RS66827B1 - Phytopreparations based on supercritical extracts of immortelle and hemp intended for slowing up visible signs of aging and procedure for obtaining - Google Patents

Description

TECHNICAL FIELD

The invention belongs to the field of obtaining medical and cosmetic phytopreparations for topical application based on supercritical extracts of immortelle (Helihrysum italicum (Roth) G. Don fil., Asteraceae) and hemp (Cannabis sativa L., Cannabaceae), which are primarily used in treatment aimed at reducing visible signs and slowing down the aging process of mature skin, thanks to an effective combination of essential ingredients for maintaining normal processes in skin cells. According to the international classification of patents, the invention can be classified into classes: SPV 9/02, A61Q19/08, A61K 36/28, A61K 31/352, A01N 6/28, C08L 5/16, A61K 8/18.

TECHNICAL PROBLEM

The process of biological aging of the organism is a natural phase in the life cycle characterized by visible changes in the structure of the skin, which are often accelerated due to the action of numerous internal and external factors, primarily free radicals and various skin inflammations. Long-term efforts of scientists around the world have been made to obtain effective preparations with improved properties for caring for mature skin and slowing down the aging process. A large number of preparations based on plant extracts, vitamins, enzymes or active substances to alleviate existing and slow down the formation of new wrinkles exist on the market and show different effectiveness in the process of revitalization and slowing down the aging process of mature skin. Market analysis revealed that there are no preparations based on a combination of immortelle (Helihrysum italicum (Roth) G.) and hemp (Cannabis sativa L., Cannabaceae) extracts, and especially no preparations with standardized extracts.

The technical problem that is solved by the present invention is the obtaining of stable and original compositions and phytopreparations for topical application with controlled release of active substances based on extracts of immortelle (Helihrjsum italicum (Roth) G.) and industrial hemp (Cannabis sativa L., Cannabaceae) that influence the maintenance of normal processes in skin cells and are effective in slowing down the visible signs of biological aging of the skin, thanks to the synergistic combination of essential ingredients.

The subject invention also solves the technical problem of obtaining a high-quality immortelle extract (Helihrysum italicum (Roth) G.), using the supercritical extraction technique. The desired profile of extracted active substances from the plant material was achieved by variations in pressure, temperature, contact time and the presence of cosolvents during the supercritical extraction process. The obtained extracts and semi-products based on the extracts of the impregnated carriers were characterized from a chemical and pharmacological point of view, in compliance with the applicable regulations and legal regulations for the purpose of application in the pharmaceutical and cosmetic industry.

The technical problem that is solved by the invention is reflected in the performance of an integrated procedure of supercritical extraction and impregnation of solid supports based on cyclic oligosaccharides obtained by extracts using supercritical carbon(IV)-oxide (nkSO2) in order to obtain stable plant semi-products and increased bioavailability during topical application. Incorporating solid carriers impregnated with plant extracts into appropriate pharmaceutical preparations in semi-solid and liquid form results in an unexpectedly effective and simple application for skin and mucous membrane changes compared to similar products, especially for inflammation and accelerated aging of the skin caused by the action of free radicals.

By formulating phytopreparations for topical application in the form of various pharmaceutical forms, a surprising improvement is achieved in slowing down the process and reducing the visible signs of skin aging. According to the invention, the preparations for topical application have unexpectedly effective properties by their composition, which avoid and overcome the shortcomings of the existing preparations for the same purpose. The technical problem that is solved by this invention is that the effective ratio of emulsifiers, emollients, water and active components is defined, as well as that the technological procedure for obtaining new preparations, intended to slow down the appearance and remove visible signs of skin aging, is fully characterized, without reducing the effectiveness of the active components.

STATE OF THE ART

Skin aging as a natural phenomenon is a complex biological process that affects all layers of the skin. The aging process of the organism is the result of a natural cycle, but also of harmful external influences, and it manifests itself on the skin already after the age of 25. There are 4 types of skin aging: internal (cell and extracellular matrix aging due to the passage of time, genetics and gravity), external (skin aging under the influence of UV rays and free radicals - photoaging, smoking, diet and bad lifestyle habits), hormonal (due to dysfunctions and aging of the hormonal system) and catabolic skin aging (due to chronic diseases). The process of skin aging is very complex, because a large number of factors influence the acceleration of this process. Skin elasticity deteriorates due to internal factors. Due to the reduction of collagen in the cells, it becomes dry and flaky, the appearance of wrinkles, pigment spots, irritations. Disturbance of the balance between new cells of the skin epithelium and dying old cells, leads to a slowdown in the growth of new and an increase in the number of dying cells and the skin becomes thinner. External factors represent an additional provocation to natural aging and have a dominant influence on the aging process of the skin, which cannot be stopped, but there are ways to delay and slow it down.

Mature skin requires intensive care to remain attractive and vital for longer. In today's information age, modern consumers are significantly educated, demanding and critically oriented towards the expected effects of the application of certain products. Modern science and medicine have achieved many significant results, providing numerous drugs, supplements and methods that work very successfully to slow down the aging process of the skin. The aim of the modern production technology of these substances is to make them reliable and effective, long-lasting and cheap, so that their use can be repeated without allergic or oncogenic effects. With the correct selection of active ingredients incorporated into the foundation, quality cosmetic preparations are obtained that can make the skin healthier and more beautiful. Manufacturers of cosmetic raw materials and products/means try to satisfy the needs of discerning consumers by designing new, innovative, multifunctional and more effective products based on natural, herbal active principles that achieve better effects on the skin. The replacement of synthetic substances with natural ones, the incorporation of natural, physiologically active substances into products intended to slow down the skin aging process and reduce the visible signs of skin aging is one of the most current trends in the pharmaceutical and cosmetic industry today. Plant extracts are used, which with their complex ingredients simultaneously act on several processes and mechanisms in the body.

Helihrysum italicum (Roth) G., Don fil., Asteraceae, immortelle

Smilje is an aromatic semi-shrub with light green leaves and golden yellow flower heads, which belongs to the Helichrysum genus and the Asteraceae family with several hundred species distributed throughout the world. Most species are bushy perennials. In the Mediterranean area, the genus Helichrysum is represented by about 25 autochthonous species (Morone-Fortunato I, Montemurro C,... Avato P. Essential oils, genetic relationships and in vitro establishment of Helichrysum italicum (Roth) G. Don ssp. italicum frorn wild Mediterranean germplasm. Industrial crops and products, 2010, 52(3):639-649). Helichrysum italicum (Roth) G. Don, ie. Mediterranean immortelle is the only species of the genus that has a significant place in cultivation and use. The Helichrysum genus is widespread in Albania, Bosnia and Herzegovina, Montenegro, France, Greece, Croatia and Italy, and also grows in North-West Africa and Asia Minor. The Mediterranean immortelle is a xerophytic species, which grows naturally on dry, sandy and rocky areas of the Mediterranean region in a wide range of altitudes between sea level and 2200 m (Nostro A, Bisignano G,... Alonzo V. Effects of Helichrysum italicum extract on growth and enzymatic activity of Staphylococcus aureus. International Journal of Antimicrobial Agents, 2001, 17:517-520), and the most famous natural site is on Corsica.

Smilje is mentioned in the works of ancient, Greek and Roman writers and naturalists as a powerful elixir that shows a regenerative effect on damaged skin. The etymological name of the genus Helichrysum comes from the Greek words Helios-sun and chrysos-gold, associating with golden-yellow flowers. Smilje also has another commonly known name, immortelle - "immortelle", because the flower remains the same in appearance after harvesting and when it dries. The first scientific research on this plant species was conducted in the 40s and 50s of the 20th century. Later research confirmed folk medicine. Metabolites isolated from immortelle have strong biological activity such as antimicrobial, anti-inflammatory, antiviral and antioxidant activity. Distillation of fresh plant material yields an essential oil with a very complex chemical composition, which shows a favorable health-medical profile in therapeutic application. In traditional medicine, decoctions and infusions of immortelle flowers and leaves are used in the treatment of colds, allergies, liver and gallbladder disorders, inflammation, infections, and skin diseases, while there are only a small number of scientific studies to validate the aforementioned effects. The use of immortelle in cosmetic preparations and dietary supplements is known, where data from clinical studies on their effectiveness are also unavailable. Thanks to its intoxicating sweet smell that has the ability to suppress the odors of other ingredients, immortelle essential oil has found cosmetic use and a reputation for anti-wrinkle, revitalizing and regenerating tired and mature skin exposed to external and internal influences. A less well-known feature of immortelle is its effect on hematomas, scars, stretch marks, and various skin inflammations.

There are phytopreparations on the market based on the supercritical extract of immortelle Helihrysum italicum (Roth) G. for the treatment of skin inflammation, slowing down the skin aging process and reducing the visible signs of skin aging, but without a precisely defined composition and testing of its effect on the skin.

"Ljbilje" d.o.o. Ljubinje from Herzegovina produces a range of "Ljekobilje" products, namely "Essential oil of immortelle", "Day cream of immortelle" and "Night vitamin cream of immortelle", "Anti-scar cream", as a combination of natural essential oils with specific properties (immortelle, lavender, tea tree, rosemary), of which essential oil of immortelle is dominant and the most effective. Distillation with water vapor produces two products - hydrolate "Imperial drops-Tinctura Helichrysum italicum" and essential oil "Imperial essential oil-Aetheroleum Helichrysum italicum" (https://ljekobilje.co.rs/).

The family manufactory on the slopes of the Homolje mountains makes the product "Nadalina cream of immortelle" from essential oil and immortelle macerate (80%) and enriched with calendula, yarrow, chamomile and raspberry seed oils, bound with beeswax (https://kremaodsmilia.com/).

The company "Royal Balm" from Rtanj mountain produces "Natural anti-wrinkle face serum" based on linseed oil, immortelle and comfrey, "Natural serum for dry and sensitive skin" based on immortelle and magnolia and "Natural anti-wrinkle cream" which contains linseed oil, immortelle flower water, wolfberry oil, comfrey and magnolia extract (https://royalbalm.rs/).

"Pure immortelle cream against wrinkles and for quick skin recovery" contains highly concentrated 98%, immortelle oil obtained by the distillation process, produced according to the GMP standard, with the addition of coconut, olives, beeswax and vitamin E (https://www.mennakozmetika.com/).

The company "Priroda lejizi" d.o.o. products "Nikkel bio elixir immortelle" based on the essence of immortelle, tangerine and orange and cold-pressed plant oils of jojoba, passionflower, virgin olive, natural vitamin E and beta carotene; "Nickel anti-wrinkle serum with immortelle" based on ceramide, hyaluronic acid, honey, immortelle and vitamin E; "Nickel rich moisturizing cream with immortelle" is based on almonds, olives, calendula, immortelle, sunflower and vitamin E, while "Nickel rich nourishing cream with immortelle" is based on almonds, ceramide, hyaluronic acid, shea butter, olives, honey, calendula, immortelle, sunflower and vitamin E (https://www.nikel.com.hr/).

Croatian company "Tinktura®" d.o.o. produces "Imfrey Balm" that revitalizes, stimulates collagen production, microcirculation, reduces wrinkles and scars (https://tinktura.com/).

"K plus" produces "Olca body lotion with immortelle" and "Olca hand cream with immortelle essential oil" with sweet almond oil, shea butter, sunflower oil, glycerin, aloe vera extract, arnica extract and immortelle essential oil.

Extraction of active components from immortelle, according to available literature data, was carried out by hydrodistillation, steam distillation and extraction using supercritical carbon(IV)-oxide (nkCO2). Among the isolated components, the dominant role is played by neryl acetate, α-pinene, α- and β-heudesmol, geranyl propanoate, limonene and camphene using organic solvents, most often ethanol, then methanol, acetone, and less often using petroleum ether, dichloromethane and diethyl ether, chloroform, n-hexane. Supercritical extracts of immortelle also contain nerol, neryl propanoate, as well as selinene and caryophyllene isomers, and heavier sesquiterpenes can also be isolated. Supercritical extracts of immortelle also have a significant content of heavier hydrocarbons (waxes) and scopoletin (compounds from the coumarin group) (Ivanovic J, Ristic M, Skala D. Supercritical CO2 extraction of Helichrysum italicum: influence of CO2 density and moisture content of plant material, Journal of Supercritical Fluids 2011, 57:129-136; Jerkovic I, Rajic M, Marijanovic Z, Bilic M, Jokic S. Optimization of supercritical CO2 extraction of dried Helichrysum italicum flowers by response surface methodology: GC-MS profiles of the extracts and essential oil, Separation Science and Technology 2016, 51(18): 2925-2931; Jokic S, Rajic M, Bilic B, Molnar M. Supercritical extraction of scopoletin from Helichrysum italicum (Roth) G. Don flowers, Phytochemical Analysis 2016, 27:290-295). Experimental values of supercritical extraction conditions in the literature for pressure range from 8-35 MPa, at a temperature of 35.86-64.14°C. Supercritical extraction from immortelle with the presence of a cosolvent was realized for the first time using sage essential oil (Maksimović S, Tadić V, Skala D, Žižovic I. Separation of phytochemicals from Helichrysum italicum: An analysis of different isolation techniques and biological activity of prepared extracts. Phytochemistry 2017, 138:9-28). Extraction from immortelle using supercritical CO2 and impregnation of starch xerogels with extract by an integrated process of extraction and impregnation using supercritical CO2 were performed at 350 bar and 40°C in order to produce biomaterials for potential oral intake of the extract. Xerogels obtained by air drying of acetogels and alcogels were used as carriers in the supercritical impregnation process. The effects of ethanol as a co-solvent, contact time, mass ratio of plant material and carrier and xerogel preparation method on the impregnation yield were examined (Maksimović S, Tadić V,... Žižović I. Utilization of the integrated process of supercritical extraction and impregnation for incorporation of Helichrysum italicum extract into com starch xerogel. Chemical Industry and Chemical Engineering Quarterly 2018, 24(2):191-200).

Active components from immortelle were also extracted using organic solvents, most suitable using ethanol, then acetone, methanol, and there are published works according to which petroleum ether, diethyl ether, dichloromethane, n-hexane were used. Ethanol proved to be a very suitable solvent for the isolation of flavonoids (apigenin, luteolin, kaempferol, naringenin, etc.) and their derivatives, phenolic acids (caffeic, ferulic, chlorogenic, caffeoylquinic, etc.), compounds from the acetophenone group, and 12-acetoxytremetone, which proved to be an active substance (Rigano D, Formisano, C, Senatore F,... Izzo A. Intestinal antispasmodic effects of Helichrysum italicum (Roth) Don ssp. italicum and chemical identification of the active ingredients, Journal of Ethnopharmacology 2013, 150(3) 901-906; Among the flavonoids isolated from the methanol extract of immortelle, the most abundant are gnaphalin, pinocembrin and tiliroside, and naringenin, kaempferol and others are also present. and their derivatives. The presence of compounds from the acetophenone group was also detected (Sala A, Carmen Recio M,... Rios JL. New acetophenone glucosides isolated from extracts of Helichrysum italicum with anti-inflammatory activity, Journal of Natural Products 2001, 64:1360-1362). Active components from the group of phenolic compounds present in methanol extracts of immortelle are phenolic acids (caffeic, chlorogenic, gallic, quinic, etc.), ursolic acid, α-terpinolene, neryl acetate and trans-caryophyllene (Tundis R, Statti GA, Conforti F,... Poli F. Influence of environmental factors on composition of volatile constituents and biological activity of Helichrysum italicum (Roth) Don (Asteraceae), Natural Product Research 2005, 19(4):379-387). In the extract obtained using diethyl ether, the presence of coumarins, flavonoids, steroids and terpenes was determined (Nostro A, Germano MP, D'Angelo N... Cannatelli A. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity, Letters in Applied Microbiology 2000, 30:379-384). 4-hydroxy-3(isopenten-2-yl)acetophenone, compounds from the group of coumarins (esculetin, scopoletin and isoscopoletin) and flavonoids (kaempferol, 3,5,7-trihydroxy-8-methoxyflavone and 3,5-dihydroxy-6,7,8-trimethoxyflavone) were isolated by extraction using chloroform from immortelle extract as an active component (Karasartov BS, Kurkin VA, Zapesochnaya GG. Coumarins and flavonoids of the flowers of Helichrysum italicum, Chemistry of Natural Compounds 1992, 28(5):504-505). Extraction from immortelle was performed using water to determine the total phenol content (Kazazic M, Djapo M, Ademovic E, Antioxidant activity of water extracts of some medicinal plants from Herzegovina region, International Journal of Pure & Applied Bioscience 2016, 4(2):85-90).

The most abundant compounds in immortelle essential oil are α-pinene at the time of the most intensive development of the plant immediately after flowering, and neryl acetate at the time of full bloom of the plant and in autumn (Politeo O. Seasonal variations of the chemical composition and biological activity of the essential oil of immortelle, Helichrysum italicum (Roth) G. Don. Master's thesis, Faculty of Natural Sciences Zagreb, 2003). Research shows that immortelle essential oil originating from France is richer in monoterpenes with oxygen (42.08%, 33.87% neryl-acetate), shows stronger antimicrobial activity against Gram-positive bacteria and fungi and higher phenol content, while oils from plants originating from Bosnia are richer in sesquiterpene hydrocarbons (39.43%, 20.84% α-pinene). Mollova S, Fidan H, Antonova D,... Stoyanova A. Chemical composition and antimicrobial and antioxidant activity of Helichrysum italicum (Roth) G. Don subspecies essential oils. Turkish Journal of Agriculture and Forestry 2020,44).

The pharmacological effect of Helichrysum italicum isolates and their components is the subject of numerous studies.

There are numerous works that investigated and confirmed the antimicrobial effect, as one of the most widespread effects of immortelle isolates and their individual components. Depending on the origin of the plant and the content of primarily terpene components, immortelle essential oil has an antibacterial effect in suppressing strains of bacteria: Staphilococcus aureus, Esccherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Campylobacter jejuni, Bacillus cereus, Klebsiella pneumoniae, Bacillus subtilis, Bacillus pumilus, Bacillus cereus, Salmonella typhimurium, Micrococcus luteus, Enterococcus cereus, Acinetobacter baumanni (Rossi PG, Berti L, Panighi J,... Muselli A. Antibacterial action of essential oils from Corsica, Journal of Essential Oil Research 2007, 19:176-182; Roussis V, Petrakis PV,... Volatile constituents of four Helichrysum species growing in Greece, Biochemical Systematics and Ecology 2000, 28:163-175; Cui H, Zhao C, Lin L on vegetables and its mechanism of action, Journal of Food Processing and Preservation 2015, 39:2663-2672; Lorenzi V, Muselli A, Bemardini AF,... Bolla J. Geraniol restores antibiotic activities against multidrug-resistant isolates from gram-negative species, Antimicrobial Agents and Chemotherapy 2009, 53(5):2209-2211).

The fungicidal effect of immortelle essential oil against fungi species: Penicillium sp., Epicoccum nigrum, Aspergillus niger, Fusarium solani, Alternaria alternate, Ascochyta rabiei and yeasts Candida albicans and Saccharomyces cerevisiae (Stupar M, Ljajevic Grbic M, Dzamic A,... Vukojevic J. Antifungal activity of Helichrysum italicum (Roth) G. Don (Asteraceae) essential oil against fungi isolated from cultural heritage objects, Archives of Biological Sciences 2014, 66(4):1539-1545; Djihane B, Wafa N, Elkhamssa S, ... Mihoub ZM.

Chemical constituents of Helichrysum italicum (Roth) G. Don essential oil and their antimicrobial activity against Gram-positive and Gram-negative bacteria, filamentous fungi and Candida albicans, Saudi Pharmaceutical Journal 2016). Terpenes isolated from the methanol extract of immortelle α-terpinolene, frara-caryophyllene and neryl acetate showed a strong effect against the fungi Pythium ultimum and Trichophyton mentagrophytes (Tundis R, Statti GA, Conforti F,... Poli F. Influence of environmental factors on composition of volatile constituents and biological activity of Helichrysum italicum (Roth) Don (Asteraceae), Natural Product Research 2005, 19(4):379-387). Ether extract of immortelle showed a strong effect against fungi A. fumigatus, Fusarium oxysporum, Microsporum canis, M. gypseum, Tricophyton mentagrophytes and yeasts C. albicans and C. tropicalis (Nostro A, German, MP, D'Angelo V, Marino A, Cannatelli MA. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity, Letters in Applied Microbiology 2000, 30:379-384). 4-hydroxy-3(isopenten-2-yl)acetophenone isolated from an immortelle extract obtained using chloroform showed a strong effect on fungi of the genus Penicillium sp., Cladosporium herbarum and Phytophora capsicci (Tomas-Barberan F, Iniesta-Sanmartin E, Tomas-Lorente F, Rumbero A. Antimicrobial phenolic compounds from three Spanish Helichrysum species, Phytochemistry 1990, 29(4): 1093-1095). Essential oil of Cmogorsky Helichrisum italicum (Roth) G. Don fil. was tested against methicillin-susceptible Staphylococcus aureus (ATCC 29213), E.coli (ATCC 25922), Candida albicans (ATCC 14053) and clinical strains of methicillin-resistant S. aureus, carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa. The results showed an unexpected fungicidal/bactericidal potential against C. albicans and A. baumannii with the help of carbapenem at a concentration of 5% v/v (Oliva A, Garzoli S, Sabatino M, Tadić V, Costantini S, Ragno R, Božović M. Chemical composition and antimicrobial activity of essential oil of Helichrysum italicum (Roth) G. Don fil. (Asteraceae) from Montenegro. Natural Product Research, 2020,34(3):445-448).

Immortelle essential oil shows an antiviral effect on tobacco mosaic virus (Bezic N, Vuko E, Ruscic M, Dunkic V, Helichrysum italicum (Roth) G. Don - essential oil composition and activity on tobacco mosaic vims infection, Journal of Plant Physiology & Pathology 2016, doi:10.4172/2329-955X.1000144). The active component arzanol, which is isolated only in the acetone extract of immortelle, prevents the replication of HIV-1-vims in infected Jurkat T-cells (Appendino G, Ottino M, Marquez N,... Munoz E. Arzanol, an anti-inflammatory and anti-Hiv-1 phloroglucinol a-rugope from Helichrysum italicum ssp. microphyllum, Journal of Natural Products 2007, 70:608-612).

The antioxidant effect of immortelle essential oil was performed using tests, most often DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (reduction of iron Fe<3+> to Fe<2+> in the presence of antioxidants). The results showed moderate antioxidant activity depending on the origin and chemical composition of the oil (Omano L, Venditti A, Sanna C,... Bianco A. Chemical composition and biological activity of the essential oil from Helichrysum microphyllum Cambess. ssp. tyrrhenicum Bacch., Brullo e Giusso growing in La Maddalena Archipelago, Sardinia, Journal of Oleo Science 2015, 64(1): 19-26; Kladar NV, Anackov GT,... Bozin BN. Biochemical characterization of Helichrysum italicum (Roth) G. Don subsp. italicum (Asteraceae): phytochemical screening, chemotaxonomy and antioxidant properties, Chemistry & Biodiversity 2015, 12:419-431. Acetophenones, isolated from the methanolic extract of immortelle, exhibit antioxidant activity (Sala A, Carmen Recio M, Schinella GR,... Rios JL. A new dual inhibitor of arachidonate metabolism isolated from Helichrysum italicum, European Journal of Pharmacology 2003, 460:219-226). Immortelle extract obtained with methanol successfully prevents enzymatic and non-enzymatic lipid peroxidation, as well as prevents the formation of superoxide radicals (Schinella GR, Toumier HA, Prieto JM,... Rios JL. Antioxidant activity of anti-inflammatory plant extracts, Life Sciences 2002, 70:1023-1033). The terpenes α-terpinolene, tram'-caryophyllene and neryl acetate, isolated from the methanol extract, showed antioxidant activity in the β-carotene/linoleic acid/Tween 40 system (Tundis R, Statti GA, Conforti F,... Poli F. Influence of environmental factors on composition of volatile constituents and biological activity of Helichrysum italicum (Roth) Don (Asteraceae), Natural Product Research 2005,19(4):379-387).

The anti-inflammatory effect of immortelle isolates was investigated for extracts obtained using organic solvents and their components in in vitro and in vivo conditions. The active components 4-hydroxy-3-(2-hydroxy-3-isopentenyl)acetophenone and benzo-γ-pyrone glucoside show a strong anti-inflammatory effect in testing 12-o-tetradecanoylphorbol 13-acetate (TRA), phospholipase A2 and carrageenan-induced mouse paw edema (Sala A, Carmen Recio M, Giner RM, Manez S, Rios JL. New acetophenone glucosides isolated ffom extracts of Helichrysum italicum with antiinflammatory activity, Journal of Natural Products 2001, 64:1360-1362; Sala A, Carmen Recio M, Schinella GR,... Rios JL. A new dual inhibitor of arachidonate metabolism isolated from Helichrysum italicum, European Journal of Pharmacology 2003, 460:219-226. The flavonoid pinocembrin showed an anti-inflammatory effect during an induced hypersensitivity reaction in sheep red blood cells (Sala A, Carmen Recio M, Schinella GR,... Rios JL. Assessment of the anti-inflammatory activity and free radical scavenging activity of tiliroside, European Journal of Pharmacology 2003, 461:53-61). In vivo tests on the skin of guinea pigs showed the effects of the flavonoids apigenin, luteolin, naringenin and their derivatives from the ethanol extract of immortelle against erythema (Facino RM, Carini M, Mariani M, Cipriani C, Anti-erythematous and photoprotective activities in guinea pigs and in man of topically applied flavonoids from Helichrysum italicum G. Acta Terapeutica 1988, 14:323-345).

Cytotoxicity of immortelle essential oil showed strong inhibitory activity tested on human malignant melanoma cells (A375 cells). The concentration sufficient to inhibit cell growth by 50% (IC50) was 16 μg/mL (Omano L, Venditti A, Sanna C,... Bianco A. Chemical composition and biological activity of the essential oil from Helichrysum microphyllum Cambess. ssp. tyrrhenicum Bacch., Brullo e Giusso growing in La Maddalena Archipelago, Sardinia, Journal of oleo science 2015, 64(l):19-26). Immortelle essential oil has minimal cytotoxicity, tested on yeasts of the S. crevisiae type, in terms of causing damage to genes of the RNR3 and RAD51 type, responsible for the metabolism and repair of deoxyribonucleic acid, and an extraordinary degree of tolerance in patients and with long-term use (Viegas DA, Palmeira-de-Oliveira A, Salgueiro L,... Palmeira-de-Oliveira R, Helichrysum italicum: From traditional use to scientific data, Journal of ethnopharmacology 2014,151:54-65).

Only one case of dermatitis has been recorded in the literature, as an allergic reaction to the presence of hydrophilic and lipophilic fractions of immortelle essential oil in a cream for the treatment of xerosis (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, 69:53-63). Immortelle essential oil did not show significant results on telomere length (the shortening of which is reflected in human aging) when tested on human K562 cells, in the presence and absence of hydrogen peroxide (Plant J. Effects of essential oils on telomere length in human cells, Medicinal & Aromatic Plants 2016, 5(2); 1-6). All the above data indicate the safety of using the immortelle isolate.

One of the most important active components of immortelle, isolated only from the acetone extract of immortelle, is arzanol, a prenylated heterodime phloroglucinol α-pyrone or 3-(3-acetyl-2,4,6-trihydroxy-5-(3-methylbut-2-en-l-yl)benzyl)-6-ethyl-4-hydroxy-5-methyl-2N-pyran-2-one. Arzanol prevents the oxidative modification of lipid components in human low-density lipoprotein (LDL) caused by the action of Cu<2+> ions, as well as the reduction of the level of polyunsaturated fatty acids. It shows strong inhibition of cholesterol autoxidation and oxidative stress induced by tert-butyl hydroperoxide in cells isolated from monkey kidney (VERO cells) and human intestinal epithelial cells (Saso-2 cells). By examining the toxicity and measuring the release of lactate dehydrogenase (LDH) in VERO cells, arzanol did not show cytotoxicity (Rosa A, Deiana M, Atzeri A,... Dessì MA. Evaluation of the antioxidant and cytotoxic activity of arzanol, a prenylated α-pyrone-phloroglucinol etherodimer from Helichrysum italicum subsp. microphyllum. Chemico-Biological Interactions 2007, 7(55(2): 117-126; Dessi MA. Protective role of arzanol against lipid peroxidation 2011, 164(l):24-32). and prostaglandin E2 in lipopolysaccharide-stimulated monocytes, isolated from human blood (Appendino G, Ottino M, Marquez,... Munoz E. Arzanol, an anti-inflammatory and anti-HIV-1 phloroglucinol a-pyrone from Helichrysum italicum ssp. microphyllum. Journal of natural products 2007, 70(4):608-612). In in vitro tests, arzanol showed a tendency to inhibit 5-lipoxygenase activity and leukotriene formation in neutrophils, as well as cyclooxygenase-1 activity and prostaglandin E2 formation. It also showed an effect against inflammation of the lung tissue of rats, induced by the action of carrageenan (Bauer J, Koeberle A, Dehm F,... Werz O. Arzanol, a prenylated heterodimeric phloroglucinyl rugope, inhibits eicosanoid biosynthesis and exhibits anti-inflammatory efficacy in vivo. Biochemical pharmacology 2011, 81(2):259-26S).

A number of patents have been filed for obtaining Helichrisum italicum extract and for new possible applications.

Patent application FR3057166 A1 protects the procedures for obtaining immortelle extract using eutectic solvents and cosmetic preparations based on the extract for topical use in the treatment of signs of aging.

The subject of application CN110314202 A is the cosmetic application of ethanolic extracts of immortelle, saffron, red and white peony, wild chrysanthemum and tangerine for the treatment of skin irregularities in the form of acne and blackheads.

The subject of protection CN107714590 A is the application of immortelle for whitening spots caused by hyperpigmentation and the method of preparing a moisturizing preparation.

Patent US10682306 V1 protects a composition for improving the health and appearance of the skin, as well as for its treatment, which contains a moisturizing mixture based on oil infusions of Calendula officinalis, Helichrisum italicum and Simphitum officinale extracts, aqueous infusions of Viola tricolor extract and at least one extract selected from Malve silvestris and Althea officinalis.

Patent US7666454 B2 protects a cosmetic preparation for the treatment of wrinkles on the skin of the face and body containing the purified essential oil of the flower tops of Helichrisum italicum by hydrodistillation or supercritical extraction using nkSO2, with about 40-70% neryl acetate and about 0.1-5% oil. In order to increase bioavailability and controlled release, immortelle oil was incorporated into nanospheres with controlled release. The cosmetic composition contains an agent that increases collagen type 1 production, additional active ingredients. Clinical tests of cosmetic formulations have confirmed the positive effect of action in the process of skin rejuvenation/rejuvenation, against free radicals, on collagen type 1 synthesis, angiogenesis, reduction of the number and depth of wrinkles.

Cannabis Sativa L., Cannabaceae, hemp

Hemp is an annual, dioecious plant that belongs to weeds. It has pronounced sexual dimorphism, i.e. male and female plants differ morphologically (Finta-Korpel'ová Z, Berenji J. Trends and achievements in industrial hemp (Cannabis sativa L.) breeding. Bulletin for Hops, Sorghum & Medicinal Plants 2007, 39:63-75). Over 700 different varieties of hemp have been described, and there are also monoecious varieties, developed to obtain fiber and seeds at the same time.

Hemp was an important source of food and fiber for thousands of years, and was also used for medicinal purposes in the cultures of Europe and Asia (Zlas J, Stark H, Seligman J,... Mechoulam R. Early medical use of cannabis. Nature 1993, 363:215; Lu, X., Clarke, R.C. The cultivation and use of hemp (Cannabis sativa L.) in ancient China. Journal of International Hemp Association 1995, 2:26-30). It is assumed that it originates from central Asia, from the northwestern part of the Himalayas. It was mostly cultivated for its fiber, the ancient Chinese used it to make ropes, clothes and paper, because hemp fiber is characterized by great strength, elasticity, durability and water resistance (Tomašić Paić A. Properties of cannabinoid receptors of the medicinal plant Cannabis sativa. Medicinski vjesnik 2012, 44(1-4): 147-162). Until the 20th century, it was massively cultivated in countries around the world, as one of the most profitable businesses (Small E, Marcus D. Hemp: a new crop with new uses for North America. Trends in new crops and new uses 2002, 284:326). Every part of industrial hemp has a use. The seeds are used in cosmetics and the food industry due to their high content of proteins and nutrients.

The stem is used in the production of fabric, twine, paper, ecological building blocks, pellets for firewood. The flower is used in the medical industry to produce oil that is used in the treatment of many diseases. The root is used to make biological cocktails that are used in agronomy. Due to a lack of information, industrial hemp has been unfairly identified as Indian hemp, which contains a significantly higher percentage of the psychoactive substance THC (Božić-Ostojić L, Antimović S, Vujčić B, Martić M. Industrial hemp-a plant of the past and the future, 8<th>International Scientific/Professional Conference, Agriculture in Nature and Environment Protection, Vukovar, Croatia, 1-3 June 2015, Croatian Soil Tillage Research Organization, 133-137). Industrial hemp contains about 0.3-1.5% delta-9-tetrahydrocannabinoid (∆<9>-TNS, THC) with a psychoactive effect, while Indian hemp, marijuana, contains about 5-35% THC. Industrial hemp has a higher concentration of cannabidiol (CBD), which reduces the psychoactive effect of THC (Goločorbin-Kon S, Pavlović N, Rašković A.,... Mikov M. Use of cannabis in medicine and pharmacy. Medicinski časopis 2015, 49(4): 130-138). Due to the identification of industrial hemp with Indian hemp in America, in 1937, the Cannabis Sativa Taxation Act was introduced, which led to a decline in hemp production after World War II, and then to a complete stop after 1958. It was only in the early nineties that interest in its production began to grow again (Rawson JM. Hemp as an agricultural commodity. DIANE Publishing, 2011). The world production of hemp seeds in 2014 was 102,954 t. The main producers of hemp seeds in 2014 were France with as much as 82.91% and China with 15.64% of the total world production (Food and Agriculture Organization of the United Nations, www.fao.org/faostat/en/#data/QC.

According to available data from the literature, the extraction of hemp seed oil was carried out using maceration, cold pressing, Soxhlet, Soxterm, ultrasonic and supercritical CO2 extraction methods (Anwar F, Latif S, Ashraf M. Analytical characterization of hemp (Cannabis sativa) seed oil from different agro-ecological zones of Pakistan. Journal of the American Oil Chemists' Society 2006, 83:323-329; Zeng Q-X, An Q, Zeng L-X, Zhu Z-W. Ultrasonic extraction of hempseed oil, Journal of food process engineering 2012, 35:76-90; Matthaus B. Oil content, tocopherol composition and fatty acid patterns of the seeds of 51 Cannabis sativa L. genotypes. The influence of process conditions on oil yield was studied only in the case of oil extraction by supercritical CO2 extraction and cold pressing (Aladić K, Jokić S, Moslavac T,...

Šubarić D. Cold Pressing and Supercritical CO2 Extraction of Hemp (Cannabis sativa) Seed Oil. Chemical and Biochemical Engineering Quarterly 2014, 28(4):481-490). During supercritical CO2 extraction, oil yield increases with increasing pressure and decreasing particle size (Tomita K, Machmudah S, Quitain AT,... Goto M. Extraction and solubility evaluation of fimctional seed oil in supercritical carbon dioxide. Journal of Supercritical Fluids 2013, 79:109-113). Very high pressure has a negative effect on oil yield, because highly compressed CO2 facilitates mutual repulsion of solute and solvent. A two-stage procedure for the extraction of oil from hemp seeds was investigated, which consisted of pressing the seeds and extracting the residual oil in the meal by supercritical CO2 extraction.

Hemp seed oil contains over 80% polyunsaturated fatty acids (PUFA) and is an extremely rich source of two essential fatty acids, linoleic acid (C18:2 omega-6) and a-linolenic acid (C18:3 omega-3). The ratio of omega-6:omega-3 (ω-6:ω-3) fatty acids in hemp oil is between 2.1 and 3:1, which is considered optimal for human health, lowers cholesterol and high blood pressure (Kunos G, Jarai Z, Batkai S,... Wagner JA. Endocannabinoids as cardiovascular modulators. Chemistry and Physics of Lipids 2000, 108:159-168), (Callaway J. Hempseed as a nutritional resource: an overview. Euphytica 2004, 140:65-72). Hemp oil contains β-caryophyllene, characterized by a cyclobutane ring and rarely found in nature, then myrcene, β-sitosterol and traces of methyl salicylate (Leizer C, Ribnicky D, Poulev A, Dushenkov S, Raskin I. The Composition of Hemp Seed Oil and Its Potential as an Important Source of Nutrition. Journal of Nutraceuticals, Functional & Medical Foods 2000, 2:35-53). Hemp seed oil contains a considerable amount of tocopherol 90 mg/100 g (γ-tocopherol 85 mg/100 g, α-tocopherol 5 mg/100 g of the total tocopherol content) (Callaway J. Hempseed as a nutritional resource: an overview. Euphytica 2004, 140:65-72). The additional nutritional value of this oil is due to the presence of 3-4% gammalinolenic acid (GLA, C18:3 omega-6) and stearidonic acid (SDA, C18:4 omega-3), which makes it superior to most comparable oils (Goldberg EM, Gakhar N,... House JD. Fatty acid profile and sensory characteristics of table eggs from laying hens fed hempseed and hempseed oil. Journal of Food Science 2012, 77; S153-S160).

There is a certain number of preparations on the market based on raw materials/active principles of natural origin, but without scientifically based and defined effectiveness testing. Hemp-based balms, creams, lotions, and ointments applied to the skin can be effective as analgesics and anti-inflammatories. Indications for which these preparations are recommended are swelling, burns, muscle pain, rashes, arthritis, as well as post-herpetic neuralgia. In states where the use of medical hemp is legal, pharmaceutical companies have developed drugs that contain natural or synthetic cannabinoids.

The first officially approved cannabinoid drug of plant origin in the world by the European Medicines Agency (EMA) is produced by the British "GW Pharmaceuticals", oromucosal spray "Sativex®" with 2.7 mg of THC and 2.5 mg of CBD in a ratio of 1:1, from Cannabis sativa L. for the treatment of multiple sclerosis and pain relief in adult patients with cancer, is registered in 27 states outside the USA (www.gwpharm.com/). The US Food and Drug Administration (FDA) has approved Greenwich Biosciences' "Epidiolex" oral solution with 100 mg/mL CBD for the treatment of epileptic seizures associated with Lennox-Gastaut syndrome (www.greenwichbiosciences.com/).

"Aspen Green" produces "Moisturizing body butter", a moisturizing body butter for skin revitalization, based on hemp plant extracts, including pure CBD, without THC.

Czech company "Palacio CZ" s.r.o. produces a range of preparations based on cold-pressed hemp oil: "Cannabis face cream", "Cannabis body balm" based on 12% hemp oil, "Cannabis flex cooling gel" and "Cannabis flex warming gel", "Cooling massage gel", "Warming massage gel", "Cannabis shower gel", "Cannabis hand cream" and "Cannabis massage gel 5%" with 5% cannabis, menthol, oil extract of chestnut, comfrey, camphor and eucalyptus.

The company "Evree" produces "Cannabis - Hand cream for dry and chapped skin with cannabis and kuki oil" and "Cannabis - Regenerative hand cream with hemp oil and papaya".

"Victoria Beauty Camco" ltd from Plovdiv, Bulgaria produces "Victoria beauty Cannabis" intensive regenerating and hydrating night cream based on hemp oil, macadamia and chamomile.

The company "AVON" produces hemp oil preparation "Cannabis sativa oil - restore & calm" soothing hand and body balm, "Cannabis sativa oil - protect & calm" hydrating day cream with SPF 20 and "Cannabis sativa oil - cleanse & calm" face cleansing oil.

The available patent documentation lists the following relevant documents:

In the patent application US20120264818 A1, preparations for topical application (solution, spray, lotion, gel, cream or ointment) are formulated with Cannabis sativa extract containing a minimum of 10 wt% tetrahydrocannibolic acid. The formulation is intended for the local treatment of methicillin-resistant infections caused by the bacterium Staphilococcus aureus (MRSA) in order to eliminate pain, inflammation, muscle tension, muscle spasms, and skin ulceration.

The subject of the application US2015086494 A1 are topical formulations for the treatment of psoriasis based on cannabis, with a concentration of CBD or THC or both greater than 2 mg/g. The formulations optionally contain therapeutically effective amounts of steroids, antiseptics, antibiotics, antifungals.

Patent EP2311475 V1 protects an improved process for the extraction of cannabinoids from plant material using liquid carbon dioxide extraction under subcritical conditions. It contains decarboxylation steps by heating the plant material to temperatures and times that ensure at least 97% conversion of acidic cannabinoids to their neutral form, while ensuring thermal decomposition of THC to CBN less than 5%, extraction and a step to reduce the proportion of unwanted materials in the extract, at a temperature of 5 to 15°C and a pressure in the range of 50 to 70 bar, and the extraction process lasts up to 10 hours.

The invention US2019054394 A1 protects the process for obtaining extracts from cannabis.

Patent application WO2017178937 protects an oil gel composition containing an essential combination of non-psychotropic phytocannabinoids from the Cannabis sativa plant: CBD, cannabidiol acid, cannabivarin and cannabigerol in combination with Olive europaea olive oil extract, Mentha arvensis leaf oil and anhydrous colloidal silicon. The composition provides the release of cannabinoids into deep tissues using a pharmaceutical patch and a system for releasing active compounds at the desired location to reduce pain and inflammation of skeletal muscles and joints caused by trauma and/or arthritis/osteoarthritis.

Patent application US10092611 V1 "Pharmaceutical composition and method of production" protects<se>procedures for obtaining cannabis extracts using supercritical fluids, as well as subsequent processing of extracts to obtain cannabis concentrates. The subject invention protects pharmaceutical dosage forms that include a concentrate (or extract) of cannabis, such as e.g. oral thin dry film based on rapidly disintegrating hydrophilic polymer and transdermal patches.

Patent EA004520 V1 protects the process of obtaining extracts containing THC, CBD and their carboxylic acids from hemp plant mass subjected to extraction with concentrated SO2 in supercritical and subcritical conditions.

Based on a detailed review of the available literature, it was determined that there are no phytopreparations based on the supercritical extract of immortelle (Helihrysum italicum (Roth) G.) and industrial hemp (Cannabis sativa L., Cannabaceae) for the treatment of skin inflammation, slowing down the process and reducing the visible signs of skin aging.

DESCRIPTION OF THE INVENTION WITH IMPLEMENTATION EXAMPLES

The essence of the invention consists of a composition based on the most effective plant raw materials, extract production technology, formulation and phytopreparation production technology based on extracts of immortelle (Helihrysum italicum (Roth) G.) and industrial hemp (Cannabis sativa L., Cannabaceae), which give the best results in terms of action for slowing down the skin aging process and reducing visible signs of skin aging. Also, the application of the pharmaceutical composition according to the invention in phytopreparations of various pharmaceutical forms (emulgel, paste, ointment, cream and gel-cream) ensures the effective action of all components and the best effect during skin treatment.

So far, research on obtaining the essential oil/extract of immortelle (Helihrysum italicum) and industrial hemp (Cannabis sativa L., Cannabaceae) has mainly focused on conventional hydrodistillation/extraction procedures. The present invention protects significantly more active fractions obtained from immortelle and industrial hemp using supercritical extraction, considering that the carriers of numerous activities are non-volatile compounds, non-polar, soluble in nkSO2. The disadvantage of conventional methods of extraction is that the extraction with an organic solvent also extracts harmful substances from the air and precipitation, such as heavy metals. Condensed gases, primarily liquid and supercritical carbon(IV)-oxide are increasingly used today for the extraction of natural bioactive compounds, especially in the food, cosmetic and pharmaceutical industries. The main advantages of this technology are reflected in the ability to easily adjust the selectivity of the solvent (condensed gas), by changing the operating pressure and temperature, as well as obtaining high-quality extracts at relatively low process temperatures (25-60°C), without traces of organic solvents. Condensed carbon(IV)-oxide has lower viscosity values than liquid and surface tension approximately equal to zero, which is why it easily diffuses into solids. The density, and thus the viscosity and diffusion rate of the concentrated carbon(IV)-oxide can be easily adjusted by changing the operating pressure and/or temperature. With the method of supercritical extraction under high pressure, with the use of carbon(IV)-oxide as a solvent, it is not possible to extract heavy metals and other harmful substances, because they are not soluble in supercritical carbon(IV)-oxide. Therefore, supercritical carbon(IV)-oxide shows a high selectivity towards active components. Accordingly, by applying the mentioned methods, extracts of immortelle and industrial hemp can be purified almost to a pure form.

Thanks to the large number of active principles mentioned in the state of the art, the use of immortelle as a strong anti-allergen, anti-inflammatory and anti-erythematous agent is known. Arzanol has been proven to be a very important compound due to its strong anti-inflammatory action, tested in vitro and in vivo, in human intestinal epithelial cells. Arzanol, as a new phloroglucinol α-pyrone, exhibits strong anti-inflammatory, anti-HIV and antioxidant activity (Kothavade PS, Nagmoti DM, Bulani VD, Juvekar AR. Arzanol, a potent mPGES-1 inhibitor: novel anti-inflammatory agent. The Scientific World Journal, 2013; Rosa A, Pollastro F, Atzeri A... Dessi MA. Protective role of arzanol against lipid peroxidation in biological systems. Chemistry and physics of lipids 2011, 164(l):24-32). With its mechanisms of action, it can be an active component in the process of neutralizing the oxidizing effect on proteins and lipids of cell membranes, and thus on the formation of free radicals, as an important factor in the process of premature aging. According to a detailed review of the available literature, arzanol could only be isolated by extraction from acetone. A special novelty of the present invention is the fact that the process of supercritical extraction of immortelle (Helihrysum italicum) successfully extracts arzanol, as one of the most valuable active components. By varying the operational parameters of the supercritical extraction, the optimal conditions for achieving unexpectedly good results were reached and arzanol was extracted for the first time using the supercritical extraction procedure with a surprising yield.

Condensed carbon(IV)-oxide represents a suitable medium for the incorporation of compounds that are soluble in it, into solid carriers. According to available data, supercritical extraction from immortelle was tested, and supercritical immortelle extract was used as an active substance for the impregnation of various solid supports in supercritical conditions (Maksimović S. Extraction from immortelle (Helichrysum italicum) and impregnation of solid supports with extract using supercritical carbon(IV)-oxide. University of Belgrade, Faculty of Technology and Metallurgy, Belgrade, 2017). The extraction of rich essential oil from immortelle was carried out at 40°C and at a pressure of 15 MPa. The cosolvent effect of sage essential oil and ethanol was examined. Supercritical extraction of components of higher molecular weight from immortelle was realized at 40°C and a pressure of 35 MPa with the addition of ethanol as a cosolvent, which caused a double extraction yield. Determination of the total content of phenols and flavonoids showed a significant increase in the total content of sesquiterpenes, coumarin derivatives and amorphene and flavonoids in the extract obtained with the addition of cosolvents, and a significant decrease in the total content of phenols and fatty acids, esters, aldehydes and alcohol. Both extracts showed moderate antioxidant activity. The influence of the ethanol cosolvent on the combined process contributed to an increase in the yield of impregnation with cotton gauze, and a decrease in the yield of impregnation of poly(propylene) fabric. The results of in vivo testing of the effect of impregnated textile materials on irritated skin showed potential for topical application. The highest impregnation yield values were achieved in the process with pure supercritical carbon(IV)-oxide, during 5 h and at a ratio of the mass of plant material to the mass of the carrier of 10.

Given that immortelle extract contains active components that are poorly soluble in water, and some active components are unstable, it is desirable to ensure their improved solubility and stability in formulated preparations by using an adequate carrier. The isolated active component arzanol is poorly soluble in water, and for long-term stability the commercial standard should be stored at -20°C (Arzanol, Product Data Sheet, Adipogen Corp.).

The innovativeness of the subject invention is in obtaining plant semi-products using the supercritical impregnation procedure, i.e. simultaneous supercritical extraction and impregnation of the carrier of natural origin with immortelle oil/extract. In order to improve the bioavailability of the active components according to the invention, carriers based on cyclic oligosaccharides were chosen for impregnation with immortelle extract. Incorporation of immortelle essential oil into the cavities of cyclodextrins or their derivatives, the active components are protected for a longer period and their improved solubility and bioavailability is enabled. Cyclodextrins are crystalline, non-hygroscopic, non-reducing, cyclic oligosaccharides, which are obtained by enzymatic transformation of starch by Bacillus macerans (Milić J, Savić S. Conventional pharmaceutical forms and modern drug carriers - characteristics and applications. In Farmaceutska medicina - selected chapters, Prostran M, Stanulović M, Marisavljević D, Đurić D. Hemofarm, Vršac, 2009). Most often, they contain six, seven or eight D-glucose residues, interconnected by an α-(1→4)-glycosidic bond, forming a macroring. They represent basket-like toroidal molecules, solid structures, with a central cavity whose size depends on the type of macroring. The inner surface of the cyclodextrin macroring cavity is hydrophobic, and the outside of the torus is hydrophilic due to the distribution of hydroxyl groups within the molecule. Cyclodextrins can form inclusion complexes with some molecules of active substances, which results primarily in improved solubility, dissolution rate and biological availability, as well as improved chemical and physical stability. Formation of complexes of poorly soluble or unstable active substances with cyclodextrins is often used in order to improve bioavailability. Cyclodextrins can affect the resorption process and biological availability of the medicinal substance by increasing the dissolution rate of the medicinal substance from the medicinal form, as well as the degree of dissolution, improving the permeability of the medicinal substance through mucous membranes and increasing stability. They are used to mask the unpleasant taste of the active substance and to convert the liquid substance into a solid state by building complexes. Hydroxylated derivatives of β- and γ-cyclodextrin are often used in commercial applications. (2-Hydroxypropyl)-β-cyclodextrin is most often used due to the adequate diameter of the cavity and the improvement of solubility, because it can change the physicochemical properties of lipophilic compounds, enable their easier diffusion through biological membranes and increase their solubility in water, the antimicrobial efficiency of chemical agents, stability and increase the release rate. Methyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, monochlorotriazinyl-β-cyclodextrin, heptakis-β-cyclodextrin and the so-called branched cyclodextrins, e.g. maltosyl-β-cyclodextrin are some of the cyclodextrin derivatives with practical application. In parenteral formulations, β-cyclodextrin (due to nephrotoxicity) and γ-cyclodextrin (because it forms visible aggregates in aqueous solutions) are not used. Natural cyclodextrins and their derivatives are used in topical and oral products.

According to the invention, α-, β- or γ-cyclodextrins and their derivatives, and/or their mixtures, are impregnated with immortelle extract (Helihrysum italicum) and incorporated into appropriate pharmaceutical forms (emulgel, pastes, ointments, creams and gel-creams), which achieves a slow/controlled release of the extract's active substances in an original way. The novelty of the invention is reflected in the application of an integrated process of extraction and incorporation of bioactive components of immortelle (Helihrysum italicum) into the cavities of cyclodextrin molecules. With a suitable choice of operational parameters of the integrated process of impregnation and extraction, optimal conditions were reached for achieving surprising results that confirmed the presence of arsanol in the final product, which was stabilized in an adequate carrier based on cyclodextrin. The integrated process proved to be a suitable method for the isolation and incorporation of bioactive components of immortelle and incorporation into cyclodextrin-based carriers.

Taking into account the increasing scientific interest in industrial hemp, as well as in extracts of this plant, one of the carriers of activity in phytopreparations is industrial hemp oil. THC-free industrial hemp seeds, as carriers of psychoactive activity, bring other pharmacologically active compounds to the fore. Industrial hemp seeds, whether oil is used or the residue that remains after the exhaustion of the oily fraction, does not contain phytoestrogens, and contains a favorable, defined ratio of ω-3 and ω-6 fatty acids, fibers, minerals, essential amino acids. Industrial hemp seeds are unique in nature for the presence of a protein - edestin globulin, which plays a role in ensuring the proper functioning of many key processes in the body, primarily in the building of antibodies (immune globulin). In the oil and the rest of the seeds after extracting the oil fraction, it is possible to have orcinol derivatives, with antibacterial, anti-inflammatory and strong antioxidant potential, which do not exhibit binding affinity for opoid receptors of the CB1 and CB2 type and do not act as psychoactive compounds.

According to the invention, industrial hemp oil with 80% PUFA is used, in which the ratio of ω-3 and ω-6 acids is 3:1, in accordance with the regulations of the European Food Safety Agency (3-5:1, EFSA, 2009). According to previous research, industrial hemp seed oil also contains γ-linoleic acid and stearidonic acid, which have only been found in a few plant sources. Studies of γ-linoleic and stearidonic fatty acids have shown that they have a beneficial effect on the skin. These compounds are powerful antioxidant agents, they stop the process of lipid peroxidation, a process responsible for the breakdown of fatty acids and their esters while simultaneously creating harmful free radicals, causing irreversible tissue damage and other pathological changes in the body. The above-mentioned pharmacological activities are necessary in the formulation of preparations intended for skin revitalization for local application.

There are no phytopreparations on the market based on a combination of supercritical extract of immortelle (Helihrysum italicum) and industrial hemp (Cannabis sativa L.) for the treatment of skin inflammation, slowing down the skin aging process and reducing the visible signs of skin aging. A review of the available data indicates that the preparations on the market are not standardized on non-flavonoid active components, the stability of the preparations has not been adequately tested, and there is no confirmation that the preparations made from the mentioned extracts provide the desired effects in the prevention of premature aging.

The technical solution also refers to the new composition of herbal extracts of immortelle (Helihrysum italicum) and industrial hemp (Cannabis sativa L.). Although the medicinal properties of the active principles of the constituent plant components are individually known for a large part from traditional application and are being scientifically confirmed more and more intensively in recent times, the proposed herbal composition is new and original. The applicability of the composition according to the invention is based on the scientifically confirmed biological properties of the active components.

For the production of high-quality phytopreparations intended for skin revitalization, a chemical analysis of plant raw materials and plant extracts is necessary in the process of formulation and quality assurance of semi-products that are incorporated into the finished product. The subject invention defines semi-finished products as components of finished formulations, with developed methods of identification and quantification of active components. It is possible to put on the market only a preparation that meets the requirements of the legal regulation, i.e. products must meet the required conditions of declared quality, efficiency and safety.

During the selection of active ingredients of natural origin, according to the invention, scientifically confirmed physiological effects of the selected medicinal plants (antimicrobial, anti-inflammatory, protective) were considered, as well as based on in vivo testing of the anti-inflammatory effect of creams incorporating the tested isolates and the safety aspect of application to healthy and artificially irritated skin. It was found that the combination of extracts of immortelle and industrial hemp according to the invention contributes to a surprising enhancement of the anti-inflammatory effect of the individual components. It is especially surprising that the combination of immortelle extracts and fatty hemp oil, according to the invention, leads to an unexpected anti-irritant effect, which is very important for application to thinned and damaged skin. In this way, the effects of the antibacterial action are combined and enhanced by the new pharmaceutical composition, providing enhanced anti-inflammatory and protective activity. Formulated preparations with a standardized content of antioxidant, anti-inflammatory active substances for local application on the skin are intended to slow down the aging process and reduce the visible signs of skin aging, given that the action of free radicals and various skin inflammations are the causes of accelerated skin aging. Smilje (Helihrysum italicum), in addition to essential oil, also contains various secondary metabolites that exhibit a strong antimicrobial, anti-inflammatory and antiviral effect, so the subject of the invention includes extracts and essential oils.

The first visible signs of aging skin are fine lines, wrinkles, loss of volume and elasticity. Structural, functional and aesthetic changes occur in the skin. Several biological processes are simultaneously present in the skin aging process. Freckles and hyperpigmentation are the result of the skin trying to protect itself from the harmful effects of the sun's rays. Free radicals damage the structure of skin cells by breaking down important skin components, such as hyaluronic acid, collagen and elastin, and cause premature aging of the skin. Over time, the skin's ability to deactivate free radicals weakens and damage occurs to all components of skin cells. Aging skin requires protection and hydration, and in old age it needs help from the outside in the form of active substances that protect it from aging. In accordance with the prevailing trends, as well as the demands and expectations of the product users, in order to improve the quality of life, the focus of research in the cosmetics industry is particularly active substances of natural origin that should reduce or delay the appearance of visible signs of skin aging, i.e. protect the skin from factors and processes that lead to premature aging. The effectiveness of preparations intended for local application implies their multifunctionality, taking into account that the changes occurring on the skin are the result of processes characterized by complicated etiologies. Dermatoses are often accompanied by dry and scaly skin, and topical application of products that significantly moisturize the skin, mostly in the form of emulsions, is a good strategy for their treatment.

A preparation for application to aging skin must meet numerous requirements, such as physical-chemical stability, as well as efficiency and safety of application (Jaksic I, Lukic M, Malenovic A, Reichl S, ...Savic S. European Journal of Pharmaceutics and Biopharmaceutics 2012, 80:164-175). That is why the choice of the substrate is essential when formulating such a product (Daniels R, Knie U. Galenics of dermal products-vehicles, properties and drug release. Journal der Deutschen Dermatologischen Gesellschaft 2007, 5(5):367-383). Conventional emulsifiers, mandatory ingredients of emulsions, can have a high potential to irritate the skin and damage its barrier (Tasić-Kostov M, Savić S. New natural sugar-based surfactants intended for stabilization of cosmetic/dermopharmaceutical vehicles: Safety and efficacy assessment. Acta medica Medianae 2015, 54(l):34-39). Contrary to the conventional ones, alkylpolyglucosides (APG) as newer, mild, biodegradable emulsifiers are attracting a lot of attention today. It is believed that, in addition to being non-irritating, they show additional positive effects when applied topically, such as moisturizing the skin. That is why it is recommended to use alkylpolyglucosides, as emulsifiers that are completely based on renewable sources and with very favorable dermatological characteristics, which are reflected in the possibility of prolonged hydration and reduced potential for alternation of skin proteins (Holmberg K. Natural surfactants. Current Opinion in Colloid & Interface Science 2001, ć(2): 148-159). Given that these are new generation emulsifiers, there is no data on their use in preparations that have plant extracts as an active component. The present invention has successfully defined the conditions for incorporating active extracts of natural origin into a formulated base with auxiliary substances and alkylpolyglucoside emulsifiers, which results in the formation of a stable cream, on the basis of which it can be concluded that creams with APG can be good excipients for incorporating natural extracts. The compatibility of the used plant extracts with alkylpolyglucosides was confirmed. From the aspect of the importance of efficiency and safety of active substances as well as excipients on the overall effects of the preparation, a satisfactory level of safety and efficiency of creams based on extracts of natural origin and new "natural"-APG emulsifiers was determined.

When choosing an excipient/substrate for preparations intended for the treatment of skin prone to irritation, it must be taken into account that the skin and soft tissues are accompanied by an inflammatory response, which results in characteristic manifestations such as erythema, edema, a feeling of warmth, pain or sensitivity, so in addition to the antimicrobial, the anti-inflammatory/anti-erythematous action of the active principles is also significant.

When formulating preparations for topical application in the treatment of skin inflammation, increasing skin hydration, whitening hyperpigmentation, reducing visible signs and slowing down the skin aging process, in addition to the selection of emulsifiers, adequate emollients (skin moisturizing agents) were selected, such as triglycerides of caprylic and caproic acids, which on the one hand soften the skin and increase its moisture, and on the other hand restore the barrier function of the skin, alleviating the symptoms accompanying skin infections.

Detailed description of the invention

Extracts and isolated essential oils were prepared from selected medicinal plants - immortelle (Helihrysum italicum) and industrial hemp (Cannabis sativa L.) for the preparation of phytopreparations, which were used for the formulation and production of appropriate phytopreparations.

In the production of phytopreparations according to the invention, the following are used:

1. extract of immortelle (Helihrysum italicum (Roth) G.) originating from the territory of Croatia.

1.1. extract of immortelle (Helihrysum italicum) obtained by the process of supercritical extraction with carbon (IV)-oxide (NKE). The extraction is carried out at a plant for the integrated process of supercritical extraction and supercritical impregnation (NKE-NKI), which consists of at least two vessels, an extractor and an adsorber, for operation under high pressure up to a maximum of 100 MPa, primarily 10-50 MPa, a pump for transporting and compressing fluids under pressure, a pump for circulating fluids under high pressure and a pressure regulator. The configuration of the HPEA (High Pressure Extraction Adsorption) 500 plant for the integrated process of supercritical extraction and supercritical impregnation, shown in Figure 1, can be applied to perform the integrated extraction-adsorption process in a batch, or/and semi-continuous mode of fluid flow under high pressure, suitable for continuous circulation of the supercritical solution through the extractor and adsorber. NKE is performed in a configuration where valves V-1, V-2, V-3 and V-9 are open while the other valves are closed. In this way, nkSO2 passes only through the extractor (not the adsorption column).

Variant solutions for obtaining immortelle extract and operational values of pressure, temperature and extraction time are as follows:

.1.1. extract of immortelle (Helihtysum italicum) obtained by the process of fractional supercritical extraction at a temperature of 31°C to 80°C, preferably from 35°C to 50°C, first at lower pressure values from 8 MPa to 25 MPa, primarily from 10 MPa to 15 MPa, until the exhaustion of the plant raw material, for a duration of 0.5 to 2 h, and then at higher pressure values from 25 MPa to 50 MPa, primarily from 30 MPa to 40 MPa, lasting from 2 to 5 h. The yield of supercritical extraction is 1-3% at lower pressure values and 1.5-4% at higher pressure values.

.1.2. immortelle extract (Helihrysum italicum) obtained by supercritical extraction at lower pressure values from 8 MPa to 25 MPa (primarily from 10 to 15 MPa), at a temperature from 31°C to 80°C, primarily from 35°C to 40°C, for a duration of 0.5 to 2 h. The yield of supercritical extraction at lower pressure is 1-3%, preferably 1.8-2.6%, and the percentage of arzanol is 3.51%.

.1.3. extract of immortelle (Helihrysum italicum) obtained by a supercritical extraction process at higher pressure values of 25 MPa to 50 MPa (primarily from 30 to 40 MPa) at a temperature of 31°C to 80°C, primarily from 35°C to 40°C, for a duration of 2 to 5 h. The yield of supercritical extraction at higher pressure values is 2-5%, preferably 2.6-3.9%, while the percentage of arzanol is 4.59%.

.1.4. immortelle extract (Helihrysum italicum) obtained by a supercritical extraction process with cosolvent at a temperature of 31°C to about 80°C, primarily from 35°C to 50°C and at a pressure of about 8 MPa to 50 MPa, primarily from 30 MPa to 40 MPa, with 96% ethanol in an amount of 5% to 30% (preferably from 5 to 10%) relative to mass C02 used for extraction from immortelle without the use of cosolvents. After completion of the experiment, ethanol was removed from the extract mixture using a rotary vacuum evaporator. The yield of extraction using cosolvent is 7.5-8.59%, and the percentage of arzanol is from 4.9% (in Table 6) to 10.34%.

.2. inclusion complex based on immortelle extract (Helihrysum italicum) and cyclic oligosaccharides, obtained by an integrated process of supercritical extraction and supercritical impregnation. The integrated process of supercritical extraction and supercritical impregnation is performed in batches on the HPEA (High Pressure Extraction Adsorption) 500 plant (Figure 1) with the circulation of supercritical SO2 through the extractor and adsorption column, without adding new amounts of fresh SO2 to the system. At the beginning of the process, in the pre-filled extractor with plant material and the adsorption column filled with cyclic oligosaccharide powder, selected from the cyclodextrin group consisting primarily of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or cyclodextrin derivatives, such as methyl-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, sulfobutylether-β-cyclodextrin, monochlorotriazinyl-β-cyclodextrin, heptakis-β-cyclodextrin or maltosyl-β-cyclodextrin) or their mixtures, at a mass ratio of 1:1 to 15:1, SO2 was introduced via a cryostat and a pump until the desired conditions were reached. After reaching operational pressure values from 8 MPa to 50 MPa, primarily between 15 MPa and 30 MPa, and temperature from 31°C to 80°C, primarily between 40°C and 60°C, circulation of nkSO2 through the plant is provided for a duration of about 2 h to about 10 h, primarily 3 h to 6 h.

The chemical analysis of obtained plant extracts of immortelle (Helihrysum italicum) originating from the territory of Croatia showed the unexpected presence of the active component arzanol in the range from about 3.5% to about 4.6%, while in the inclusion complex the content of arzanol is min.

0.0075% (table 7). Namely, according to all available literature data with the results of chemical analysis of immortelle from Croatia, the presence of arzanol is not mentioned anywhere. The chemical analysis of the extracts shows that the effective choice of process parameters, as well as the addition of ethanol as a cosolvent, had a positive effect on the selectivity of supercritical CO2 according to the content of the active component arzanol.

. extract/fatty oil of industrial hemp (Cannabis sativa L.) without tetrahydrocannabinol - commercial product, obtained by the process of supercritical extraction with carbon(IV)-oxide and additionally purified, with a defined content of polyunsaturated fatty acids (PUFA), 80%, in which the optimal ratio of linoleic (LA) and linolenic (LNA) acid is 3:1 and with a source of vitamin E (100-150 mg/100g).

Adopting the scientifically confirmed position that the best effect is achieved through synergism, herbal products based on immortelle extract (Helihrysum italicum) (obtained by the process of supercritical extraction and the integrated process of supercritical extraction and supercritical cyclodextrin impregnation) and hemp oil, were used as intermediate products in the formulation of preparations for topical application. Incorporation of immortelle extract into cyclodextrin-based carriers was applied with the aim of protecting the active components from harmful environmental influences, such as oxidation, degradation, heat, light, evaporation and moisture (Cheirsilp B, Rakmai J. Inclusion complex formation of cyclodextrin with its guest and their applications. Biology, Engineering and Medicine, 201 b, 2(1) 1-6). Within the inclusion complexes, the chemical and biological properties of complex active principles are improved and better solubility and stability are ensured (Cameiro SB, Costa Duarte FÍ, Heimfarth L, Siqueira Quintans JDS,... Neves de Lima ÁA. Cyclodextrin-dmg inclusion complexes: In vivo and in vitro approaches. International journal of molecular sciences 2019, 20(3):642). Eh vivo and in vitro tests showed increased stability and slower degradation of flavonoids, a lower and controlled rate of release of the cyclodextrin phytocomplex compared to the pure extract (Spada G, Gavini E, Cossu M, Rassu G, Carta A, Giunchedi P. Evaluation of the effect of hydroxypropyl-β-cyclodextrin on topical administration of milk thistle extract. Carbohydrate polymers 2013, 92(l):40-47).

Formulated preparations based on a combination of extracts of immortelle (Helihrysum italicum) and hemp according to the invention, after topical application, exhibit a strong effect on the skin, while they do not lead to an unwanted antibacterial effect in the form of skin damage, i.e. skin protective factor. The stated concept according to the invention was not found in the available literature.

None of the known procedures defines the simultaneous one-step obtaining of a pure product with a stable and appropriate content of arzanol, from the aspect of efficiency and safety of its topical application, which is another main characteristic of the invention.

The invention protects the recipe for the range of phytopreparations, which maximizes the effectiveness of the ingredients, prolonged release and synergism, and which gives the best effects when applied to the skin. Formulations of products for topical application with industrial hemp and immortelle (Helihrysum italicum) extract incorporated into cyclodextrin-based carriers using the nkSO2 technique in order to stabilize and homogenize them, ensure the slowing down of the skin aging process and the mitigation of visible signs of skin aging, with a satisfactory safety profile in the form of skin microbiota damage and irritation reactions.

The obtained extracts of immortelle (Helihrysum italicum) (pure and incorporated in cyclodextrin-based carriers) and industrial hemp (Cannabis sativa L.) with a high, standardized content of bioactive molecules, were used for the production of pharmaceutical and cosmetic preparations according to prescribed recipes and with the use of usual pharmaceutical supplements. The selection of active ingredients of natural origin, according to the invention, was carried out on the basis of scientifically confirmed physiological actions of the medicinal herb immortelle (Helihrysum italicum) and industrial hemp (Cannabis sativa L., Cannabaceae) (antimicrobial, anti-inflammatory, protective), the results of in vitro testing of the antimicrobial potential of their extracts/essential oils and their combinations, as well as on the basis of in vivo testing of the anti-inflammatory action of preparations incorporating the tested isolates and the safety aspect of their application to healthy and artificially irritated skin. Pharmaceutical compositions have a soothing effect on painful places and accelerate the process of tissue recovery. Antioxidants present in the extracts help to eliminate toxins and harmful substances, facilitate the supply of cells with oxygen and nutrients, speed up venous circulation, enable better tissue blood flow and speed up the recovery process. Also, they help strengthen blood vessels, prevent them from bursting, which gives the skin elasticity and a healthy appearance.

The subject of the invention are pharmaceutical compositions containing 1.00%-2.00% of immortelle extract (Helihrysum italicum) obtained by supercritical extraction with carbon(IY)-oxide (pure or incorporated in a cyclodextrin-based carrier), 1.00%-2.50% of industrial hemp extract/fatty oil (Cannabis sativa L., Cannabaceae) and a suitable excipient for the appropriate pharmaceutical form up to 100%. Pharmaceutical forms, according to the invention, include semi-solid preparations (emulgels, gels, creams, gel-creams, pastes, ointments).

Emulgels, as modern carriers of active substances, are emulsions (oil in water or water in oil) in which gels are incorporated. They are obtained by mixing an emulsion and a dispersed gelling agent. Emulsions in themselves represent a system with controlled release of the active substance because the active substance, dispersed in the internal phase, must diffuse through the external phase to reach the skin and be absorbed in it. Gels form lattices that trap particles of the active phase. Therefore, emulgels possess properties of both emulsions and gels, and represent good systems for controlled delivery of active substances. Also, emulgels have a number of other advantages such as easy application, good thermodynamic stability, can serve as good carriers for active substances and act as penetration enhancers.

For the preparation of emulgel-type preparations according to the invention, table 1 lists the composition of active components and suitable excipients, their content (in %) and their role in the formulated emulgel. Emulgels with immortelle extract and hemp oil are effective in increasing skin hydration, reducing transepidermal water loss from the skin, as well as skin whitening potential. After long-term use, they do not cause irritation or redness of the skin and maintain the pH of the skin within physiological limits. Emulgels have shown excellent potential for increasing skin hydration.

Creams are multiphase preparations consisting of a fatty (lipophilic) and aqueous phase. They are intended for use on the skin and certain mucous membranes. Creams speed up venous circulation and enable better tissue blood supply and speed up the recovery process. They help strengthen blood vessels, prevent them from bursting, which gives the skin elasticity and a healthy appearance. Facilitating the nutrition of cells with oxygen and nutrients, these preparations contribute to maintaining the health of the skin and mucous membranes in people who have a tendency to develop varicose veins and hemorrhoids.

According to the invention, for the preparation of a cream-type preparation, table 2 lists the composition of active components and suitable excipients, their content (in %) and their role in the formulated cream.

Ointments are semi-solid preparations for application on the skin and are intended for local or transdermal application. They consist of a single-phase substrate in which solid substances or liquids can be dispersed. Lipophilic fats can absorb a small amount of water. Fats that emulsify water can absorb a larger amount of water and thus build water-in-oil or oil-in-water emulsions, depending on the emulsifiers they contain. Hydrophilic ointments are preparations composed of ointments that are mixed with water. For the preparation of ointment-type preparations according to the invention, Table 3 lists the composition of active components and suitable excipients.

Pastes are semi-solid preparations that contain a large part of small, solid substances dispersed (suspended) in the substrate. Table 4 lists the composition of active components and suitable excipients for the preparation of paste-type preparations according to the invention.

Gel-creams are multiphase systems that contain a fatty phase (lipophilic) and an aqueous phase (hydrophilic), whose optimal viscosity is achieved by adding a gelling agent.

The composition of active components and suitable excipients for making gel-cream according to the invention is listed in table 5.

The final preparations were characterized from a chemical point of view. The stability of the product and its safety profile were tested, and its effectiveness was determined when applied to healthy volunteers (in vivo testing).

All safety and effectiveness tests were performed in accordance with the current European regulation - "Regulation (EG) Na 1223/2009 of the European Parliament" and performed (in vivo) tests on healthy volunteers in accordance with the guidelines resulting from the application of this regulation.

The novelty of the subject invention in relation to the available state of the art is represented by: → new products/pharmaceutical compositions based on plant extracts from the immortelle Helihrysum italicum (Roth) G. Don fil., Asteraceae) and hemp (Cannabis sativa L., Cannabaceae), → new products/pharmaceutical compositions based on hemp (Cannabis sativa L,) and inclusion complexes of the extract of the immortelle Helihrysum italicum and carriers based on cyclodextrin,

→ new products for topical application of a combination of hemp and immortelle extracts to slow down the process and reduce the visible signs of aging of mature skin,

→ new semi-products - inclusion complexes of Helihrysum italicum extract and carriers based on cyclic oligosaccharides, increased bioavailability when applied topically

→ a new procedure for obtaining inclusion complexes of Helihrysum italicum extract and a carrier based on cyclodextrin,

→ new products/phytopreparations in the form of emulgels, creams, gel-creams, ointments and pastes.

The advantages of the improved procedure of supercritical extraction of immortelle in relation to the results published in the state of the art, primarily in relation to the work of Maksimović (Maksimović S. 2017. Extraction from immortelle (Helichrysum italicum) and impregnation of solid supports with the extract using supercritical carbon(IV)-oxide, University of Belgrade, Faculty of Technology and Metallurgy, Belgrade) is that the active component arzanol is not isolated in the extract from the territory of Croatia, under conditions supercritical extraction at 40°C and pressures of 15 MPa and 35 MPa (yield 2.7±0.5 without cosolvent and 5.9±0.7 with 10% ethanol). By adequately varying the conditions of pressure, temperature and contact time of supercritical extraction, with and without the addition of a co-solvent, an immortelle extract with an unexpected, surprising arzanol content and an improved yield is obtained.

The advantages of the integrated process of supercritical extraction and impregnation of solid supports based on cyclic oligosaccharides obtained with immortelle extract using supercritical carbon(IV)-oxide according to the present invention are:

- exhaustive extraction of the desired bioactive substances in the extract (high yield),

- obtaining very pure products without the presence of organic solvents after the extraction phase to a trace concentration,

The advantages of formulated preparations for topical application based on extracts of immortelle Helihrysum italicum and hemp Cannabis sativa L. according to the invention are as follows:

- defined effective ratio of emulsifiers, emollients, water and active components,

- acceptable formulations of phytopreparations for safe use for most users,

- unexpectedly effective and simple applications for skin and mucous membrane changes compared to similar products, especially for inflammation and accelerated aging of the skin caused by the action of free radicals.

- surprising increase in skin hydration, whitening hyperpigmentation, slowing down the process and reducing visible signs of skin aging.

DESCRIPTION OF DRAFT PICTURES

Figure 1. Schematic of the HPEA 500 high-pressure extraction and impregnation plant; V-1 to V-9: valves for high pressure operation.

Figure 2. Curves of supercritical extraction from immortelle (y-extraction yield,%, x-consumption of SO2 per mass of plant material, g/g): a) cumulative curve of fractional NKE at 15 MPa, 35 MPa and at 40°C; b) curve at 10 MPa and 40°C, c) curve at 30 MPa and at 40°C, d) curve at 35 MPa and at 40°C.

Figure 3. HPLC chromatogram of immortelle extract (Helihrysum italicum (Roth) G.) originating from the territory of Croatia (without and with added cosolvent), detection at λ=280 nm.

Figure 4. Increase in skin hydration expressed in % compared to initial values.

In order to facilitate the understanding of the subject matter of the present invention, examples are given below which explain the various stages in more detail, but in no way limit it.

FEASIBILITY EXAMPLES

Example 1: Method of fractional supercritical extraction with carbon (IV)-oxide for obtaining

liquid extract of immortelle (Helihrysum italicum (Roth) G.)

The extraction of immortelle (Helihrysum italicum (Roth) G.) originating from the territory of Croatia was carried out at the laboratory plant HPEA 500 for the integrated process of supercritical extraction and supercritical impregnation, presented in Figure 1. The main parts of the plant are the extractor, adsorption column, pressure regulator and gear pump. NKE is performed in a configuration where valves V-1, V-2, V-3 and V-9 are open while the other valves are closed. In this way, nkSO2 passes only through the extractor (not the adsorption column). After placing the plant material in the extractor, liquid CO2 was introduced from a siphon bottle using a high-pressure liquid pump (MiltonRoy, France), previously passing through a cryostat to prevent its evaporation. After reaching the desired pressure and temperature values, a continuous flow of CO2 through the system is ensured until the plant material is exhausted. Separation of the extract from the solvent was carried out in a glass test tube (separator) at the exit from the system. CO2 was released from the system into the atmosphere, and its consumed mass was measured using a scale on which the bottle was placed. The pressure in the system is maintained using a backpressure regulator. The extraction was performed at a temperature of 31-80°C, primarily 35.5-50°C, first at a lower pressure of 8-25 MPa, primarily 10-14.9 MPa until the plant raw material was exhausted, for a duration of 0.5-2 h, and then at an elevated pressure of 25-50 MPa, primarily 30-40 MPa, for 2-5 h. After the expiration of the set circulation time, the system was decompressed for 10 min. The experimental extraction yield of the extraction was 1.23% at lower pressure and 1.87% at elevated pressure. Data on extraction yields and experimental conditions are shown in Table 6. Supercritical extraction kinetics from immortelle were monitored and supercritical extraction curves are shown in Figure 2a.

Example 1.1. Supercritical carbon (IV)-oxide extraction procedure for obtaining liquid extract of immortelle (Helihrysum italicum) at lower pressure

The extraction of immortelle (Helihrysum italicum (Roth) G.) from the territory of Croatia was carried out on the HPEA 500 laboratory plant for the integrated NKE-NKI process, according to the description given in example 1. The pressure in the system was maintained at a value of 10 MPa, and the temperature of the system was 40°C, for a duration of 0.82 h. The experimental extraction yield was 1.84% (Table 6). The kinetics were monitored and the supercritical extraction curves from immortelle are shown in Figure 2b.

Example 1.2. Supercritical carbon (IV)-oxide extraction procedure for obtaining liquid extract of immortelle (Helihrysum italicum (Roth) G.) at higher pressure

The extraction of immortelle (Helihrysum italicum (Roth) G.) from the territory of Croatia was carried out on the HPEA 500 laboratory plant for the integrated NKE-NKI process according to the description given in example 1. The pressure in the system was maintained at a value of 34.9 MPa at a temperature of 40°C, for 4.05 h. The experimental yield was 3.37% (Table 6). The supercritical extraction curves from immortelle are shown in Figures 2c and 2d.

Example 1.3. Method of supercritical extraction with carbon (IV)-oxide for obtaining liquid extract of immortelle (Helihrysum italicum (Roth) G.) with kosolovent

Supercritical extraction from immortelle (Helihrysum italicum (Roth) G.) from the territory of Croatia with kosolovent was carried out on the laboratory plant HPEA 500 for the integrated process NKE-NKI according to the description given in example 1. The pressure in the system was maintained at a value of 34.9 MPa, ethanol was added in the amount of 5, 10, 15, 20 and 30 mass% (preferably 10 mass%) in relation to the mass of CO2 used for extraction from immortelle without the use of cosolvents. After the experiment was completed, ethanol was removed from the extract mixture using a rotary vacuum evaporator. The yield of extraction of immortelle with the use of cosolvent ethanol was 7.72-8.59. It was not possible to construct supercritical extraction curves from immortelle with the addition of a cosolvent, because during the experiments with the extract, ethanol was also extracted in a glass test tube.

Example 2: Integrated procedure of supercritical extraction and supercritical impregnation of α-cyclodextrin with immortelle extract (Helihrysum italicum) - obtaining the inclusion complex α-cyclodextrin: immortelle extract

The integrated procedure of supercritical extraction and supercritical impregnation was carried out in batches on the HPEA 500 laboratory plant for the integrated NKE-NKI process (Figure 1) with the circulation of supercritical CO2 through the extractor and the adsorption column, without adding new amounts of fresh CO2 to the system. At the beginning of the process, the extractor is filled with plant material, and the adsorption column is filled with α-cyclodextrin powder (mass ratio 10:1) (20 g of immortelle and 2 g of α-cyclodextrin), CO2 (500 g) is introduced via a cryostat and a pump until the desired conditions are reached. After reaching the operational values of pressure and temperature, the circulation of nkCO2 through the plant was ensured for 5 h, by starting the gear pump and closing the valve V-l. After the expiration of the specified circulation time (impregnation time), the system was decompressed for 10 min. Supercritical impregnation of α-cyclodextrin powder with immortelle extract resulted in a yield of 4.29%, while the theoretical extraction yield during the integrated NKE-NKI process was 3.44%.

Example 3: Integrated procedure of supercritical extraction and supercritical impregnation of β-cyclodextrin with immortelle extract (Helihrysum italicum) - obtaining the inclusion complex β-cyclodextrin: immortelle extract

The integrated procedure of supercritical extraction and supercritical impregnation was performed in batches on the laboratory plant HPEA (High Pressure Extraction Adsorption) 500 (Figure 1) with the circulation of supercritical CO2 through the extractor and the adsorption column, without adding new amounts of fresh CO2 to the system. At the beginning of the process, the extractor is filled with plant material, and the adsorption column is filled with β-cyclodextrin powder (mass ratio 20:1) (40 g of immortelle and 2 g of β-cyclodextrin), CO2 (500 g) is introduced via a cryostat and a pump until the desired conditions are reached. After reaching the operational values of pressure and temperature, the circulation of nkCO2 through the plant was ensured for 4.5 h, by starting the gear pump and closing the valve V-l. After the expiration of the specified circulation time (impregnation time), the system was decompressed for 10 min. The yield of supercritical impregnation of β-cyclodextrin with immortelle extract is 4.29%.

Example 4: Integrated procedure of supercritical extraction and supercritical impregnation of γ-cyclodextrin with immortelle extract (Helihrysum italicum) - obtaining the inclusion complex γ-cyclodextrin: immortelle extract

The integrated procedure of supercritical extraction and supercritical impregnation was carried out in batches on the laboratory plant HPEA (High Pressure Extraction Adsorption) 500 (Figure 1) with the circulation of supercritical CO2 through the extractor and the adsorption column, without adding new amounts of fresh CO2 to the system. At the beginning of the process, the extractor is filled with plant material, and the adsorption column with γ-cyclodextrin powder (mass ratio 1:1) (2 g of immortelle and 2 g of γ-cyclodextrin), then CO2 (500 g) is introduced via a cryostat and a pump until the desired conditions are reached. After reaching the operational values of pressure and temperature, the circulation of nkCO2 through the plant was ensured for 3 h, by starting the gear pump and closing the valve V-l. After the specified impregnation time, the system was decompressed for 10 minutes. The yield of supercritical impregnation of γ-cyclodextrin powder with immortelle extract is 3.16%.

Example 5: Integrated procedure of supercritical extraction and supercritical impregnation of (2-hydroxypropyl)-β-cyclodextrin with immortelle extract (Helihrysum italicum) - obtaining the inclusion complex (2-hydroxypropyl)-β-cyclodextrin: immortelle extract

The integrated procedure of supercritical extraction and supercritical impregnation was carried out in batches on the HPEA 500 laboratory plant for the integrated NKE-NKI process (Figure 1) with the circulation of supercritical CO2 through the extractor and the adsorption column, without adding new amounts of fresh CO2 to the system. At the beginning of the process, the extractor is filled with plant material, and the adsorption column is filled with (2-hydroxypropyl)-β-cyclodextrin powder (mass ratio 1:5) (2 g immortelle and 10 g (2-hydroxypropyl)-β-cyclodextrin)), then CO2 (500 g) is introduced via a cryostat and a pump until the desired conditions are reached. After reaching the operational values of pressure and temperature, the circulation of nkCO2 through the plant was ensured for 3 hours, by starting the gear pump and closing the valve V-l. After the expiration of the specified circulation time (impregnation time), the system was decompressed for 10 min. The yield of supercritical impregnation of (2-hydroxypropyl)-β-cyclodextrin with immortelle extract was 8.85%.

Example 6: Integrated procedure of supercritical extraction and supercritical impregnation of methyl-β-cyclodextrin with immortelle extract (Helihrysum italicum) - obtaining the inclusion complex methyl-β-cyclodextrin: immortelle extract

The integrated procedure of supercritical extraction and supercritical impregnation was carried out in batches on the HPEA 500 laboratory plant for the integrated NKE-NKI process (Figure 1) with the circulation of supercritical CO2 through the extractor and the adsorption column, without adding new amounts of fresh CO2 to the system. At the beginning of the process, CO2 (500 g) was introduced through the cryostat and pump until the desired conditions were reached in the pre-filled extractor with plant material and the adsorption column with methyl-β-cyclodextrin powder (mass ratio 1:20) (2 g of immortelle and 40 g of methyl-β-cyclodextrin). After reaching the operational values of pressure and temperature, the circulation of nkCO2 through the plant was ensured for 4 h, by starting the gear pump and closing the valve V-l. After the expiration of the specified circulation time (impregnation time), the system was decompressed for 10 min. The yield of supercritical impregnation was 9.58%.

Example 7. HPLC analysis of the chemical composition of the obtained immortelle extracts

The results of the examination of the chemical composition of the obtained extracts of immortelle (Helihrysum italicum (Roth) G.), collected on the territory of Croatia, are shown in Table 7, and Figure 2 shows the HPLC chromatogram of the obtained extracts (without and with the addition of cosolvents). For the separation of components in extracts of immortelle (Helihrysum italicum (Roth) G.) under different conditions of supercritical extraction by the HPLC method, a mixture of SNSl3:MeON in the ratio 7:3 was used as the eluent, the retention time (without and with the addition of cosolvents) was Rt=89.33 min, and detection was performed at a wavelength of λ=280 nm. The obtained data show the surprising presence of arzanol in the range from about 3.50% to about 4.60% (table 7), which according to the literature was the only possible isolation from acetone immortelle from Croatia, the presence of arzanol is not mentioned anywhere. The chemical analysis of the extracts shows that the addition of ethanol as a cosolvent and the effective choice of process parameters positively influenced the selectivity of supercritical CO2 according to the content of the active component arzanol.

Example 8. Procedure for obtaining emulgel with a combination of hemp oil and immortelle extract

Sodium benzoate (0.15-3.50g) was dissolved in a mixture of propylene glycol (2.50-5.00g) and water in a beaker. Carbopol Ultrez 10 (0.45-l.00g) was gradually added over the surface. The ingredients of the water phase were heated to 75°C and added gradually to the fat phase (caprylic/capric triglycerides (3.00-5.00g), polysorbate 80 (1.00-2.50g), shea butter (1.00-3.50g), tocopherol acetate (0.10-0.50g) and butylhydroxytoluene (0.01-0.02g)) which was preheated to 70°C, with mixing with a propeller rotary mixer at 500 revolutions per minute. At the emulgel temperature of 40°C, a 10% sodium hydroxide solution (1.50-3.00g) was added to the formulation, and after vigorous mixing, the active components were added: hemp oil (1.00-2.50g) and immortelle extract (1.00-2.00%). Mixing was continued until the emulgel cooled down and reached room temperature.

Example 8.1. Method of obtaining emulgel with a combination of hemp oil and the inclusion complex α-cyclodextrin: immortelle extract

Sodium benzoate (0.15-3.50g) was dissolved in a mixture of propylene glycol (2.50-5.00g) and water in a beaker. Carbopol Ultrez 10 (0.45-l.00g) was gradually added over the surface. The ingredients of the water phase were heated to 75°C and added gradually to the fat phase (caprylic/capric triglycerides (3.00-5.00g), polysorbate 80 (1.00-2.50g), shea butter (1.00-3.50g), tocopherol acetate (0.10-0.50g) and butylhydroxytoluene (0.01-0.02g)) which was preheated to 70°C, with stirring by a propeller rotary mixer (500 rpm). At the emulsification temperature of 40°C, a 10% sodium hydroxide solution (1.50-3.00g) was added to the formulation, and after vigorous mixing, the active components were added: hemp oil (1.00-2.50g) and the inclusion complex α-cyclodextrin: immortelle extract (with the content of incorporated immortelle extract l.00-2.00g). Mixing was continued until the emulgel cooled down and reached room temperature.

Example 8.2. Method of obtaining emulgel with a combination of hemp oil and the inclusion complex β-cyclodextrin: immortelle extract

Sodium benzoate (0.15-3.50g) was dissolved in a mixture of propylene glycol (2.50-5.00g) and water in a beaker. Carbopol Ultrez 10 (0.45-l.00g) was gradually added over the surface. The ingredients of the water phase were heated to 75°C and added gradually to the fat phase (caprylic/capric triglycerides (3.00-5.00g), polysorbate 80 (1.00-2.50g), shea butter (1.00-3.50g), tocopherol acetate (0.10-0.50g) and butylhydroxytoluene (0.01-0.02g)) which was preheated to 70°C, with stirring by a propeller rotary mixer (500 rpm). At an emulsification temperature of 40°C, a 10% sodium hydroxide solution (1.50-3.00g) was added to the formulation, and after vigorous mixing, the active components hemp oil (1.00-2.50g) and the inclusion complex β-cyclodextrin: immortelle extract, with the content of immortelle extract 1.00-2.00g, were added. Mixing was continued until the emulgel cooled down and reached room temperature.

Example 8.3. Method of obtaining emulgel with a combination of hemp oil and inclusion complex γ-cyclodextrin: immortelle extract

Sodium benzoate (0.15-3.50g) was dissolved in a mixture of propylene glycol (2.50-5.00g) and water in a beaker. Carbopol Ultrez 10 (0.45-l.00g) was gradually added over the surface. The ingredients of the water phase were heated to 75°C and added gradually to the fat phase (caprylic/capric triglycerides (3.00-5.00g), polysorbate 80 (1.00-2.50g), shea butter (1.00-3.50g), tocopherol acetate (0.10-0.50g) and butylhydroxytoluene (0.01-0.02g)) which was preheated to 70°C, with stirring by a propeller rotary mixer (500 rpm). At an emulsification temperature of 40°C, a 10% sodium hydroxide solution (1.50-3.00g) was added to the formulation, and after vigorous mixing, the active components were added: hemp oil (1.00-2.50g) and inclusion complex γ-cyclodextrin: immortelle extract, with immortelle extract content 1.00-2.00%. Mixing was continued until the emulgel cooled down and reached room temperature.

Example 8.4. Method of obtaining emulgel with a combination of hemp oil and inclusion complex (2-hydroxypropyl)-β-cyclodextrin: immortelle extract

Sodium benzoate (0.15-3.50g) was dissolved in a mixture of propylene glycol (2.50-5.00g) and water in a beaker. Carbopol Ultrez 10 (0.45-l.00g) was gradually added over the surface. The ingredients of the water phase were heated to 75°C and added gradually to the fat phase (caprylic/capric triglycerides (3.00-5.00g), polysorbate 80 (1.00-2.50g), shea butter (1.00-3.50g), tocopherol acetate (0.10-0.50g) and butylhydroxytoluene (0.01-0.02g)) which was preheated to 70°C, with stirring by a propeller rotary mixer (500 rpm). At the emulgel temperature of 40°C, a 10% sodium hydroxide solution (1.50-3.00g) was added to the formulation, and after vigorous mixing, the active components were added: hemp oil (1.00-2.50g) and the inclusion complex (2-hydroxypropyl)-β-cyclodextrin: immortelle extract, with immortelle extract content of 1.00-2.00%. Mixing was continued until the emulgel cooled down and reached room temperature.

Example 8.5. The process of obtaining emulgels with a combination of hemp oil and cyclodextrin inclusion complexes with immortelle extract

Sodium benzoate (0.15-3.50g) was dissolved in a mixture of propylene glycol (2.50-5.00g) and water in a beaker. Carbopol Ultrez 10 (0.45-l.00g) was gradually added over the surface. The ingredients of the water phase were heated to 75°C and added gradually to the fat phase (caprylic/capric triglycerides (3.00-5.00g), polysorbate 80 (1.00-2.50g), shea butter (1.00-3.50g), tocopherol acetate (0.10-0.50g) and butylhydroxytoluene (0.01-0.02g)) which was preheated to 70°C, with stirring by a propeller rotary mixer (500 rpm). At the emulgel temperature of 40°C, a 10% sodium hydroxide solution (1.50-3.00g) was added to the formulation, and after vigorous mixing, the active components were added: hemp oil (1.00-2.50g) and the inclusion complex α-cyclodextrin: immortelle extract and/or β-cyclodextrin: immortelle extract and/or γ-cyclodextrin: immortelle extract, and/or (2-hydroxypropyl)-β-cyclodextrin: immortelle extract and/or methyl-β-cyclodextrin: immortelle extract (with a total content of incorporated immortelle extract 1.00-2.00g). Mixing is continued until the emulgel cools down and reaches room temperature.

Example 9. Procedure for obtaining creams with a combination of extracts of immortelle and hemp

Potassium sorbate (0.10-1.00g) is first dissolved in purified water (up to 100g) and the solution is heated to a temperature of 70-72°C. The components of the fatty phase (cetostearol (1.00-4.00g), caprylic and caproic acid triglycerides (3.00-8.00g), white petrolatum (1.00-5.00g), liquid paraffin (0.10-4.00g), C12-14 alkyl polyglucoside/C12-14 alcohol (4.00-10.00g), cocoa glucoside/alcohol are weighed separately. of coconut (l.00-5.00g)), are heated to the same temperature (until all components dissolve, i.e. until the mass becomes completely clear). The fatty phase is then added in a thin stream to the aqueous phase with stirring (800 rpm) for 10 min, then the speed is reduced to 500 rpm and stirring continues for another 15 min. The mixing speed is then reduced to 400 rpm, until the mass cools down. At 35°C, extracts (1.00-2.00g) and hemp (1.00-2.50g) are added to the finished cream. The mass is mixed at a mixer speed of 500 rpm until the cream cools down to room temperature.

Example 9.1. The procedure for obtaining creams with a combination of hemp extract and cyclodextrin inclusion complexes with immortelle extract

Potassium sorbate (0.10-1.00g) is previously dissolved in purified water (up to 100g) and the solution is heated to a temperature of 70-72°C. The components of the fatty phase (cetostearol (1.00-4.00g), caprylic and caproic acid triglycerides (3.00-8.00g), white petrolatum (1.00-5.00g), liquid paraffin (0.10-4.00g), C12-14 alkyl polyglucoside/C12-14 alcohol (4.00-10.00g), cocoa glucoside/alcohol are weighed separately. of coconut (l.00-5.00g), are heated to the same temperature (until the mass becomes completely clear). Then, the fat phase is added in a thin stream while stirring (800 rpm), and then the speed is reduced to 500 rpm and the mixing speed is then reduced to 400 rpm 35°C, the extract is added to the finished cream hemp (l.00-2.50g) and inclusion complex α-cyclodextrin: immortelle extract and/or β-cyclodextrin: immortelle extract and/or γ-cyclodextrin: immortelle extract, and/or (2-hydroxypropyl)-β-cyclodextrin: immortelle extract and/or methyl-β-cyclodextrin: immortelle extract (with a total content of immortelle extract l.00-2.00g). The mass is mixed at a mixer speed of 500 rpm until the cream cools to room temperature.

Example 10. Procedure for obtaining ointment with a combination of extracts of immortelle and hemp

To make 100 g of grease, first dissolve potassium sorbate (0.10-0.50 g) in purified water (25.00-45.0 g), add propylene glycol (2.00-5.50 g) and heat the solution to a temperature of 70-72°C.

The components of the fat phase are measured separately (cetostearol 10.00-20.00g, caprylic and caproic acid triglycerides 10.00-27.00g, white vaseline 15.00-25.00g, liquid paraffin 7.00-15.50g, polysorbate 60 2.00-6.00g, sorbitan stearate 2.00-6.00g, isopropyl myristate 5.00-15.50g) are heated to the same temperature (until all components dissolve, ie until the mass becomes completely clear). The fat phase is then added in a thin stream to the aqueous phase with stirring (800 rpm) for 10 minutes, then the speed is reduced to 500 rpm and stirring is continued for another 15 minutes. The mixing speed is then reduced to 400 rpm, until the mass cools down. At 35°C, extracts of immortelle (1.00-2.00g) and hemp (1.00-2.50g) are added to the mass. The mass is stirred at a speed of 500 rpm until it cools down to room temperature.

Example 10.1. The procedure for obtaining fat with a combination of hemp extract and cyclodextrin inclusion complexes with immortelle extract

To make 100 g of grease, first dissolve potassium sorbate (0.10-0.50 g) in purified water (25.00-45.0 g), add propylene glycol (2.00-5.50 g) and heat the solution to a temperature of 70-72°C. The components of the fat phase are measured separately (cetostearol 10.00-20.00g, caprylic and caproic acid triglycerides 10.00-27.00g, white vaseline 15.00-25.00g, liquid paraffin 7.00-15.50g, polysorbate 60 2.00-6.00g, sorbitan stearate 2.00-6.00g, isopropyl myristate 5.00-15.50g) are heated to the same temperature (until all components dissolve, ie until the mass becomes completely clear). The fat phase is then added in a thin stream to the aqueous phase with stirring (800 rpm) for 10 minutes, then the speed is reduced to 500 rpm and stirring is continued for another 15 minutes. The mixing speed is then reduced to 400 rpm, until the mass cools down. At 35°C, hemp extract (1.00-2.50g) and the inclusion complex α-cyclodextrin: immortelle extract and/or (β-cyclodextrin: immortelle extract and/or γ-cyclodextrin: immortelle extract, and/or methyl-β-cyclodextrin: immortelle extract and/or (2-hydroxypropyl)-β-cyclodextrin: immortelle extract (with the total content of incorporated immortelle extract) are added to the mass. l.00-2.00g) The mass is mixed at a speed of 500 rpm until it cools down to room temperature.

Example 11. Procedure for obtaining a paste with a combination of immortelle extract and hemp extract

To make 100 g of paste, potassium sorbate (0.10-2.50g) is first dissolved in purified water (30.00-55.00g), propylene glycol (5.50-8.00g) is added and the solution is heated to a temperature of 70-72°C.

The components of the fat phase are measured separately (cetostearol 2.50-10.00g, triglycerides of caprylic and caproic acids 5.00-12.00g, white vaseline 5.50-10.00g, liquid paraffin 5.00-8.50g, polysorbate 60 2.00-6.00g, sorbitan monostearate 2.00-6.00g, isopropyl myristate 5.00-15.00g) are heated to 70-72°C (until all components dissolve, ie until the mass becomes completely clear). The fat phase is then added in a thin stream to the aqueous phase with stirring (800 rpm) for 10 minutes, then the speed is reduced to 500 rpm and stirring is continued for another 15 minutes. Then gradually add ZnO from 15.00-27.00g and talc from 3.00-7.00g. The mixing speed is reduced to 400 rpm, until the mass cools down. At 35°C, extracts are added to the mass: immortelle extract (1.00-2.00g) and hemp (1.00-2.50g).

Example 11.1. The procedure for obtaining a paste with a combination of hemp extract and cyclodextrin inclusion complexes with immortelle extract

To make 100 g of paste, potassium sorbate (0.10-2.50g) is first dissolved in purified water (30.00-55.00g), propylene glycol (5.50-7.00g) is added and the solution is heated to a temperature of 70-72°C. The components of the fat phase are measured separately (cetostearol 2.50-10.00g, triglycerides of caprylic and caproic acids 5.00-12.00g, white vaseline 5.50-10.00g, liquid paraffin 5.00-8.50g, polysorbate 60 2.00-6.00g, sorbitan monostearate 2.00-6.00g, isopropyl myristate 5.00-15.00g) are heated to the same temperature (until all components dissolve, ie until the mass becomes completely clear). The fat phase is then added in a thin stream to the aqueous phase with stirring (800 rpm) for 10 minutes, then the speed is reduced to 500 rpm and stirring is continued for another 15 minutes. Then gradually add ZnO in the amount of 15.00-27.00g and talc in the amount of 3.00-7.00g. The mixing speed is then reduced to 400 rpm, until the mass cools down. At 35°C, hemp extract (1.00-2.50g) and inclusion complex α-cyclodextrin: immortelle extract and/or β-cyclodextrin: immortelle extract and/or γcyclodextrin: immortelle extract, and/or (2-hydroxypropyl)-β-cyclodextrin: immortelle extract and/or methyl-β-cyclodextrin: immortelle extract (with the total content of incorporated immortelle extract) are added to the mass. l.00-2.00g).

Example 12. Procedure for obtaining a gel-cream with a combination of extracts of immortelle and hemp

To make 100 g of gel-cream in purified water (25.0-75.0g), potassium sorbate (0.10-2.50g) is previously dissolved and glycerol (2.50-9.00g), propylene glycol (1.00-8.00g), ethanol (1.00-5.50g) and polysorbate 20 (0.50-6.00g) are added. After dissolving all the components, the gelling agent (carbomer) is spread over the surface in the amount of 0.15-2.00g. The solution is heated to a temperature of 70-72°C. The components of the fatty phase are measured separately (cetostearol 2.50-4.00g, triglycerides of caprylic and caproic acids 2.00-8.00g, white vaseline 0.50-3.00g, liquid paraffin 0.10-1.50g, C12-14 alkyl polyglucoside/C12-14 alcohol 2.00-7.00g, coco glucoside/alcohol coconut 0.50-3.00g), heat to 70-72°C (until all components dissolve, i.e. until the mass becomes completely clear). The fatty phase is then added in a thin stream to the aqueous phase with stirring (800 rpm) for 10 minutes, then the speed is reduced to 500 rpm and stirring is continued for another 15 minutes. Then a gelling agent (10% sodium hydroxide solution 0.10-0.50g) is added. The mixing speed is reduced to 400 rpm until the mass cools down. At 35°C, extracts of immortelle (1.00-2.00g) and hemp (1.00-2.50g) are added to the finished cream. The mass is mixed at a speed of 500 rpm until the gel-cream cools down to room temperature.

Example 12.1. The procedure for obtaining a gel-cream with a combination of hemp extract and cyclodextrin inclusion complexes with immortelle extract

To make 100g of gel-cream, potassium sorbate (0.10-2.50g) is previously dissolved in purified water (25.0-75.0g) and glycerol (2.50-9.00g), propylene glycol (1.00-8.00g), ethanol (1.00-5.50g) and polysorbate 20 (0.50-6.00g) are added. After dissolving all the components, the gelling agent (carbomer) is spread over the surface in the amount of 0.15-2.00g. The solution is heated to a temperature of 70-72°C. The components of the fat phase are measured separately (cetostearol 1.00-4.00g, triglycerides of caprylic and caproic acids 2.00-8.00g, white vaseline 0.50-3.00g, liquid paraffin 0.10-1.50g, C12-14 alkyl polyglucoside/C12-14 alcohol 2.00-7.00g, coco glucoside/coconut alcohol walnuts 0.50-3.00g), are heated to the same temperature (until all components dissolve, ie until the mass becomes completely clear). The fatty phase is added in a thin stream to the aqueous phase with stirring (800 rpm) for 10 minutes, then the speed is reduced to 500 rpm and stirring is continued for another 15 minutes. Then a gelling agent (10% sodium hydroxide solution 0.10-0.50g) is added. The mixing speed is then reduced to 400 rpm, until the mass cools down. At 35°C hemp extract (1.00-2.50g) and inclusion complex are added to the finished cream: α-cyclodextrin: immortelle extract and/or β-cyclodextrin: immortelle extract and/or γ-cyclodextrin: immortelle extract, and/or (2-hydroxypropyl)-β-cyclodextrin: immortelle extract and/or methyl-β-cyclodextrin: immortelle extract (with incorporated immortelle extract) 1.00-2.00g). The mass is mixed at a speed of 500 rpm until the gel-cream cools down to room temperature.

Example 13: In vivo test protocol

Testing the safety and efficacy of the formulated emulgel with extracts of the immortelle Helihrysum italicum (Roth) G. and industrial hemp (Cannabis sativa L., Cannabaceae) according to the invention lasted 30 days. Healthy volunteers participated in the study, who according to the instructions applied active emulgel and placebo emulgel every day in the morning and in the evening for 28 days on precisely determined places on the volar side of the forearm. Biophysical parameters of the skin (electrical capacitance, transepidermal water loss (TEGV), melanin index (MI), erythema index (EI) and pH value) were measured after 7, 14 and 28 days of application, i.e. 2 days after stopping the application of emulgels, in order to monitor the duration of the effect after stopping the application. All measurements were taken in the morning, before the morning application. Changes in biophysical parameters of the skin were measured at the site of application of active emulgel, placebo emulgel as well as at the untreated control site. The measured values were compared with the basal values (measured the day before the start of emulgel application), their changes over time were monitored, and the changes of parameters at the site of application of the active emulgel were compared with the placebo and untreated control site. Multi Probe Adapter MPA®9 and corresponding probes Mexameter® MH 18, Tewameter and Comeometer® CM 825 (produced by Courage & Khazaka Electronic GmbH, Germany) were used. All measured data are presented as mean value ± standard deviation, and then statistically processed using the IBM SPSS Statistics 20 program.

Example 13.1: Effect of emulgel application on the electrical capacitance of the skin

The electrical capacitance test provides data on the state of hydration of the surface layers of the skin. During the test, there was a gradual increase in the hydration of the skin, i.e. increasing values of the electrical capacitance of the skin of the subjects treated with active emulgel or placebo were measured (Figure 4). The increase was statistically significant in both cases (p<0.01).

Compared to the basal values, in the places treated with active emulgel there was an increase in electrical capacitance by 10.43 (37.87%) after 14 days and by 16.73 (58.24%) after 28 days, when the average measured value of electrical capacitance was 44.83±9.26. After stopping applying the active cream, a slight drop in skin hydration was observed in the subjects and the average measured electrical capacitance 2 days after the last application was 40.02±7.73, which is certainly still higher than the measured basal values by 11.69 (41.26%).

An increase in electrical capacitance was also measured on the skin of subjects treated with placebo emulgel, and after the 14th day it was 6.50 (20.86%) and after the 28th day it was 9.27 (29.75%). As in the case of the area lubricated with active emulgel, in this area too, after the lubrication was stopped, the hydration decreased. After 28 days of emulgel application, the hydration in the active emulgel applied site was higher than in the placebo-treated site.

At the untreated control site, there was no statistically significant change in electrical capacitance during the entire study period (p>0.05).

In relation to the other places, the lowest values of electrical capacitance were measured, which indicates that the application of active emulgel and placebo led to an increase in skin hydration in the subjects. The increase in the moisture content of placebo-treated skin is probably due to the emollients present in the emulgel. The greatest increase in moisture was observed on the skin coated with active emulgel, and it can be assumed that this may be due to the influence of active substances.

Example 13.2: Effect of emulgel application on transepidermal water loss - in vivo study

A constant decrease in transepidermal water loss (TEGV), which is statistically significant (p<0.01), indicates that smearing with active emulgel or placebo in the subjects led to an improvement in the barrier function of the skin, which was also confirmed by the ANOVA test. There was no statistically significant change in TEGV at the untreated control site (p>0.05). The decrease in TEGV in the place treated with active emulgel after 14 days was 2.03±4.49 (16.26%) and after 28 days 3.64±4.04 (29.13%). After stopping emulgel application, there was a slight increase in TEGV. A decrease in TEGV also occurred on the skin of subjects treated with a placebo, but this decrease during the entire study was less than on the skin treated with active emulgel. TEGV values were highest at the untreated control site at all times.

Example 13.3: Effect of emulgel application on the melanin index

The obtained results of testing the effect of emulgel application on melanin index during 30 days are shown in table 8. On the skin treated with active emulgel or placebo, there was a statistically significant decrease in melanin index, which indicates the potential of these emulgels in whitening hyperpigmentation. In contrast to the initial value of 129.04±29.62, after 28 days of applying the active cream, the average measured value of the melanin index in the place treated with the active emulgel was 116.60±25.05, while 2 days after the cessation of applying the active cream, the MI value increased to 119.04±27.95.

The ANOVA test showed that compared to the untreated control, there was no statistically significant reduction in MI, indicating that despite active emulgel and placebo alone can whiten hyperpigmentation, normal skin lubricated with active emulgel or placebo was not significantly whitened compared to untreated skin.

Example 13.4; The effect of emulgel application on the erythema index - an in vivo study

The erythema index (EI) did not change statistically significantly during the study, which is probably due to the discolouration of the active emulgel.

Example 13.5: Effect of emulgel application on pH value - in vivo study

In relation to the average measured initial values, there was an increase in the pH value in the places treated with placebo and active emulgel (p<0.05). However, all measured pH values are within the recommended limits. Although both active emulgel and placebo led to an increase in pH value, this increase was not statistically significant compared to the change in pH value at the untreated control site, which means that long-term use of active emulgel or placebo does not lead to changes in pH value beyond normal limits.

Claims (9)

Patent claims 1. Inclusion coreplex of carriers based on α-cyclodextrin impregnated with immortelle extract Helichrysum itcilicum (Roth) 0, Don fil., Asteraceae, originating from the territory of Croatia, with arzanol content in the inclusion complex min. 0.0075% obtained by applying an integrated process of supercritical extraction and supercritical impregnation with supercritical carbon(IV)-oxide. 2. Inclusion complex according to patent claim 1, characterized in that the yield of supercritical impregnation of Helichrysum italicum immortelle extract on a carrier based on α-cyclodextrin is min. 4.29%. 3. The method of obtaining the inclusion complex according to patent claims 1 and 2, characterized by the fact that the integrated process of supercritical extraction from the plant material of the immortelle Helichrysum italicum and supercritical impregnation of the carrier based on α-cyclodextrin with the extract is carried out in a batch or semi-continuous mode with the circulation of supercritical carbon(IV)-oxide and the extract through at least one vessel for work under high pressure in which the extraction process and at least one vessel for work under high pressure in which the impregnation process takes place simultaneously at a pressure of 8 MPa to 50 MPa, primarily from 15 MPa to 30 MPa and a temperature from 31°C to 80°C, primarily from 40°C to 60°C, during a time of 2 to 10 h, primarily from 3 to 6 h. 4. The method according to patent claim 3, characterized in that the mass ratio of the immortelle plant material Helichrysum italicum and the carrier based on α-cyclodextrin is from 1:1 to 15:1. 5. The method according to patent claim 3, characterized in that the mass ratio of the immortelle Helichrysum italicum plant material and the carrier based on α-cyclodextrin is 10:1. 6. The composition, indicated by the fact that it contains an inclusion complex of a carrier based on α-cyclodextrin impregnated with the extract of immortelle Helichrysum italicum according to patent claims 1-2 and commercial fatty oil of industrial hemp Cannabis sativa L., without tetrahydrocannabinol and with a defined content of polyunsaturated fatty acids of 80%, in which the optimal ratio of linoleic and linolenic acids is 3:1. 7. A product in the form of an emulgel, indicated by the fact that it contains 1.00-2.50% hemp oil of Cannabis sativa L. without tetrahydrocannabinol with 80% polyunsaturated fatty acids and a ratio of linoleic and linolenic acid 3:1 and 1.00-2.00% of immortelle extract Helihrysum italicum, incorporated in a carrier based on α-cyclodextrin according to patent claims 1-2, and usual pharmaceutical additives for forming pharmaceutical-technological form, a mixture with 0.15-3.50% sodium benzoate in purified water with 5.0 g glycerol, 2.50-5.00% propylene glycol and 0.45-1.00% Carbopol Ultreza 10, added to the fat phase with 3.00-5.00% triglycerides of caprylic and caproic acids, 1.00-2.50% polysorbate 80, 1.00-3.50% shea butter, 0.10-0.50% tocopherol acetate and 0.01-0.02% butylhydroxytoluene. 8. Use of the composition according to patent claim 6 for increasing hydration, whitening hyperpigmentation, reducing visible signs and slowing down the aging process of the skin. 9. Use of emulgel according to patent claim 7 to increase hydration, whiten hyperpigmentation, reduce visible signs and slow down the skin aging process.