ITPI20110101A1 - HELICHRYSUM EXTRACT IN JOJOBA OIL AND COMPOSITIONS BASED ON SUCH EXTRACT, IN PARTICULAR, TO TREAT SKIN CONDITIONS - Google Patents

Description

Description of the industrial invention entitled: â € œEXTRACT OF HELICHRYSUM IN JOJOBA OIL OR WAX AND COMPOSITIONS BASED ON THIS EXTRACT, IN PARTICULAR, TO TREAT SKIN CONDITIONSâ €,

DESCRIPTION

Scope of the invention

The present invention relates to an extract of a plant of the genus Helichrysum (helichrysum) in jojoba oil and a method for producing this extract.

Furthermore, the invention relates to a medicament, medical device or cosmetic containing an extract of a plant of the genus Helichrysum in jojoba oil suitable for the treatment of diseases and / or particular conditions of the skin of a human being or an animal. .

The invention also refers to a composition containing such an extract, having an insect repellent effect.

Antecedents of the invention

In the past, many attempts have been made to develop synthetic drugs or remedies of natural origin for various skin conditions. However, extracts obtained from different types of plants, or from different parts of the same plant, often differ greatly in composition and effects.

In particular, the use in herbal medicine and cosmetics of compositions containing derivatives of plants of the genus Helichrysum and jojoba wax is known.

Helichrysum

The Helichrysum genus belongs to the Asteraceae family and includes about six hundred species of flowering plants. Various species of helichrysum are known which contain substances potentially useful for treating skin diseases, for example substances with antioxidant power. Among these species, Helichrysum italicum or angustifolium, which grows spontaneously in southern Europe, finds particular use. In particular, in Italy it is common in the center, in the south and in the islands. It grows in rocky places, up to about 800 meters in height. It has a bushy habit, green-silver in color, and reaches a height of 30-40 cm. It blooms from late spring to early autumn. The flowers are gathered in flower heads with an external envelope consisting of dark yellow petals and a central conical receptacle that houses the individual flowers, which have a light yellow color and are tubular in shape. Helichrysum italicum contains various types of substances such as volatile compounds, flavonoids, terpenoids and others. Other species belonging to the genus Helichrysum that contain substances potentially useful for treating skin diseases are, for example, H. frigidum, H. litoreum, H. melitense, H. montelinasanum, H. nebrodense, H. rupestre (var. Errerae) , H. rupestre (var. Melitense), H. rupestre (var. Messerii), H. rupestre (var. Pendulum), H. rupestre (var. Rupestre), H. saxatile, H. siculum, H. stoechas.

Chrysin and other flavonoids

Also known are the antioxidant, anti-inflammatory, antimicrobial properties of flavonoids such as Crisina, Galangina, Pinocembrina, known to be present in many plants of the genus Helichrysum.

More in detail, Chrysin (5,7-Dihydroxy-2-phenyl-4H-benzo [b] pyran-4-one) can attenuate apoptosis, the production of reactive substances with oxygen (ROS), and also the expression of cyclooxygenase 2 (COX-2) induced by UVB and UVA. Chrysin predominantly reverses the down-regulation of aquaporin 3 (AQP-3) by UVB. It predominantly reverses the activation of JNK and weakly inhibits the activation of p38 caused by UVA and UVB. The topical application of Chrysin is followed by an effective percutaneous absorption and does not cause skin irritation. Overall, the results demonstrated significant benefits of Chrysin on the protection of keratinocytes against lesions induced by cytotoxic factors such as UVA and UVB. See Wu, Nan-Lin et al., Chrysin Protects Epidermal Keratinocytes from UVA- and UVB-Induced Damage, Journal of Agricultural and Food Chemistry, 2011 59 8391â € “8400.

FR 2906716 describes the use of Chrysin in cosmetic compositions for the treatment of oily skin, and its effectiveness in inhibiting the proliferation of human sebocytes.

US 2005/0049206 describes a group of flavonoids, and extracts containing them, which have pharmaceutical properties useful in the therapy of fibrotic diseases to protect or treat or repair fibrotic lesion tissues.

European Journal of Medicinal Chemistry, 46, 1, 2011, 393-398 treats Chrysin and its analogues as immunosuppressive agents and potentially useful in diseases such as psoriasis.

Volatile compounds of Helichrysum

WO 2007/066305 describes analgesic effects in the human or animal body of a composition containing one or more sesquiterpenes.

Trans-2-decenal ((E) -2-Decenal) and nonanal aldehydes have insect repellent properties. See, for example, US 2007/0154504, JP 2006/327957, which describes the use against Dermatophagoides farinae, and US 6,524,605 which describes compositions comprising a monoterpenoid or a sesquiterpenoid effective for repelling arthropods such as cockroaches, mosquitoes, ticks, spiders , and others.

In addition, trans-2-decenal and nonanal aldehydes are classified in the CosIng database as cosmetic ingredients, in particular as fragrance agents.

Jojoba

Jojoba oil or wax is obtained from jojoba seeds or Simmondsia chinensis, a shrub-like plant from some desert areas of North America. Jojoba oil is present in the seeds, in a percentage by weight normally higher than 50%. See e.g. D.J. Undersander, et al., Jojoba. Alternative Field Crops Manual, 1990.

Jojoba wax has a structure very similar to human sebum, with which it is completely miscible. Because of this, it is tolerated by human skin much better than other vegetable oils, and does not clog pores. By not evaporating, it provides longer lasting hydration than water-based moisturizing formulations. Furthermore, it is chemically very stable, does not go rancid and does not lose antioxidant agents even after prolonged storage.

Products based on helichrysum and jojoba

An example of Helichrysum composition in jojoba wax is found in the homeopathic product â € œEczema no moreâ €. However, the type of helichrysum used is not known. Furthermore, it seems that essential oil of helichrysum is used, that is a product obtained by steam distillation of helichrysum.

Another example is found in ITPI2001A000056, which describes a method of producing extracts of Helichrysum italicum in jojoba wax, in which helichrysum flower heads are macerated in a jojoba oil, in particular with a Helichrysum: oil ratio between 0, 2 and 0.3. Maceration can be followed by pressing and adding an essential oil of chamomile. However, the chemical composition of the product obtained is not specified, nor that of the starting materials used.

The presence of substances from helichrysum and possibly jojoba oil in preparations for the treatment of skin conditions is also reported in publications KR20110023257, US2004258783A1, WO2008053246A1, GB2472379, EP2018892.

Insect repellent substances

Also known are substances which have a repellent effect for insects. These substances are useful in areas, such as tropical areas, where insects are carriers of dangerous diseases such as malaria, West Nile fever, dengue fever, and others.

As known, the skin comprises a thin superficial layer, the epidermis, which provides resilience and barrier properties, and is made up of many cell types. Keratinocytes make up the majority of cells in the epidermis, in the case of man 75-80%.

In numerous diseases and / or conditions involving the skin it may be desirable to stimulate the proliferation of keratinocyte cells in the epidermis.

In fact, the skin is equipped with regeneration and repair mechanisms that intervene both physiologically and in the presence of particular conditions such as lesions. It is known that the proliferation mechanisms of keratinocytes are involved in numerous diseases and / or skin conditions, such as skin aging, burns, in particular sunburns, and others.

There is therefore a need for a new medicament or cosmetic or medical device that is effective for treating skin diseases and / or conditions such as those listed above. In particular, there is a need for a new medicament or cosmetic or medical device that is capable of stimulating the proliferation of keratinocytes.

There is also a need for a new medicament or cosmetic or medical device that has an insect repellent effect.

Summary of the invention

It is therefore the purpose of the invention to provide an extract of a plant of the genus Helichrysum in jojoba oil that allows to treat various skin conditions, such as skin aging, and burns of various types, more effectively than products and extracts. based on currently known Helichrysum.

It is also the purpose of the invention to provide an extract of a plant of the genus Helichrysum in jojoba oil that allows to stimulate the proliferation of keratinocyte cells of the epidermis more effectively than occurs with the aforementioned known products and extracts, for treat skin conditions.

It is also an object of the invention to provide such an extract which has a lower tendency to cause skin sensitization and / or allergic phenomena than products of the known art.

It is also an object of the present invention to provide a helichrysum extract in jojoba oil which is effective in repelling various types of insects.

It is also an object of the present invention to provide a method for the preparation of such an extract of helichrysum.

These and other purposes are achieved by a method for preparing an extract of a plant of the genus Helichrysum in jojoba oil or wax, this method comprising the steps of:

∠’preparation of a part of the plant chosen from the upper areas of the plant, in particular the flowering tops;

∠’predisposition of a jojoba oil having an acidity higher than 0.3, expressed as an acid number;

â ’contact of said part of the plant of the genus Helichrysum with jojoba oil in conditions of extraction;

∠’recovery of the extract obtained in the contact phase.

By upper aerial parts we mean the non-woody parts of the plant, namely in particular the flower heads, the apical stems, and the first leaves.

The acid number is defined as the amount of potassium hydroxide (KOH) in milligrams that is needed to neutralize one gram of oil.

It has indeed been surprisingly discovered that a more acidic jojoba oil, and therefore considered poorer for many applications of this oil, has a high solubilizing power for flavonoids. In other words, it favors the extraction of flavonoids, increasing their concentration values in the extracts. At the same time, extracts formulated from an acidity oil chosen from those indicated are of better quality, as they have an unexpectedly low level of allergens.

In particular, the acidity is higher than 0.32, more particularly it is higher than 0.36.

This method allows to obtain an extract of a plant of the genus Helichrysum in jojoba oil characterized by the fact that it comprises a higher content of Chrysin (5,7-Dihydroxy-2-phenyl-4H-benzo [b] pyran-4-one) to 80 mg per kg of fatty acids with twenty carbon atoms and having only one unsaturation.

Preferably, the chrysin content is higher than 84 mg / kg of these fatty acids with 20 carbon atoms and a single unsaturation, more preferably it is higher than 104 mg / kg of such fatty acids. Fatty acids with 20 carbon atoms having only one unsaturation form from about 65% to about 80% of jojoba oil.

In fact, the inventors have observed that an extract of helichrysum in jojoba oil is rich in the following flavonoids: Crisina, Galangina, Pinocembrina. As anticipated, the antioxidant, anti-inflammatory and antimicrobial properties of these flavonoids are known.

Furthermore, the inventors have surprisingly found that an extract of helichrysum in jojoba oil that is particularly rich in flavonoids such as Chrysin, Galangin, Pinocembrina, has properties that stimulate the growth of keratinocytes, regardless of the action of cytotoxic factors such as UV radiation.

In the characterization of the extract reference is made to Chrysin, instead of the total content of flavonoids, since Chrysin is the most abundant flavonoid, followed by Galangina and Pinocembrina. However, it is observed that the quantities of these three flavonoids are correlated with each other, in other words there is a substantial proportionality between the concentration of Galangina and Pinocembrina with respect to the concentration of Chrysin. Furthermore, the analytical method for determining the concentration of Chrysin alone leads to less variable results, and therefore more reliable, than the method for quantifying all flavonoids.

This method also allows to obtain an extract of a plant of the genus Helichrysum in oil or in jojoba wax, characterized by a weight ratio of limonene (S - (-) - 4-isopropenyl-1-methyl-1-cyclohexene; R - (+) - 4-isopropenyl-1-methyl-1-cyclohexene) compared to β-selinene ((3S, 4aR, 8aS) -8a-methyl-5-methylidene-3-prop-1-en-2- il-1,2,3,4,4a, 6,7,8-octahydronaphthalene) less than 0.4.

Preferably, the limonene: βselinene weight ratio is lower than 0.08 and even more preferably this weight ratio is lower than 0.06.

Also in this case, to characterize the presence of limonene, β-selinene was chosen as the reference as it is the most abundant component of the volatile fraction of the extract, and thus the method for determining the amount of limonene provides more reliable results. The limonene: β-selinene ratio can be determined as the ratio between the areas of the respective peaks of a trace obtained with a chromatographic analysis.

The inventors have in fact observed that the volatile part of a Helichrysum extract in jojoba wax is particularly rich in: β-selinene; trans-2-decenal; Î ± selinene; AR-curcumene; nonanal, whose analgesic effects are known, as anticipated. Surprisingly, on the contrary, and unlike the essential oil, this extract is particularly low in limonene, a well-known allergenic agent. Furthermore, the inventors have found that the Helichrysum extract in jojoba contains much less limonene than the Helichrysum essential oil.

As anticipated, some of the substances of the volatile part, for example trans-2-decenal, AR-curcumene, nonanal and octanal, are also repellent for insects.

Therefore, the extract according to the invention has a lower tendency to cause skin sensitization and / or allergic phenomena compared to known products.

Furthermore, the extract according to the invention makes it possible to create formulations to treat specific skin conditions that benefit from a stimulating action on the proliferation of keratinocytes that are also hypoallergenic and analgesic.

In particular, the plant of the genus Helichrysum belongs to the Helichrysum italicum species, in particular Helichrysum italicum subspecie italicum, Helichrysum italicum subspecie microphyllum, Helichrysum italicum subspecie serotinum, Helichrysum italicum italicum subspecie italicum variant, Helichrysum italicum variant typic , Helichrysum italicum HyperCreso variant, Helichrysum italicum subspecie italicum Corsican variant.

In particular, the plant of the genus Helichrysum belongs to the Helichrysum stoechas species.

In particular, the plant of the genus Helichrysum belongs to the Helichrysum arenarium species.

As will be described below, the enrichment of compounds such as flavonoids, terpenoids and others, indicated above, with respect to the known Helichrysum extracts and known products based on Helichrysum and jojoba oil, can be obtained by selecting the appropriate part of the plant that is subjected to extraction, in particular using the upper aerial parts, more particularly the flowers, as material from which to obtain the extract.

In addition, or alternatively, enrichment in the compounds indicated above can be obtained by appropriately choosing jojoba oil, in particular by choosing an oil having an acidity higher than 0.3.

In addition, or alternatively, the enrichment in the compounds indicated above can be obtained by performing the extraction, i.e. the contact between the plant of the genus Helichrysum and jojoba oil, with a dynamic operation, for example a percolation and / or centrifugation operation.

In particular, the plant of the genus Helichrysum belongs to a species chosen from:

Helichrysum italicum, in particular Helichrysum italicum subspecie italicum, Helichrysum italicum subspecie microphyllum, Helichrysum italicum subspecie serotinum, Helichrysum italicum subspecie siculum, Helichrysum italicum variant pseudysum italicum, variant Helichrysum italicum variant, Helichrysum italicum variant, Helichrysum italicum variant, Helichrysum italicum variant italicum Corsican variant;

∠’Helichrysum stoechas;

∠’Helichrysum arenarium.

The method can comprise a step of adding pharmaceutically and / or cosmetically acceptable components suitable for increasing the solubility of chrysin and other flavonoids, these components being selected, in particular, from propylene glycol, glycerin and cyclodextrins.

Enrichment in useful active ingredients such as the above mentioned flavonoids can be obtained by choosing the extraction method. The contact phase between the part of Helichrysum italicum and jojoba oil can be performed with any extraction technique known in the art. Advantageously, the contact phase is performed with a technique chosen between maceration and a dynamic technique, in which the jojoba oil is in motion with respect to a mass of helichrysum, chosen between percolation and centrifugation.

Preferably, the contact step is performed with a percolation technique. In fact, with the percolation technique an extract is obtained that has at the same time a higher content of flavonoids and a lower content of limonene compared to the extracts obtainable from the same plant and with the same oil, and by carrying out the other steps of the method under the same conditions. operational.

Advantageously, the contact step is carried out at a contact temperature between 25 ° C and 90 ° C, in particular between 50 ° C and 85 ° C, more particularly between 70 ° C and 80 ° C. In this way it is possible to further raise the solvent power of jojoba oil in relation to the flavonoids present in helichrysum, without compromising its characteristics. In particular, in this way it is possible to obtain concentrations of flavonoids higher than the products of the known art also by carrying out the contact phase in static conditions, for example in maceration conditions.

Advantageously, the contact phase is performed with a quantity of helichrysum between 150 and 350 g on 1000 ml of jojoba oil, in particular between 200 and 320 g on 1000 ml of jojoba oil, more particularly between 220 and 300 g on 1000 ml of jojoba oil.

Advantageously, the step of preparing said part of the plant to be used for extraction comprises a step of drying said part of the plant. Another advantage of using dried aerial parts consists in obtaining an extract substantially free from aqueous humidity. This allows to exploit the antioxidant character of some components of jojoba oil, which do not require the use of preservation compounds such as synthetic antioxidants.

Advantageously, the step of preparing said part of the plant to be used for extraction comprises a step of carrying out an herbal tea cut, that is, a crushing step.

In particular, the extract of the invention can be obtained from only dried Helichrysum flowers, rather than from other parts of the plant. For example, the flowers of Helichrysum italicum preferably come from a spontaneous Italian plant and are collected during the balsamic period.

An advantage of using upper aerial parts and / or a jojoba oil with a high solubilizing power for flavonoids is that an extract is obtained whose volatile fraction includes:

∠’high quantities of flavonoids such as Crisina, Galangina, Pinocembrina, obtaining an extract whose properties stimulating the growth of keratinocytes have been mentioned previously, and in particular,

∠’high quantities of Chrysin, which is a flavonoid whose known photoprotective and cytoprotective properties for keratinocytes have been previously indicated;

∠’high quantities of Galangina and Pinocembrina which are known to be antioxidants, anti-inflammatory and antimicrobial;

∠’high amounts of β-selinene and / or Î ± -selinene and / or trans-2-decenal and / or AR-curcumene and / or nonanal and / or octanal, which are known to repel insects;

- low amounts of limonene, which is an allergen.

The recovery phase of the extract can be performed with any method known in the art, in particular by a technique chosen from pressing, hydraulic pressing, filtration, centrifugation, or a combination of these techniques.

The extract can be used as obtained from the previous phases of processing, or the method can also include a post-treatment phase of the extract recovered in the recovery phase of the extract. In particular, a post-treatment phase can be:

∠’a dilution phase, for example to restore the volume of jojoba oil initially used in the extract;

∠’a sterilization phase;

∠’a phase of further extraction;

∠’a phase of separation of a component.

Regardless of whether one of these post-treatment phases is carried out, the method can also include a phase for adding substances of various kinds. For example, a phase may be chosen between adding further active ingredients.

The method can also include a stage of packaging the Helichrysum extract in a form suitable for distribution.

The Helichrysum italicum extract of the invention can be used as an active ingredient in a medicament or cosmetic, as well as it can be used in a medical device.

Although the Helichrysum italicum extract of the invention can be used as such, it can be incorporated into a medicament or cosmetic with the addition of any pharmaceutically or cosmetically acceptable excipient, for example a filler, a thickener, or other excipient known in the pharmaceutical art or in the cosmetic preparation art.

The invention therefore also includes a medicament or a cosmetic or a medical device containing the extract described above obtained from a plant of the genus Helichrysum in jojoba oil, or an extract obtained with the method described above, to treat diseases and / or conditions for which stimulation of keratinocyte proliferation is useful.

In particular, this medicament or cosmetic or medical device is suitable for treating:

∠’wounds, including cuts, grazes, abrasions, insect bites, stab wounds, gunshot wounds, surgical wounds, etc .;

∠’burns, including burns from ultraviolet radiation, sun burns, radiotherapy burns, open flame burns, burns from contact with hot bodies, burns from acid substances, burns from caustic substances, etc .;

∠’skin ulcers, including pressure sores, sores due to diabetes, etc .;

∠’scars, including post-operative scars, scars due to excessive accumulation of connective tissue as a result of tissue damage, atrophic scars, acne scars, etc .;

∠’skin aging, including aging due to age, aging due to ultraviolet radiation, aging due to environmental pollution, aging due to atmospheric factors, aging due to hormonal factors, etc .;

∠’stretch marks, including stretch marks due to pregnancy, stretch marks due to marked changes in body weight, stretch marks due to hormonal factors, etc; ∠’wrinkles, including expression lines, etc.

Due to the properties of the flavonoids and compounds present in the volatile fraction of the extract, the medicament or cosmetic or medical device can show beneficial effects such as antimicrobial, antibacterial, protective effects against ultraviolet radiation, anti-inflammatory, antioxidants, analgesics, sebum ( re) balancing, i.e. in the treatment of oily seborrhea, in the prevention of the formation of fibrotic lesional tissues, in the prevention of lesions due to psoriasis.

The medicament of the invention can be formulated in any form suitable for any route of administration. The preferred route of administration is any topical route of administration.

The preparation of the medicament or cosmetic can be carried out by means of known pharmaceutical or standard cosmetic techniques. Such techniques are described, for example, in Remington's Pharmaceutical Science, 18th edition, edited by Gennaro et al., 1980, Mack Publishing Co., Easton, PA.

Advantageously, the composition comprises an essential oil. In particular, this essential oil is chosen from: Roman chamomile essential oil; essential oil of matricaria chamomile; carnation essential oil; tea tree essential oil; lavender essential oil; essential oil of oregano, or mixtures thereof. In particular, Roman chamomile, in addition to giving a more pleasant aroma to the composition, has an anti-inflammatory power that enhances the effectiveness of the Helichrysum extract, with a synergistic effect.

The use of the extract described above for the preparation of such a medicament or cosmetic or medical device also falls within the scope of the invention.

An insect repellent composition containing the extract described above from a plant of the genus Helichrysum also falls within the scope of the invention.

The use of the extract described above for the preparation of a repellent composition for insects and / or mites also falls within the scope of the invention.

Brief description of the drawings

The invention will be illustrated below with the following description of its embodiments, given by way of non-limiting example, with reference to the attached drawings in which:

∠’Figures 1a-1d show the analysis of an essential oil of Helichrysum Italicum;

∠’Figure 2 shows a chromatographic profile of the essential oil of Figures 1a-1d;

∠’Figure 3 is a micrograph of three chromatographic analysis plates on a thin layer of samples of Helichrysum italicum extracts in jojoba oil;

∠’Figure 4 shows the trend of the percentage proliferation of keratinocytes in a culture medium in the presence of various concentrations of three extracts of Helichrysum Italicum in jojoba oil, and an essential oil of Helichrysum italicum;

∠’Figure 5 shows the positive correlations between the quantities of some compounds found in extracts of Helichrysum Italicum in jojoba oil and the percentage of proliferation on keratinocyte cells with extract at a concentration of 1%.

EXAMPLES

1. Chemical analysis of jojoba oils

The jojoba oils or waxes used are characterized by the chemical-physical analysis parameters shown in Table 1.

- Table 1 - METHOD VALUE

Appearance Clear liquid

Color (Gardner) 15 max.

Slight odor

Specific gravity. 20 ° C, g / ml

Refractive index, 20 ° C

Acid number

Iodine number

Equivalent of saponification

Peroxide number, meq / kg

Unsaponifiable fraction,%

Composition of acids

fats (% of total a.g.)

2. Materials used and conditions of the extraction phase. Example 1

Extraction by maceration. The following operating parameters were respected:

∠’temperature: ambient;

∠’total duration of maceration: 28 days;

∠’acidity of jojoba oil = 0.38;

∠’amount of oil used = 1000 ml;

∠’type of plant used: dried Helichrysum italicum, herbal tea cut;

∠’amount of plant used = 250 g;

∠’ratio of helichrysum: oil = 0.25 g / ml;

∠’acidity of jojoba oil = 0.38;

∠’Helichrysum ratio: oil = 0.25.

Example 2

Extraction by percolation in a 1 liter micro extractor without double piston, therefore with percolation by simple fall of the oil through a bed of helichrysum, and without external recirculation of the oil. The following operating parameters were respected:

∠’temperature: ambient;

∠’pressing time = 3 minutes;

∠’depression time = 6 minutes;

∠’number of cycles = 88;

∠’total time of the cycles: 792 minutes;

∠’acidity of jojoba oil = 0.38;

∠’amount of oil used = 1100 ml;

∠’type of plant used: dried Helichrysum italicum, herbal tea cut;

∠’amount of plant used = 275 g;

∠’ratio of helichrysum: oil = 0.25 g / ml.

Example 3

Extraction by forced percolation in a Timatic model 5p rapid extractor, equipped with double piston. The oil is drawn and recirculated in the extraction chamber. The following operating parameters were respected:

∠’temperature: ambient;

∠’number of forced percolations: 5, with external recirculation activated;

∠’pressing time = 3 minutes;

∠’depression time = 6 minutes;

∠’operating pressure = 8.0 bar;

∠’total extraction time = 891 minutes;

∠’number of cycles = 99;

∠’acidity of jojoba oil = 0.2;

∠’Amount of oil used = 6000 ml;

∠’type of plant used: dried Helichrysum italicum, herbal tea cut;

∠’amount of plant used = 800 g;

Helichrysum ratio: oil = 0.13 g / ml.

3. Analysis of the flavonoids of the extracts

The analysis of the flavonoids was carried out by HPLC by the company Chelab (Resana, TV, Italy) under the constraint of confidentiality. The detailed description of the analytical procedure is available from that company. The results of the analysis are reported in Tables 2a, 2b, 2c, respectively for the extracts of examples 1, 2 and 3.

- Table 2a - Analysis of the flavonoids of the extract of example 1

Sum of quantified flavonoids 225.8 mg / kg - Table 2b - Analysis of flavonoids in the extract of example 2

Sum of quantified flavonoids 241.9 mg / kg - Table 2c - Analysis of flavonoids in the extract of example 3

Sum of quantified flavonoids 132.4 mg / kg

4. Gas chromatographic analysis

of the volatile fraction of the extracts

The analysis of the volatile fraction was performed by gas chromatography by Chelab (Resana, TV, Italy).

In particular, the composition study of the volatile fraction, that is of the â € œactiveâ € flavoring fraction, was carried out by means of GC / MS and GC / FID analysis of the extracts obtained. Different but complementary extraction techniques were applied as regards the type of information provided. This was aimed at defining the best analytical protocol to be used for the analysis of the aromatic profile.

The protocol that provides for the analysis of extracts obtained by SDE has proved to be the most suitable for the characterization and comparison of samples, as it guarantees better reproducibility and better sensitivity towards some important compounds.

The composition of the volatile fractions of the extracts of Examples 1, 2 and 3 is reported in Table 3, with reference to the compounds considered to be the most significant. The relative distribution values of the listed compounds, evaluated as percentage areas of the gas chromatographic trace, have been normalized with respect to the total sum of the volatile compounds considered.

By way of comparison, Figures 1a-1d and 2 show the analysis of the essential oil of helichrysum used as a reference, purchased by Daniele Patrizia Erboristeria Balaina Lu Capruleddu, Luogosanto, Olbia Tempio, Sardinia, Italy.

Also, as reported in Parasitol. Res. (2010) 107, 1455â € “146, a chromatographic study on helichrysum essential oil samples revealed that the main constituents are:

∠’Neril acetate 25.3%;

∠’Î ± -Pinene 14.5%;

∠’Limonene 12.3%;

∠’γ-Curcumene 8.7%;

∠’Neril propionate 6.4%;

∠’Nerolo 5.2%,

in accordance with the data of Figures 1a-1d and 2.

From the data in table 3 and Figures 1a-1d and 2, it can be seen that the composition of the essential oil is extremely different from the composition of the jojoba wax extract of examples 1, 2 and 3.

- Table 3 - Composition of the volatile fraction of the extracts Example 1 Example 2 Example 3 5. Analysis by thin layer chromatography of the extracts (Examples 1 and 2)

A qualitative analysis by thin layer chromatography (TLC) was performed of samples of the extracts of Helichrysum italicum in jojoba oil of examples 1 and 2, and in other oils, to identify any differences.

Samples analyzed

∠’I â €" Example 1 (maceration);

∠’II â €" Example 2 (percolation by gravity);

∠’III - Jojoba wax alone.

In Figure 3, relating to the TLC profile, sample I (Example 1) is indicated as â € œDâ €, sample II (Example 2) as â € œEâ €, and sample III as â € œGâ €.

The samples were analyzed after treatment of 2 drops of extract with 5 drops of methanol, vortexing for 10 seconds and leaving the two phases to stratify. The methanol phase was subjected to TLC, as indicated below:

∠’stationary phase: TLC Silica Gel F254 plates, Merck, Code 1.05554.0001;

∠’mobile phases: 1) 100% dichloromethane; 2) dichloromethane / n-hexane 50% / 50%;

∠’deposit of samples: in quantities of 2 µl or 3 µl by means of microcapillaries;

â ’visualization of the spots: by irradiation with UV light at 254 nm and 365 nm, and then by spraying the plate with a 5% vanillin solution in concentrated sulfuric acid.

As Figure 3 shows, a comparison between samples I, II and III was performed to identify the differences between leachate, macerate and jojoba wax alone. The presence of a spot 30 highlighted in the box only in sample II (leachate) indicates that the leachate contains a component in much higher concentrations than the macerate. This component is absent in jojoba oil alone (sample III), therefore it is believed that it comes from helichrysum. This spot 30 is the only one that exhibits a slight fluorescence at 254 nm.

6. Examples of wording

Oleolite formulation

Helicrysum italicum extract; Simmondsia Chinensis seed oil; Anthemis Nobilis oil.

Shampoo-shower formulation

Water, Sodium cocoanfoacetate, Cocammdopropyl Betaine, Acrylates C / 10-30 acrylate crossipolimer, Simmondsia Chinensis seed oil, Helicrysum italicum extract, Anthemis Nobilis oil, Methylisothiazolinone, Methylchloroisothiazolinone, Lactic acid, Sodium hydroxide.

Solid soap formulation

Cocos Nucifera, Water, Elaeis Guinensis, Olea Europea, Sodium Hydroxide, Simmondsia Chinensis Seed Oil, Anthemis nobilis Oil, Helicrysum italicum Extract, Tricticum Vulgare

Face-body cream formulation

Water, Butyrospermum parkii (Galam butter), Stearic Acid, Glycerin, Simmondsia Chinensis oil, Helicrysum italicum extract, methylglucose dioleate, caprylic capric triglyceride, hydrogenated beaver oil, Phenoxyethanol, Methyl paraben, Butyl, Propyl, Isopammide , Tocopheryl acetate, Anthemis nobilis oil.

7. Effect of extracts on cell proliferation of dermal keratinocytes

The test substances were evaluated in a range from 1% v / v to 0.0001% v / v.

Human keratinocytes (HEK, Human Epidermis Keratinocyte, ECACC) were grown in Complete Keratinocyte Growth Medium (ECACC) according to the supplier's instructions, and were used for the assay between the first and sixth pass.

The assay was performed using the Roche Diagnostic Cell Proliferation Reagent WST-1 kit. The assay was performed according to the supplier's instructions.

More specifically, the cells were seeded in a 96-well plate for absorbance reading. Cell density ranged from 2,000 to 8,000 cells per well in a volume of 100 microliters per well. The test substance had a concentration within the range defined above. In one control, no product was added to the cells.

The tests were performed in quadruplicate, in addition a blank was associated with the medium without the product to be tested (control) and with the product to be tested, at the concentration defined above in single.

A first phase of cell incubation lasted 18 hours. Subsequently, the reagent was added (10 µl per well) and the incubation continued for another 2 hours, or for another 4 hours. At the end of the incubation, an absorbance reading was performed for 1 s at a wavelength of 450 nm in a Perkin Elmer Victor 2 Wallac microplate reader.

The results are shown in the graphs of Figures 4 and 5, in which cell proliferation is expressed as percentage proliferation (%), defined as:

Proliferation% = [(absorbance of treated cells - absorbance of blank) / (absorbance of control - absorbance of blank)] x 100

With reference to Figures 4 and 5, the extract of example 1, which gave a good stimulation of cell proliferation on keratinocytes, has a chrysin content of 123 mg / kg of oil. Consequently, a threshold could be 123x0.65 = 80 mg / kg of oil. Similarly, for the extract of example 2 a preferred threshold value could be 130x0.65 = 84 mg / kg of oil and an even more preferred value could be 130x0.80 = 104 mg / kg of oil.

From the graph shown in Figure 4 it can be seen that Example 3 does not involve any significant stimulation of the proliferation of keratinocytes at all tested concentrations. On the contrary, Examples 1 and 2, in particular the latter, stimulate the proliferation of keratinocytes in a significantly higher way than the control value, at least at the two highest concentrations tested. At lower concentrations, ie working with a more diluted extract, the proliferation values are similar to those of the control.

A 100% proliferation value is considered as a control.

The different behavior of Example 3 with respect to Examples 1 and 2 can be explained by referring to the different chemical composition, in particular to the different content of the flavonoids Crisina, Galangina and Pinocembrina, as reported in Tables 2a, 2b, 2c. The content of these flavonoids in Example 3 is significantly lower than that of Examples 1 and 2.

In independent experiments, not reported, jojoba wax showed no significant influence on keratinocyte cell proliferation. Therefore the effect on the keratinocytes depends on the helichrysum and not on the jojoba.

The graph of Figure 5 shows all the positive correlations between the quantities of the compounds determined in the samples of the extracts of Examples 1, 2 and 3, and the percentage of proliferation on HEK cells with extract at a concentration of 1%. These results are also reported in Table 4.

- Table 4 -

Growth Sample% with SEM extract

at a concentration of 1%

Example 3 111 6

Example 2 188 3

Essential oil -2 1

Example 1 146 8

It can be observed that such positive correlations occur for the three flavonoids Galangina, Pinocembrina and Crisina, for the terpenes Î ± -selinene and β-selinene and for curcumene. Conversely, no positive correlations were found for aldehydes.

Claims (10)

CLAIMS 1. A method of preparing an extract of a Helichrysum in jojoba oil, called a method comprising the steps of: ∠’preparation of a part of the plant chosen from the upper areas of the plant, in particular the flowering tops; ∠’predisposition of a jojoba oil having an acid number greater than 0.3; â ’contact of said part of said Helichrysum italicum plant with said jojoba oil in extraction conditions; ∠’recovery of said extract obtained in said contact phase, in particular said extract having a chrysin content per kg of fatty acids with 20 carbon atoms having a single unsaturation higher than 80 mg and / or said extract comprising limonene and βselinene having a weight ratio of limonene with respect to said l β-selinene less than 0.4, in particular, said plant of the genus Helichrysum belongs to a species chosen from: ∠’Helichrysum italicum, in particular Helichrysum italicum subspecie italicum and / or Helichrysum italicum subspecie microphyllum; ∠’Helichrysum stoechas; ∠’Helichrysum arenarium. A method according to claim 1, wherein said jojoba oil has an acid number greater than 0.32, more particularly an acid number greater than 0.36. 3. A jojoba oil extract obtained from a plant of the genus Helichrysum, called jojoba oil comprising fatty acids with 20 carbon atoms having only one unsaturation, said extract comprising a predetermined Chrysin content characterized by the fact that said Chrysin content, per kg of fatty acids with 20 carbon atoms having a single unsaturation, is higher than 80 mg. 4. An extract as per claim 3, in which said Chrysin content is higher than 84 mg per kg of fatty acids with 20 carbon atoms with a single unsaturation, more particularly said Chrysin content is higher than 104 mg / kg of fatty acids with 20 carbon atoms with only one unsaturation. 5. A jojoba oil extract obtained from a plant of the genus Helichrysum in jojoba oil, said extract comprising limonene and β-selinene characterized in that said limonene has a weight ratio with respect to said β-selinene lower than 0.4. 6. An extract according to claim 5, wherein said weight ratio is lower than 0.08, in particular said weight ratio is lower than 0.06. 7. A method according to claim 1, wherein said contact step is performed with a percolation or maceration technique, preferably percolation, in particular, said contact step is carried out by keeping said jojoba oil at a contact temperature between 25 ° C and 90 ° C, more particularly between 50 ° C and 85 ° C, even more particularly between 70 ° C and 80 ° C, in particular, said step of preparing a part of said plant comprises a step of drying said part of said plant. 8. A medical or cosmetic composition comprising an extract according to any one of claims 3 to 6, and / or an extract obtained by the method according to any of claims 1, 2 and 7, for the treatment and / or prevention of a disease and / or condition for which stimulation of keratinocyte cell proliferation is required, said disease and / or condition being chosen from: ∠’wounds, in particular cuts, grazes, abrasions, insect bites, stab wounds, gunshot wounds, surgical wounds; ∠’burns, in particular burns from ultraviolet radiation, sun burns, radiotherapy burns, burns from open flame, burns from contact with hot bodies, burns from acid substances, burns from caustic substances; ∠’skin ulcers, in particular bedsores, sores due to diabetes; ∠’scars, in particular postoperative scars, scars due to excessive accumulation of connective tissue following tissue damage, atrophic scars, acne scars; ∠’skin aging, in particular aging due to age, aging due to ultraviolet radiation, aging due to environmental pollution, aging due to atmospheric factors, aging due to hormonal factors; ∠’stretch marks, in particular stretch marks due to pregnancy, stretch marks due to marked changes in body weight, stretch marks due to hormonal factors; ∠’wrinkles, especially expression lines. 9. A composition according to claim 8, comprising an essential oil, in particular said essential oil being selected from: essential oil of Roman chamomile; essential oil of matricaria chamomile; carnation essential oil; tea tree essential oil; lavender essential oil; essential oil of oregano, or mixtures thereof. An insect repellent composition comprising an extract according to any one of claims 3 to 6, and / or an extract obtained by a method according to any one of claims 1,2 and 7.