i.e. in a drug:water ratio of 1 :8 for 1 .0 hour. The boiled Helichrysum italicum product was then separated by decantation, filtration and pressing. About 900 ml of boiling decoction were obtained from this operation.

2bb. The drug Helichrysum italicum cut for infusion (250 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was boiled in an open container with 2 liters of water, i.e. in a drug:water ratio of 1 :8. for 1 .5 hours. The boiled Helichrysum italicum product was then separated by decantation, filtration and pressing. About 900 ml of boiling decoction were obtained from this operation.

2bc. The drug Helichrysum italicum cut for infusion (250 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was boiled in an open container with 2 liters of water, i.e. in a drug:water ratio of 1 :8. for 2.5 hours. The boiled Helichrysum italicum product was then separated by decantation, filtration and pressing. About 900 ml of boiling decoction were obtained from this operation.

The boiled Helichrysum italicum product was then stored or dried as described hereinafter.

2c. Preparation of dry boiled Helichrysum italicum product of the invention

The Helichrysum italicum product obtained from 2aa, 2ab or 2ac or 2ba, 2bb or 2bc was dried by placing it lying on a surface in such a way as to form a layer of thickness of less than 1 cm in a warm environment (30-50 °C), without direct contact with sunlight for 2 weeks. Starting from 250 g of Helichrysum italicum subjected to hydrodistillation (2aa), (2ab), (2ac) or boiling in an open container (2ba), (2bb), (2bc), about 240 g of dry Helichrysum italicum product were obtained.

2d. Preparation of the boiling Helichrysum italicum decoction of the invention The drug Helichrysum italicum cut for infusion (250 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was subjected to hydrodistillation for 2.5 hours with 2 liters of water, with a drug:water ratio of 1 :8. Alternatively, the drug Helichrysum italicum cut for infusion (250 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was boiled in an open container with 2 liters of water, i.e. in a drug:water ratio of 1 :8. for 2.5 hours. The boiled Helichrysum italicum product was then separated by decantation, filtration and pressing. About 900 ml of boiling decoction were obtained from this operation, which was stored frozen, in this case in portions of 100-200 ml.

2e. Preparation of the hydroalcoholic Helichrysum italicum extract called "Extract 3"

The drug Helichrysum italicum cut for infusion (40 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was placed in the Clevenger apparatus for hydrodistillation 500 ml of distilled water were added. The hydrodistillation was started and allowed to continue for 2 hours. The mixture consisting of the boiled Helichrysum italicum product and the boiling decoction was placed in a flask and evaporated to dryness. 280 ml of absolute EtOH were added to the residue. The maceration was continued for 7 days at room temperature in the dark. After the indicated time, the resulting extract was filtered, pressed and evaporated to dryness in a rotary evaporator at 40 °C and at reduced pressure.

2f. Preparation of the hydroalcoholic Helichrysum italicum extract called "Extract 17febA"

The drug Helichrysum italicum cut for infusion (40 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was subjected to hydrodistillation with water (500 ml) acidified to pH = 4 with powder citric acid or, alternatively, it was boiled in an open container. The boiling was continued for 2 hours, after which the mixture was concentrated almost to dryness. Ethanol at 95° in a 1 :3 ratio at room temperature was added to the mixture concentrated to dryness, consisting of the boiled Helichrysum italicum product and the decoction, and maceration was continued for 21 days. After the indicated time, the resulting extract was filtered, pressed and evaporated to dryness in a rotary evaporator at 40 °C and at reduced pressure. 2g. Preparation of the hydroalcoholic Helichrysum italicum extract called "Extract 17febC"

The drug Helichrysum italicum cut for infusion (10 g), consisting of winter Helichrysum italicum harvested in the geographical area of Vecchiano (Pisa, Italy), was subjected to hydrodistillation or, alternatively, was boiled with water (125 ml) in an open container. The boiling was continued for 30 minutes, after which the mixture was concentrated almost to dryness. Ethanol at 90° in a 1 :3 ratio at room temperature was added to the mixture consisting of the boiled Helichrysum italicum product and the decoction, and maceration was continued for 21 days. After the indicated time, the resulting extract was filtered, pressed and evaporated to dryness in a rotary evaporator at 40 °C and at reduced pressure. 2h. Preparation of the hydroalcoholic Helichrysum italicum extract called "Extract 7febA"

The drug Helichrysum italicum cut for infusion (40 g) consisting of dry Helichrysum italicum cultivated in Sardinia in the farm Erboristeria Officinale di Daniele Patrizia was subjected to hydrodistillation with water (500 ml) or, alternatively, it was boiled in an open container. The boiling was continued for 2 hours, after which the mixture was concentrated almost to dryness. Ethanol at 95° in a 1 :3 ratio at room temperature was added to the mixture consisting of the boiled Helichrysum italicum product and the decoction, and maceration was continued for 21 days. After the indicated time, the resulting extract was filtered, pressed and evaporated to dryness in a rotary evaporator at 40 °C and at reduced pressure.

2i. Preparation of the Helichrysum italicum extract in jojoba wax, called "Macerated oil 7A"

1 g of dry boiled Helichrysum italicum product obtained from Example 2c was macerated with 7 g of jojoba wax for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2I. Preparation of the alcoholic Helichrysum italicum extract called "Extract 13" 10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of alcohol at 95° for 3 months at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2m. Preparation of the hydroalcoholic Helichrysum italicum extract "Extract 14" 10 g of dry boiled Helichrysum italicum product of Example 2c admixed with 40 ml of boiling decoction of Example 2d were macerated with 80 g of alcohol at 95° for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2n. Preparation of the glycerinated Helichrysum italicum extract "Extract 9" 10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of glycerin (Zeta Farmaceutici) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed. 2p. Preparation of the hydroglycerinated Helichrysum italicum extract "Extract 10" 10 g of dry boiled Helichrysum italicum product of Example 2c admixed with 40 ml of boiling decoction of Example 2d were macerated with 80 g of glycerin (Zeta Farmaceutici) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2q. Preparation of the glycolic Helichrysum italicum extract "Extract 1 1 "

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of propylene glycol (Sigma Aldrich) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2r. Preparation of the hydroglycolic Helichrysum italicum extract "Extract 12"

10 g of dry boiled Helichrysum italicum product of Example 2c admixed with 40 ml of boiling decoction of Example 2d were macerated with 80 g of propylene glycol (Sigma Aldrich) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2s. Preparation of the Helichrysum italicum extract in jojoba wax "Macerated oil 1 "

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of jojoba wax (I Provenzali, cold squeezed) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2t. Preparation of the macerated oil Helichrysum italicum extract in almond oil "Macerated oil 2"

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of almond oil (Aboca, cold squeezed, food use) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2u. Preparation of the macerated oil Helichrysum italicum extract in hemp oil "Macerated oil 3"

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of hemp oil (Prodigi della Terra, cold squeezed, organic, food use) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2v. Preparation of the macerated oil Helichrysum italicum extract in corn germ oil "Macerated oil 4"

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of corn germ oil (Crudigno, cold squeezed, organic, food use) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2w. Preparation of the macerated oil Helichrysum italicum extract in extra virgin olive oil "Macerated oil 5"

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of extra virgin olive oil (Accademia Olearia, cold squeezed, food use) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2x. Preparation of the macerated oil Helichrysum italicum extract in sunflower seeds oil "Macerated oil 6"

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of sunflower seeds oil (Sabo, cold squeezed, organic, food use) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2y. Preparation of the macerated oil Helichrysum italicum extract in sesame oil "Macerated oil 7"

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of sesame oil (Sabo, cold squeezed, organic, food use) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed.

2z. Preparation of the macerated oil Helichrysum italicum extract in peanut seeds

011 "Macerated oil 8"

10 g of dry boiled Helichrysum italicum product of Example 2c were macerated with 80 g of peanut seeds oil (Conad, food use) for 1 month at room temperature and in the dark. After the indicated time, the resulting extract was filtered and pressed. Example 3: determination of the allergen content

The content of allergens, specifically linalool, eugenol and limonene, was determined for the following products:

reference product of Example 1 a: drug Helichrysum italicum cut for infusion reference product of Example 1 b: Helichrysum italicum decoction

reference product of Example 1 c: macerated oil in reference jojoba wax reference product of Example 1 e: OTI Helichrysum italicum mother tincture Invention product of Example 2c: dry boiled Helichrysum italicum product product of the invention of Example 2e: hydroalcoholic Helichrysum italicum extract "Extract 3"

product of the invention of Example 2f: hydroalcoholic Helichrysum italicum extract "Extract 17febA"

product of the invention of Example 2g: hydroalcoholic Helichrysum italicum extract "Extract 17febC"

product of the invention of Example 2h: hydroalcoholic Helichrysum italicum extract "Extract 7febA"

They were analyzed for their content of known fragrance allergens from Helychrisum italicum such as limonene, linalool and eugenol.

This determination was carried out at LabAnalysis Sri, Casanova Lonati (Pavia, Italy) according to the protocol described hereinafter.

Protocol:

The determination of the analytes limonene, linalool and eugenol was carried out using the HeadSpace solid phase microextraction technique and gas chromatography analysis associated with a mass spectrometer (HS-SPME-GC- MS). Calibration curves are constructed for the quantification of the analytes. Preparation of standards for the construction of the calibration curves

For solid samples, standards were dissolved in methanol to prepare stock solutions containing respectively 5, 10, 20, 2, 50 and 100 mg/l of each analyte (Sigma Aldrich). 100 mg/l of a chlorobenzene-d5 solution (internal standard) 1000 mg/l were added to each of these solutions. 10 μΙ of each stock solution were taken and placed in vials with a 20 ml head space, sealed immediately with a cap fitted with a septum. Standards containing 20, 50, 100, 200, 500 and 1000 ng of each analyte and 1000 ng of chlorobenzene-d5, respectively, were thus prepared.

Each standard was incubated at 40 °C for 30 minutes, after which an SPME fiber was introduced through the vial septum and exposed in the head space. The exposure was continued for 30 minutes at 40 °C, then the fiber was placed in the injector of a GC-MS for instrumental analysis.

For liquid samples, the standards were dissolved in 50% ethanol/water at concentrations of 1 , 2, 5, 10 mg/l. Of each solution, 5 ml were placed in a head space vial and 25 μΙ of a dichlorobenzene-d5 (internal standard) 1000 mg/l solution were added. The sealed vial was incubated at 40 °C for 30 minutes, after which an SPME fiber was introduced through the vial septum and exposed in the head space. The exposure was continued for 30 minutes at 40 °C, then the fiber was placed in the injector of a GC-MS for instrumental analysis.

The construction of the calibration curves was carried out by forced linear regression through the origin by the least-squares method, interpolating the response factors obtained from the ratio between the area of the analytes and the area of the internal standard.

Conditions:

- injector: splitless 240 °C;

- column: SUPELCOWAX™ 10, 60m x 0.25mm x Ο.δμιτι film thickness;

- carrier gas: He, 1 .2 ml/min;

- oven temperature: initial isotherm 40 °C for 1 minute, 10 °C/min up to 280 °C, final isotherm for 10 minutes;

- detector: mass spectrometer with El source, full scan 35 m/z-400.

Preparation of the samples

The solid samples, respectively of the products indicated above in examples 1 a (drug Helichrysum italicum cut for infusion), 2c (dry boiled Helichrysum italicum product), 2e (hydroalcoholic Helichrysum italicum extract "Extract 3"), 2f (hydroalcoholic Helichrysum italicum extract "Extract 17febA"), 2g (hydroalcoholic Helichrysum italicum extract "Extract 17febC") and 2h (hydroalcoholic Helichrysum italicum extract "Extract 7febA") were prepared by weighing an amount ranging from 20 to 500 mg, depending on the sample availability, in a head space vial. 10 μΙ of a chlorobenzene-d5 100 mg/l solution were added and the vials were sealed immediately. Each sample was incubated at 40 °C for 30 minutes, after which an SPME fiber was introduced through the vial septum and exposed in the head space. The exposure was continued for 30 minutes at 40 °C, then the fiber was placed in the injector of a GC-MS for instrumental analysis. The liquid samples respectively of the products indicated above in examples 1 b (Helichrysum italicum decoction) and 1 e (OTI Helichrysum italicum mother tincture) were prepared by taking 5 ml and placing them in a head space vial. 25 μΙ of dichlorobenzene-d5 1000 mg/l were added. The vial was sealed and incubated at 40 °C for 30 minutes, after which an SPME fiber was introduced through the septum and exposed in the head space. The exposure was continued for 30 minutes at 40 °C, then the fiber was placed in the injector of a GC-MS for instrumental analysis.

The following Table 1 shows the concentration of the three allergens limonene, linalool and eugenol for each product analyzed.

Table 1 : concentration of Helichrysum italicum allergens

The table above shows that the products of invention had a particularly low allergen content, in most cases less than that of the reference products.

Example 4: determination of the phenolic/polyphenolic antioxidant compounds The total polyphenol content, as well as the content of the following phenols and polyphenols: gallic acid, 4-hydroxybenzoic acid, epicatechin, caffeic acid, chlorogenic acid, benzoic acid, rutin and quercetin, were determined for the following products:

Protocol

The samples of drug Helichrysum italicum cut for infusion and of dry Helichrysum italicum product were prepared by extracting 5 g of sample with 100 ml of methanol in ultrasounds for 30 minutes, then continuing the extraction at room temperature overnight. An aliquot of each extract in methanol was then analyzed by HPLC-UV, under the following conditions:

- column: SunFire C18, 2.1 mm x 100 mm, 3.5 μιτι film;

- injected volume: 15 μΙ;

- eluent A: acidic water (2% acetic acid);

- eluent B: acetonitrile/acidic water (0.5% acetic acid) 1 :1 ;

- flow rate: 1 .2 ml/min;

- elution gradient: 0 to 2 minutes 90% A, 15 minutes at 0% A, 5 minutes 0% A; - UV detector: 280 nm.

The various extracts were prepared by extracting about 20 mg of sample in 2 ml of methanol in ultrasounds. The resulting extracts were filtered and subjected to HPLC-UV analysis in the above conditions. The Helichrysum italicum decoction 5 was diluted in methanol before analysis.

The polyphenols searched using external reference standards were: 4- hydroxybenzoic acid, gallic acid, epicatechin, caffeic acid, chlorogenic acid, benzoic acid, rutin, quercitin. The compounds searched and optionally contained in the samples were quantified following the analysis of a standard solution of 0 about 50 mg/l containing all the above compounds, so as to determine the response factor of each analyte to be then used for quantification. Any other compounds found were quantified using the response factor of quercetin.

The results obtained are shown in the following table 2.

Table 2

Total 1 .14 4.68 2.28 0,224 0.0552 29.6 20.4 18.2 19.5 polyphenols

mg/l mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

Total 1 1400 468000 22800 2240 552 296000 204000 182000 195000 polyphenols

These results show that the products of the invention have a particularly high total content of polyphenols, in most cases higher than the reference products. Moreover, each product of the invention has a peculiar profile (fingerprint) of 5 specific phenolic and polyphenolic compounds. Note that the sample of Example 2c of the invention has a lower total polyphenol content than the untreated drug Helichrysum italicum. This indicates that a certain loss of polyphenols occurs during the deterpenization step a), which however is much lower than the (desired) reduction of volatile substances and allergens, resulting in a very0 favorable overall ratio between polyphenol content and allergene/volatile substance content, particularly for a dermatological use of Helichrysum italicum- based products.

Example 5: evaluation of the ratio between polyphenols/phenols and allergens In fact, as is apparent from the following table 3, the object of the invention was5 achieved since the products of the invention are characterized by a very high ratio of polyphenols/phenols compared to allergens, eugenol, limonene and linalool, certainly higher than the reference products. The higher this ratio, the more favorable the therapeutic index of the Helichrysum italicum product. 0 Table 3

decoction

Ex. 1 e: OTI Helichrysum >1600 >1723 >1400

italicum mother tincture

Ex. 1 c: Macerated oil in 329 103 1 15

reference jojoba wax

Ex. 2e: extract 3 >209286 >225385 >183125

Ex. 2f: extract 17febA 60000 >170000 >127500

Ex. 2g: extract 17febC >202222 >227500 >182000

Ex. 2h: extract 7febA >21 6667 >243750 >195000

Table 3 shows that the products of invention (Ex. 2e, 2f, 2g and 2h) have a ratio of total content of polyphenols/phenols with respect to allergen content that is higher than the reference products. To be noted, the intermediate product of invention 2c has a ratio of total content of polyphenols/phenols with respect to allergen content that is higher than the starting drug (Ex. 1 a), meaning that the boiling step of the present method successfully decreased the allergen content leaving nearly unchanged the polyphenol/phenol content.

Example 6: evaluation of the effects and of the antioxidant properties of the products of the invention and those of reference

The following product samples were prepared:

- Samples with a known antioxidant activity:

galangin, chrysin, caffeic acid, chlorogenic acid, gallic acid, lipoic acid, tocopherol, ascorbic acid (all bought from Sigma-Aldrich)

- Samples of reference products, in particular the following ones:

reference product of Example 1 b: Helichrysum italicum decoction

reference product of Example 1 c: macerated oil in reference jojoba wax reference product of Example 1 e: OTI Helichrysum italicum mother tincture

- Samples of products of the invention:

product of the invention of Example 2e: hydroalcoholic Helichrysum italicum extract "Extract 3"

product of the invention of Example 2f: hydroalcoholic Helichrysum italicum extract "Extract 17febA" product of the invention of Example 2g: hydroalcoholic Helichrysum italicum extract "Extract 17febC"

The power of the antioxidant/free radical scavenging activity, hereinafter referred to as "antioxidant action" for simplicity, was evaluated for the above samples through assay with the radical DPPH.

Protocol

In this assay, a chemical test was used where the decay of the radical diphenyl picrylhydrazyl (DPPH) in the absence or in the presence of antioxidants is evaluated. This radical, in fact, is used to test the ability of substances to act as scavengers of free radicals. DPPH in solution has a purple color which changes into yellow-colorless when this radical extracts a hydrogen atom from a radical scavenger to give rise to the reduced form DPPH-H.

The assay was performed in 96-well microplates (Tissue Culture Plate 96-well flat bottom cell+ of SARSTEDT) according to the following procedure. 20 μΙ of sample solutions under examination were placed into each well of a microplate, named "antioxidant reaction cell", at the desired concentration in the reaction medium, which consists in one case of ethanol and in one case of phosphate buffered saline (PBS). Then, 20 μΙ of an ethanol solution of 200 μΜ DPPH were added, obtaining a final concentration of DPPH in the antioxidant reaction cells of 100 μΜ. Immediately after the addition of the radical DPPH, a photographic survey of the antioxidant reaction cells was carried out as explained hereinafter in more detail. 20 minutes after the first addition of DPPH, another 20 μΙ of 200 μΜ ethanol solution of the same reagent were then added, obtaining a final concentration of DPPH of 133 μΜ in the antioxidant reaction cells. The microplate was subjected to a second photographic survey, after which it was kept at rest at room temperature in the dark for 24 hours. After such a time, another 20 μΙ of an ethanol solution of 200 μΜ DPPH were finally added, obtaining a final concentration of DPPH of 150 μΜ in the antioxidant reaction cells. The microplate was then subjected to a third and final photographic survey. The assay with the radical DPPH for antioxidant activity, which uses a photographic survey, was previously validated by a similar assay associated with spectrophotometric detection, which is a common and consolidated method. An excellent correlation, in terms of EC50 (Mg/ml) and Hill coefficient, was observed in the two techniques.

Photographic survey

The microplate was photographed using a Samsung Galaxy Ace 2 smartphone with built in 5.0 megapixel camera.

Then, the photographs of the microplate were imported into the GIMP 2.6.10. software and processed as follows:

- rectangular selection of area of the microplate,

- selection of the blue channel,

- tone adjustment to the maximum and minimum brightness,

- analysis of the single reaction cells of the microplate using the elliptical selecrion tool,

- registration of the mean values of the three-color blue channel in its lower tone peak in the histogram representing the number of pixels compared to the brightness,

- calculation of the logarithm of the absolute three-color blue value recorded in the preceding step,

- normalization with respect to a reference positive control (100% of antioxidant activity, consisting of the sample under examination to which ascorbic acid was added at a concentration in the assay of 20 mg/ml) and to a reference negative control (0% of antioxidant activity, consisting of the reaction medium alone, i.e. ethanol or PBS). The parameter thus obtained was defined as "DPPH blue intensity".

Quantification of the antioxidant activity power

The samples were evaluated at various concentrations obtained following serial dilutions and the antioxidant activity was expressed in terms of ECso (Mg/ml) and Hill coefficient calculated from the dose-response curve with variable slope generated by interpolation with a non-linear regression model (GraphPAD Prism software version 4.00 (GraphPAD, San Diego, CA, USA)). "Dose" means the concentration of the test compound expressed as -log (mg/ml), whereas "response" means the value of the "DPPH blue intensity" parameter. This quantification was carried out for each of the 3 DPPH concentrations in the assay (100, 133 and 150 μΜ).

Then, the ECso (Mg/ml) and Hill coefficient parameters of the dose-response curves were calculated, which allowed giving a quantitative estimate of the antioxidant activity of each of the samples tested. Such ECso values, expressed as g/ml, and of Hill coefficient for each compound with known antioxidant activity tested in the assay with the radical DPPH at concentrations of 100 μΜ, 133 μΜ and 150 μΜ are shown in Table 4, while the values obtained for the Helichrysum italicum products, both of reference and of the invention, are shown in Table 5.

Table 4: ECso (Mg/ml) and Hill coefficient parameters for compounds with known antioxidant activity obtained by assay for determining the antioxidant activity with the radical DPPH used at the concentrations of 100 μΜ, 133 μΜ and 150 μΜ, by dissolving the compounds in PBS and carrying out a photographic survey of the antioxidant reaction cells.

Table 5: ECso (Mg/ml) and Hill coefficient parameters for the Helichrysum italicum products, both of reference and of the invention, obtained by assay for determining the antioxidant activity with the radical DPPH used at the concentrations of 100 μΜ, 133 μΜ and 150 μΜ, by dissolving the compounds in

PBS and carrying out a photographic survey of the antioxidant reaction cells.

The evaluation of the ECso values for each compound with known antioxidant activity (Table 4) shows that the compounds ascorbic acid, caffeic acid, chlorogenic acid, gallic acid, chrysin and galangin have a much more powerful antioxidant activity than tocopherol and lipoic acid. This is in accordance with the data found in the literature. In particular, galangin, chlorogenic acid and ascorbic acid stand out among the most active compounds. The Helichrysum italicum products of the invention have an antioxidant activity comparable to, and in some cases even higher than, compounds with a known antioxidant activity such as chrysin, galangin, caffeic acid and chlorogenic acid, known to be contained in Helichrysum italicum itself. This may indicate a possible presence of synergism between the various antioxidant components present in the extracts of the invention, when present simultaneously.

Example 7: long-term stability of the antioxidant/reducinq effect of Helichrysum italicum extracts in jojoba and in PBS, evaluated by assay with DPPH

The stability of the antioxidant effect over time was evaluated for the following samples:

- macerated oil obtained by placing Extract 3 (Example 2e) in contact with jojoba wax at a ratio of 10 mg of Extract 3 in 1 ml of jojoba wax.

- macerated oil 7 A (Example 2i)

- Extract 3 dissolved in PBS at a concentration of 3 g/ml

- jojoba wax

- PBS

- ascorbic acid

Each sample was prepared at time 0 and stored in Eppendorf tubes at room temperature in the dark for 6 months.

Protocol

The assay was carried out in 0.5 ml microtubes (SARSTEDT). In more detail, the assay was carried out according to the following procedure. 5 μΙ of sample under examination were placed in a microtube, called "antioxidant reaction cell", at a double concentration with respect to the final concentration of the product in the assay (2x) in jojoba or in PBS. Then, 10 μΙ of an ethanol solution of 200 μΜ DPPH were added, followed by photographic survey as described in Example 5. This assay was carried out on samples at to (immediately after dispersion of the extracts in the carrier) and after 3 months and 6 months of storage.

Evaluation of the antioxidant activity

The antioxidant activity is considered as:

- antioxidant, if the substance showed a complete discoloration in a manner similar to the reference positive control (100% antioxidant activity)

- partially antioxidant, if the substance showed a partial discoloration compared to the reference positive control (100% antioxidant activity)

- non-antioxidant, if the substance showed no discoloration compared to the reference negative control (0% antioxidant activity, consisting of jojoba wax or

PBS alone).

The following Table 6 shows the trend of the antioxidant activity over time, particularly at to (immediately after dispersion of the extracts in the carrier), at t1 = 3 months and at t2 = 6 months, of the samples Extract 3 in jojoba wax, Extract 3 in PBS, Macerated oil 7A, jojoba wax, PBS and ascorbic acid.

Table 6

^*negative control

^**positive control, prepared at the time of use The results in the above Table 6 show that the antioxidant activity of the products of the invention remained stable over time up to 6 months from the preparation of the extracts.

Example 8: preliminary sensory evaluation of the fragrance of Helichrysum italicum extracts

The following samples were used for the preliminary olfactory sensory evaluation: reference product of Example 1 a: drug Helichrysum italicum cut for infusion reference product of Example 1 b: Helichrysum italicum decoction

reference product of Example 1 c: macerated oil in reference jojoba wax reference product of Example 1 e: OTI Helichrysum italicum mother tincture product of the invention of Example 2e: hydroalcoholic Helichrysum italicum extract "Extract 3"

product of the invention of Example 21 : alcoholic Helichrysum italicum extract "Extract 13"

product of the invention of Example 2m: hydroalcoholic Helichrysum italicum extract "Extract 14"

product of the invention of Example 2n: glycerinated Helichrysum italicum extract "Extract 9"

product of the invention of Example 2p: hydroglycerinated Helichrysum italicum extract "Extract 10"

product of the invention of Example 2q: glycolic Helichrysum italicum extract "Extract 1 1 "

product of the invention of Example 2r: deterpenized hydroglycolic Helichrysum italicum extract "Extract 12"

product of the invention of Example 2s: Helichrysum italicum extract in jojoba oil "Macerated oil 1 "

product of the invention of Example 2t: macerated oil Helichrysum italicum extract in almond oil "Macerated oil 2"

product of the invention of Example 2u: macerated oil Helichrysum italicum extract in hemp oil "Macerated oil 3"

product of the invention of Example 2v: macerated oil Helichrysum italicum extract in corn germ oil "Macerated oil 4"

product of the invention of Example 2w: macerated oil Helichrysum italicum extract in extra virgin olive oil "Macerated oil 5" product of the invention of Example 2x: macerated oil Helichrysum italicum extract in sunflower seeds oil "Macerated oil 6"

product of the invention of Example 2y: macerated oil Helichrysum italicum extract in sesame oil "Macerated oil 7"

product of the invention of Example 2z: macerated oil Helichrysum italicum extract in peanut seeds oil "Macerated oil 8"

Protocol

The preliminary sensory analysis was carried out by two operators by evaluating the intensity of the smell and the presence of any particular olfactory notes. The test was carried out on the products as such and, in the case of extracts, after applying a small amount thereof on the skin of the forearm and waiting until the evaporation of the extraction solvent, if any.

Table 7 shows comments on the intensity and any olfactory notes detected.

Table 7. Preliminary evaluation of the intensity of the smell and any olfactory notes detected for Helichrysum italicum products both of reference and of the invention

Helichrysum italicum

Extract 17febC weak smell of dried fruits (walnuts) and jam with very weak scent of Helichrysum italicum

Extract 7febA weak smell of dried fruits (walnuts) and jam with very weak scent of Helichrysum italicum

Extract 13 weak and sweet smell of dried fruits

(raisins) with very weak scent of Helichrysum italicum

Extract 14 weak and sweet smell of dried fruits

(raisins) with very weak scent of Helichrysum italicum

Extract 9 weak smell of dried fruits (walnuts) with very weak scent of Helichrysum italicum

Extract 10 smell similar to Extract 9, but slightly more intense

Extract 1 1 weak smell of dried fruits (walnuts) with very weak scent of Helichrysum italicum

Extract 12 weak smell of dried fruits (walnuts) with very weak scent of Helichrysum italicum

Macerated oil 1 the predominant smell is that of jojoba wax, combined with a very weak scent of dried fruit and Helichrysum italicum

Macerated oil 2 a scent of dried fruit and Helichrysum italicum is combined with the smell of almond oil Macerated oil 3 the predominant smell is the quite intense one of hemp oil combined with a very weak scent of dried fruit

Macerated oil 4 the predominant smell is that of corn germ oil, combined with a very weak scent of dried fruit and Helichrysum italicum

Macerated oil 5 the predominant smell is that of olive oil combined with a very weak scent of dried fruit

Macerated oil 6 the predominant smell is that of sunflower seeds oil combined with a very weak scent of dried fruit

Macerated oil 7 the predominant smell is the intense one of sesame oil combined with a very weak scent of dried fruit

Macerated oil 8 a scent of dried fruit and Helichrysum italicum is combined with the smell of peanut oil

Results

These preliminary results showed that the products of the invention have a softer smell intensity compared to the reference products; moreover, each product of the invention has its own specific olfactory note that is pleasant. The olfactory notes resemble little smelling dried fruits (walnuts, chestnuts), except in the case where has alcohol is present as extraction solvent, in which case there are hints of sweeter dried fruits (raisins, plums). Generally, the olfactory notes of Helichrysum italicum and/or fruit in macerated oils are so faint as to be prevailed over by the smell of the same oils used, except in the case of almond and peanut oils.

Example 9: evaluation of the efficacy in the treatment of atopic dermatitis in pediatric subjects

The sample tested was Macerated oil 1 of Example 2s.

Description of the study

A case study carried out on a pediatric (4 years old) subject with non- impetiginized atopic dermatitis treated for 3 days without success with emollients (jojoba wax) was carried out.

Treatment with Macerated oil 1 led to the complete regression of symptoms (redness and itching) of atopic dermatitis and to the restoration of normal skin conditions, following two applications per day for two days on the affected area (face). In previous episodes, conditions of the same extent in the same subject, i.e. wherein treatment with emollient jojoba wax alone had not been effective, had been treated successfully with topical cortisone.

Results

In this case, the Macerated oil 1 showed higher efficacy than jojoba wax and comparable to cortisone products.

Claims

1 . A process for the preparation of an extract of Helichrysum italicum comprising the steps of:

4) extracting the boiled Helichrysum italicum product or the dry product of Helichrysum italicum with a solvent selected from the group consisting of water, ethanol, propylene glycol, glycerin, jojoba wax and derivatives thereof, a vegetable oil and mixtures thereof.

2. The process of claim 1 , wherein the boiling step 1 ) is carried out for a time period in the range from 1 hours to 1 .5 hours.

3. The process of claim 1 or 2, wherein the boiling step 1 ) is carried out through hydrodistillation or through boiling in an open container.

4. The process of claim 3, wherein the boiling step 1 ) is carried out in acidic environment, preferably in the range of pH from 2 to 6.

5. The process according to anyone of claims 1 -4, wherein the separation step 2) of the aqueous solution from the boiled Helichrysum italicum product is carried out through filtration and pressing.

6. The process according to anyone of claims 1 -5, wherein the drying step 3) is carried out in a suitable oven.

7. The process according to anyone of claims 1 -6, wherein the solvent or the solvent mixture of step 4) is selected from the group consisting of water/ethanol, ethanol, water/propylene glycol, propylene glycol, water/glycerin, glycerin, jojoba wax and its derivatives and a vegetal oil.

8. An extract of Helichrysum italicum obtainable from the process of anyone of claims 1 -7.

9. The extract according to claim 8, wherein said extract or the Helichrysum italicum product of step 1 ) show a ratio of the amount of total polyphenols/phenols with respect to the amount of limonene higher than 10,000, a ratio of the amount of total polyphenols/phenols with respect to the amount of linalool higher than 10,000 and a ratio of the amount of total polyphenols/phenols with respect to the amount of eugenol higher than 10,000.

10. The extract according to claim 8, wherein said extract or the Helichrysum italicum product of step 1 ) show a ratio of the amount of total polyphenols/phenols with respect to the amount of limonene higher than 50,000, a ratio of the amount of total polyphenols/phenols with respect to the amount of linalool higher than 50,000 and a ratio of the amount of total polyphenols/phenols with respect to the amount of eugenol higher than 50,000.

1 1 . A cosmetic composition comprising the extract of Helichrysum italicum of anyone of claims 8-10 and a cosmetically acceptable carrier.

12. A pharmaceutical composition comprising the extract of Helichrysum italicum of anyone of claims 8-10 and a pharmaceutically acceptable carrier.

13. An extract of Helichrysum italicum according to anyone of claims 8-10 for use in the treatment of atopic dermatitis.

14. A food or a food supplement comprising the extract of Helichrysum italicum according to anyone of claims 8-10 and, optionally, a food-grade carrier.